Pharmaceutical Manufacturing Handbook Production and Processes

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PHARMACEUTICAL MANUFACTURING HANDBOOK Production and Processes

SHAYNE COX GAD, PH.D., D.A.B.T. Gad Consulting Services Cary, North Carolina

A JOHN WILEY & SONS, INC., PUBLICATION



SHAYNE COX GAD, PH.D., D.A.B.T. Gad Consulting Services Cary, North Carolina

A JOHN WILEY & SONS, INC., PUBLICATION

Copyright © 2008 by John Wiley & Sons, Inc. All rights reserved Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate percopy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permission. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com. Library of Congress Cataloging-in-Publication Data is available. ISBN: 978-0-470-25958-0

Printed in the United States of America 10 9 8 7 6 5 4 3 2 1

CONTRIBUTORS

Susanna Abrahmsén-Alami, AstraZeneca R&D Lund, Lund, Sweden, Oral Extended-Release Formulations James Agalloco, Agalloco & Associates, Belle Mead, New Jersey, Sterile Product Manufacturing Fakhrul Ahsan, Texas Tech University, Amarillo, Texas, Nasal Delivery of Peptide and Nonpeptide Drugs James Akers, Akers Kennedy & Associates, Kansas City, Missouri, Sterile Product Manufacturing Raid G. Alany, The University of Auckland, Auckland, New Zealand, Ocular Drug Delivery; Microemulsions as Drug Delivery Systems Monique Alric, Université d’Auvergne, Clermont-Ferrand, France, Recombinant Saccharomyces Cerevisiae as New Drug Delivery System to Gut: In Vitro Validation and Oral Formulation Sacide Alsoy Altinkaya, Izmir Institute of Technology, Urla-Izmir, Turkey, Controlled Release of Drugs from Tablet Coatings Maria Helena Amaral, University of Porto, Porto, Portugal, Vaginal Drug Delivery Anil Kumar Anal, Living Cell Technologies (Global) Limited, Auckland, New Zealand, Controlled-Release Dosage Forms Gavin Andrews, Queen’s University Belfast, Belfast, Northern Ireland, Effects of Grinding in Pharmaceutical Tablet Production Sophia G. Antimisiaris, School of Pharmacy, University of Patras, Rio, Greece, Liposomes and Drug Delivery

vi

CONTRIBUTORS

Robert D. Arnold, The University of Georgia, Athens, Georgia, BiotechnologyDerived Drug Product Development C. Scott Asbill, Samford University, Birmingham, Alabama, Transdermal Drug Delivery Maria Fernanda Bahia, University of Porto, Porto, Portugal, Vaginal Drug Delivery Bernard Bataille, University of Montpelier 1, Montpellier, France, Tablet Design Gerald W. Becker, SSCI, West Lafayette, Indiana, Biotechnology-Derived Drug Product Development; Regulatory Considerations in Approval of Follow-On Protein Drug Products B. Wayne Bequette, Rensselaer Polytechnic Institute, Troy, New York, From Pilot Plant to Manufacturing: Effect of Scale-Up on Operation of Jacketed Reactors Erem Bilensoy, Hacettepe University Faculty of Pharmacy, Ankara, Turkey, Cyclodextrin-Based Nanomaterials in Pharmaceutical Field Stéphanie Blanquet, Université d’Auvergne, Clermont-Ferrand, France, Recombinant Saccharomyces Cerevisiae as New Drug Delivery System to Gut: In Vitro Validation and Oral Formulation Gary W. Bumgarner, Samford University, Birmingham, Alabama, Transdermal Drug Delivery Isidoro Caraballo, University of Sevilla, Seville, Spain, Tablet Design Stephen M. Carl, Purdue University, West Lafayette, Indiana, BiotechnologyDerived Drug Product Development; Regulatory Considerations in Approval of Follow-On Protein Drug Products Sudhir S. Chakravarthi, University of Nebraska Medical Center, College of Pharmacy, Omaha, Nebraska, Biodegradable Nanoparticles D.F. Chowdhury, University of Oxford, Oxford, United Kingdom, Pharmaceutical Nanosystems: Manufacture, Characterization, and Safety Barbara R. Conway, Aston University, Birmingham, United Kingdom, Solid Dosage Forms José das Neves, University of Porto, Porto, Portugal, Vaginal Drug Delivery Osama Abu Diak, Queen’s University Belfast, Belfast, Northern Ireland, Effects of Grinding in Pharmaceutical Tablet Production Brit S. Farstad, Instititue for Energy Technology, Isotope Laboratories, Kjeller, Norway, Radiopharmaceutical Manufacturing Dimitrios G. Fatouros, School of Pharmacy and Biomedical Sciences, Portsmouth, England, Liposomes and Drug Delivery Jelena Filipoviç-Grčič, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia, Nasal Powder Drug Delivery

CONTRIBUTORS

vii

Eddy Castellanos Gil, Center of Pharmaceutical Chemistry and University of Havana, Havana, Cuba; University of Sevilla, Seville, Spain; University of Montpelier 1, Montpellier, France, Tablet Design Anita Hafner, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia, Nasal Powder Drug Delivery A. Atilla Hincal, Hacettepe University Faculty of Pharmacy, Ankara, Turkey, Cyclodextrin-Based Nanomaterials in Pharmaceutical Field Michael Hindle, Virginia Commonwealth University, Richmond, Virginia, Aerosol Drug Delviery Bhaskara R. Jasti, University of the Pacific, Stockton, California, Semisolid Dosages: Ointments, Creams, and Gels Yiguang Jin, Beijing Institute of Radiation Medicine, Beijing, China, Nanotechnology in Pharmaceutical Manufacturing David Jones, Queen’s University Belfast, Belfast, Northern Ireland, Effects of Grinding in Pharmaceutical Tablet Production Anne Juppo, University of Helsinki, Helsinki, Finland, Oral Extended-Release Formulations Paraskevi Kallinteri, Medway School of Pharmacy, Universities of Greenwich and Kent, England, Liposomes and Drug Delivery Gregory T. Knipp, Purdue University, West Lafayette, Indiana, BiotechnologyDerived Drug Product Development; Regulatory Considerations in Approval of Follow-On Protein Drug Products Anette Larsson, Chalmers University of Technology, Göteborg, Sweden, Oral Extended-Release Formulations Beom-Jin Lee, Kangwon National University, Chuncheon, Korea, Pharmaceutical Preformulation: Physiochemical Properties of Excipients and Powders and Tablet Characterization Xiaoling Li, University of the Pacific, Stockton, California, Semisolid Dosages: Ointments, Creams, and Gels David J. Lindley, Purdue University, West Lafayette, Indiana, BiotechnologyDerived Drug Product Development Roberto Londono, Washington State University, Pullman, Washington, Liquid Dosage Forms Ravichandran Mahalingam, University of the Pacific, Stockton, California, Semisolid Dosages: Ointments, Creams, and Gels Kenneth R. Morris, Purdue University, West Lafayette, Indiana, BiotechnologyDerived Drug Product Development; Regulatory Considerations in Approval of Follow-On Protein Drug Products Erin Oliver, Rutgers, The State University of New Jersey, Piscataway, New Jersey, Biotechnology-Derived Drug Product Development; Regulatory Considerations in Approval of Follow-On Protein Drug Products

viii

CONTRIBUTORS

Iván Peñuelas, University of Navarra, Pamplona, Spain, Radiopharmaceutical Manufacturing Omanthanu P. Perumal, South Dakota State University, Brookings, South Dakota, Role of Preformulation in Development of Solid Dosage Forms Katharina M. Picker-Freyer, Martin-Luther-University Halle-Wittenberg, Institute of Pharmaceutics and Biopharmaceutics, Halle/Saale, Germany, Tablet Production Systems Satheesh K. Podaralla, South Dakota State University, Brookings, South Dakota, Role of Preformulation in Development of Solid Dosage Forms Dennis H. Robinson, University of Nebraska Medical Center, College of Pharmacy, Omaha, Nebraska, Biodegradable Nanoparticles Arcesio Rubio, Caracas, Venezuela, Liquid Dosage Forms Maria V. Rubio-Bonilla, Research Associate, College of Pharmacy, Washington State University, Pullman, Washington, Liquid Dosage Forms Ilva D. Rupenthal, The University of Auckland, Auckland, New Zealand, Ocular Drug Delivery Maria Inês Rocha Miritello Santoro, Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil, Packaging and Labeling Helton M.M. Santos, University of Coimbra, Coimbra, Portugal, Tablet Compression Raymond K. Schneider, Clemson University, Clemson, South Carolina, CleanFacility Design, Construction, and Maintenance Issues Anil Kumar Singh, Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil, Packaging and Labeling João J.M.S. Sousa, University Compression

of

Coimbra,

Coimbra,

Portugal,

Tablet

Shunmugaperumal Tamilvanan, University of Antwerp, Antwerp, Belgium, Progress in Design of Biodegradable Polymer-Based Microspheres for Parenteral Controlled Delivery of Therapeutic Peptide/Protein; Oil-in-Water Nanosized Emulsions: Medical Applications Chandan Thomas, Texas Tech University, Amarillo, Texas, Nasal Delivery of Peptide and Nonpeptide Drugs Gavin Walker, Queen’s University Belfast, Belfast, Northern Ireland, Effects of Grinding in Pharmaceutical Tablet Production Jingyuan Wen, The University of Auckland, Auckland, New Zealand, Microemulsions as Drug Delivery Systems Hui Zhai, Queen’s University Belfast, Belfast, Northern Ireland, Effects of Grinding in Pharmaceutical Tablet Production

CONTENTS

PREFACE SECTION 1 1.1

xiii MANUFACTURING SPECIALTIES

Biotechnology-Derived Drug Product Development

1 3

Stephen M. Carl, David J. Lindley, Gregory T. Knipp, Kenneth R. Morris, Erin Oliver, Gerald W. Becker, and Robert D. Arnold

1.2

Regulatory Considerations in Approval on Follow-On Protein Drug Products

33

Erin Oliver, Stephen M. Carl, Kenneth R. Morris, Gerald W. Becker, and Gregory T. Knipp

1.3

Radiopharmaceutical Manufacturing

59

Brit S. Farstad and Iván Peñuelas

SECTION 2 ASEPTIC PROCESSING

97

2.1

99

Sterile Product Manufacturing James Agalloco and James Akers

SECTION 3 3.1

FACILITY

From Pilot Plant to Manufacturing: Effect of Scale-Up on Operation of Jacketed Reactors

137

139

B. Wayne Bequette ix

x

3.2

CONTENTS

Packaging and Labeling

159

Maria Inês Rocha Miritello Santoro and Anil Kumar Singh

3.3

Clean-Facility Design, Construction, and Maintenance Issues

201

Raymond K. Schneider

SECTION 4 4.1

NORMAL DOSAGE FORMS

Solid Dosage Forms

233 235

Barbara R. Conway

4.2

Semisolid Dosages: Ointments, Creams, and Gels

267

Ravichandran Mahalingam, Xiaoling Li, and Bhaskara R. Jasti

4.3

Liquid Dosage Forms

313

Maria V. Rubio-Bonilla, Roberto Londono, and Arcesio Rubio

SECTION 5 5.1

NEW DOSAGE FORMS

345

Controlled-Release Dosage Forms

347

Anil Kumar Anal

5.2

Progress in the Design of Biodegradable Polymer-Based Microspheres for Parenteral Controlled Delivery of Therapeutic Peptide/Protein

393

Shunmugaperumal Tamilvanan

5.3

Liposomes and Drug Delivery

443

Sophia G. Antimisiaris, Paraskevi Kallinteri, and Dimitrios G. Fatouros

5.4

Biodegradable Nanoparticles

535

Sudhir S. Chakravarthi and Dennis H. Robinson

5.5

Recombinant Saccharomyces cerevisiae as New Drug Delivery System to Gut: In Vitro Validation and Oral Formulation

565

Stéphanie Blanquet and Monique Alric

5.6

Nasal Delivery of Peptide and Nonpeptide Drugs

591

Chandan Thomas and Fakhrul Ahsan

5.7

Nasal Powder Drug Delivery

651

Jelena Filipović-Grčić and Anita Hafner

5.8

Aerosol Drug Delivery

683

Michael Hindle

5.9

Ocular Drug Delivery

729

Ilva D. Rupenthal and Raid G. Alany

5.10

Microemulsions as Drug Delivery Systems Raid G. Alany and Jingyuan Wen

769

CONTENTS

5.11

Transdermal Drug Delivery

xi

793

C. Scott Asbill and Gary W. Bumgarner

5.12

Vaginal Drug Delivery

809

José das Neves, Maria Helena Amaral, and Maria Fernanda Bahia

SECTION 6 TABLET PRODUCTION 6.1

Pharmaceutical Preformulation: Physicochemical Properties of Excipients and Powers and Tablet Characterization

879

881

Beom-Jin Lee

6.2

Role of Preformulation in Development of Solid Dosage Forms

933

Omathanu P. Perumal and Satheesh K. Podaralla

6.3

Tablet Design

977

Eddy Castellanos Gil, Isidoro Caraballo, and Bernard Bataille

6.4

Tablet Production Systems

1053

Katharina M. Picker-Freyer

6.5

Controlled Release of Drugs from Tablet Coatings

1099

Sacide Alsoy Altinkaya

6.6

Tablet Compression

1133

Helton M. M. Santos and João J. M. S. Sousa

6.7

Effects of Grinding in Pharmaceutical Tablet Production

1165

Gavin Andrews, David Jones, Hui Zhai, Osama Abu Diak, and Gavin Walker

6.8

Oral Extended-Release Formulations

1191

Anette Larsson, Susanna Abrahmsén-Alami, and Anne Juppo

SECTION 7 7.1

ROLE OF NANOTECHNOLOGY

Cyclodextrin-Based Nanomaterials in Pharmaceutical Field

1223 1225

Erem Bilensoy and A. Attila Hincal

7.2

Nanotechnology in Pharmaceutical Manufacturing

1249

Yiguang Jin

7.3

Pharmaceutical Nanosystems: Manufacture, Characterization, and Safety

1289

D. F. Chowdhury

7.4

Oil-in-Water Nanosized Emulsions: Medical Applications

1327

Shunmugaperumal Tamilvanan

INDEX

1367

PREFACE

This Handbook of Manufacturing Techniques focuses on a new aspect of the drug development challenge: producing and administering the physical drug products that we hope are going to provide valuable new pharmacotherapeutic tools in medicine. These 34 chapters cover the full range of approaches to developing and producing new formulations and new approaches to drug delivery. Also addressed are approaches to the issues of producing and packaging these drug products (that is, formulations). The area where the most progress is possible in improving therapeutic success with new drugs is that of better delivery of active drug molecules to the therapeutic target tissue. In this volume, we explore current and new approaches to just this issue across the full range of routes (oral, parenteral, topical, anal, nasal, aerosol. ocular, vaginal, and transdermal) using all sorts of forms of formulation. The current metrics for success of new drugs in development once they enter the clinic (estimated at ranging from as low as 2% for oncology drugs to as high as 10% for oral drugs) can likely be leveraged in the desired direction most readily by improvements in this area of drug delivery. The Handbook of Manufacturing Techniques seeks to cover the entire range of available approaches to getting a pure drug (as opposed to a combination product) into the body and to its therapeutic tissue target. Thanks to the persistent efforts of Michael Leventhal, these 34 chapters, which are written by leading practitioners in each of these areas, provide coverage of the primary approaches to these fundamental problems that stand in the way of so many potentially successful pharmacotherapeutic interventions.

xiii

SECTION 1 MANUFACTURING SPECIALTIES

1.1 BIOTECHNOLOGY-DERIVED DRUG PRODUCT DEVELOPMENT Stephen M. Carl,1 David J. Lindley,1 Gregory T. Knipp,1 Kenneth R. Morris,1 Erin Oliver,2 Gerald W. Becker,3 and Robert D. Arnold4 1

Purdue University, West Lafayette, Indiana Rutgers, The State University of New Jersey, Piscataway, New Jersey 3 SSCI, West Lafayette, Indiana 4 The University of Georgia, Athens, Georgia 2

Contents 1.1.1 Introduction 1.1.2 Formulation Assessment 1.1.2.1 Route of Administration and Dosage 1.1.2.2 Pharmacokinetic Implications to Dosage Form Design 1.1.2.3 Controlled-Release Delivery Systems 1.1.3 Analytical Method Development 1.1.3.1 Traditional and Biophysical Analytical Methodologies 1.1.3.2 Stability-Indicating Methodologies 1.1.3.3 Method Validation and Transfer 1.1.4 Formulation Development 1.1.4.1 Processing Materials and Equipment 1.1.4.2 Container Closure Systems 1.1.4.3 Sterility Assurance 1.1.4.4 Excipient Selection 1.1.5 Drug Product Stability 1.1.5.1 Defining Drug Product Storage Conditions 1.1.5.2 Mechanisms of Protein and Peptide Degradation 1.1.5.3 Photostability 1.1.5.4 Mechanical Stress 1.1.5.5 Freeze–Thaw Considerations and Cryopreservation 1.1.5.6 Use Studies 1.1.5.7 Container Closure Integrity and Microbiological Assessment 1.1.5.8 Data Interpretation and Assessment Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad Copyright © 2008 John Wiley & Sons, Inc.

3

4 1.1.6

1.1.7

1.1.1

BIOTECHNOLOGY-DERIVED DRUG PRODUCT DEVELOPMENT

Quality by Design and Scale-Up 1.1.6.1 Unit Operations 1.1.6.2 Bioburden Considerations 1.1.6.3 Scale-Up and Process Changes Concluding Remarks References

INTRODUCTION

Although the origins of the first biological and/or protein therapeutics can be traced to insulin in 1922, the first biotechnology-derived pharmaceutical drug product approved in the United States was Humulin in 1982. In the early stages of pharmaceutical biotechnology, companies that specialized primarily in the development of biologicals were the greatest source of research and development in this area. Recent advances in molecular and cellular biological techniques and the potential clinical benefits of biotechnology drug products have led to a substantial increase in their development by biotechnology and traditional pharmaceutical companies. In terms of pharmaceuticals, the International Conference on Harmonization (ICH) loosely defines biotechnology-derived products with biological origin products as those that are “well-characterized proteins and polypeptides, their derivatives and products of which they are components, and which are isolated from tissues, body fluids, cell cultures, or produced using rDNA technology” [1]. In practical terms, biological and biotechnology-derived pharmaceutical agents encompass a number of therapeutic classes, including cytokines, erythropoietins, plasminogen activators, blood plasma factors, growth hormones and growth factors, insulins, monoclonal antibodies, and vaccines [1]. Additionally, short interfering and short hairpin ribonucleic acids (siRNA, shRNA) and antisense oligonucleotide therapies are generally characterized as biotechnologyderived products. According to the biotechnology advocacy group, The Biotechnology Industry Organization (BIO), pharmaceutical-based biotechnology represents over a $30 billion dollar a year industry and is directly responsible for the production of greater than 160 drug therapeutics and vaccines [2]. Furthermore, there are more than 370 biotechnology-derived drug products and vaccines currently in clinical trials around the world, targeting more than 200 diseases, including various cancers, Alzheimer’s disease, heart disease, diabetes, multiple sclerosis, acquired immunodeficiency syndrome (AIDS), and arthritis. While the clinical value of these products is well recognized, a far greater number of biotechnology-derived drug products with therapeutic potential for life-altering diseases have failed in development. As the appreciation of the clinical importance and commercial potential for biological products grows, new challenges are arising based on the many technological limitations related to the development and marketing of these complex agents. Additionally, the intellectual property protection of an associated agent might not

INTRODUCTION

5

provide a sufficient window to market and regain the costs associated with the discovery, research, development, and scale-up of these products. Therefore, to properly estimate the potential return on investment, a clear assessment of potential therapeutic advantages and disadvantages, such as the technological limitations in the rigorous characterization required of these complex therapeutic agents to gain Food and Drug Administration (FDA) approval, is needed prior to initiating research. Clearly, research focused on developing methodologies to minimize these technological limitations is needed. In doing so we hypothesize the attrition rate can be reduced and the number of companies engaged in the development of biotechnology-derived products and diversity of products will continue to expand. Technological limitations have limited the development of follow-on, or generic biopharmaceutical products that have lost patent protection. In fact, the potential pitfalls associated with developing these compounds are so diverse that regulatory guidance concerning follow-on biologics is relatively obscure and essentially notes that products will be assessed on a case-by-case basis. The reader is encouraged to see Chapter 1.2 for a more detailed discussion concerning regulatory perspectives pertaining to follow-on biologics. Many of the greatest challenges in producing biotechnology-derived pharmaceuticals are encountered in evaluating and validating the chemical and physical nature of the host expression system and the subsequent active pharmaceutical ingredient (API) as they are transferred from discovery through to the development and marketing stages. Although this area is currently a hotbed of research and is progressing steadily, limitations in analytical technologies are responsible for a high degree of attrition of these compounds. The problem is primarily associated with limited resolution of the analytical technologies utilized for product characterization. For example, without the ability to resolve small differences in secondary or tertiary structure, linking changes to product performance or clinical response is impossible. The biological activity of traditional small molecules is related directly to their structure and can be determined readily by nuclear magnetic resonance (NMR), X-Ray crystallography (X-ray), mass spectrometry (MS), and/or a combination of other spectroscopic techniques. However, methodologies utilized for characterizing biological agents are limited by resolution and reproducibility. For instance, circular dichroism (CD) is generally considered a good method to determine secondary structural elements and provides some information on the folding patterns (tertiary structure) of proteins. However, CD suffers from several limitations, including a lower resolution that is due in part to the sequence libraries used to deconvolute the spectra. To improve the reliability of determining the secondary and tertiary structural elements, these databases need to be developed further. An additional example is the utility of two-dimensional NMR (2D-NMR) for structural determination. While combining homonuclear and heteronuclear experimental techniques can prove useful in structural determination, there are challenges in that 2D-NMR for a protein could potentially generate thousands of signals. The ability to assign specific signals to each atom and their respective interactions is a daunting task. Resolution between the different amino acids in the primary sequence and their positioning in the covalent and folded structures become limited with increasing molecular weight. Higher dimensional techniques can be used to improve resolution; however, the resolution of these methods remains limited as the number of amino acids is increased.

6


Understanding the limitations of the analytical methodologies utilized for product characterization has led to the development of new experimental techniques as well as the refined application of well-established techniques to this emerging field. Only through application of a number of complementary techniques will development scientists be able to accurately characterize and develop clinically useful products. Unfortunately, much of the technology is still in its infancy and does not allow for a more in-depth understanding of the subtleties of peptide and protein processing and manufacturing. For instance, many of the analytical techniques utilized for characterization will evaluate changes to product conformation on the macroscopic level, such as potential denaturation or changes in folding, as observed with CD. However, these techniques do not afford the resolution to identify subtle changes in conformation that may either induce chemical or physical instabilities or unmask antigenic epitopes. Further limiting successful product development is a lack of basic understanding as to critical manufacturing processes that have the potential to affect the structural integrity and activity of biopharmaceuticals. As with traditional small molecules, stresses associated with the different unit operations may affect biopharmaceutical products differently. In contrast to traditional small molecules, there is considerable difficulty in identifying potentially adverse affects, if any, that a particular unit operation may have on the clinically critical structural elements of a drug. Considering that many proteins exhibit a greater potential for degradation from shear stress, it is particularly important to assess any negative effects of mixing, transport through tubing, filtration, and filling operations. Essentially all unit operations for a given manufacturing process could create enough shear stress to induce minor structural changes that could lead to product failure. The difficulty is establishing what degree of change will have an impact on the stability, bioactivity, or immunogenic potential of the compound. Unfortunately, unless exhaustive formulation development studies are conducted, coupled with a comprehensive spectrum of analytical methodologies, these effects may not be readily evident until after scale-up of the manufacturing process or, worse yet, in the clinical setting. Moreover, modeling shear and stress using fluid dynamic structurally diverse molecules is a foreboding task. Extending these models to validate process analytical technologies (PAT) and incorporate critical quality by design (QbD) elements in the development process for a collection of biopharmaceuticals would be largely hindered by the daunting nature of the task at hand. The use of biological systems to produce these agents results in additional variability. Slight changes in nutrient profile could affect growth patterns and protein expression of cultured cells. Furthermore, microbial contamination in the form of viruses, bacteria, fungi, and mycoplasma can be introduced during establishment of cell lines, cell culture/fermentation, capture and downstream processing steps, formulation and filling operations, or drug delivery [3]. Therefore, establishing the useful life span of purification media and separation columns remains a critical issue for consistently producing intermediates and final products that meet the defined quality and safety attributes of the product [4]. In short, understanding the proper processability and manufacturing controls needed has been a major hurdle that has kept broader development of biopharmaceutical products relatively limited. Notwithstanding the many technological hurdles to successfully develop a pharmaceutically active biotechnology product, they offer many advantages in terms of

FORMULATION ASSESSMENT

7

therapeutic potency, specificity, and target design (not generally limited to a particular class or series of compounds). This is an iterative approach, whereby every new approved compound, new lessons, and applications to ensure successful product development are realized, thereby adding to our knowledge base and facilitating the development of future products. This chapter will discuss some of the fundamental issues associated with successful biopharmaceutical drug product development and aims to provide an understanding of the subtleties associated with their characterization, processing, and manufacturing.

1.1.2


In order to select the most appropriate formulation and route of administration for a drug product, one must first assess the properties of the API, the proposed therapeutic indication, and the requirements/limitations of the drug and the target patient population. Development teams are interdisciplinary comprised of individuals with broad expertise, for example, chemistry, biochemistry, bioengineering, and pharmaceutics, that can provide insight into the challenges facing successful product development. As such, knowledge gained through refinement of the API manufacturing process through to lead optimization is vital to providing an initial starting point for success. Information acquired, for example, in the way of analytical development and API characterization, during drug discovery or early preclinical development that can be applied to final drug product development may contribute to shorter development times of successful products. The host system utilized for API production is critical to the production of the final product and will determine the basic and higher order physicochemical characteristics of the drug. Typically biopharmaceuticals are manufactured in Escherichia coli as prokaryotic and yeast and Chinese hamster ovary (CHO) cells as eukaryotic expression systems [5]. While general procedures for growth condition optimization and processing and purification paradigms have emerged, differences in posttranslational modifications and host–system related impurities can exist even with relatively minor processing changes within a single production cell line [5]. Such changes have the potential to alter the biopharmaceutical properties of the active compound, its bioactivity, and its potential to elicit adverse events such as immunogenic reactions. These properties will be a common theme as they could potentially play a major role in both analytical and formulation development activities. During the process of lead optimization, characterization work is performed that would include a number of parameters that are critical to formulation and analytical development scientists. The following information is a minimalist look at what information should be available to support product development scientists: • • • • • •

Color Particle size and morphology (for solid isolates) Thermoanalytical profile (e.g., Tg for lyophiles) Hygroscopicity Solubility with respect to pH Apparent solution pH

8

BIOTECHNOLOGY-DERIVED DRUG PRODUCT DEVELOPMENT • • • •

Number and pKa of ionizable groups Amino acid sequence Secondary and tertiary structural characteristics Some stability parameters with respect to pH Temperature Humidity Light Mechanical stress Oxygen sensitivity Impurity profile Misfolded/misaligned active Potential isoforms Expression system impurities Potency [median inhibitory concentration (IC50)] Animal Pharmacokinetic/Pharmacodynamic (PK/PD) and Tox profiles

•

• •

All of the above information will prove invaluable in determining the potential methods for rational drug delivery. Particular attention should be paid to the relative hygroscopicity of the API, of course, any stability information, as well as the impurity profile and ADMET (absorption, distribution, metabolism, excretion, and toxicity) information. In short, the more information that is available when development activities are initiated, the easier it is to avoid common pitfalls and make development decisions more rationally.

1.1.2.1

Route of Administration and Dosage

Biologics are traditionally very potent molecules that may require only picomolar blood concentrations to elicit a therapeutic effect. Given that the amount of drug required per dosage will be commensurate with the relative potency of the molecule, small concentrations are generally required for any unit dose. Biopharmaceuticals typically have large molecular weights relative to conventional pharmaceutical agents, which may be increased further by posttranslational modifications. The pharmacokinetics (ADMET) of biotechnology products have been reviewed elsewhere [6], but generally they have short circulating half-lives [7]. As such, biological products are most often delivered parenterally and formulated as solutions, suspensions, or lyophilized products for reconstitution [8, 9]. However, one must first ascertain the potential physiological barriers to drug delivery and efficacy before assessing potential routes of administration. These barriers may include actual physical barriers, such as a cell membrane, that could restrict the drug from reaching its site of action or chemical barriers, including pH or enzymatic degradation. Based on current drug delivery approaches, the proteinaceous nature of biological products limits their peroral delivery due to their susceptibility to proteases and peptidases present in the gastrointestinal tract as well as size limitations for permeating through absorptive enterocytes [10].


9

Difficulties in peroral delivery have stimulated researchers to explore alternate delivery mechanisms for biologics, such as through the lungs or nasal mucosa [11, 12]. Further, advances in technology and our understanding of the mechanisms limiting oral delivery of biotechnology products have led to innovative drug delivery approaches to achieve sufficient oral bioavailability. However, no viable products have successfully reached the market [13]. As a result of the technological limitations inherent in biopharmaceutical delivery, these compounds are largely delivered parenterally through an injection or implant. When assessing the potential routes of administration, one must consider the physicochemical properties of the drug, its ADMET properties, the therapeutic indication, and the patient population, some of which are discussed below. Table 1 provides a list of some of those factors that must be addressed when determining the most favorable route of administration and the subsequent formulation for delivery. Ideally the route of administration and subsequent formulation will be optimized after identifying critical design parameters to satisfy the needs of patients and health care professionals alike while maintaining the safety and efficacy of the product. Parenteral administration is the primary route of delivering biopharmaceutical agents (e.g., insulin); however, issues associated with patient compliance with administration of short-acting molecules are a challenge. Yet, the risk-to-benefit ratio must be weighed when determining such fundamental characteristics of the final dosage form. For instance, a number of biopharmaceutical compounds are administered subcutaneously, but this route of parenteral administration exhibits the highest potential for immunogenic adverse events due to the presence of Langerhans cells [14]. A compound’s immunogenic potential is related to a host of factors, both

TABLE 1 Factors That Determine Route of Administration Site of action Therapeutic indication Dosage Potency/biological activity Pharmacokinetic profile Absorption time from tissue vs. IV Circulating half-life Distribution and elimination kinetics Toxicological profile Immunogenic potential Patient population characteristics Disease state Pathophysiology Age Pharmacodynamic profile Onset and duration of action Required clinical effect Formulation considerations Stability Impurity profile

10


patient and treatment related; however, if an alternate, potentially safer route of administration is available, it may be prudent to consider it. Other factors, such as the frequency of dosing (especially into an immune organ such as the skin) and the duration of treatment, can also dramatically increase the potential for immunogenic reactions [14]. Many of the factors that contribute to the immunogenic potential of biopharmaceuticals, such as impurities, degradation products, and native antigenic epitopes, can be mitigated through altering the physicochemical properties of the drug (e.g., pegylation [15, 16], acylation [17, 18], increased glycosylation to mask epitopes [19]) or changing the characteristics of the formulation [20, 21]. In reality, the pharmaceutical industry has done a good job of recognizing the potential implications of immunogenic reactions and readily embraced technologies that can either mask or eliminate potential antigenic epitopes. However, additional research is needed to further identify and remove immunogenic epitopes. 1.1.2.2

Pharmacokinetic Implications to Dosage Form Design

Biological agents are generally eliminated by metabolism into di- and tripeptides, amino acids, and smaller components for subsequent absorption as nutrients or clearance by the kidney, liver, or other routes. Renal elimination of peptides and proteins occur primarily via three distinct mechanisms. The first involves the glomerular filtration of low-molecular-weight proteins followed by reabsorption into endocytic vesicles in the proximal tubule and subsequent hydroysis into small peptide fragments and amino acids [22]. Interleukin 11 (IL-11) [23], IL-2 [24], insulin [25], and growth hormone [26] have been shown to be eliminated by this method. The second involves hydrolysis of the compound at the brush border of the lumen and subsequent reabsorption of the resulting metabolites [6]. This route of elimination applies to small linear peptides such as angiotensin I and II, bradykinin, glucagons, and leutinizing hormone releasing hormone (LHRH) [6, 27, 28]. The third route of renal elimination involves peritubular extraction from postglomerular capillaries and intracellular metabolism [6]. Hepatic elimination may also play a major role in the metabolism of peptides and proteins; however, reticuloendothelial elimination is by far the primary elimination route for large macromolecular compounds [29]. Biopharmaceutical drug products are subject to the same principles of pharmacokinetics and exposure/response correlations as conventional small molecules [6]. However, these products are subject to numerous pitfalls due to their similarity to nutrients and endogenous proteins and the evolutionary mechanisms to break them down or prevent absorption. The types of pharmacokinetic-related problems that a biotechnology drug development team may encounter range from lack of specificity and sensitivity of bioanalytical assays to low bioavailability and rapid drug elimination from the system [6]. For example, most peptides have hormone activity and usually short elimination half-lives which can be desirable for close regulation of their endogenous levels and function. On the other hand, some proteins such as albumin or antibodies have half-lives of several days and formulation strategies must be designed to account for these extended elimination times [6]. For example, the reported terminal half-life for SB209763, a humanized monoclonal antibody against respiratory syncytial virus, was reported as 22–50 days [30]. Furthermore, some peptide and protein products that persist in the bloodstream exhibit the potential for idiosyncratic adverse affects as well as increased immunogenic poten-

ANALYTICAL METHOD DEVELOPMENT

11

tial. Therefore, the indication and formulation strategy can prove crucial design parameters simply based on clearance mechanisms. 1.1.2.3

Controlled-Release Delivery Systems

Given that the majority of biopharmaceutical products are indicated for chronic conditions and may require repeated administrations, products may be amenable to controlled-release drug delivery systems. Examples include Lupron Depot (leuprolide acetate), which is delivered subcutaneously in microspheres [31], and Viadur, which is implanted subcutaneously [32]. Various peptide/protein controlled delivery systems have been reviewed recently by Degim and Celebi and include biodegradable and nondegradable microspheres, microcapsules, nanocapsules, injectable implants, diffusion-controlled hydrogels and other hydrophilic systems, microemulsions and multiple emulsions, and the use of iontophoresis or electroporation [33]. These systems offer specific advantages over traditional delivery mechanisms when the drug is highly potent and if prolonged administration greater than one week is required [5, 33]. However, each of these systems has its own unique processing and manufacturing hurdles that must be addressed on a case-by-case basis. These factors, coupled with the difficulties of maintaining product stability, limit the widespread application of these technologies. However, the introduction of postapproval extended-release formulations may also provide the innovator company extended patent/commercial utility life and, as such, remains a viable option for postmarketing development. A current example of this is observed in the development of a long-acting release formulation of Amylin and Eli Lilly’s co-marketed Byetta product.

1.1.3 ANALYTICAL METHOD DEVELOPMENT The physical and chemical characterization of any pharmaceutical product is only as reliable as the quality of the analytical methodologies utilized to assess it. Without question, the role of analytical services to the overall drug product development process is invaluable. Good analytical testing with proper controls could mean the difference between a marketable product and one that is eliminated from development. Analytical methodologies intended for characterization and/or assessment of marketed pharmaceutical products must be relevant, validatable, and transferable to manufacturing/quality assurance laboratories. 1.1.3.1 Traditional and Biophysical Analytical Methodologies Typically, there are a handful of traditional analytical methodologies that are utilized to assess the physical, chemical, and microbiological attributes of smallmolecule pharmaceutical products. While many of these testing paradigms can still be utilized to assess biopharmaceuticals, these molecules require additional biophysical, microbiological, and immunogenic characterization as well. In brief, analytical methodologies should evaluate the purity and bioactivity of the product and must also be suitable to assess potential contaminants from expression systems as well as different isoforms and degradation products of the active. Biophysical

12


methodologies allow for assessment of the structural elements of the product with respect to its activity. Such assessments include structural elements, such as the folding of the molecule, and also encompass potential posttranslational modifications and their impact on structure. A list of typical analytical parameters and methodologies utilized to assess those parameters can be found in Table 2. The impact of a molecule’s biophysical characteristics on its clinical efficacy should be readily quantifiable. With respect to rational drug design, it is also extremely important to minimize external factors that may influence the formation of any adverse response. One such factor is the presence of degradation products and drug-related impurities that may be responsible for an immune response. One such industrial example is granulocyte-macrophage colony-stimulating factor [GMCSF, or Leukine (sargramostim), by Berlex Co.], which is produced as a recombinant protein synthesized and purified from a yeast culture, Saccharomyces cerevisiae. As expected, the expression system has an impact on the final product: sargramostim, manufactured from S. cerevisiae, yields an O-glycosylated protein, while molgramostim (Leucomax), synthesized using an E. coli expression system, is nonglycosylated [34]. The E. coli–derived product exhibited a higher incidence of adverse reactions in clinical trials and never made it to the market. With respect to the drug product, the immunogenic reactions included [34, 35]: TABLE 2 Analytical Methodologies and Their Utility for API and Drug Product Characterization Parameter Assessed

Methodologies

Appearance

Visual appearance, colorimetric assays, turbidity

Purity, degradation products and related substances

GPC/SEC-HPLC, RPHPLC, gel electrophoresis, immunoassays, IEF, MS, CD, CE

Molecular weight determination

GPC/SEC-HPLC, gel electrophoresis, multiangle laser light scattering (MALLS), laser diffraction Biological activity (direct or indirect)

Potency

pH

Potentiometric assays

Utility Simple determination of physical stability, i.e., are there particles in solution, is the solution the correct color/turbidity? Is the container closure system seemingly intact? Gives a general idea of the relative purity of the API and the drug product. Are there impurities related to the expression system? Are there alternate API isoforms present? Can degradation products be distinguished from the active component(s)? Is the product a single molecular weight or polydisperse? Is the molecular weight dependent on posttranslational modifications? Does the compound have reproducible in vitro activity and can this be correlated to in vivo? Is the product pH labile or do pH changes affect potency is such ways that are not evident in other assays, i.e., minimal degradation and/or unfolding?

ANALYTICAL METHOD DEVELOPMENT

13

TABLE 2 Continued Parameter Assessed Primary structural elements

Secondary structural elements

Methodologies Protein sequencing, N-term degradation (Edman degradation), peptide mapping, amino acid composition, 2D-NMR CD, 2D-NMR, in silico modeling from AA sequence

Tertiary structural elements

Disulfide content/position, CD

Agglomeration/ aggregation

Subvisual and visual Particle size analysis, immunogenicity RP-HPLC, gel electrophoresis, AEHPLC, CE, MALDI-MS, ES-MS, enzyme arrays Karl Fischer, TGA, NIR

Carbohydrate analysis

Water content (lyophilized products) Immunogenic potential

Surface plasmon resonance, ELISA, immunoprecipitation

Sterility

Membrane filtration

Bacterial endotoxins

Limulus amebocyte lysate (LAL)

Container closure integrity

Dye immersion, NIR, microbial ingress/sterility

Utility Verifies primary amino acid sequence and gives preliminary insight into activity.

Secondary structural elements result from the primary sequence and help define the overall conformation (3D folding) of the compound. Determines correct folding and overall integrity of the 3D product. Qualitative determination for denaturation potential. Also correlates to immunogenic potential. Indicator of physical instability. Also gives an indication of immunogenic potential. Ensures proper posttranslational modifications and carbohydrate content. Indicator of hydrolytic potential and process efficiency. Methodologies generally only give positive/negative indicators of immunogenic potential. In vitro methodologies do not always correlate to in vivo. Indicator of microbial contaminants from manufacturing operations. Gives an idea of processing contaminants and potentially host organism contaminants. Demonstrates viability of container closure system over the life of the product.

Abbreviations: gel permeation chromatography (GPC), size exclusion chromatography (SEC), highperformance, or high-pressure, liquid chromatography (HPLC), reverse phase (RP), isoelectric focusing (IEF), mass spectrometry (MS), circular dichroism (CD), capillary electrophoresis (CE), nuclear magnetic resonance (NMR), anion exchange (AE), matrix-assisted laser desorption ionization (MALDI), electrospray ionization (ES), thermogravimetric analysis (TGA), near infrared (NIR), enzyme-linked immunosorbent assay (ELISA)

14


1. Formation of antibodies which bind and neutralize the GM-CSF 2. Formation of antibodies which bind but do not affect the efficacy of GM-CSF 3. Antibody formation against proteins not related to GM-CSF, but to proteins from the expression system (E. coli) 4. Antibodies formed against both product- and non-product-related proteins 5. No antibody formation This example clearly illustrates not only the range of clinical manifestations with respect to antibody formation to drug therapy but also how the choice of an expression system can affect the final product. In this example, the expression system was responsible for the adverse events reported. This finding is certainly clinically relevant considering the homologous product, sargramostim, has been on the U.S. market for quite some time. The above example also gives an indication of the relative importance of carbohydrate analysis. Without question, protein glycosylation is the most complex of all posttranslational modifications made in eukaryotic cells, the importance of which cannot be underestimated. For many compounds, glycosylation can readily affect protein solubility (as influenced by folding), protease resistance, immunogenicity, and pharmacokinetic/pharmacodynamic profiles (i.e., clearance and efficacy) [36]. Typical analytical methodologies used to assess carbohydrate content are also listed in Table 2. 1.1.3.2

Stability-Indicating Methodologies

Analytical methodologies that are specific to the major analyte that are also capable of separating and quantifying potential degradation products and impurities, while simultaneously maintaining specificity and accuracy, are deemed stability indicating. Traditional stability-indicating high-performance liquid chromatography (HPLC) methodologies for small molecules are developed and validated with relative ease. Typically, the stability-indicating nature of an analytical method can be demonstrated by subjecting the product to forced degradation in the presence of heat, acid, alkali, light, or peroxide [37]. If degradation products are sufficiently well resolved from the active while maintaining specificity and accuracy, the method is suitable. In contrast to small molecules, there is no one “gold standard” analytical methodology that can be utilized to determine the potential degradation products and impurities in the milieu that may constitute a biopharmaceutical drug product. Furthermore, a one-dimensional structure assessment (e.g., in terms of an absorption spectrum) does not give any indication of the overall activity of the product, as is the case with traditional small molecules. Thus, the stability assessment of biopharmaceuticals will typically comprise a multitude of methodologies that when taken together give an indication of the stability of the product. The overall goal is to assess the structural elements of the compound as well as attempt to determine the relative quantities of potential degradation products, as well as product isoforms and impurities, that are inherent to the expression systems utilized for API manufacture. However, it is still advised that bioactivity determinations are made at appropriate intervals throughout the stability program, as discussed below. Furthermore, any biopharma-

FORMULATION DEVELOPMENT

15

ceutical stability program should also minimally include an evaluation of the in vitro immunogenicity profile of the product with respect to time, temperature, and other potential degradative conditions. 1.1.3.3

Method Validation and Transfer

Analytical method validation is the process by which scientists prove that the analytical method is suitable for its intended use. Guidances available on validation procedures for some traditional analytical methodologies [38] can be adapted to nontraditional methodologies. The United States Pharmacopeia (USP) and National Formulary (NF) do provide some guidance on designing and assessing biological assays [39], as does the U.S. FDA [40]. Essentially, validation determines the acceptable working ranges of a method and the limitations of that method. At a minimum the robustness, precision, and accuracy of quantitative methodologies should be determined during support of API iteration and refinement, while at the very least the robustness of qualitative methodologies should be assessed. Of particular importance for successful analytical method validation is ensuring that the proper standards and system suitability compounds have been chosen and are representative or analogous to the compound to be analyzed and traceable to a known origin standard, such as the National Institute of Standards and Technology (NIST) or USP/NF. If a reference standard from an “official” source is not available, in-house standards may be used provided they are of the highest purity that can be reasonably obtained and are thoroughly characterized to ensure its identity, strength, quality, purity, and potency. Methods developed and validated during the product development phase are routinely transferred to quality control or contract laboratories to facilitate release and in-process testing of production batches. Ensuring that method transfer is executed properly, with well-defined and reproducible system suitability and acceptance criteria, is the responsibility of both laboratories. Experiments should consist of all those parameters assessed during method validation and should include an evaluation of laboratory-to-laboratory variation. This information will give an idea of the reliability of the methodology and equipment used under the rigors of largescale manufacturing.

1.1.4


The previous sections have highlighted some of the limitations and difficulties in developing biotechnology-derived pharmaceuticals. Although there are major technological limitations in working with these products, their synthesis and manufacturing are significantly more reproducible compared to naturally derived biologics. Determining the most appropriate route of administration and subsequent formulation is dependent on a number of factors, including the product’s indication, duration of action, pharmacokinetic parameters, stability profile, and toxicity. As mentioned previously, biopharmaceuticals are typically delivered parenterally, and thus we will focus on those studies required to successfully develop a parenteral formulation of a biopharmaceutical agent. The goal of formulation development is to design a dosage form that ensures the safety and efficacy of the product through-

16


out its shelf life while simultaneously addressing the clinical needs of both the patient and caregivers to ensure compliance. Formulation development is truly a balancing act, attempting to emphasize the benefits of the therapy and patient compliance while maximizing drug efficacy and minimizing toxicity. As such, a number of studies are required to properly design and develop a formulation, many of which are discussed below. 1.1.4.1

Processing Materials and Equipment

An important factor in the quality and reproducibility of any formulation development activity is the materials utilized for formulating and processing studies. In addition, the choice of container closure systems for the API and the formulation needs to be considered carefully to provide maximum product protection and optimal stability. Variability between small- and larger scale development stages may also be significant depending on the API and materials involved during process scale-up. It is important to conduct process development studies utilizing equipment representative of what will be used for large-scale production, if possible. Implementing this design approach will enable at least some limited dimensional analysis, allowing for early identification of critical design parameters, thereby facilitating scale-up or permitting earlier attrition decisions and cost savings. Regardless, it is important to consider the chemical composition and material properties of every manufacturing component that may contact the drug product. For instance, processing vessels may be made of glass, glass-lined steel, or bare steel, while stir paddles used to ensure homogeneity made be manufactured of a number of different materials. In short, any manufacturing unit that could potentially come into intimate contact with either the formulation or the API should be demonstrated to be compatible with the product, including sampling instruments, sample vials, analytical and processing tubing, and so forth. Material incompatibility could result in something as simple as unexplained analytical variability due to a loss of drug through adsorptive mechanisms to something as serious as a loss of bioactivity or an increase in immunogenic potential. Therefore, equipment design and materials would ideally be consistent from formulation development through to scale-up and process validation; however, this may not be readily feasible. As such, determining the chemical and physical compatibility of each piece of processing equipment with the API is critical to maintaining the physical and chemical attributes of the product. Furthermore, such studies help eliminate potential sources of experimental variability and give a better indicator as to the relative technological hurdles to successful product development. Material compatibility protocols must be clearly defined and require that analytical methodologies be suitable for their intended use. Typically, product purity methods and cleaning methodologies utilized to determine residual contaminating product on processing equipment are used for compatibility studies as they are sufficiently sensitive and rugged to accurately determine product content in the presence of a multitude of potential confounding factors. This is particularly important when assessing potential metal, glass, and tubing compatibilities. Compatibility is a function not only of the product’s intimate contact with surrounding materials but also of the contact time and surface area with these equipment. As such, protocols should be designed to incorporate expected real-world conditions the product will


17

see when in contact with the material. For instance, temperature, light, and mechanical stimulation should mimic usage conditions, although study duration should include time intervals that surpass expectations to estimate a potential worst case. These factors should all be considered when examining potential process-related stability. 1.1.4.2

Container Closure Systems

The ICH guideline for pharmaceutical development outlines requirements for container closure systems for drugs and biologics [41]. The concept paper prepared for this guidance specifically states that “the choice of materials for primary packaging should be justified. The discussion should describe studies performed to demonstrate the integrity of the container and closure. A possible interaction between product and container or label should be considered” [42]. In essence, this indicates that the container closure system should maintain the integrity of the formulation throughout the shelf life of the product. In order to maintain integrity, the container closure system should be chosen to afford protection from degradation induced by external sources, such as light and oxygen. In addition to the primary container, the stability of the product should also be examined in the presence of IV administration components if the product could be exposed to these conditions (see Section 1.1.5.6). Understanding the potential impact of product-to-container interactions is integral to maintaining stability and ensuring a uniform dosage. For example, adsorption of insulin and some small molecules has been demonstrated to readily occur in polyvinyl chloride (PVC) bags and tubing when these drugs were present as additives in intravenous (IV) admixtures [43]. In addition to their use in large-volume parenterals and IV sets, thermoplastic polymers have also recently found utility as packaging materials for ophthalmic solutions and some small-volume parenterals [43]. However, there are many potential issues with using these polymers as primary packaging components that are not major concerns with traditional glass container closure systems, including [44]: 1. Permeation of vapors and other molecules in either direction through the wall of the plastic container 2. Leaching of constituents from the plastic into the product 3. Sorption (absorption and/or adsorption) or drug molecules or ions on the plastic material These concerns largely preclude the utility of thermoplastic polymers as the primary choice of container closure system for protein and peptide therapeutics, although the formulation scientist should be aware of the potential advantages of these systems, such as the ease of manufacturability and their cost. These systems are also finding greater utility in intranasal and pulmonary delivery systems. Parenterally formulated biopharmaceuticals are typically packaged in glass containers with rubber/synthetic elastomeric closures. Pharmaceutical glass is composed primarily of silicon dioxide tetrahedron which is modified with oxides such as sodium, potassium, calcium, magnesium, aluminum, boron, and iron [45]. The USP classifies glass formulations as follows:

18


Type I, a borosilicate glass Type II, a soda–lime treated glass Type III, a soda–lime glass NP, a soda–lime glass not suitable for containers for parenterals The tendency of peptides to adsorb onto glass surfaces is well known and a major concern in the pharmaceutical industry. This is especially important when the dose of the active ingredient is relatively small and a significant amount of drug is adsorbed to these surfaces. In addition, the leaching of atoms or elements in the glass’s silicate network into solution is also a potential issue. This is especially important for terminally heat sterilized products where oxide additives included in the silicate network are relatively free to migrate/leach, resulting in increased solution pH, reaction catalysis, and so on [45]. As such, only type 1 treated glass is traditionally used for parenterally administered formulations, where these alkaline-rich phases in the glass have been eliminated, thus decreasing the potential for container closure system interactions. Additional approaches, including surface treatment with silicone (siliconization), have also been developed to minimize the interaction of biotechnology products with free silanols (Si–OH) [46]. Elastomeric closures are typically used for syringe and vial plungers and closures. For vials, elastomers provide a soft and elastic material that can permit the entry of a hypodermic needle without loss of the integrity [45]. For syringes, the closures not only provide a permeation barrier but also allow for a soft gliding surface facilitating plunger movement and drug delivery. Elastomeric polymers, however, are very complex materials composed of multiple ingredients in addition to the basic polymers, such as vulcanizing agents, accelerators, activators, antioxidants, fillers, lubricating agents, and pigments [45]. As leaching of these components into solution is a potential issue, the compatibility of the drug formulation with the closures must be studied early during the formulation development process. The choice and type of elastomeric closure depends on the pH and buffer, if any preservatives are present, the sterilization method, moisture vapor/gas protection, and active compatibility [47]. In addition, the problem of the additives in rubber leaching into the product can be reduced by the coating with specific polymers such as Teflon [48]. Container closure systems required for implantable devices are further restricted by the fact that they are required to be compatible with the formulation over the intended shelf life and therapeutic application time as well as being biocompatible. This means that the system not only must afford protection to and contain the formulation but also cannot cause any potential adverse effects, such as allergy. Typically, implantable systems are composed of biocompatible metals, such as titanium or polymers such as polyethylene glycol or polylactic-co-glycolic acid.

1.1.4.3

Sterility Assurance

Maintaining the sterility of biopharmaceutical products is especially important due to the relative potency and their innate potential for immunogenic reactions. Further, the biochemical nature of these compounds enables them to serve as potential nutrients for invading organisms. Methods for sterilizing small molecules include


19

heat terminal sterilization, terminal filtration coupled with aseptic processing techniques, ultraviolet (UV) and gamma irradiation, ethylene oxide exposure (for containers and packaging only), and electron beam irradiation. While terminal heat sterilization is by far the most common sterilization technique, it normally cannot readily be utilized for peptide or protein formulations due to the potential effects of heat and pressure on the compound’s structure [48]. Furthermore, irradiation can affect protein stability by cross-linking the sulfur-containing and aromatic residues, resulting in protein aggregation [49]. To overcome these issues, sterile filtration coupled with aseptic processing and filling is the preferred manufacturing procedure for biopharmaceuticals. Garfinkle et al. refer to aseptic processing as “those operations performed between the sterilization of an object or preparation and the final sealing of its package. These operations are, by definition, carried out in the complete absence of microorganisms” [50]. This highlights the importance of manufacturing controls and bioburden monitoring during aseptic processes. Newer technologies such as isolator technology have been developed to reduce human intervention, thereby increasing the sterility assurance. These technologies have the added benefit of facilitating aseptic processing without construction of large processing areas, sterile suites, or gowning areas [50]. Even the most robust monitoring programs do not ensure the sterility of the final formulation. As such, aseptically processed formulations are traditionally filtered through a retentive final filter, which ensures sterility. Coupled with proper component sterilization, traditionally by autoclaving, these processes ensure product sterility. However, filtration is a complex unit operation that can adversely affect the drug product through increased pressure, shear, or material incompatibility. Therefore, filtration compatibility must be assessed thoroughly to demonstrate both product compatibility, and sufficient contaminant retention [51]. Parenteral Drug Association (PDA) technical report 26 provides a thorough systematic approach to selecting and validating the most appropriate filter for a sterilizing filtration application [51]. 1.1.4.4

Excipient Selection

Pharmaceutical products are typically formulated to contain selected nonactive ingredients (excipients) whose function is to promote product stability and enable delivery of the active pharmaceutical ingredient(s) to the target site. These substances include but are not limited to solubilizers, antioxidants, chelating agents, buffers, tonicity contributors, antibacterial agents, antifungal agents, hydrolysis inhibitors, bulking agents, and antifoaming agents [45]. The ICH states that “the excipients chosen, their concentration, and the characteristics that can influence the drug product performance (e.g. stability, bioavailability) or manufacturability should be discussed relative to the respective function of each excipient” [42]. Excipients must be nontoxic and compatible with the formulation while remaining stable throughout the life of the product. Excipients require thorough evaluation and optimization studies for compatibility with the other formulation constituents as well as the container/closure system [52]. Furthermore, excipient purity may be required to be greater than that listed in the pharmacopeial monograph if a specific impurity is implicated in potential degradation reactions (e.g., presence of trace metals) [48].

20


One of the critical factors in excipient selection and concentration is the effect on preferential hydration of the biopharmaceutical product [53, 54]. Preferential hydration refers to the hydration layers on the outer surface of the protein and can be utilized to thermodynamically explain both stability enhancement and denaturation. Typical excipients used in protein formulations include albumin, amino acids, carbohydrates, chelating and reducing agents, cyclodextrins, polyhydric alcohols, polyethylene glycol, salts, and surfactants. Several of these excipients increase the preferential hydration of the protein and thus enhance its stability. Cosolvents need to be added in a concentration that will ensure their exclusion from the protein surface and enhance stability [54]. A more comprehensive review of excipients utilized for biopharmaceutical drug products is available elsewhere [48]. Buffer Selection In addition to maintaining solution pH, buffers serve a multitude of functions in pharmaceutical formulations, such as contributing toward overall isotonicity, preferential hydration of proteins and peptides, and serving as bulking agents in lyophilized formulations. The buffer system chosen is especially important for peptide and proteins that have sensitive secondary, tertiary, and quaternary structures, as the overall mechanisms contributing to conformational stabilization are extremely complex [48]. Furthermore, a protein’s propensity for deamidation at a particular pH can be significant, as illustrated by Wakankar and Borchardt [55]. This study illustrated stability concerns with peptides and proteins at physiological pH in terms of asparagine (Asn) deamidation and aspartate (Asp) isomerization, which can be a major issue with respect to circulating half-life and potential in vivo degradation. This study and others also provide insight into predicting potential degradative mechanisms based on primary and secondary structural elements allowing for formulation design with these pathways in mind. Selecting the appropriate buffer primarily depends on the desired pH range and buffer capacity required for the individual formulation; however, other factors, including concentration, effective range, chemical compatibility, and isotonicity contribution, should be considered [56]. Some acceptable buffers include phosphate (pH 6.2–8.2), acetate (pH 3.8–5.8), citrate (pH 2.1–6.2, pK 3.15, 4.8, and 6.4), succinate (pH 3.2–6.6, pK 4.2 and 5.6), histidine (pK 1.8, 6.0, and 9.0), glycine (pK 2.35 and 9.8), arginine (pK 2.18 and 9.1), triethanolamine (pH 7.0–9.0), trishydroxymethylaminomethane (THAM, pK 8.1), and maleate buffer [48]. Additionally, excipients utilized solely for tonicity adjustment, such as sodium chloride and glycerin, may not only differ in ionic strength but also could afford some buffering effects that should be considered [52]. Preservatives In addition to those processing controls mentioned above (Section 3.1.4.3), the sterility of a product may be maintained through the addition of antimicrobial preservatives. Preservation against microbial growth is an important aspect of multidose parenteral preparations as well as other formulations that require preservatives to minimize the risk of patient infection upon administration, such as infusion products [52]. Aqueous liquid products are prone to microbial contamination because water in combination with excipients derived from natural sources (e.g., polypeptides, carbohydrates) and proteinaceous active ingredients may serve as excellent media for the growth [57]. The major criteria for the selection of an appropriate preservative include efficiency against a wide spectrum of micro-

DRUG PRODUCT STABILITY

21

organisms, stability (shelf life), toxicity, sensitizing effects, and compatibility with other ingredients in the dosage form [57]. Typical antimicrobial preservatives include m-cresol, phenol, parabens, thimerosal, sorbic acid, potassium sorbate, benzoic acid, chlorocresol, and benzalkonium chloride. Cationic agents such as benzalkonium chloride are typically not utilized for peptide and protein formulations because they may be inactivated by other formulation components and their respective charges may induce conformational changes and lead to physical instability of the API. Further, excipients intended for other applications, such as chelating agents, may exhibit some antimicrobial activity. For instance, the chelating agent ethylenediaminetetraacetic acid (EDTA) may exhibit antimicrobial activity, as calcium is required for bacterial growth. Identifying an optimal antimicrobial preservative is based largely on the effectiveness of that preservative at the concentration chosen. In short, it is not enough to assess the compatibility of the preservative of choice with the API and formulation and processing components. There also needs to be a determination of whether the preservative concentration is sufficient to kill certain standard test organisms. The USP presents standard protocols for assessing the relative efficacy of a preservative in a formulation using the antimicrobial effectiveness test (AET) [58]. Briefly, by comparing the relative kill efficiency of the formulation containing varying concentrations of the preservative, the formulator can determine the minimal concentration required for preservative efficacy and design the formulation accordingly.

1.1.5 1.1.5.1

DRUG PRODUCT STABILITY Defining Drug Product Storage Conditions

From a regulatory standpoint, the primary objective of formulation development is to enable the delivery of a safe and efficacious drug product to treat and/or mitigate a disease state throughout its proposed shelf life. The efficacy and in many cases the safety of a product are directly related to the stability of the API, both neat and in the proposed formulation under processing, storage, and shipping conditions as well as during administration. As such, the concept of drug stability for biotechnologyderived products does not change substantially from that of small molecules, although the level of complexity increases commensurate with the increased complexity of the APIs in question and the formulation systems utilized for their delivery. Stability study conditions for biotechnology-derived APIs and their respective drug products are not substantially different from those studies conducted for small molecules. Temperature and humidity conditions under which to conduct said studies are outlined in ICH Q1A(R2), which incorporates ICH Q1F, stability study conditions for zones III and IV climactic conditions [59]. Additional guidance specific to conducting stability studies on biopharmaceutical drug products is given in ICH Q5C [1]. However, the intention of ICH Q5C is not to outline alternate temperature and humidity conditions to conduct primary stability studies; rather it provides guidance with respect to the fact that the recommended storage conditions and expiration dating for biopharmaceutical products will be different from product to product and provides the necessary flexibility in letting the applicant determine

22

BIOTECHNOLOGY-DERIVED DRUG PRODUCT DEVELOPMENT T 80

60

40

20

4

5

6

7

8

9

pH

FIGURE 1 Empirical phase diagram for ricin toxin A-chain generated using CD molar ellipticity at 208 nm, ANS fluorescence, and intrinsic Trp fluorescence intensity data. Labels indicate the state of the protein within the same region of color based on evaluation of a compilation of data sets. (Reproduced with permission from ref. 62.)

the proper storage conditions for their respective product. Furthermore, this document provides general guidance in directing applicants in the types of analytical methodologies that may be used and direction on how to properly assess the stability of these complex molecules [1]: Assays for biological activity, where applicable, should be part of the pivotal stability studies. Appropriate physicochemical, biochemical and immunochemical methods for the analysis of the molecular entity and the quantitative detection of degradation products should also be part of the stability program whenever purity and molecular characteristics of the product permit use of these methodologies.

One recent approach to aid in defining the design space for protein and peptide therapeutics has been to create empirical phase diagrams indicating the relative stability of compounds based on altering conditions and assessing conformational changes via a compilation of analytical techniques (Figure 1) [60–62]. These empirical phase diagrams can be generated based on pH, temperature, salt concentration, and so on, and, although seemingly laborious at first glance, could provide invaluable information in defining the extremes to which a compound may be subjected without altering its conformation. For instance, if an empirical phase diagram determines the safe temperature range for a compound is up to 35 °C and an excursion occurs to 33 °C, this information would give the stability scientist a guideline as to the appropriate course of action. Under the traditional testing paradigm of ICH Q1A, where stability testing is limited to 25, 30, and 40 °C, one may not know the compound’s upper transition temperature to induce conformational changes. If the information is not already available, then additional excursion studies may need to be conducted to assimilate this information and take the appropriate course of action. 1.1.5.2

Mechanisms of Protein and Peptide Degradation

The inherent heterogeneity of peptide and protein drug substances results in their relative sensitivity to processing, storage, and handling conditions as well as a mul-

DRUG PRODUCT STABILITY

TABLE 3

23

Potential Degradative Mechanisms of Peptides and Proteins

Chemical degradative mechanisms

Physical degradative mechanisms

Degradative Mechanism

Site of Occurrence

Oxidation Peptide bond hydrolysis N-to-O migration α- to β-Carboxy migration Deamidation Acylation Esterification/carboxylation Unfolding Aggregation

Intrachain disulfide linkages Met, Trp, Tyr AA backbone Ser and Thr Asp and Asn Asn and Gln α-Amino and ε-amino group Glu, Asp, and C-term Partial unfolding of tertiary structure Aggregation of subunits could result in precipitation Adsorption to processing equipment and container closure systems

Adsorption Source: Modified from Crommelin et al. [5].

titude of other factors. Most importantly, this heterogeneity results in a whole host of potential degradative mechanisms, some of which are compiled in Table 3 and include chemical instability pathways such as oxidation, hydrolysis of side chains and potentially the peptide backbone, and deamidation of Asn and Gln side chains. Also, physical instability manifesting in the form of protein unfolding, formation of intermediate structures, aggregation, and adsorption to the surfaces of containers and other equipment can be a major technical hurdle in developing any biopharmaceutical and may or may not be related to chemical instability [63]. Further complicating matters is that instability can potentially manifest in various ways and may or may not be detectable by any one method. Taken together, however, the compilation of methodologies utilized for stability assessment should give a good approximation as to the degradative mechanisms of the compound in its respective formulation. Further, bioactivity and immunogenicity assays should play integral roles in assessing the relative stability of any biopharmaceutical compound. Briefly stated, the chemical and physical stability of products is extraordinarily difficult to assess and will not be belabored here as good reviews on this topic are readily available in the literature [63, 64]. 1.1.5.3

Photostability

In certain cases, exposure of pharmaceutical compounds to UV and visible light could result in electronic excitation, termed vertical transition, that could ultimately result in light-induced degradation. The ICH guideline Q1B [65] is a reference on how to conduct photostability stress testing for pharmaceutical compounds. In brief, compounds are exposed to an overall illumination of not less than 1.2 million lux hours and an integrated near-UV energy of not less than 200 Wh/m2 [65]. These requirements are in addition to normal stability stress testing and require the additional caveat that analytical methodologies are suitable to also detect photolytic degradation products, as discussed above. A comprehensive discussion of small-molecule photolytic degradative mechanisms is available for further review [66].

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1.1.5.4

Mechanical Stress

Regulatory guidance on appropriate methods to evaluate the effect of shear stress and process-handling stability studies is not available. However, these studies are integral in determining the relative stability of the product with respect to mechanical stresses introduced during development and manufacturing. Although not typically recognized as a major degradative pathway for most small-molecule dosage forms, the introduction of mechanical stress is recognized as a major challenge in the formulation of semisolids and can potentially induce physical instability of biopharmaceuticals, although the extent of this effect is currently unknown. For example, processing shear may influence the protein’s outer hydration shell, altering the stabilizing energy provided from preferential hydration and resulting in the exposure of internal, nonpolar residues. This may facilitate aggregation if enough shear force is provided. Alternately, the shear energy required to force unfolding has been studied but has not been related to the fluid dynamic shear experienced during processing. Therefore, stress studies should include meticulous controls in the form of temperature, light and humidity, and fluid dynamic shear as a function of time. Data from these studies could be incorporated into empirical phase diagrams, and/ or response surfaces, to help further define the design space for the active and finished drug product. Understanding the effects of stress introduced during the manufacturing processing of biopharmaceutical products could facilitate the selection of appropriate PAT tools and QbD incorporation in the development of these products. Clearly, there is a considerable need for research in this area, and until the extent of the possible effects are understood, this lack of knowledge poses an unknown risk and prevents adequate risk assessment for biopharmaceutical development activities consistent with ICH Q9. 1.1.5.5

Freeze–Thaw Considerations and Cryopreservation

The rapid or continuous freezing and thawing of protein products could contribute significantly to instability of the API. Such studies are typically designed to assess the implications of potential transport and handling conditions. These conditions include not only the manufacturing processing, storage, and shipment to warehouses and pharmacies but also subsequent pharmacy storage and patient handling [52]. Unpredictable and somewhat modest temperature fluctuations could easily induce degradation or conformational changes that may reduce bioactivity or expose antigenic epitopes [5]. These effects could also be a result of altered preferential hydration at the surface of the peptide or protein through salting-out effects induced by rapid freezing, which could easily denature the product [67]. 1.1.5.6

Use Studies

Stability of biopharmaceutical compounds should also be determined under conditions that mimic their normal usage. For instance, the stability of reconstituted lyophilized products should be assessed with respect to time and temperature and, if applicable, light and mechanical stimuli. Likewise, the stability of a compound included in implantable devices and controlled-release microsphere formulations should be determined over the course of its required use, under conditions which mimic the heat, moisture, light, and enzymatic physiological conditions to which it

QUALITY BY DESIGN AND SCALE-UP

25

will be implanted. Such studies should also determine the release profile of the compound over these specified conditions. Drug products intended for IV administration are generally dosed as an initial bolus followed by a slow infusion. Consequently, admixture studies of the compound in potential IV fluids, such as 0.9% (w/v) saline, 5% (w/v) dextrose, and Ringer’s solution, should also be assessed to determine the relative stability of the compound in this new environment. These studies are critical as the formulation dynamic that protected and stabilized the compound has now been altered dramatically with dilution. This environmental change could potentially impact the preferential hydration of the compound as well as directly induce conformational changes based on the diluent chosen and the compound’s potential degradative mechanism(s). Additional contributing factors to instability in admixture solutions could be due to changes in pH, mechanical mixing of the compound in the IV bag, adsorption of the compound to the bag itself (which is typically polymeric), or IV sets used for administration, as well as an increased potential for oxidative degradation. The suitability of analytical methodologies should also be determined in the presence of these additional analytes. 1.1.5.7

Container Closure Integrity and Microbiological Assessment

Ensuring that parenteral pharmaceuticals maintain their sterility over the course of their shelf life is an integral part of any stability assessment [68]. Parenteral dosage forms must be free from microbiological contamination, bacterial endotoxins, and foreign particulate matter. Selection of the adequate sterile manufacturing process has been briefly discussed above. Determining the microbiological integrity of the product over its shelf life also gives an indication of the relative quality of the container closure system chosen for the formulation. Compendial sterility and endotoxin testing are often used for this purpose; however, sampling is dependent on a statistical evaluation of the batch size, unit fill volume, and method of product sterilization [68]. Additionally, since these tests are destructive, it would be impossible to test an entire stability batch to ensure viability of a container closure system. Other nondestructive tests have been developed to determine the integrity of a container’s closure system [69]. These tests could also serve as a surrogate indicator of product manufacturing quality over time. 1.1.5.8

Data Interpretation and Assessment

Interpretation of primary stability data for determining expiration dating and primary storage conditions has been outlined by ICH Q1E [70]. This guidance document delineates broad methodologies for interpreting primary and accelerated stability data and extrapolation of said data for determining expiry dating. Of course, expiry dating cannot be made without reference to specifications for those primary stability-indicating parameters assessed, which is discussed below. Traditionally, stability assessments performed during preformulation will give an indication of the potential storage conditions as well as allow for extrapolation of accelerated stability studies to kinetic degradation rates. Typically this is done through Arrhenius manipulations. However, as one would expect, these analyses are not readily useful for biopharmaceutical products, as there is rarely a linear correlation between

26


temperature and the compound’s degradative rate. This is primarily due to the complex and often competing degradative mechanisms as well as the potential for so-called molten globule intermediate phases. In spite of these limitations, ICH Q5C does provide relevant guidance in illustrating the flexibility required for determining storage conditions, as these products usually require a very narrow temperature condition to maintain optimal stability. Further guidances may be needed to enhance uniformity in testing methodology and enable the utilization of validated PAT methodologies.

1.1.6 1.1.6.1

QUALITY BY DESIGN AND SCALE-UP Unit Operations

Unit operations are defined as the individual basic steps in a process that when linked together define the process train and result in the final product. In practical terms, a unit operation is often defined as an individual step that is carried out on one piece of equipment. Typical biopharmaceutical API unit operations may include fermentation or bioreactor processes, cell separation through centrifugation or microfiltration, virus removal or inactivation, cell lysis and inclusion body precipitation, product refolding, and purification steps [71]. Conversely, those unit operations for drug product manufacturing procedures would be similar to those seen in the manufacture of a small molecule of comparable dosage form, namely mixing, fluid transfer, sterile filtration, dose filling, lyophilization, and so on. Of course, unit operations will be dependent on the manufacturing process for the specific dosage form, but careful preformulation and characterization studies will enable relatively straightforward process design and ease subsequent scale-up activities. Modeling of unit operations for both small and large molecules is a recognized gap in our ability to achieve QbD. The application of accepted engineering methods to the problem is the subject of active research. 1.1.6.2

Bioburden Considerations

Bioburden refers to the amount of microbial flora that can be detected on an item, on a surface, or in a solution [68]. As mentioned previously, microbial contamination and bioburden are especially important for biotechnology-derived parenteral products since these products are typically capable of supporting microbial growth. Special care should be taken to ensure not only that the final packaged product does not contain microbial contamination but also that manufacturing equipment is also free from contamination. Monitoring bioburden and determining potential levels of microbial contamination on equipment surfaces are particularly important with respect to the material being evaluated. In general, bioburden counts in parenteral solutions are obtained by conducting the total aerobic counts and total yeast and mold counts as specified in the USP microbial limits test (61) or an equivalent test [72]. In addition, membrane filtration of larger than specified volumes may also be used to detect any microbial contamination when sample results are expected to contain a negligible number of microbial flora or in the presence of potential confounding factors, such as antimicrobial

QUALITY BY DESIGN AND SCALE-UP

27

preservatives [68, 72]. It is important to note that the presence of a high bioburden count can present an endotoxin contamination problem, as whole microbial cells and spores can be removed by sterilizing grade filtration (0.2 μm), while endotoxins are not [68]. These issues also underscore the importance of cleaning methods and their respective validation as well as assessing relevant product contamination on manufacturing equipment. 1.1.6.3

Scale-Up and Process Changes

The FDA defines process validation as “establishing documented evidence that provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined quality attributes” [73]. While validation studies are typically performed at full scale, in most cases scale-down or laboratory-scale models were used to initially develop the manufacturing process. Consequently, scale-down process precharacterization and characterization studies are considered crucial to successful process validation for both API and drug product manufacturing schemes [74]. Although they do require qualification work and a significant commitment of time and resources, characterization studies provide significant insight into the critical process and control parameters for each unit operation as well as improved success rates for process validation due to a better, more complete understanding of the process [74]. In engineering terms, characterization studies identify the critical parameters useful for dimensional analysis that enable successful process scale-up. While the above explanation attempts to simplify the scale-up process, it is not meant to trivialize it. In fact, scale-up is probably the most difficult manufacturing challenge for traditional small molecules, let alone biopharmaceuticals. Issues such as homogeneous mixing, bulk product holding and transfer, and sterile filtration could all be potentially compounded due to the increased scale and introduced stress. However, a QbD approach to rational drug design should enable simplified process scale-up and validation. This is only true if experimental design approaches have been utilized to identify the design space for the processes involved in the production of the molecule. This is also where the greatest benefit of developing empirical phase diagrams early in development could materialize. Essentially, the QbD approach identifies the quality attributes of the product based on scientific rationale as opposed to attempting to fit the proverbial square peg into a round hole through a trial-and-error approach. This rational design approach goes further to identify the limiting factors of each unit operation and provides the means of attempting to correlate how each unit operation affects the final product quality attributes. In order to initiate a successful QbD program, the first step is to identify those process parameters that are essential to product quality and develop well-validated analytical methodologies to monitor those parameters. In short, the process involves identification of the potential design space for production of the molecule and confirmation that design space through rational, deliberate experimentation. Ideally, process monitoring should be done in real time to minimize production time and if possible online; however, this may not always be the case or even necessary depending upon the relative duration of the process to the test. Recognizing potential quality metrics earlier in the development process could also potentially facilitate

28


greater flexibility during product development and subsequent process characterization [74]. Certainly, manufacturing site-specific differences could also potentially introduce variability into processes. It is for this reason that site-specific personnel training, process/technology transfer and validation, and stability assessments are required to ensure product quality. By definition, a process designed under the auspices of QbD should enable a degree of process knowledge that allows for controlled process changes without affecting the final product or requiring regulatory approval. For immediate- and controlled-release solid dosage products, SUPAC guidelines provide direction on the studies to conduct to determine the impact of a process change. Although there is some regulatory guidance available for biological products (e.g., “Changes to an Approved Application for Specified Biotechnology and Specified Synthetic Biological Products” or “FDA Guidance Concerning Demonstration of Comparability of Human Biological Products, Including Therapeutic Biotechnology-Derived Products”), process changes need to be evaluated on a case-by-case basis. The comparative analysis of process changes should also be evaluated with respect to defined product specifications. PAT will be invaluable in determining the potential impact of process changes. While stability is often the main metric for small-molecule drug product, bioactivity and immunogenicity will need to be added metrics for biopharmaceuticals. Therefore, any process change should be approached subjectively and care should be taken to validate the relative impact on the safety and efficacy of the product.

1.1.7

CONCLUDING REMARKS

Although the goals are the same, developing biotechnology molecules presents challenges that are unique compared to the development of conventional small molecules. The innate complexity of the molecular and macromolecular structures requires three dimensionally viable stability assays and understanding. The complexity of possible physiological responses and interactions requires an enhanced understanding of the formulation and processing stresses to identify the minor but critical changes that result in product unacceptability. A key to addressing these challenges is the development of analytical techniques with the sensitivity and reliability to detect and monitor such changes and to provide data to another gapclosing activity—modeling unit operations. Also the need to develop meaningful kinetic models is obvious to everyone involved in the development of both large and small molecules. Linking this type of information to the major efforts in the discovery arena is a necessary step to bringing the products of the future to market. The use of biotechnology products is increasing exponentially and many opportunities exist to improve their development. The first step may be defining rational biotechnology-derived drug “developability” standards that can be assessed during preclinical/early development testing. Such a tiered approach based upon the potential risk, the confidence in methodology, and benefit has of course been a proven strategy for small molecules, and a preliminary version applicable to biotechnology drug products is likely possible today given the topics discussed in this chapter.

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ACKNOWLEDGMENTS The authors would like to thank The School of Pharmacy and Pharmaceutical Sciences, the Department of Industrial and Physical Pharmaceutics of Purdue University and the National Institutes of General Medical Sciences (R01-GM65448) for their financial support. REFERENCES 1. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Q5C: Quality of biotechnological products: Stability testing of biotechnological/biological products, Nov. 30, 1995. 2. Biotechnology Industry Organization. Available: http://www.bio.org/, accessed Jan. 10, 2007. 3. Nims, R., Presene, E., Sofer, G., Phillips, C., and Chang, A. (2005), Adventitious agents: Concerns and testing for biopharmaceuticals, in Rathore, A. S., and Sofer, G., Eds., Process Validation in Manufacturing of Biopharmaceuticals: Guidelines, Current Practices, and Industrial Case Studies, Taylor and Francis, Boca Raton, FL. 4. Rathore, A. S., and Sofer, G. (2005), Life span studies for chromatography and filtration media, in Rathore, A. S., and Sofer, G., Eds., Process Validation in Manufacturing of Biopharmaceuticals: Guidelines, Current Practices, and Industrial Case Studies, Taylor and Francis, Boca Raton, FL. 5. Crommelin, D. J. A., Storm, G., Verrijk, R., de Leede, L., Jiskoot, W., and Hennink, W. E. (2003), Shifting paradigms: Biopharmaceuticals vs. low molecular weight drugs, Int. J. Pharm., 266, 3–16. 6. Tang, L., Persky, A. M., Hochhaus, G., and Meibohm, B. (2004), Pharmacokinetic aspects of biotechnology products, J. Pharm. Sci., 93(9), 2184–2204. 7. Roberts, M. J., Bentley, M. D., and Harris, J. M. (2002), Chemistry for peptide and protein pegylation, Adv. Drug Deliv. Rev., 54, 459–476. 8. Frokjaer, S., and Otzen, D. (2005), Protein drug stability: A formulation challenge, Nat. Rev., 4, 298–306. 9. Niu, C., and Chiu, Y. (1998), FDA perspective on peptide formulation and stability issues, J. Pharm. Sci., 87, 1331–1334. 10. Washington, N., Washington, C., and Wilson, C. G. (2001), Physiological Pharmaceutics: Barriers to Drug Absorption, 2nd ed., Taylor and Francis, New York. 11. Hussain, A., Arnold, J. J., Khan, M. A., and Ashan, F. (2004), Absorption enhancers in pulmonary protein delivery, J. Controlled Release, 94, 15–24. 12. Alpar, H. O., Somavarapu, S., Atuah, K. N., and Bramwell, V. W. (2005), Biodegradable mucoadhesive particulates for nasal and pulmonary antigen and DNA delivery, Adv. Drug Deliv. Rev., 57, 411–430. 13. Thanou, M., Verhoef, J. C., and Junginger, H. E. (2001), Chitosan and its derivatives as intestinal absorption enhancers, Adv. Drug Deliv. Rev., 50, 91–101. 14. Schellekens, H. (2002), Immunogenicity of therapeutic proteins: Clinical implications and future prospects, Clin. Ther., 24, 1720–1740. 15. Bhadra, D., Bhadra, S., Jain, P., and Jain, N. K. (2002), Pegnology: A review of PEG-ylated systems, Pharmazie, 57, 5–29. 16. Matthews, S. J., and McCoy, C. (2004), Peginteferon α2a: A review of approved and investigational uses, Clin. Ther., 26, 991–1025.

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17. Knudsen, L. B., Nielsen, P. F., Huusfeldt, P. O., Johansen, N. L., Madsen, K., Pedersen, F. Z., Thogersen, H., Wilken, M., and Agerso, H. (2000), Potent derivatives of glucagonlike peptide-1 with pharmacokinetic properties suitable for once daily administration, J. Med. Chem., 43, 1664–1669. 18. Foldvari, M., Attah-Poku, S., Hu, J., Li, Q., Hughes, H., Babiuk, L. A., and Kruger, S. (1998), Palmitoyl derivatives of interferon α: Potential for cutaneous delivery, J. Pharm. Sci., 87, 1203–1208. 19. Egrie, J.C., and Browne, J. K. (2001), Development and characterization of novel erythropoiesis stimulating protein (NESP), Nephrol. Dial. Transplant., 16(Suppl 3), 3–13. 20. Haselbeck, A. (2003), Epoetins: Differences and their relevance to immunogenicity, Curr. Med. Res. Opin., 19, 430–432. 21. Hochuli, E. (1997), Interferon immunogenicity: Technical evaluation of interferon-α2A, J. Int. Cytokine Res., 17, S15–S21. 22. Maack, T., Johnson, V., Kau, S. T., Figueiredo, J., and Sigulem, D. (1979), Renal filtration, transport, and metabolism of low-molecular-weight proteins: A review, Kidney Int., 16, 251–270. 23. Takagi, A., Masuda, H., Takakura, Y., and Hashida, M. (1995), Disposition characteristics of recombinant human interleukin-11 after a bolus intravenous administration in mice, J. Pharmacol. Exp. Ther., 275, 537–543. 24. Anderson, P. M., and Sorenson, M. A. (1994), Effects of route and formulation on clinical pharmacokinetics of interleukin-2, Clin. Pharmacokinet., 27, 19–31. 25. Rabkin, R., Ryan, M. P., and Duckworth, W. C. (1984), The renal metabolism of insulin, Diabetologia, 27, 351–357. 26. Johnson, V., and Maack, T. (1977), Renal extraction, filtration, absorption, and catabolism of growth hormone, Am. J. Phsiol., 233, F185–F196. 27. Carone, F. A., and Peterson, D. R. (1980), Hydrolysis and transport of small peptides by the proximal tubule, Am. J. Physiol., 238, F151–F158. 28. Carone, F. A., Peterson, D. R., and Flouret, G. (1982), Renal tubular processing of small peptide hormones, J. Lab. Clin. Med., 100, 1–14. 29. Braeckman, R. (2000), Pharmacokinetics and pharmacodynamics of protein therapeutics, in Reid, R., Ed., Peptide and Protein Drug Analysis, Marcel Dekker, New York. 30. Meissner, H. C., Groothuis, J. R., Rodriguez, W. J., Welliver, R. C., Hogg, G., Gray, P. H., Loh, R., Simoes, E. A., Sly, P., Miller, A. K., Nichols, A. I., Jorkasky, D. K., Everitt, D. E., and Thompson, K. A. (1999), Safety and pharmacokinetics of an intramuscular monoclonal antibody (SB 209763) against respiratory syncytial virus (RSV) in infants and young children at risk for severe RSV disease, Antimicrob. Agent Chemother., 43, 1183–1188. 31. Prescribing information for Lupron Depot®. Manufactured for TAP Pharmaceuticals, Inc., Lake Forest, IL 60045. 32. Prescribing information for Viadur®. Manufactured by Alza Corporation, Mountain View, CA 94043. 33. Degim, I. T., and Celebi, N. (2007), Controlled delivery of peptides and proteins, Curr. Pharm. Des., 13, 99–117. 34. Sylvester, R. K. (2002), Clinical applications of colony-stimulating factors: A historical perspective, Am. J. Health-Syst. Pharm., 59, s6–s12. 35. Dorr, R. T. (1993), Clinical properties of yeast-derived versus Escherichia coli-derived granulocyte-macrophage colony-stimulating factor, Clin. Ther., 15, 19–29. 36. Jenkins, N., Shah, P. M., and Buckberry, L. D. (2000), Carbohydrate analysis of glycoproteins and glycopeptides, in Reid, R., Ed., Protein and Peptide Drug Analysis, Marcel Dekker, New York.

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1.2 REGULATORY CONSIDERATIONS IN APPROVAL OF FOLLOW-ON PROTEIN DRUG PRODUCTS Erin Oliver,1 Stephen M. Carl,2 Kenneth R. Morris,2 Gerald W. Becker,3 and Gregory T. Knipp1 1

Rutgers, The State University of New Jersey, Piscataway, New Jersey Purdue University, West Lafayette, Indiana 3 SSCI, West Lafayette, Indiana 2

Contents 1.2.1

1.2.2

1.2.3

1.2.4

1.2.5

1.2.6

Introduction 1.2.1.1 Emergence of Biotechnology Industry 1.2.1.2 Challenges Facing “Biogenerics” History of Biologics Regulation in United States 1.2.2.1 Early Biologics Regulation (1800s–1990s) 1.2.2.2 Modern Biologics Regulation (1990s–Today) Regulatory Classification of Proteins 1.2.3.1 Definitions and Key Terminology 1.2.3.2 Application of Definitions to Proteins: Is It a Drug or a Biologic? 1.2.3.3 Regulatory Approval Path for Proteins Regulation of Generic Drugs 1.2.4.1 History of Generic Drug Legislation in United States 1.2.4.2 Approval Process for Generic Drugs 1.2.4.3 Application of Generic Regulations to Biologics Legal Arguments Related to Follow-On Proteins 1.2.5.1 Constitutionality of 505(b)(2) Process for Drugs 1.2.5.2 Constitutionality of 505(b)(2) Process for Follow-On Proteins 1.2.5.3 Applicability of 505(j)(1) or ANDA Process to Biogenerics 1.2.5.4 Current Rules Relating to Bioequivalence of Generic Drugs 1.2.5.5 Statutory Authority Scientific Issues Related to Follow-On Proteins (Data Requirements) 1.2.6.1 “Sameness” as per Orphan Drug Regulations 1.2.6.2 “Sameness” as per Postapproval Change Guidances

Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad Copyright © 2008 John Wiley & Sons, Inc.

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34

REGULATORY CONSIDERATIONS IN APPROVAL

1.2.7

Proposed Regulatory Paradigm: Case Studies 1.2.7.1 Case Study 1: Fortical [Calcitonin-Salmon (rDNA Origin)] 1.2.7.2 Case Study 2: Omnitrope [Somatropin (rDNA Origin)] 1.2.7.3 Case Study 3: Generic Salmon Calcitonin Summary and Conclusions References

1.2.8

1.2.1

INTRODUCTION

The ongoing need to provide the U.S. population with cost-effective pharmacological therapies has led to an emergent public health initiative in this country, namely for generic versions of therapeutic proteins. Greater access to generic drugs was made possible by the passage of the 1984 Drug Price Competition & Patent Term Restoration Act, commonly referred to as Hatch–Waxman. Generics have historically afforded considerable savings to the American consumer in need of prescription medication. Ten years after the Hatch–Waxman amendments, the Congressional Budget Office estimated that purchasers saved a total of $8–$10 billion on prescriptions at retail pharmacies by substituting generic drugs for their brand-name counterparts in 1994 [1]. To put those numbers in the context of today’s pharmaceutical landscape, a recent report issued by the U.S. Department of Health and Human Services estimates that generic drugs constitute 63% of the total prescription medicines sold in the United States [2]. This same report suggests that generic drugs cost approximately 11% of the total cost of branded pharmaceuticals (on a per-dose basis). At the same time, the development and use of therapeutic proteins have increased dramatically, with more than 850 biotechnology drug products and vaccines currently in trials [3]. Further, it is estimated that by the year 2010 nearly one-half of all newly approved medicines will be of biological origin [4]. The industrial financial incentives for the pursuit of follow-on biologics (heretofore termed biogenerics) are substantial with sales of biotechnology medicines in the United States rising 17% to approximately $30 billion in 2005 and growing at an annual rate of about 20% thereafter [3]. Not unexpectedly, the U.S. Food and Drug Administration (FDA) is experiencing mounting pressure to progress the cause of biogenerics. In a letter dated February 10, 2006, Senators Henry Waxman and Orrin Hatch (authors of the original “generic” legislation) urged the FDA to develop and implement clear guidelines for the approval of follow-on biological products for certain well-characterized proteins like insulin and human growth hormone (HGH) [5]. Additionally, recent litigation has compelled the FDA to take action on a pending drug application for a follow-on protein (FOP) drug product [Omnitrope, somatropin (recombinant DNA, rDNA origin)] [6]. A significant barrier to the emergence of “biogenerics” is the absence of a clear, efficient abbreviated pathway for approval. This hurdle is linked to significant scientific and legal issues in the United States in terms of how proteins are classified (drug vs. biologic) and subsequently regulated as well as how “generics” are tradi-

HISTORY OF BIOLOGICS REGULATION IN UNITED STATES

35

tionally defined in terms of equivalence and substitutability. However, an examination of the vast array of biologicals on the market today reveals that not all proteins are created equal. This range of complexity may provide an opportunity for stepwise progress on the regulatory front. This chapter presents the background to this multifaceted issue and examines the key regulatory challenges facing biogenerics today. An appropriate regulatory paradigm for the approval of FOPs is proposed and supported though a discussion of recent case studies. 1.2.1.1

Emergence of Biotechnology Industry

The explosion of scientific advances over the last quarter century has spawned the biotechnology industry and whole new classes of therapeutic agents for the treatment and prevention of disease. In October of 1982, the FDA approved the first protein-based therapeutic created by DNA technology in the form of Humulin (recombinant insulin). Developed by Eli Lilly & Co., with technical assistance from Genentech, Humulin is indicated for the treatment of diabetes. At the time, the use of recombinant technology was somewhat limited to the production of smaller, nonglycosylated proteins such as insulin (51 amino acids) and HGH (191 amino acids) using bacterial hosts. The seminal discovery by Columbia’s Richard Axel of the process of cotransformation enabled complex protein production and glycosylation and thus spurred the emergence of the modern biotechnology industry [7]. The phenomenal growth observed in the biotechnology sector is notable in terms of the extraordinary number and diversity of therapeutic peptides and proteins that have been developed within a period of only about 20 years. Examples of therapeutic proteins in current use include cytokines, clotting factors, vaccines, and monoclonal antibodies, as illustrated in Table 1 [8]. As presented in Table 2, many of these “early” biotechnology products have reached the end of their period of patent exclusivity [4–9]. Thus, it is appropriate to now consider the next steps in the “life cycle” of these products as potential generic drugs. 1.2.1.2

Challenges Facing “Biogenerics”

The diversity and complexity of biologic molecules that drive their utility as therapeutic agents also contribute to the difficulty in classifying them as pharmacological entities, namely, whether they are drugs or biologics. This difficulty in classification is of profound importance since there are fundamental differences in how the FDA regulates drugs and biologics. To appreciate the current challenges facing the pharmaceutical and biotechnology industry, it is informative to review the historical background associated with the classification and regulation of biologics in the United States, particularly in the context of the nation’s evolving drug regulation system.

1.2.2


Due to the scientific limitations of the early to mid-1900s, significant differences existed between the approaches taken to manufacture and analyze biologics and

36


TABLE 1 Examples of Therapeutic Peptide and Protein Molecules Currently Marketed in United States Peptides

Nonglycosylated proteins

Glycosylated proteins

Monoclonal antiobodies

Antibiotics: bacitracin, bleomycin, gramicidine, capreomycin Hormones: corticotropin, glucagon, gonadrolein HCl, leuprolide acetate, histrelin acetate, oxytocin, secretin, goserelin acetate, vassopressin Others: polymixin B, eptifibatide, cyclosporine Interleukins: andresleukin (IL-1), denileukin diftitox (fusion, proteinIL-2+ DT), anakinra (IL-2) Interferons: interferon alpha-n1, interferon alpha-n3, interferon alpha2a, peg interferon alfa-2b, interferon alfacon-1, Interferon alpha-2b, interferon beta-1b, interferon gamma-1b, Enzymes/inhibitors: anistreplase, asparaginase, lactase, trypsin, alpha1 proteinase inhibitor, urokinase, deoxyribonuclease, fibrinolysin, chymotrypsin, pancreatin, papain, urokinase Growth factors/hormones: Filigrastim pegfilgrastim, somatropin, becaplermin, somatrem, menotropins Antithrombotic agents: thrombin, fibrinogen, hirudin, hirulog, fibrin Others: insulin, gelatin, prolactin, albumin (human), hemoglobin, collagen Interferon beta-1a Antithrombotic agents: alteplase, drotrecogin alfa, antithrombin III Antianemic: darbopoetin alfa, erythropoietin Growth hormones: follitropin alpha, follitropin beta, chorionic gonadotropin (Human) Immuno globulins (IG): pertusssis IG, rabies IG, tetanus IG, hepatitis B IG, varicella zoster IG, rho(D) IG, normal immune globulin, lymphocyte anti-thymocyte, IB (equine) Coagulation factors: factor VII antihemophilic factor, factor IX (human, recombinant) Factor VIII (others): etanercept (CSF), sargramostim (TNF) avciximab, alemtuzamub, basiliximab, gentuzumab, satumomab, inflixibam, palivizumab

drugs. This reality led to the creation of separate and distinct regulatory pathways for drugs and biologics. As noted earlier, the developments in analytical chemistry and improvements in process technologies have, in recent times, blurred the lines between drug and biologic drug development. In the current era of pharmaceutical development and standards harmonization, one might question the continued need for two distinct pathways. Recognizing the shifting paradigm of drug development, the history of biologics regulation is discussed below in two parts: early history and present day. 1.2.2.1

Early Biologics Regulation (1800s–1990s)

This country’s earliest experience with biologics dates back to the infectious scourges of the late 1800s and early 1900s when epidemics of typhoid, yellow fever, smallpox, diphtheria, and tuberculosis were being battled by new advances in immunology. The discovery and development of vaccines and antitoxins led to the creation of a


TABLE 2

Patent Expiration Dates for U.S. Marketed Biologics

Brand Name

Generic Name

Indication

Company

Humulin Nutropin Abbokinase Ceredase Cerezyme Streptase Intron A Serostim Humatrope Geref

Recombinant insulin Somatropin Eudurase urokinase Alglucerase Imiglucerase Streptokinase IFN-α-2b Somatropin Somatropin Sermorelin

Eli Lilly Genentech Abbott Genzyme Genzyme AstraZeneca Biogen/Roche Serono Eli Lilly Serono

Synagis

Palivizumab

Novolin Protropin

Human insulin Somatrem

TNKase

Tenecteplase TNK-tPA IFN-γ-1b

Diabetes Growth disorders Ischaemic events Gaucher disease Gaucher disease Ischaemic events Hepatitis B and C AIDS wasting Growth disorders Growth hormone deficiency Respiratory syncytial virus Diabetes Growth hormone deficiency Acute myocardial infarction Chronic granulomatous disease; malignant osteoporosis Acute myocardial infarction HIV Anemia

Actimmmune

Activase, Alteplase Proleukin Epogen, Procrit, Eprex Neupogen

37

tPA IL-2 Erythropoietin

Filgrastim (G-CSF)

Anemia, leukemia, neutropenia

Patent Expiry

Novo Nordisk Genentech

Expired Expired Expired Expired Expired Expired Expired Expired Expired Expired (2004) Expired (2004) 2005 2005

Genentech

2005

InterMune

2005, 2006, 2012

Genentech

2005, 2010

Chiron Amgen

2006, 2012 2013

Amgen

2015

Abbott

Note: Based on our search of available patent sites for only the reference product. IFN-Interferon; tPA-Tissue Plasminogen Activator, IL-interleukin; HIV-Human Immunodeficiency Virus; G-CSF- Granulocyte-Colony Stimulating Factor; TNKase- Tenecteplase.

whole new “biopharmaceutical” industry. As demand increased, the pharmaceutical manufacturers responded and in turn supplanted the government’s role in the public supply of vaccines (per Vaccine Act of 1813) [10]. Unfortunately, the commercialization of vaccines by smaller, less experienced, and likely less scrupulous manufacturers led to problems. Similar to the history of drug regulation, early advances in biologics regulation could be characterized as responsive rather than proactive. Change often occurred following tragedy and the result of government’s attempt to respond. Some of the key milestones of early biologics regulation are summarized in Table 3. The following years saw many administrative changes in terms of the specific governmental agency responsible for regulating biologics, but with few substantive changes to the regulations themselves.

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TABLE 3 1901

1902

1906

1919

1937 1938

1941

1944

Key Milestones in Early Biologics Regulation

Ten children died in St. Louis from administration of tetanus-contaminated diphtheria antitoxin. In this case, no safety testing had been performed prior to use. Biologics Control Act (BCA) signed into law: • Authorizing the regulation of commercial viruses, serums, toxins, and analogous products • Requiring the licensure of biologics manufacturers and establishments • Providing governmental inspectional authority • Making it illegal for the commercial distribution of product not manufactured and labeled in accordance with the act Pure Food and Drug Act enacted (precursor of modern-day drug regulation). Lack of mention of biologics as a class effectively represents first distinction between drug and biologic regulation. BCA amended: • Required reporting of changes in equipment, manufacturing processes, personnel; establishment of formal quality control procedures; and submission of samples for regulatory inspection and approval for release • Recognized potential that slight changes to manufacturing conditions (raw materials, process, personnel, etc.) could have significant and adverse effect on product quality • Required strict control of input (environment and manufacturing conditions) rather than end-stage testing of quality attributes due to limitations in analytical methodology to detect these effects Elixir sulfanilamide, containing the poisonous solvent diethylene glycol, kills 107, many of whom are children. Food, Drug and Cosmetic Act (FDCA) enacted: • Established concept of “new drugs” requiring proof of safety prior to marketing • Required submission of an investigational new Drug (IND) application prior to clinical use of an experimental drug in humans • Required approval of a new drug application (NDA) prior to commercial sale of drugs • Granted federal government power of seizure of misbranded or adulterated drugs • Defined “drugs” comprehensively; not excluding potential of “biologics” to function as drugs • Approximately 300 deaths and injuries result from distribution of sulfathiazole tablets tainted with the sedative phenobarbital. • Insulin Amendment passed to require FDA testing/certification of purity and potency. Public Health Service (PHS) Act enacted to consolidate and codify previous biologics laws: • Outlined licensing requirements for biologics—for both product (product licensing application, or PLA) and establishment where the product was manufactured (establishment licensing application, or ELA) • Required submission of samples of each manufactured lot of all biologicals for government testing and certification prior to commercial release • Required sponsors to own all of manufacturing facilities, effectively eliminating multiparty or contract manufacturing


1.2.2.2

39

Modern Biologics Regulation (1990s–Today)

Whereas early biologics regulation was grounded by technical limitations, modern biologics regulation is driven by tremendous advances in scientific knowledge. Development of analytical tools and techniques has dramatically increased the ability to characterize proteins and substantiate the structure, composition, and function of the therapeutic molecule. These advances enable the detection of small differences in molecular weight; elucidation of primary, secondary, and tertiary protein structures; detection and quantification of posttranslational modifications (i.e., patterns of glycosylation); and improved understanding of structure–function relationships and potential immunogenic responses. Simultaneously, developments in the fields of pharmaceutical and biotechnological manufacturing have greatly improved process efficiency and control. This recent technological evolution has had a direct impact on biologics regulation as reflected below in several key events: •

•

•

•

In 1995, the FDA agreed to eliminate lot testing requirements for certain highly characterized products once the company’s ability to consistently manufacture product of acceptable quality was established. In 1996, the FDA and Congress dismantled the dual-licensing process, requiring the submission of a single BLA (biologics license application), making the content and format of a biologics application similar to that required for new drug applications (NDAs). In 1996, the Center for Biologics Evaluation and Research (CBER) liberalized its definition of “legal manufacturer” and eliminated many of the barriers to cooperative, multiparty manufacturing arrangements. In 1997, Congress passed a noteworthy piece of legislation affecting modern pharmaceutical regulation in the Food and Drug Modernization Act (FDAMA). Among the many goals of the act was to harmonize the drug and biologic approval processes.

In fact, current pharmaceutical/regulatory initiatives appear to extract the best practices from biologic and drug approaches which can apply equally to both classes of products: •

•

•

The Quality Systems Approach and GMPs for the 21st Century, two initiatives being pursued by the FDA for drugs and devices, emphasize the utility of building quality into the process, consistent with the strict control of “input factors” seen in early biologic regulation. Initiatives such as Process Analytical Technologies build on the concept of conventional drug product testing using increasingly sophisticated analytical techniques to provide continuous process monitoring and finished-product quality assurance of multiple pharmaceutical dosage forms. The current global initiative to harmonize electronic submission format and content requirements effectively creates one standard data package for drugs or biologics. Thus, the eNDA (electronic new drug application) or eBLA (electronic biologics license application) will eventually be replaced by the eCTD (electronic common technical document).

40


1.2.3

REGULATORY CLASSIFICATION OF PROTEINS

Despite the blurring of lines between drugs and biologics, there remain two different mechanisms to bring protein drug products to the U.S. marketplace. The choice of approval framework is dependent on the protein’s classification as a drug or biologic. The history of this regulatory distinction is rooted in the technical differences between small-molecule drugs and macromolecular biologics. Traditionally, drugs were characterized as having well-defined chemistry. Conversely, biologics were large, complex macromolecules whose active moiety defied characterization and quantitation. By necessity, different means of assuring the safety and efficacy of these therapeutic products were required at the time. The modern-day consequence is a legal system that distinguishes between proteins as drugs and proteins as biologics. The distinction is based on statutory definitions as well as historical precedent and has implications in terms of the approval pathways for original and follow-on products. 1.2.3.1

Definitions and Key Terminology

Drugs are defined by the U.S. Food and Drug Act [FD&C Act, 21 U.S.C. 321(g)(1)]by function as any article Federal Food, Drug and Cosmetic Act (a) intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease in man or animals and (b) intended to affect the structure or function of the body [11]. Biologics as a class may be regulated as drugs but are defined within the Public Health Service Act [PHSA, 42 U.S.C. 262(a)] by category as “a virus, therapeutic serum, toxin, antitoxin, vaccine, blood, blood component or derivative, allergenic product, or analogous product, or arsphenamine (or any other trivalent organic arsenic compound), applicable to the prevention, treatment, or cure of diseases or injuries of humans” [12]. A cursory examination of these definitions reveals that they are not mutually exclusive, leading to confusion about how to appropriately and consistently apply them. This point is illustrated when one reviews the history of how the FDA has categorized and subsequently regulated these drugs and biologics as shown below. 1.2.3.2 Application of Definitions to Proteins: Is It a Drug or a Biologic? The answer to this fundamental question is not straightforward and has evolved over time. Historically, some natural-source-derived proteins such as insulin, hyaluronidase, menotropins, and Human Growth Hormone (HGH) have been regulated as drugs. While other natural-source-proteins such as blood factors were regulated as biologics. When recombinant proteins and monoclonal antibodies began development in the 1970s–1980s, these were regulated as follows: 1. By the Center for Drug Evaluation and Research (CDER) under the Food, Drug and Cosmetic Act (FDCA) as drugs when they were hormones such as insulin, HGH, and parathyroid hormone (PTH) derivatives 2. By the CBER under the PHSA as biologics when they were cytokines or blood factors such as factor VIII for hemophilia

REGULATION OF GENERIC DRUGS

TABLE 4

41

FDA Center Regulatory Responsibility for Therapeutic Biological Products

CDER

CBER

Monoclonal antibodies (in vivo use) Proteins intended for therapeutic use: Cytokines (e.g., interferons) Enzymes (e.g., thrombolytics) other novel proteins except those assigned to CBER Immunomodulators (nonvaccine, nonallergenic) Growth factors, cytokines, some hormones and monoclonal antibodies intended to mobilize, stimulate, decrease, or otherwise alter the production of hematopoietic cells in vivo

Cellular product, including products composed of human, bacterial, or animal cells Vaccines Allergenic extracts Antitoxins, antivenoms, venoms Blood, blood components, plasmaderived products (e.g., albumin, immunoglobulins, clotting factors, fibrin sealants, proteinase inhibitors), recombinant and transgenic versions of plasma derivatives

As other recombinant proteins and monoclonal antibodies came under development, the CBER held primary responsibility for this review, with the CDER retaining responsibility for hormones such as insulin and HGH. However, in 2003 all therapeutic proteins were transferred from the CBER to the CDER. This reassignment of review responsibility did not impact the legal classification of these protein products, such that the Center for Drug Evaluation and Research assumed responsibility for the review and approval of biologics approved under Section 351 of the PHSA. The basic distribution of these therapeutic biologics to the respective FDA center is reflected in Table 4; however, many of the current complex biotechnology-derived products do not fit neatly into accepted definitions and require case-by-case classification [13]. 1.2.3.3

Regulatory Approval Path for Proteins

The relevance of the preceding discussion becomes important with the understanding that therapeutic proteins classified as drugs are governed under a different set of laws than those classified as biologics. Drugs are approved via submission of NDAs under Section 505 of the FD&C Act, while biologics are supported by BLAs under the PHSA. These two approval paths are similar in terms of application content, that is, requirement of complete reports of clinical safety and efficacy data to support approval. However, only the drug regulation, that is, Section 505 of the FD&C Act, has been amended to outline an abbreviated approval mechanism for generic products.

1.2.4 1.2.4.1

REGULATION OF GENERIC DRUGS History of Generic Drug Legislation in United States

In 1984, Congress responded to America’s need for safe, affordable medicines by passing a pivotal piece of legislation, The Drug Price Competition and Patent Term

42


Restoration Act (Hatch–Waxman amendments). The intent of this act was to effectively balance the need to encourage pharmaceutical innovation with the desire to accelerate the availability of lower cost alternatives to approved drugs. The act also sought to eliminate unnecessary or redundant clinical testing to protect patients (reduce the number of patients in need receiving placebo in controlled clinical trials) and conserve industry and agency resources. To accomplish the goal of faster to market, cheaper alternatives, the amendments stipulated the following [14]: •

•

For Innovator Companies The act encouraged continued innovation, research, and development activities by providing manufacturers with meaningful incentives in the form of patent protection/restoration and marketing exclusivity, thus allowing them to recoup some of their investments. For Generic Companies The act provided access to certain innovator information without the threat of legal action via patent infringement suits (safe harbor provisions), allowing generics the opportunity to prepare for market introduction prior to the expiration of patent/exclusivity terms. This effectively limited the period of innovator exclusivity to the statutory timelines.

1.2.4.2 Approval Process for Generic Drugs The act served as a boon to the generic industry by paving the path to abbreviated and accelerated drug approvals. From a legal perspective, the Hatch–Waxman amendments modified Section 505 of the FD&C Act to create two new abbreviated approval pathways (see Table 5) [14]. In essence, the abbreviated NDA (ANDA) and 505(b)(2) processes allow generic manufacturers the ability to rely on what is already known about the drug and refer to the agency’s finding of safety and efficacy for the innovator. For an ANDA, the generic product must meet certain criteria related to bioequivalence and product sameness. However, a 505(b)(2) application often describes a drug with substantial differences to the innovator (which would seem more closely related to FOPs). 1.2.4.3 Application of Generic Regulations to Biologics A central question is “Do biologics fall under the provisions of the Hatch–Waxman Act?” Since the Hatch–Waxman Act specifically amended the FD&C Act, biologics TABLE 5

Description of NDA Approval Mechanisms

Traditional path

Abbreviated path

Abbreviated path

1. 505(b)(1)—Application that contains full reports of investigations of safety and effectiveness to which sponsor has right of reference (stand-alone NDA) 2. 505(b)(2)—Application that contains full reports of investigations of safety and effectiveness, where the sponsor relies on studies conducted by someone else to which the sponsor does not have right of reference 3. 505(j)(1)—Abbreviated new drug application (ANDA) containing information to show the product is a duplicate of an already approved drug product

LEGAL ARGUMENTS RELATED TO FOLLOW-ON PROTEINS

43

approved via a BLA under the PHSA are not covered by this legislation nor does the PHSA have similar provisions for biogenerics. However, those few therapeutic proteins approved via Section 505 of the FDCA as NDAs are covered by the Hatch–Waxman amendments and thus are legally considered appropriate for filing a 505(b)(2) or 505(j)(1) application. For simple, well-characterized peptides and proteins regulated under Section 505 of the FD&C Act, mechanisms are already in place to bring FOPs to the market. In fact, several FOPs have already been approved by the FDA, including GlucaGen (glucagon recombinant for injection), Hylenex (hyaluronidase recombinant human), Hydase and Amphadase (hyaluronidase), Fortical (calcitonin salmon recombinant) Nasal Spray, and Omnitrope [somatropin (rDNA origin)] [15]. Further details related to the latter two are presented in the discussion of actual case studies.

1.2.5

LEGAL ARGUMENTS RELATED TO FOLLOW-ON PROTEINS

The legal arguments regarding the approval of biogenerics relate to several different aspects of drug/biologics law. 1.2.5.1

Constitutionality of 505(b)(2) Process for Drugs

The agency’s authority to grant approval of drugs via the 505(b)(2) process has previously been challenged by several companies. The nature of these challenges has questioned the FDA’s right to use proprietary information of the innovator in support of another company’s drug approval. Recall that the 505(b)(2) process allows a company to use data for which it does not have right of reference (i.e., another company’s safety and efficacy data) in support of its own application. The FDA’s long-standing interpretation of the statute seems firm and well founded in precedent since over 80 applications for drugs have been approved via the 505(b)(2) route since its inception with indications ranging from cancer pain to Attention Deficit Disorder (ADD) [16]. 1.2.5.2

Constitutionality of 505(b)(2) Process for Follow-On Proteins

The constitutionality issues related to FOPs are similar to those mentioned above for drugs, namely protection of proprietary information and intellectual property rights. Some critics opine that issues unique to FOPs create additional legal hurdles. For example, the rules pertaining to the disclosure of safety and effectiveness information are different for biologics licensed under the PHSA and drugs approved under the FDCA. When the rules were originally written (1974), it was thought that safety and effectiveness for one biologic would not support the licensure of another. So these data were deemed not to be protected trade secrets and could be publicly disclosed immediately after issuance of the biologic’s license [see 21 CFR 601.51(e), 1974]. However, since this language applies strictly to the PHSA, it has no bearing on discussions related to the 505(b)(2) process. In other public challenges, opponents argue that the unique and complex nature of biologics and the close relationship between their method of preparation and clinical attributes require that the FDA use and disclose the manufacturing methods

44


and process information contained in an innovator’s application. Further, this use and disclosure would violate Trade Secret and Constitutional Law (Fifth Amendment “taking clause”) [17, 18]. The concept of “the product is the process” may have been applicable to early biologics, but current capabilities allow the chemical, biologic, and functional comparison of well-characterized protein drugs. The follow-on manufacturer need not necessarily utilize the identical method of manufacture or proprietary technology to reproduce a follow-on biologic with similar clinical safety and efficacy. Additionally, it is important to distinguish between the regulatory requirements for approval of an actual generic protein (duplicate of innovator; see discussion below) and those associated with a 505(b)(2), which requires a showing of similarity between two products. Any differences between the two would need to be adequately supported by bridging studies and appropriate clinical and/or nonclinical data. The FDA has confirmed this interpretation in its response to petitions filed regarding FOPs (both in general and targeted to specific applications). The FDA has clearly said, “the use of the 505(b)(2) pathway does not entail disclosure of trade secret or confidential commercial information, nor does it involve unauthorized reliance on such data” [18]. 1.2.5.3 Applicability of 505(j)(1) or ANDA Process to Biogenerics Biogenerics per se, that is, protein drug products approved via 505(j)(1), would need to demonstrate their bioequivalence to the innovator protein. However, due to their complexity and heterogeneity, the classical biopharmaceutical principles upon which the current ratings of therapeutic equivalence are based do not apply in their current language to complex macromolecules. For example, due to the nature and complexity of an immunogenic response, one concern would be if traditional bioequivalence appropriately addresses the complex safety issues associated with biologics. 1.2.5.4

Current Rules Relating to Bioequivalence of Generic Drugs

The list of approved drug products with therapeutic equivalence (Orange Book) was originally intended as an information source to states seeking formulary guidance [19]. The list provides the FDA’s recommendations as to which generic prescription drug products are acceptable substitutes for innovator drugs. The term innovator is used to describe the reference listed drug, or RLD [21 CFR 314.94(a)(3)], upon which an applicant (generic) relies in seeking approval of its ANDA. In layman’s terms the RLD describes the original NDA-approved drug and is often referred to as the “pioneer” drug. Under the Drug Price Competition and Patent Term Restoration Act of 1984, manufacturers seeking approval to market a generic drug need to submit data to the FDA demonstrating that their proposed drug product is bioequivalent to the pioneer (innovator) drug product. A major premise underlying the 1984 law is that bioequivalent drug products are therapeutically equivalent, will produce the same clinical effect and safety profile as the innovator product, and are therefore, interchangeable [19]. So how would FOPs be classified using conventional definitions of bioequivalence? To answer this question, it is necessary to review current legal definitions of bioequivalence terms [19]:

LEGAL ARGUMENTS RELATED TO FOLLOW-ON PROTEINS •

•

•

45

Two products are bioequivalent in “ the absence of a significant difference in the rate and extent to which the active ingredient or active moiety in pharmaceutical equivalents or pharmaceutical alternatives becomes available at the site of drug action when administered at the same molar dose under similar conditions in an appropriately designed study” [21 CFR 320.1(e)]. An appropriately designed comparison could include (1) pharmacokinetic (PK) studies, (2) pharmacodynamic (PD) studies, (3) comparative clinical trials, and/or (4) in vitro studies. Pharmaceutical equivalents are those drug products which are formulated to contain the same amount of active ingredient in the same dosage form to meet the same (compendial or other applicable) standards of quality. Pharmaceutical alternatives are drug products that contain the same therapeutic moiety, or its precursor, but not necessarily in the same amount or dosage form. Drug products are considered to be therapeutic equivalents only if they are pharmaceutical equivalents and if they can be expected to have the same clinical effect and safety profile when administered to patients under the conditions specified in the labeling. Although pharmaceutical alternatives may ultimately be proven bioequivalent, given their differences they are not automatically presumed to be.

Given these definitions, FOPs would likely be considered pharmaceutical alternatives if one presumes that pioneer and follow-on proteins are identical at a precursor stage, prior to potential post-translational modification. This presumption may also be consistent with the similarity standard the agency applies to ascertain orphan drug status (see discussion in Section 1.2.6). Follow-on proteins cannot be considered to be therapeutic equivalents since they are not pharmaceutical equivalents and cannot be expected to have the same clinical effect and safety profile in the absence of testing. This assertion is supported by the following: •

•

The potential impact of how posttranslational modifications, such as glycosylation, can directly impact protein conformation and subsequently affect biological activity, including the overall safety and efficacy of the drug product. An underlying premise of bioequivalence assessments is a clearly defined pharmacokinetic/pharmacodynamic relationship; however, the relation between blood levels and effect is less clearly established for proteins [20].

Consequently, within the current regulatory framework, FOPs are unique products that may be “similar” but are not the same as innovator proteins, consistent with their approval via a 505(b)(2) pathway. This interpretation is supported by the FDA’s designation of Omnitrope as having a BX rating in the Orange Book. The code BX in the Orange Book refers to drug products for which the data are insufficient to determine therapeutic equivalence as compared to a therapeutic rating of A indicative of interchangeability. This concept of similarity is also consistent with the definitions proposed by the European Agency for the Evaluation of Medicinal Products (EMEA) for generic versions of proteins [21]: Bio-similar products: second and subsequent versions of biologics that are independently developed and approved after a pioneer has developed an original version. Bio-similar products may or may not be intended to be molecular copies of the innovator’s product; however, they rely on the same mechanism of action and therapeutic indication.

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1.2.5.5

Statutory Authority

Unlike the FDCA, which affords therapeutic protein drugs the legal pathway of abbreviated drug approval for a FOP, the PHSA currently has no similar provisions. Such a pathway for approval or licensure of FOP products under the PHSA would require new legislation and recent congressional developments suggest that work is underway to create this statutory pathway. Legislation proposed on September 29, 2006, by U.S. Representative Henry Waxman (D-CA) and Senator Charles Schumer (D-NY) seeks to amend the PHSA to authorize the FDA to approve abbreviated applications for biologic products that are “comparable” to previously approved (brand name) biologic products. Entitled The Access to Life-Saving Medicine Act, this bill outlines a process by which the FDA could determine, on a product-by-product basis, the studies necessary to demonstrate comparability of a FOP product to a brand name product and assure its safety and effectiveness. The act allows for an applicant to seek interchangeability with a brand name product, recognizing that the extent of data to support such a designation must be discussed with the FDA. To encourage the development of interchangeable products, the bill would authorize tax incentives and periods of marketing exclusivity. The bill would also seek to create an improved process to facilitate early resolution of patent disputes which might otherwise delay competition [22]. 1.2.6 SCIENTIFIC ISSUES RELATED TO FOLLOW-ON PROTEINS (DATA REQUIREMENTS) The challenge of FOPs demonstrating similar quality, safety, and efficacy to the innovator product relates to the poor predictability of physicochemical characteristics and biologic activity. For example, there are several different interferon-α and erythropoietin α and β products currently on the market. These variants are characterized by differences in sequence, glycosylation pattern, and in vitro measures of specific activity; however, their clinical safety and efficacy profiles are considered similar [20]. In contrast, different formulations of insulin and growth hormone containing the same active ingredient exhibit significant differences in bioavailability [20]. Additionally, the inability to adequately predict immunogenic responses from in vitro data or animal studies remains a concern. The answer to the challenge is that generic manufacturers must go through a similar process of in-depth characterization, including identification of critical structural elements of the product (structure/function) when developing a FOP. Although the regulatory standards for demonstrating similarity are currently undefined, some insight can be gleaned from consideration of FDA expectations in terms of granting orphan drug status to similar proteins and assessing postapproval Chemistry, Manufacturing and Controls (CMC) changes for innovator proteins. 1.2.6.1 “Sameness” as per Orphan Drugs Regulations The Orphan Drug Act of 1983 was implemented in response to the government’s concern that viable treatments for rare diseases were not being explored due to excessive costs of drug development in comparison to the relatively small popula-

SCIENTIFIC ISSUES RELATED TO FOLLOW-ON PROTEINS

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tion of potential users (and sales). Orphan drugs are (a) those used to treat rare diseases, defined by the act as affecting RCP =100%

3 0.0

1

19540 13027 6514

51.5 103.0 154.5 206.0 O F Distance (mm)

0 0.0 O

51.5 103.0 154.5 206.0 F Distance (mm)

FIGURE 9 Typical chromatograms for 99mTc-MDP. The left strip and chromatogram are obtained with ITLC-SG in sodium acetate. The right strip and chromatogram are obtained in methyl ethyl ketone (MEK). When combining these, any free pertechnetate (99mTcO4−) and/or hydrolyzed 99mTc can be detected. Thus the fraction representing 99mTc-MDP (RCP) can be calculated.

one test method, for instance, TLC and HPLC, to get a complete picture of the different radiochemical impurities. Alternatively, one can use one chromatographic method consisting of a constant stationary phase but varying the mobile phase (solvent). An example is the radiochemical purity test of 99mTc-methylenediphosphate (MDP), a radiolabeled phosphate used in bone scintigraphy. When using two TLC systems, one with sodium acetate as a solvent and one with methyl ethyl ketone (MEK) as a solvent, the different 99mTc compunds in the product can be determined. A small aliquot of the radiopharmaceutical preparation is spotted on an instant thin-layer chromatography (ITLC) strip. The strip is dipped into the chromatography flask while keeping the spot above the solvent. During the chromatography process, the different components of the sample distribute differently in the ITLC strip, depending on the solubility and polarity of the components. In systems like this, each component is characterized by an Rf value, defined as the ratio of the distance traveled by the component to the distance the solvent front has advanced from the original point of application of the test material. The distribution of the radioactive components on the strips can be monitored by use of an appropriate device for measuring radioactivity and printed in a chromatogram. Figure 9 shows typical chromatograms for 99mTc-MDP in the TLC systems described above. Chemical Purity The chemical purity of a radiopharmaceutical is the fraction of the material in the desired chemical form. Chemical impurities may arise from the breakdown of the material either before or after labeling. Chemical impurities may also arise from the manufacturing process, such as aluminum in a 99mTc eluate, coming from the aluminum column on the generator. Residuals of solvent from the radiopharmaceutical synthesis are also considered as chemical impurities. If the chemical impurity is present before labeling, the result may be undesirable labeled molecules. Furthermore, chemical impurities may cause a toxic effect. Highperformance liquid chromatography and gas chromatography (GC) are important methods for determination of chemical impurities in a radiopharmaceutical.

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Sterility and Pyrogen Testing Sterility indicates the absence of any viable bacteria or microorganisms in a radiopharmaceutical preparation. Hence, sterility testing is performed to prove that radiopharmaceuticals are essentially free of viable microorganism. The test for microbial contamination of these products is best carried out with filter methods. It is a great advantage to incubate only the filters instead of the radioactive solutions. The test is performed according to the Ph.Eur/USP monograph on Sterility tests [13, 14], but with an important modification. Small batch sizes, typical for radiopharmaceuticals, make it necessary to use smaller test volumes than required in the monographs. Also the risk for radiation exposure supports this modification. All radiopharmaceuticals for human administration are required to be pyrogen free. Also the tests for apyrogenicity must be modified when applied for these products. The classical rabbit test for pyrogens was never a convenient test for parenteral radiopharmaceuticals. Practical problems due to radioactive rabbits and the need for larger test volumes made this a difficult task. Today, the Limulus amebocyte test (LAL) is the method of choice and has been accepted by the Ph. monographs for many years. This test is normally done within an hour, compared to several days for the rabbit test. However, even the LAL test may be too time consuming for the very short lived PET radiopharmaceuticals. Hence, less time consuming methods are in progress and will probably improve this situation. Meanwhile, it is accepted that the test for apyrogenicity, like the sterility test is for most radiopharmaceuticals, is finished after release of the most short lived radiopharmaceuticals. Bubble Point Testing of Filters Parenteral radiopharmaceuticals that are not terminally sterilized must undergo a sterile filtration process as part of the aseptic production procedure. Although the supplier certifies the filters used, they must be checked for integrity after use to assure that there has been no leakage during the filtration. The integrity of the filter may be demonstrated by bubble point testing. In this test, the filter is placed and monitored under controlled pressure. When the test is done on wet filters, the pressure needed to push gas through the filter is defined as the bubble point. A filter with given pore width has a corresponding bubble point value. Most frequently, sterile filtration is performed by 0.22-μm filters; hence the bubble point is about 3–4 bars. However, the filter supplier should specify the bubble point valid for a specific filter. Since this is an in-process test, special caution must be given to radiation protection. The test equipment should be placed within a closed and shielded unit and a system should be in place to collect any radioactive spill from the test. When the filter integrity test fails, the sterile filtration process must be rejected. Visual Inspection of Finished Product As part of the quality control, all parenterals will be subject to an inspection for the possible content of particles. Visual inspection of radiopharmaceuticals is more complicated than for other pharmaceuticals, as radiation protection guidelines strongly discourage any direct eye contact with radioactive sources. Normally, the visual inspection of a radiopharmaceutical is performed by placing the vial on a rotating station connected to a camera. The station is properly shielded, and the operators can study the solution on a distant screen.

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1.3.5.4 Validation and Control of Equipment and Procedures Preventive maintenance, calibration, and qualification programs should be operated to ensure that all facilities and equipment used in the manufacture of radiopharmaceuticals are suitable and qualified (GMP, Annex 3). Special emphasis should be put on critical equipment for handling of radiopharmaceuticals, such as dose calibrators that are used to check the accuracy of the dispensing of patient doses. Particular programs are outlined for checking the dose calibrator, including constancy, accuracy, linearity, and geometry. The general principles of validation outlined in the GMP regulations are valid for radiopharmaceuticals as well as for other pharmaceuticals. All validation activities should be planned and clearly defined and documented in a validation master plan (VMP). Special emphasis should be given on the validation of aseptic processes in the production of radiopharmaceuticals. Studies, including media fill tests, must be performed and recorded to demonstrate maintenance of sterility throughout the production process. This is particularly important since most radiopharmaceuticals are dispatched and used before the sterility test is finished. 1.3.5.5

Stability Aspects of Radiopharmaceuticals

As discussed already, radiopharmaceuticals are exposed to stability problems, particularly when radiolabeled compounds are involved. Decomposition of labeled compounds by radiolysis depends on the specific activity of the radioactive material, the energy of the emitted radiation, and the half-life of the radionuclide. Particles, such as α and β radiation, are more damaging than γ rays, due to their short range and local absorption in matter. The stability of a compound is time dependent on exposure to light, change in temperature, and radiolysis. The longer a compound is exposed to these conditions, the more it will tend to break down. Stabilizers such as ascorbic acid and benzyl alcohol may be added to inhibit or delay the decompostion. Many preparations are stored in the dark under refrigeration to slow down the degradation of the material [2]. The expiry date of a radiopharmaceutical is based upon data from stability studies designed to demonstrate the described effects on the product after storage. Hence, for most stability studies on radiolabeled compounds, the radiochemical purity and pH are the most important physiochemical parameters to study. Moreover, for parenteral radiopharmaceuticals, a stability study also has to demonstrate the maintenance of sterility and apyrogenicity after storage. 1.3.6 EXTEMPORANEOUS PREPARATION OF RADIOPHARMACEUTICALS An extemporaneous preparation is defined as a product which is dispensed immediately after preparation and not kept in stock [10]. Hence, many radiopharmaceuticals could fall into this category due to their limited shelf life. The use of extemporaneous preparation should be limited to situations where there is no product with marketing authorization (MA) available. This could be prepared based upon a prescription for a named patient (magistral preparation) or a production based upon a formula and prepared on a regular basis. The latter is a

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common situation for many radiopharmaceuticals. For radiopharmaceuticals with short half-lives or rare indications, no sizable commercial market exists. Consequently, no pharmaceutical company will be prepared to obtain a MA for a product that will not yield a profit due to these limitations. Still, there is a need from a medical point of view to have such products available. For radiopharmaceuticals incorporating radionuclides with a physical half-life of only a few minutes, only local production is feasible. They are therefore prepared in hospital pharmacies or laboratories and supplied for individual or small numbers of patients on a daily basis. The extemporaneous preparation of radiopharmaceuticals is regulated on a national level, and hence this regulation may differ from country to country. The Pharmaceutical Inspection Convention (PIC/S) has drafted a guide to good practices for preparations of medicinal products in pharmacies [10], valid for medicinal products that do not have a MA, prepared extemporaneously or for stock. For medicinal products prepared to a larger extent or for use in clinical trials, industrial GMPs are applicable. Although the suggested guide outlines a general principle according to GMP, different requirements are particularly evident when it comes to documentation and quality control testing. There is also a discussion about the grades of background environment needed for production, with a differentiation between products with shelf lives less than or longer than 24 h [10]. While aseptic manufacturing according to industrial GMP has to be performed in grade A with a grade B background, this proposed guide opens for a relaxation to this. For an aseptic preparation of a product with a shelf life of less than 24 h, using a biohazard safety cabinet (BSC), the background environment may be grade D. Even for products with a shelf life longer than 24 h, an extensively documented procedure may allow grade C in background, as long as grade B clothing is worn. In general, the referred draft guide is based much upon a risk related approach and is graduated, depending on the size and type of prepared medicinal products. As to documentation for extemporaneous prepared products, the proposed guide set as a minimum requirement to specify the name, strength, and expiry date of the product. If a product is prepared for a single patient (magistral production), it is assumed that no end product testing will be required. For radiopharmaceuticals, though, the activity in each dose must be measured before administration. Chemical and microbiological quality control is not required for products that have a shelf life of 24 h or less, provided that frequent process validation is performed. In addition, chemical and microbiological information must be available to justify the shelf life for the product. For products that are prepared extemporaneously at a regular basis or even for a limited stock, a product specific documentation (product file) is needed. This will include specifications, instructions, and records but also a pharmaceutical assessment of safety data, toxicity, biopharmaceutical aspects, stability, and product design. The product file should also include a product review as soon as a product is used repeatedly or over longer periods. Furthermore, the drafted guide suggests that the level of end-product testing for those products will depend on the associated risk connected to the scale of operation, shelf life of the product, frequency of preparation, as well as type of product (parenterals, orals) and type of facility where the product has been prepared.

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Independent of which regulation applies at a national level to extemporaneous or magistral preparation of radiopharmaceuticals, the patients should be entitled to expect that these products are prepared accurately, are suitable for use, and will meet the expected standards for quality assurance. Pharmacists involved in this kind of production must ensure that they and any other staff involved are competent to undertake the tasks to be performed and that the requisite facilities and equipment are available [11]. As for other radiopharmaceutical production, systems must be in place to ensure the operator safety due to handling of radioactive materials. All involved staff must have sufficient training in radiation safety issues, in addition to training in GMP. REFERENCES 1. Britton, K. (1996), Radiopharmaceuticals for the future, Curr. Dir. Radiopharma. Res. Dev. (Ed. by Stephen Mather), viii. Developments in Nuclear medicine, Vol XXX. London, UK. 2. Saha, G. B. (1998), Fundamentals of Nuclear Pharmacy, 4th ed., Springer, Heidelberg, Germany. 3. Alexoff, D. L. Automation for the synthesis and application of PET radiopharmaceuticals, BNL-68614 Officinal File Copy. 4. Bremer, P. O. (1995), Aseptic production of radiopharmaceuticals, in Aseptic Pharmaceutical Manufacturing, Vol. II, Application for the 1990s, Interpharm, Michael J. Groves and Ram Murty, pp. 153–180. 5. Nordic Council on Medicines. (1989), Radiopharmacy: Preparation and Control of Radiopharmaceuticals in Hospitals, NLN Publications No. 26, Uppsala, Sweden. 6. European Commision (2003), EU Guide to Good Manufacturing Practice, Annex 1 and 3, Brussels, Belgium, October 8. 7. Dabbah, R. (1995), Controlled environments in the pharmaceutical and medical products industry: A global review from regulatory, compendial, and industrial perspectives, in Aseptic Pharmaceutical Manufacturing, Vol. II, Application for the 1990s, Interpharm, Michael J. Groves and Ram Murty, pp. 11–40. 8. Lee, M. C., PET and PET/CT are the fastest growing imaging modalities worldwide, paper presented at the 5th International Conference on Isotopes (5ICI), Brussels, Belgium, Apr. 25–29, 2005. 9. Zalutsky, M. R., Pozzi, O., and Vaidyanatha, G., Targeted radiotherapy with alpha particle emitting radionuclides, paper presented at the International Symposium on Trends in Radiopharmaceuticals (ISTR-2005), Vienna, Austria, Nov. 14–18, 2005. 10. Pharmaceutical Inspection Convention (2006, Aug.), PIC/S guide to good practices for preparation of medicinal products in pharmacies, PE 010-1 (Draft 2), Geneva, Switzerland. 11. Standards for good professional practice (2000), Pharm. J. 265(7109), 233. 12. Kowalsky, R. J., and Falen, S. W. (2004), Radiopharmaceuticals in Nuclear Pharmacy and Nuclear Medicine, American Pharmacists Association, Forrester Center, WV.

FURTHER READINGS European Commision. (2006), EU Guide to Good Manufacturing Practice, Annex 3; draft proposal, Brussels, Belgium, Apr. 12.

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Rootwelt, K. (2005), Nukleærmedisin, 2nd ed. Gyldendal Norsk Forlag AS, Oslo, Norway. Schwochau, K. (2000), Technetium: Chemistry and Radiopharmaceutical Applications, VCH Verlagsgesellschaft Mbh, Weinheim, Germany. Welch, M. J., and Redvanly, C. S., Eds. (2002), Handbook of Radiopharmaceuticals, Wiley, Hoboken, NJ.

SECTION 2 ASEPTIC PROCESSING

2.1 STERILE PRODUCT MANUFACTURING James Agalloco1 and James Akers2 1

Agalloco & Associates, Belle Mead, New Jersey Akers Kennedy & Associates, Kansas City, Missouri

2

Contents 2.1.1 2.1.2

2.1.3

2.1.4 2.1.5

Introduction Process Selection and Control 2.1.2.1 Formulation and Compounding 2.1.2.2 Primary Packaging 2.1.2.3 Process Objectives Facility Design 2.1.3.1 Warehousing 2.1.3.2 Preparation Area 2.1.3.3 Compounding Area 2.1.3.4 Aseptic Compound Area (If Present) 2.1.3.5 Aseptic Filling Rooms and Aseptic Processing Area 2.1.3.6 Capping and Crimping Sealing Areas 2.1.3.7 Sterilizer Unload (Cooldown) Rooms 2.1.3.8 Corridors 2.1.3.9 Aseptic Storage Rooms 2.1.3.10 Lyophilizer Loading and Unloading Rooms 2.1.3.11 Air Locks and Pass-Throughs 2.1.3.12 Gowning Rooms 2.1.3.13 Terminal Sterilization Area 2.1.3.14 Inspection, Labeling, and Packaging Aseptic Processing Facility Alternatives 2.1.4.1 Expandability Utility Requirements 2.1.5.1 Water for Injection 2.1.5.2 Clean (Pure) Steam 2.1.5.3 Process Gases 2.1.5.4 Other Utilities


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2.1.6

Sterilization and Depyrogenation 2.1.6.1 Steam Sterilization 2.1.6.2 Dry-Heat Sterilization and Depyrogenation 2.1.6.3 Gas and Vapor Sterilization 2.1.6.4 Radiation Sterilization 2.1.6.5 Sterilization by Filtration Facility and System: Qualification and Validation Environmental Control and Monitoring 2.1.8.1 Sanitization and Disinfection 2.1.8.2 Monitoring Production Activities 2.1.9.1 Material and Component Entry 2.1.9.2 Cleaning and Preparation 2.1.9.3 Compounding 2.1.9.4 Filling 2.1.9.5 Stoppering and Crimping 2.1.9.6 Lyophilization Personnel Aseptic Processing Control and Evaluation 2.1.11.1 In-Process Testing 2.1.11.2 End-Product Testing 2.1.11.3 Process Simulations Terminal Sterilization Conclusion Appendix References Additional Readings

2.1.7 2.1.8

2.1.9

2.1.10 2.1.11

2.1.12 2.1.13

2.1.1

INTRODUCTION

The manufacture of sterile products is universally acknowledged to be the most difficult of all pharmaceutical production activities to execute. When these products are manufactured using aseptic processing, poorly controlled processes can expose the patient to an unacceptable level of contamination. In rare instances contaminated products can lead to microbial infection resulting from products intended to hasten the patient’s recovery. The production of sterile products requires fastidious design, operation, and maintenance of facilities and equipment. It also requires attention to detail in process development and validation to ensure success. This chapter will review the salient elements of sterile manufacturing necessary to provide acceptable levels of risk regarding sterility assurance. Commensurate with the criticality associated with sterile products, the global regulatory community has established a substantial number of the basic requirements that firms are expected to adhere to in the manufacture of sterile products. The most extensive of these are those defined by the Food and Drug Administration

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(FDA) in its 2004 Guideline on Sterile Drug Products Produced by Aseptic Processing and the European Agency for the Evaluation of Medicinal Products (EMEA) Annex 1 on Sterile Medicinal Products [1, 2]. Substantial additional information is available from the International Organization for Standardization (ISO), the Parenteral Drug Association (PDA), and the International Society for Pharmaceutical Engineering (ISPE) (see Appendix) [3]. The organizations have provided a level of practical, experience-based detail not found in the regulatory documents, thereby better defining practices that are both compliant with regulatory expectations and based upon rational, evidence-based science and engineering. Consideration of patient risk associated with pharmaceutical production emerged largely from regulatory impetus, by which the regulatory community stated its intended goal to structure its inspectional process using patient safety as a major focus in determining where to allocate their inspectional and review resources. Emanating from the International Conference on Harmonization (ICH) efforts to produce a harmonized approach to pharmaceutical regulation, risk-based compliance has been adopted in Europe, Japan, and the United States [4, 5]. Sterile products, especially those made by aseptic processing, have been properly identified as a high priority by the global regulatory community. Several risk analysis approaches have been developed that can help the practitioner review practices with the goal of minimizing risk to the patient [6–8].

2.1.2


The production of sterile products is profoundly impacted both by formulation and the selection of primary packaging components. Design parameters for a facility and selection of appropriate manufacturing technologies for the product require that the formulation process and packaging components be chosen and evaluated in advance. 2.1.2.1

Formulation and Compounding

The vast majority of parenteral formulations are solutions requiring a variety of tankage, piping, and ancillary equipment for liquid mixing (or powder blending), filtration, transfer, and related activities. Suspensions, ointments, and other similar products, including the preparation of the solutions for lyophilized products, can be manufactured in the same or very similar equipment. The scale of manufacturing can vary substantially, with the largest batches being well in excess of 5000 L (typically for large-volume parenteral production), down to less than 50 mL for radiopharmaceuticals or biologicals customized for a particular patient. The majority of this equipment is composed of 300 series austenitic stainless steel, with tantalum or glass-lined vessels employed for preparation of formulations sensitive to iron and other metal ions. The vessels can be equipped with external jackets for heating and/or cooling and various types of agitators, depending upon the mixing requirements of the individual formulation. In many facilities, a variety of tank sizes are available for use. Larger facilities may have the high-capacity tanks permanently installed and permanently connected to process utilities. Smaller vessels are generally mobile and positioned in individual processing booths or rooms as needed.

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After sterilizing filtration (or sterilization by heat or other means), comparably sized vessels are sometimes utilized to contain the product prior to and during the filling process. These holding vessels are often steam sterilized along with the connecting piping prior to use. There are a number of firms that fill directly from the compounding vessel using in-line filtration eliminating the intermediate vessel. When this approach is used, a small moist-heat-sterilized surge tank or reservoir tank may be required, particularly with modern time–pressure filling systems. This practice may reduce initial facility and equipment cost but places additional constraints on operational flexibility. The use of disposable equipment for compounding and holding of sterile formulations is coming into greater use. This eliminates the cleaning of vessels prior to reuse, but confirmation of material compatibility is required. Disposable equipment is often used with products manufactured in small to moderate volumes, and while reducing initial equipment expenses disposable equipment also results in contaminated waste, which cannot be recycled or reused and must be treated appropriately. Aseptic compounding as required for suspensions and other formulations in which open-vessel processes are required mandate an ISO 5 environment providing ideally >400 air changes/hour in which these steps can be performed with minimal opportunity for adventitious contamination. This could be accomplished using a protective curtain and a unidirectional flow hood (UFH) or other more evolved designs such as a restricted access barrier (RABs) system or an isolator (technologies that provide a higher level of employee separation from the area in which materials are handled can get by with lower air exchange rates). All activities requiring opening of processing lines such as sampling or filter integrity testing should be performed using similar protective measures. The preparation of sterile suspensions requires a facility/equipment design capable of safe addition of sterile solids to a liquid vehicle and is conventionally performed using a specifically designed processing area to minimize contamination potential. Comparable and greater complexity is generally required for creams, ointments, emulsions, and the increasingly common liposome formulations. Some sterile powder formulations (these are predominantly, but not exclusively, antibiotics) may require sampling, mixing, milling, and subdivision activities similar to those found in oral powder manufacturing. The facilities and equipment utilized for these products is substantially different from that used for liquids, and the production area bears little resemblance to that utilized for liquids. These materials are received sterile and must be processed through sterilized equipment specifically intended for powder handling in a fully aseptic environment with ISO 5 protection over all open container activities. 2.1.2.2

Primary Packaging

The primary package for parenteral formulations provides protection to the sterile materials throughout the shelf life. The components of the primary package are every bit as important to contamination control and hence safety of the finished product as the formulation itself, and their preparation must be given a comparable level of consideration. The most commonly used container is glass; vials are still the most common, although increasingly prefilled syringes are chosen. Glass ampoules are still seen. However, although convenient from a manufacturing perspective, the


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difficulty involved in opening ampoules while at the same time avoiding problems with glass particulate or microbial contamination has reduced their popularity. The use of plastic containers (as vials, ampoules, or syringes) is increasingly common given their reduced weight and resistance to breakage. Blow-fill seal (BFS) and form-fill seal (FFS) are utilized for the filling of numerous ophthalmic and other noninjectable formulations in predominantly low-density polyethylene (LDPE) containers. With the exception of ampoules and BFS/FFS, an elastomeric closure system is also necessary to seal the containers. Some delivery systems (i.e., prefilled syringes, multichamber vials, and others may require more than one elastomeric component to operate properly. In the case of vials, an aluminum crimp is applied to secure the closure to the vial. Prefilled syringes may require the preparation and assembly of additional components such as needles, needle guards, stoppers, diaphragms, or plungers, depending on the specifics of the design. Lyophilization is required to ensure the stability of some formulations and requires the use of closures that allow venting of the container during the freeze-drying process. Full seating of the closure is accomplished within the lyophilizer using moving shelves to seat the closure. Glass is ordinarily washed prior to sterilization/depyrogenation to reduce contamination with foreign material prior to filling. In aseptic fill processes, the glass is then depyrogenated using dry heat. This can be accomplished using either a continuous tunnel (common for larger volumes and high-speed lines) or a dry heat oven (predominantly for small batches). The depyrogenation process serves to sterilize the glass at the same time, and thus the glass components must be protected postprocessing. This is generally accomplished by short-term storage in an ISO 5 environment often accompanied by covering within a lidded tray. There are suppliers that offer depyrogenated glass vials and partially assembled syringes in sealed packages for filling at a customer’s site. In this instance, the supplier assumes responsibility for the preparation, depyrogenation, and aseptic packaging. Glass ampoules are available presealed and depyrogenated; the end user has merely to open, fill, and reseal the syringe under appropriate conditions. Plastic components (whether container or closure) can be sterilized using steam, ethylene oxide, hydrogen peroxide, or ionizing radiation. The γ irradiation is accomplished off-site by a subcontractor with appropriate expertise as these methods are considered the province of specialists because of the extreme health hazards directly related to the sterilization method. Electron beam sterilization may also be done by a contractor, although compact lower energy electron beam systems have been introduced that allow sterilization in-house. Steam sterilization is ordinarily performed in house, though many common components are becoming available presterilized by the supplier. Preparation steps prior to sterilization vary with the component and the methods used to produce the component. Rubber components are washed to reduce particles, while this is less common with plastic materials. Syringes vary substantially in design details and can be aseptically assembled from individual components. However, increasingly, these are supplied as presterilized partial assemblies in sealed containers. The BFS and FFS are unique systems in that the final container is formed as a sterile container just prior to the aseptic filling step. The BFS requires careful control over the endotoxin content of the LDPE (and other polymeric materials) beads used to create the containers as well as the melting conditions utilized to form them.

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The FFS utilizes in-line sterilization/drying of the film prior to shaping of the containers. 2.1.2.3

Process Objectives

The production of parenteral products requires near absolute control over microorganisms. Endotoxin contamination is a serious health concern, particularly among neonates and infants and also requires a high level of control and validation. Additionally, the control of foreign matter, including particles and fibers of various types, is also vitally important to end-user safety. Assuring appropriate control over these potential contaminants requires careful attention to several factors: facility design, equipment selection, sterilization procedures, cleaning regimens, management of personnel, and the process details associated with compounding, filling, and sealing of product containers. Each of these will be discussed in detail.

2.1.3

FACILITY DESIGN

To provide control of microbial, pyrogen, and particles controls over the production environment are essential. The facility concerns encompass the entire building, but the most relevant components are those in which production materials are exposed to the environment. 2.1.3.1 Warehousing Environmental protection of materials commences upon receipt where samples for release are taken from the bulk containers. Protection of the bulk materials is accomplished by the use of ISO 7 classified environments for sampling. All samples should be taken aseptically, which mandates unidirectional airflow and full operator gowning. This practice is mandated by current good manufacturing practice (CGMP) and assures that sampling does not introduce contaminants to the materials that will be used in the production. Where central weighing/subdivision of active ingredients and excipients are performed, similar protection is provided for identical reasons. The expectation is that these measures reduce the potential for contamination ingress into materials that have yet to receive any processing at the site. Materials and components that are supplied sterile are received in this area, but samples are often packaged separately by the supplier to eliminate the need for potentially invasive sampling of the bulk containers. Where so-called delivery samples are used, it is critical that these samples are known to be fully representative of the production process. Additionally, where sterility or bioburden control of sampled materials is critical, thought must be given to the methods used to reseal the containers to ensure that moisture levels, bioburden levels, or in the case of sterile products sterility assurance are not compromised. 2.1.3.2

Preparation Area

The materials utilized for production of sterile processes move toward the filling area through a series of progressively cleaner environments. Typically, the first step

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is transfer into an ISO 8 [Class 100,000, European Union (EU) Grade D] environment in which the presterilization preparation steps are performed. Wooden pallets and corrugated materials should always be excluded from this zone (and any classified environment), and transfers of materials are performed in air locks designed to reduce the potential for particle ingress and to a lesser extent microbial ingress. Preparation areas provide protection to materials and components for a variety of activities: component washing (glass, rubber, and other package components), cleaning of equipment (product contact fill parts, process tools, etc.), and preassembly/ wrapping for sterilization. In some facilities, this area is also utilized to support compounding operations in which case process utensils, small containers, and even portable equipment will be cleaned and prepared for sterilization. Careful attention must be given to material flow patterns for clean and dirty equipment to prevent cross contamination. In larger facilities, the equipment wash room may be a separate room proximate to the preparations area with defined flows for materials and personnel. Ideally, materials should move through the facility in a unidirectional fashion, with no cross over of any kind. The preparations area typically includes storage areas where clean and wrapped change parts, components, and vessels can be held until required for use in the fill or compounding areas. (Just-in-time practices are desirable for all parenteral operations to avoid extensive and extended storage of materials in the higher classified fill or compounding areas.) The preparations area is ordinarily located between the warehouse and the filling/compounding areas and connected to each of those by material/equipment air locks. Preparation areas are supplied with high-efficiency particulate air (HEPA) filters (remote-mounted HEPAs are commonplace). The common design requirement is more than 20 air changes per hour, turbulent airflow (see below), and temperature and relative humidity controlled for personnel comfort. As in any clean room area designed for total particulate control, the air returns should be low mounted. Wall and ceiling surfaces should be smooth, easily cleaned, and tolerant of localized high humidity. Floors should be typically monolithic with integral drains to prevent standing water. Common utilities are water for injection, deionized water, compressed air, and clean/plant steam. Clean-in-place (CIP) and sterilizein-place (SIP) connections may be present if the prep area supports compounding as well. Ordinarily, present within the preparation area are localized areas of ISO 5 unidirectional airflow (Class 100) utilized to protect washed components prior to sterilization and/or depyrogenation. These areas are not aseptic and should not be subjected to the more rigorous microbial expectations of aseptic processing. They are designed to reduce/eliminate the potential for particle contamination of unwrapped washed materials. Operators accessing these protective zones wear gloves at all times when handling materials. Operators in the preparations area are typically garbed in low particle uniforms (or suits) with shoe, hair, and beard covers. The use of latex or other gloves is required when contacting washed components. Sterilized gowns and three-stage gowning facilities are not required to enter or work in this ISO 8 environment. Gowns are generally donned within a single-stage airlock, which is maintained at a pressure slightly negative to the ISO 8 working environment. Separate personnel entry/exit are not typically necessary for this lower classified environment.

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Equipment within the preparations area varies with the practices of the firm and can include manual or ultrasonic wash/rinse sinks; single or double door automated parts washers; batch or continuous glass washers; stopper washers for closure components; CIP/SIP stations; equipment wrap areas (as described above); and staging areas for incoming (prewash) components, dirty equipment, and cleaned components/equipment. An adjacent classified storage area(s) may be present in larger facilities to accommodate the full variety of change parts and equipment that is not in immediate use. Where the preparations area also supports compounding, it may include additional equipment such as pH meters, filter integrity apparatus, and the like in support of those operations. (Note: Where compounding requires aseptic conditions for rigorous control of bioburden, as is the case for unpreserved biologics and other contamination-sensitive products, it is best to provide separate entry for compounding. The moisture level and hence contamination potential in a typical preparation area is unsuitable for entry into an aseptic compounding area). Depending on the scale of the operation, the preparations area may include the loading areas for both sterilizers and ovens. In high-throughput operations where the use of tunnels for glass depyrogenation is more prevalent, glass washers and tunnels for each filling line may be in separate ISO 8 rooms accessed from the preparations area. 2.1.3.3

Compounding Area

The manufacture of parenteral solutions is ordinarily performed in ISO 7 (Class 10,000, EU Grade C) controlled environments in which localized ISO 5 unidirectional flow hoods are utilized to provide greater environmental control during material addition. These areas are designed to minimize the microbial, pyrogen, and particle contributions to the formulation prior to sterilization. Depending upon the scale of manufacture, this can range from small containers (up to 200 L) (disposable containers are coming into use for these applications), to portable tanks (up to 600 L) to large fixed vessel (10,000 L or more have been used) in which the ingredients are formulated using mixing, heating, cooling, or other unit operations. Smaller vessels are placed or rolled onto scales, while fixed vessels are ordinarily mounted on weigh cells. The vessels may be equipped for temperature and pressure measurement instruments, as mandated by process requirements. Compounding areas often include equipment for measuring mass and volume of liquid and solid materials including, for example, graduated cylinders, and scales of various ranges, transfer and metering pumps, homogenizers, prefilters, and a variety of other liquid/powder handling equipment. Liquid handling may be accomplished by single-use flexible hose, assemblies of sanitary fittings, or some combination thereof. A range of smaller vessels to be used for the addition of formulation subcomponents or excipients to the primary compounding tank may be required as well. Because parenteral formulations can include aqueous and nonaqueous vehicles, suspensions, emulsions, and other liquids, the capabilities of the compounding area may vary. Agitators can be propeller, turbine, high shear, or anchor designs depending upon the requirements of the products being manufactured, and it is not uncommon to find examples of each in larger facilities. It is preferable to perform as much of the process as possible while the formulated liquid is nonsterile to ease sterilization requirements, although precautions to prevent microbial and endotoxin contamination are important risk abatement features.

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The formulation area is customarily a combination of open floor space, adjacent to three-sided booths and individual processing rooms in which the ingredients are handled and individual batches are produced. Walls and ceiling materials are selected to be impervious to liquids and chemical spills and are easy to clean. Floors in these areas are monolithic and should be sloped (at 1–3 : 100) to drains with appropriate design elements and control procedures to eliminate backflow potential (regulatory bans on drains in classified areas are focused on protecting aseptic environments and are inappropriate for nonsterile compounding areas). Pit scales should be avoided in new installations; floor-mounted scales intended for cleaning underneath the base are preferable. Compounding areas are supplied with HEPA filters (ceiling-mounted terminal HEPAs are more common, though central supply is possible in areas of low contamination risk). The common design requirement is more than 50–60 air changes per hour, turbulent airflow (see below), with temperature and relative humidity for personnel comfort. Air returns may be at or near floor level, with localized extraction provided as necessary to minimize dusting of powder materials. Where substantial heat is generated from processing or sterilization, a ceiling or high wall return may be more appropriate. Wall and ceiling surfaces are smooth, easy to clean, and tolerant of localized high humidity. Floors are typically monolithic with integral drains to prevent standing water. Common utilities are water for injection, deionized water, nitrogen, compressed air, clean/plant steam, and heating and cooling media for the fixed and portable tanks. Water for injection use points are often equipped with sanitizable heat exchangers for operator safety. Cleaning of the fixed vessels and portable tanks is accomplished using either manual sequenced cleaning procedures or more commonly with a CIP system. Cleaning of other items can be accomplished in a wash area accessed from the compounding area or in a common wash room incorporating both filling and compounding equipment. Sterilization of the nonsterile processing equipment and vessel is often provided for as an option, even where it is not routinely required to control product bioburden. Where production volumes or physical location dictate, the compounding area may have a separate preparations area from that utilized to support filling operations. Personnel working in the compounding area typically wear a coverall (which may be sterilized for contamination control as required), with head/beard covers, as well as dust masks and sterile gloves. Additional personnel protective equipment may be necessary for some of the materials being processed. A fresh gown should be donned upon each entry into the compounding area. Separate gowning/degowning rooms should be provided to minimize cross-contamination potential for personnel working with different materials. As nonsterile compounding areas are often ISO 6–7 environments but are not aseptic, the more rigorous contamination controlling designs required of aseptic gowning areas (see below) are somewhat reduced. 2.1.3.4 Aseptic Compounding Area (If Present) Where products are filled using in-line filtration direct to the filling machine, an aseptic compounding area may not be present. In those instances the final sterilizing filter will be located in the fill room. Products that are held/processed in sterilized vessels prior to filling require an aseptic compounding area. This is typically an ISO 7 in environment with localized

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ISO 5 unidirectional flow present where open-product containers or aseptic operations are conducted. Some products may require larger ISO 5 suites with full HEPA coverage rather than the more common ISO 5 clean booth design. Fixed vessels in this area are cleaned and sterilized in situ, while portable vessels are typically relocated to the wash area for cleaning. Sterilization of portable vessels may be accomplished at an SIP station in the aseptic core, compounding, or preparations areas. When accomplished outside the aseptic processing area, resterilization of the connecting lines may be appropriate. Filters for sterilization of solutions from compounding to holding vessels are typically located in this environment as well, with sterilization by either SIP or sterilization in an autoclave. The use of integrated, programmable logic controlled (PLC) filter skids with automatic CIP/SIP and filter integrity testing is frequently seen for contamination sensitive products. Depending upon the formulations being produced, additional sterilized processing equipment may be present in this area for use in the process. This can include in-line homogenizers, static mixers, and colloid mills. Where sterile powders are produced, the aseptic compounding processes can include blending, milling, and subdivision equipment. Aseptic compounding areas typically require a means to introduce sterile equipment, tubing, and other items, so access to a sterilizer is desirable. The aseptic compounding area may be contiguous to the aseptic filling suites. If it is not, separate gowning areas must be provided for personnel as well as separate air locks/passthroughs (see below). Personnel working in aseptic compounding wear full aseptic garb: sterile gown, hood, face mask, goggles, foot covers, and gloves. Adaptations may be necessary for potent/toxic compounds to assure operators are properly protected from hazardous materials. Gowning areas are ordinarily shared with aseptic filling, but where they are not shared a comparable design, albeit on a smaller scale, is appropriate. The facility design features match that of the aseptic filling room/aseptic processing areas described in greater detail below. Utility services would mimic those utilized in the nonsterile compounding area that is usually adjacent (next to or above) to the aseptic compounding area. Temperature and humidity should be controlled to similar levels as those required for aseptic filling. Since CIP/SIP systems tend to generate heat and humidity, sufficient capacity must be available to control temperatures to approximately 18–20°C and 300°C) to increase processing speed and include a cooling zone that facilities discharge at or near room temperature. Typically, heating of the glass to 300°C or more for 3 or more minutes will result in much greater than the three-log endotoxin reduction required in current industry standards. The air inside the tunnel is HEPA filtered, and newer designs allow for dry-heat sterilization of the cooling zone as an added protective measure. Tunnels must be positioned with some care as they ordinarily will terminate into a fill room. A pressure differential between the cooling zone of the tunnel and the fill room is critical for proper operation of the tunnel. The pressure differential must conform to the requirements stipulated by the tunnel manufacturer. It is not necessary to have a >12.5 PA (particulate air) differential between the in-feed side of the heating zone of the tunnel and the exit side of the cooling zone. It has been suggested by

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some that, since the in-feed side of the tunnel is typically in ISO 7 or 8 space, a greater differential is required; however, this is not true since the cooling zone is ISO 5, and the heating zone is certain to be sterile and is also ISO 5 in terms of particulate air quality. Their in-feed is often direct from a glass washer, which may be remote from the main preparations area utilized for washing, wrapping, and sterilizer loading. 2.1.6.3

Gas and Vapor Sterilization

The sterilization of materials using noncondensing gases (ethylene oxide, chlorine dioxide, or ozone) or condensing vapors such as hydrogen peroxide is a supplementary process intended for items that cannot be exposed to heat. The utilization of gas/ vapor designs is coming into increased use as a supportive technology for isolation technology for presterilized items such as syringes and stoppers that must be introduced into the isolators aseptic zone. Air locks using these agents can be utilized in similar fashion for the supply of materials to manned clean rooms. Control over agent concentration or injection mass, relative humidity, and temperature may be required for these systems. There are different types of vapor processes available, and users should generally follow the cycle development strategy suggested by the manufacturer of the equipment they have chosen. Specific temperature and humidity ranges may be required for some vapor processes to assure appropriate efficacy [25, 26]. 2.1.6.4

Radiation Sterilization

The use of radiation within a parenteral facility would have been considered unthinkable prior to the start of the twenty-first century. While γ irradiation is typically a contracted service provided off-site, electron beam sterilization advances can make the installation of an in-house (and generally an in-line) system a real possibility. An in-line system would be utilized similarly to the gas/vapor systems described above for treatment of external surfaces for entry into either a clean room or isolator-based aseptic processing facility. The use of this same technology for terminal sterilization is also possible [1]. Association for the Advancement of Medical Instrumentation (AAMI)/ISO 11137 provides widely accepted guidance on the development and validation of radiation sterilization processes. 2.1.6.5

Sterilization by Filtration

Filters are utilized to sterilize liquids and gases by passage through membranes that retain microorganisms by a combination of sieve retention, impaction, and attractive mechanisms [27]. In contrast with the other forms of sterilization that are destructive of the microorganisms, filters rely on separation of the undesirable items (microorganisms as well as nonviable particles) from the fluid. Because filtration requires passage of the fluid from the “dirty” (upstream) side of filter to the clean (downstream) side of the filter, the downstream piping and equipment must be both “clean” and sterile prior to the start of the filtration process. This will ordinarily require the use of SIP procedures or sterilization followed by aseptic assembly. Sterilizing filtration of parenterals is a complex and often inadequately considered subject, and numerous controls are required on the filter, fluid, and sterilizing/

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operating practices employed. PDA Technical Reports 26 and 40 can be instructive in understanding the relevant concerns [28, 29].

2.1.7

FACILITY AND SYSTEM: QUALIFICATION AND VALIDATION

Facilities for the manufacture of sterile products require the qualification/validation of the systems/equipment and procedures utilized for that production. Each system described above and others with a direct/indirect impact on the quality of the products being produced should be placed into operation using a defined set of practices. The general approach is described below, and best practices include the development of traceable documentation from project onset. The preferred approach begins during a project’s conceptual design phase where provisions for meeting the CGMP expectations and user requirement specifications establishing the technical basis for the processes are first defined. This is commonly followed by the validation master planning exercise in which the user requirement specifications are used as a basis for the development of acceptance criteria for process control studies. This effort should be accompanied by an analysis of risk that considers product attributes, target patient population, as well as technical and compliance requirements. Detailed design follows in which the specifics of the various systems are refined. Construction of the facility and fabrication of the process equipment follows and a variety of controls are necessary during these activities to satisfy user requirements for compliance of the various elements of the facility. Typically, factory acceptance testing (FAT) will be done on all key process equipment, usually at the manufacturer’s plant site; much of the information gathered during FAT can be referenced in the qualification activities to follow. Physical completion is followed by a well-defined step termed commissioning in which construction and fabrication errors and omissions are addressed. Site acceptance testing of installed process equipment may be done in parallel with facility commissioning. Formal qualification of the facility ensues in which the installed systems and equipment are evaluated for their conformance to the design expectations. The very last steps in this process are variously termed performance qualification. Detailed discussion of these subjects is not possible within the constraints of this chapter, however the qualification/validation of equipment, systems, and processes has been extensively addressed in the literature [30].

2.1.8

ENVIRONMENTAL CONTROL AND MONITORING

Confirmation of appropriate conditions for aseptic processing and its supportive activities is required by regulation. In the highest air quality environment utilized for aseptic processing, ISO 5, there is a general expectation that the air and surfaces be largely free of microbial contamination and the number of particles be within defined limits (less than 3500 particles greater than 0.5 μm/m3). Proving the complete absence of something is an impossible requirement, so the usual expectation is that 99+% of all samples taken from this most critical environment be free of detectable microorganisms. The minimum monitoring expectations for these environments as defined by the regulators are consistently attainable in nearly all instances,


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especially those with lesser expectations. This is accomplished by proper design, periodic facility disinfection, and measures to control the ingress of microorganisms and particles for materials entering each environment from adjacent less clean areas [31]. 2.1.8.1

Sanitization and Disinfection

Disinfection is customarily performed by gowned personnel during nonoperating periods using such agents as phenolics, quaternary ammonium compounds, aldehydes, and other nonsporicidal agents. The frequency of treatment varies with the ability of the facility to maintain the desired conditions between disinfection. Sporicidal agents such as dilute hydrogen peroxide or bleach are reserved for those occasional periods when control over the spore population warrants and is often employed after lengthy maintenance shutdowns or at the end of construction. Isolation technology replaces the manual disinfection with reproducible decontamination with a sporicidal agent and thus assures a superior level of environmental control as compared to manned environments. The manual treatments fall short of this level of control due to the uncertainties of the manual procedure and recontamination of the environment as a consequence of the very personnel and activities utilized to disinfect it. To mitigate these weaknesses, automatic sporicidal disinfection of manned clean spaces has been developed by multiple vendors. Disinfection of the less critical environments is accomplished in the same manner albeit on a less frequent interval befitting their higher allowable levels of microorganisms. 2.1.8.2

Monitoring

Aseptic environments are subject to a variety of monitoring systems including air, surface, and personnel monitoring for viable microorganisms and for nonviable particles. Environmental monitoring programs are often developed during the qualification of a new facility using a multiphase approach. Methods for the monitoring and expectations for performance have been extensively discussed in the literature and will only be addressed briefly in the context of this chapter [1, 2, 31, 32]. In general, the frequency and intensity of monitoring and concern for cleanliness increases as the product progresses from preparation steps (typically in ISO 7/8 environments) to more important activities (nonsterile compounding in ISO 6) and ultimately into the aseptic core (aseptic compounding and filling in ISO 5). Sampling site and time selection should be a balance between the need to collect meaningful data and avoidance of sampling interventions that could adversely (and inadvertently) impact product quality. Microbiological sampling must always be done by well-trained staff utilizing careful aseptic technique. This will both minimize risk to the product and also improve the reliability of the data by reducing the likelihood of false-positive results. Air Sampling The relative cleanliness of air in the most critical environment is assessed using passive sampling systems such as settle plates or estimated volumetrically using active air samplers. Active air samplers should be designed to be isokinetic in operation to avoid disruptions to unidirectional airflow. Considerable variability has been reported among the several sampling methods employed for

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active air sampling, and there are also reports that active air sampling may have advantages in terms of sensitivity. Passive sampling using settle plates can be a useful adjunct in critical areas with limited access and where an active sampler might interfere with airflow or entail a worrisome intervention risk. It must be recognized that attempts to support the “sterility” of the cleanest aseptic environments (those in ISO 5) by aggressive sampling may have exactly the opposite effect. Sampling too frequently will increase process contamination risk by causing critical interventions that are best avoided within these very clean environments. As personnel are the greatest single source of microbial contamination and conduct the sampling, sampling intensity should be carefully considered. There is no value to taking air samples beyond those required to assess the relative cleanliness level within the environment. Surface Sampling Surfaces in the classified environments are monitored using a variety of methods but most commonly with contact plates (on smooth surfaces) or swabs (for irregular surfaces). Surface sampling in aseptic environments (ISO 5/6) is typically performed after the completion of the process to avoid the potential for adventitious contamination of the production materials as a consequence of sampling activities during the process. Fortunately, studies indicate that contamination does not build up during typical processing operations in modern clean rooms. Sampling with these materials may leave a trace of media or water on the sampled surface, and cleaning of the surface immediately after sampling is commonplace. Sampling of product contact surfaces (i.e., fill needles, feeder bowls, etc.) should only be performed after completion of the process, and the results of this testing should not be considered as an additional sterility test on the products. As in any form of manual environmental sampling, the risk of contamination by samplers during the processing of a sample makes the data less than completely reliable. Sampling of surfaces such as walls and floors should not be overdone because with good attention to aseptic technique they should be of little concern relative to actual process risk. Sampling on these surfaces is probably most useful in assessing ongoing changes in microflora and to confirm the adequacy of the disinfection program. Personnel Sampling The monitoring of personnel gown surfaces is an adaptation of surface sampling in which samples are taken from surfaces on the operator. In ISO 5 environments, this ordinarily entails the gloved hands and perhaps forearms. As with any other sampling of a critical surface (the gloved hand is often in closest proximity to sterile product contact surfaces and sterilized components), the sampling should be performed at the conclusion of the aseptic activity. Sampling during the midst of the process risks contamination of the product and should be avoided. Sampling of other aseptic gown surfaces is ordinarily restricted to gowning certification or postmedia fill testing, where more aggressive sampling can sometimes be informative. Whenever a gowned individual is sampled, the sample should be taken in the background environment (not ISO 5), and the individual should immediately exit and regown before continuing any further activity in the aseptic core area. Sampling of personnel in less critical environments can be useful; however, meeting regulatory expectations in these areas is ordinarily straightforward. Recommended contamination levels often distinguish among the different room classification levels found within clean rooms. While this may seem reasonable, it is not completely


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logical since operators often move frequently between these different levels of classification during the conduct of their work. Total Particulate Monitoring Confirming the ability of the facility’s heating, ventilation, and air-conditioning (HVAC) system to maintain the appropriate conditions throughout (to the extent practical) the classified environments is most easily accomplished using electronic total particle counters that can provide near immediate feedback on conditions during production operations. Total particle samples can be taken automatically, using permanently installed probes oriented into the unidirectional airflow. As such, they can be positioned proximate to critical activities to reaffirm the continued quality of the air in the vicinity of the sterile materials and surfaces. Manual total particulate air sampling can be a dangerous intervention and therefore if required should be timed so as to minimize risk to product. Attempts to correlate total particle counts with microbial counts have proven difficult. Correlations are only meaningful when the source of foreign material is personnel since people are the only source of airborne contamination within an aseptic processing area. When personnel are the only source of particulate, the ratio between viable and nonviable particles have been consistently found to be >1000 : 1, which means that in ISO 5 environments even relatively large total particulate count excursions would typically contribute microbial contamination that fell far below the limit of detection. Process equipment can and often does contribute airborne particulate matter but not detectable levels of microbial contamination. Also, microbial sampling is highly variable with respect to sensitivity, accuracy, precision, and limit of detection making correlations, particularly in rooms of highest air quality. So, it might seem logical to think that particle excursions are indicative of coincident microbial excursions especially in the cleaner environments (ISO 5) where the aseptic process takes place. It is common practice for firms to interrupt their aseptic processes when atypical total particulate excursions are observed so that the scientists and engineers can determine the source of the foreign material. Monitoring frequency and expectations in the less critical environments is always reduced relative to the critical aseptic environments. Where firms have introduced unidirectional air systems in preparations and compounding areas for particle control, there is often the temptation to expect these areas to meet the same microbial limits that these locations might attain in the aseptic core. This temptation should be resisted to avoid unnecessary sampling and deviations associated with expecting these environs to meet the conditions of aseptic areas where sanitization frequency, background environment, and most importantly personnel gowning are far superior to that found in the less clean locales [33]. Housekeeping An important component of environmental control are the housekeeping activities utilized to clean the facility external to the controlled environments. Aseptic operations utilize a series of protective environments to protect the sterile field. Controls on the surrounding unclassified areas are an important part of the overall control scheme for sterile manufacturing. These unclassified areas support sterile operations in a variety of ways, and it is important to conduct activities therein that assist in the environmental control. Routine housekeeping, periodic sanitization, and even occasional environmental monitoring may be appropriate to

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assure that microbial and particle loads on items, equipment, and personnel entering the classified environments is appropriately controlled.

2.1.9

PRODUCTION ACTIVITIES

The preparation of sterile materials requires execution of a number of supportive processes that together constitute the manufacturing process. They are intended to control bioburden, reduce particle levels, remove contaminants, sterilize, and/or depyrogenate. Nearly all of these activities occur within the controlled environments and are subject to qualification/validation. 2.1.9.1

Material and Component Entry

Prior to the start of any production activity, materials and components must be transferred from a warehouse environment into a classified environment. For most items this will necessitate removal from boxes or cartons, transfer to a nonwooden pallet, and passage through an air lock which serves as the transfer system between the controlled and uncontrolled environments. Often components are contained within plastic bags within a box or carton, and in some cases there are multiple bag layers to facilitate disinfection and passage through air locks into different zones of operation within the aseptic area. The firm may utilize an external disinfection of the materials in conjunction with this transfer. The concern is for minimization of particles and bioburden on these as yet unprocessed items in order to protect the controlled environment. Raw materials may be weighed in a weigh area in which they are transferred to plastic bags and/or noncorrugate containers prior to the transfer. The weighing area provides ISO 7 or better conditions, and may be a dedicated portion of the warehouse proper; in a central weighing/dispensing area; or in a location contiguous to the compounding area. Sterile ingredients are never opened anywhere other than an aseptic environment and must be handled aseptically at all times including sampling and processing of samples. 2.1.9.2

Cleaning and Preparation

Once the container component items have been introduced into the preparations area, they must be readied for sterilization/depyrogenation. For many items this consists of washing/rinsing processes designed to remove particles and reduce bioburden and endotoxin levels. The application of silicone suspensions for glass or closure materials is sometimes employed to provide lubrication allowing smoother feeding of components or dispensing (elimination of product accumulation on vial). Following the cleaning, items for sterilization are dried, wrapped, and staged/stored for steam sterilization. Washed containers are either placed in trays or boxes for depyrogenation in ovens or are directly loaded into dry-heat tunnels. It is common practice to protect all washed items with ISO 5 air from the completion of washing, through either wrapping or placement into a sterilizer or oven for passage into the aseptic area. The intention is to avoid foreign matter that could result in contamination of product.


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It is increasingly common for components to be supplied by the vendor in a ready-to-sterilize condition (washed and pretreated as necessary). Some items are available in a ready-to-use configuration with the supplier providing sterile and pyrogen-free components. The use of supplier-prepared items eliminates the need for preparation activities at the fill site and requires modification of material in-feed practices relative to on-site prepared items. The process equipment (portable tanks, valves, fill needles, etc.) and consumable materials (filters, hoses, gaskets, etc.) are prepared using a variety of methods. Portable tanks are subjected to CIP (and perhaps SIP as well) in the preparation area. Smaller items are disassembled (if necessary) and cleaned either manually or in a cabinet washer. After cleaning they are wrapped and staged/stored prior to sterilization. Tubing should not be reused; its preparation typically consists of flushing with WFI followed by cutting to the required length. It is best to preassemble fill sets with tubing, filters, and fill needles/pumps and then wrap them in preparation for sterilization. This process obviates poststerilization assembly steps and therefore mitigates contamination risk. These steps may be performed in ISO 5 environments to reduce total particulate contamination on the items. There are items that must be transferred into the aseptic processing area that cannot be treated within a sterilizer/oven. These include portable tanks, electronic equipment, and containers of sterile materials (ready-to-use items, sterile powders, environmental monitoring media, etc.). Air locks, pass-throughs, and similar designs are employed in which the exterior surfaces of the items are disinfected. The disinfection process may be completed by personnel outside and/or inside the aseptic area depending upon the specifics of the design. At the completion of the cleaning process, the items should be free of contaminating residues including traces of prior products, free of endotoxin, and well-controlled in terms of total particulate and microbial levels. This level of control would be appropriate regardless of whether the items, equipment, or components are to be sterilized or not. Sterilization, other than by relatively high temperature dry heat, has only a modest impact on endotoxin levels; cleaning provides the only means to control endotoxin for materials and equipment that is sterilized by other means. 2.1.9.3

Compounding

Fixed equipment in the compounding area (nonaseptic or aseptic) is cleaned in place. This eliminates traces of prior products, particles, and pyrogens. Sterilization in place is required for the aseptic fixed equipment and is sometimes employed for the nonaseptic equipment as well as a bioburden control measure. Fixed transfer lines must be cleaned and sterilized as well, and this is accomplished independently or in conjunction with the vessels. The reuse of hoses and tubing is discouraged as cleaning and extractables cannot be confirmed beyond a single use. The preparation of the product is performed within a classified environment with careful attention to the batch record, especially for time limits and appropriate protection of materials during handling to guard against all forms of contamination. This is proper for nonsterile compounding to minimize contamination prior to filtration/sterilization and is required for aseptic compounding activities. Barrier designs and other means of physically separating the worker from the product are recommended as a minimum even in nonaseptic compounding. As compounding may

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expose the worker to a variety of potent/toxic materials, the use of personnel protective equipment may be required. In extreme cases, the use of containment system may be required to protect the compounding operator. Where the compounding is nonaseptic, careful control over the environment, materials, and equipment is still appropriate to reduce viable/nonviable levels and to reduce the potential for endotoxin. Time limits should be imposed on manufacturing operations for additional control over microorganisms and thus microbial toxins. Once the materials have been sterilized, interventions near either the formulation or product contact surfaces/parts should be minimized. Direct handling of these materials should only be done with sterilized tools or implements; nonsterile objects, such as operator gloves, should never directly contact a sterilized surface. Sampling, filter integrity testing, process connection, and other activities should all be designed to eliminate the need for personnel exposure to sterile items. Aseptic compounding is often a required activity for sterile products that cannot be filter sterilized. The preparation of the sterile solids for use in these formulations is outside the scope of this chapter, but it is often acknowledged as the most difficult of all pharmaceutical processes to properly execute. Handling these materials at the fill site is performed using ISO 5 environments, and the use of closed systems is preferred [34]. 2.1.9.4

Filling

Aseptic filling is performed in ISO 5 environments, and a variety of approaches are utilized with the technology choice largely dependent upon the facility design, batch size, and package design. Older plants utilize manned clean rooms in which aseptically gowned personnel operate the filling equipment: performing the setup, supplying components, making any required adjustments, and conducting the environmental monitoring. As human operators are directly or indirectly responsible for essentially all microbial contamination, aseptic filling operations are increasingly designed to minimize the potential for operator contamination to enter the critical environment. Barriers of various sophistication and effectiveness are employed to increase the protection afforded to sterile materials. The most evolved of the cleanroom designs are RAB systems in which personnel interventions are restricted to defined locations. Many newer facilities utilize isolation technology in which the filling environment is fully enclosed and personnel contamination is completely avoided. Filling designs for syringes and ampoules differ only with respect to the details of component handling and closure design. However, it is wise not to underestimate the influence of both component quality and component handling reliability on contamination control in aseptic processing. Components that minimize the need for intervention and equipment that is rather tolerant of component variability will result in better contamination control performance. Aside from these distinctions, the range of filling technologies previously described is also possible. The filling of plastic containers is accomplished using two very different approaches. Pre-formed containers can be sterilized in bulk, introduced into the aseptic suite via air locks, oriented (unscrambled), and filled. Blow-fill-seal prepares sterile bottles (most often LDPE) on line just prior to filling and sealing.


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Filling of suspensions, emulsions, and other liquids may require slightly different filling designs to assure uniformity of dose in each container. Ointments and creams are sometimes filled at elevated temperatures to improve their flow properties through the delivery and filling equipment. These are ordinarily filled into presterilized plastic tubes that have largely replaced aluminum tubes for these formulations. Powders are typically filled in vials using equipment specifically engineered for that purpose. An inerting gas (typically nitrogen, but other gases can be utilized) may be added to the headspace of the container to protect formulations that are oxygen sensitive. If the product is particularly sensitive to oxygen, purging may be done in the empty container prior to filling and again immediately after filling. Products may also be filled in an isolator under a nitrogen atmosphere if required. Products that require inert gas purging will also generally require inert gas for pressurization of tanks to provide motive force to drive the product through the filter(s) and into the filling reservoir. 2.1.9.5

Stoppering and Crimping

If the product is not freeze dried, the primary closure or “stopper” is applied shortly after completion of the filling process to better assure the sterility of the contents. When the product is to be lyophilized, the stopper may be partially inserted after filling and be fully seated after completion of the lyophilization cycle. Alternatively, the container could be left open and a stopper applied after completion of the drying. Crimping is the act of securing the closure to the vial. It must be performed with sufficient uniform downward force to assure the container is properly secured. Too little downward force results in inadequately secured closures, while excessive force can result in container breakage. The force contributed by the crimp roller may be controllable as well. Applying the closure to syringes, ampoules, and other containers usually differs in methodology from the approaches used for vials, but the objective is identical to secure the container’s contents fully assuring the product’s critical quality attributes (especially sterility) are maintained throughout its shelf life. 2.1.9.6

Lyophilization

Lyophilization (or freeze-drying) is a process utilized to convert a water-soluble material filled into a container to a solid state by removal of the liquid while frozen. The process requires the use of deep vacuums and careful control of temperatures. By conducting the process under reduced pressure, the water in the container converts from ice directly to vapor as heat is applied and is removed from the container by the vacuum. The dissolved solids in the formulation cannot undergo this phase change and remain in the container. At the completion of the cycle, the container will be returned to near atmospheric pressure; stoppers are applied or fully seated and crimped as described above. Lyophilization is particularly common with biological materials whose stability in aqueous solution may be relatively poor. The time period in solution and the temperature of the solution are kept at a specified low temperature to prevent product degradation [35].

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As partially stoppered but unsealed containers must be transferred to the lyophilizer from the fill line, various designs have been utilized to protect the containers during this transit. Among the common alternatives utilized are the following: •

• • •

Placement of the lyophilization in the wall of the fill room to allow for direct loading Battery-operated unidirectional airflow carts to a remote lyophilizer ISO 5–protected conveyors with single shelf loading Transfer utilizing isolator technology

The use of trays for supporting the containers during the transfer, loading, lyophilization, and unloading steps was at one time common. The major problem with the use of trays for this purpose was the heat/handling-related distortion of the tray bottom that impacted the uniformity of the heating process in the freeze dryer. This was overcome by the use of trays with bottoms that were removed after loading and reinserted after completion of the drying. The current preference is for the placement of the containers directly on the shelf eliminating the trays entirely. This is accomplished by single height loading/unloading of the individual shelves with various pusher designs. The use of thermocouples to monitor product temperature inside selected vials with the lyophilizer is still the prevalent practice. The utility of this data is questionable and the current trend is to eliminate this “requirement” as soon as possible to better assure sterility of the unsealed vials by eliminating placement of the thermocouples. The lyophilizer chamber and condenser should be cleaned with a CIP system after each batch to prevent cross-contamination and, after cleaning, both should be sterilized. If a slot door loading system is utilized, periodic opening of a full door in the lyophilizer may be required to remove stoppers and glass that may have fallen.

2.1.10

PERSONNEL

Aseptic processing in the pharmaceutical industry is almost entirely dependent upon the proficiency of the personnel assigned to this most critical of all activities. The operators must be able to consistently aseptically transfer sterile equipment and materials in a manner that avoids contamination of those materials [1]. This is no mean feat given the contamination continuously released by personnel and the prevailing need for personnel for execution of the process activities. Personnel proficiency in aseptic operations must be firmly established before they are allowed to conduct critical aseptic process steps. Operators must master a number of relevant skills in order to be declared competent. The usual progression is from classroom training (CGMP, microbiology, sterilization, etc.) to relevant practical exercises (aseptic media transfers, aseptic gowning rehearsals) and ultimately to the core aseptic skills required (aseptic gowning certification, aseptic

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assembly/technique) using a growth medium. Through this approach the operator gradually acquires the necessary skills to be a fully qualified member of the production staff. Training/qualification of personnel is an ongoing requirement and must be repeated periodically to assure the skills are maintained. Continuing evaluation of operator qualification is accomplished using written examinations, practical challenges, documented observation, and participation in process simulation trials. There is general acknowledgment of the risk associated with heavy reliance on personnel for aseptic processing. This has fostered much of the innovative designs for aseptic filling such as RABS and isolators where personnel are largely removed from the critical environment. The future will undoubtedly witness aseptic technologies where human interaction with sterile materials has been eliminated.

2.1.11 ASEPTIC PROCESSING CONTROL AND EVALUATION The preparation of any pharmaceutical product requires controls over the production operations to assure the end result is a product that meets the required quality attributes. The methods utilized for this control are supported by formalized validation studies in which proof of consistency is demonstrated by appropriately designed experiments. The definition of appropriate operating parameters is the primary objective of the development activities and is further confirmed during scale-up to commercial operations. The validation supports that the routine controls applied to the process are appropriate to assure product quality [36]. This is typically accomplished in formalized validation activities in which expanded sampling/testing of the product materials is performed to substantiate their uniformity and suitability for use [30]. 2.1.11.1

In-Process Testing

The sampling and testing of in-process materials during the course of the manufacturing process can confirm that essential conditions have been provided. This is appropriate in preparation, compounding, and filling activities. Sampling in preparation processes can confirm the absence of particles, proper siliconization levels, and cleanliness of equipment to assure that production items and equipment are suitable for use. Samples for microbiological quality, must, as previously mentioned, always be done by fully gowned staff under ISO 5 conditions using excellent aseptic techniques. During compounding, in-process testing can confirm proper pH, dissolution of materials, bioburden, and potency prior to filling. Filling operations can be monitored for fill volume (weight), headspace oxygen, and particles. These activities can all be automated to reduce interventions. These are typical examples of in-process controls utilized to assure acceptability of the process while it is underway. In the event of an abnormal result, corrective measures could be applied before further processing. The validation effort supports that these control measures are sufficient to assure product quality, when met during production operations. The sample intervals, sizes, and locations for in-process testing are chosen to enhance the validation. The tolerance limits are usually tightened relative to the release requirements to further assure that no out-of-tolerance materials are produced.

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2.1.11.2

End-Product Testing

Upon completion of the process, samples are taken to establish that the batch meets the final product specifications defined for release. Predefined sampling plans are utilized to obtain representative samples of the entire batch, the prior validation effort having assured through an expanded sampling effort that the process provides a uniform product. End-product sampling often suffers from the inability to link an anomalous result with a specific portion/segment of the batch. If the validation is insufficiently rigorous, an out-of-specification result will ordinarily result in rejection of the batch and little opportunity to take effective corrective action. The FDA has been supportive of the use of process analytical technologies (PATs) as an improvement on end-product testing [37]. These are intended to act as on-line indicators of critical product attributes enabling immediate corrective action and preventing the production of off-specification materials. This approach is common in the continuous process industries where feedforward controls are often employed. Their application to the more batch-oriented pharmaceutical/biotechnology industry is an acknowledgment that this approach can assure product quality more fully than a sampling-based approach. The PAT applications are still relatively few in number, but their utility in lieu of traditional quality methods is certainly promising. The preceding relates solely to product quality attributes, based upon chemical or physical requirements. Assurance of sterility, the most critical of all the quality components for an aseptically filled sterile product relies on the following: •

• • •

The validation of the various sterilization processes for preparation of materials, equipment, and formulations The design of the aseptic manufacturing process and facility The establishment and maintenance of a proper processing environment Most importantly, the proficiency of the operating personnel directly involved with the aseptic process

There is no direct means to evaluate the cumulative capability of these measures. We infer success in aseptic processing through the evaluation of indirect measures of performance: air pressure differentials, total particle counts, viable monitoring results, and end-product sterility testing. The enormous challenge of aseptic processing is that none of the in-process or end-product testing results can prove that the attribute of sterility is attained with a high degree of certainty. Therefore, we rely on validation and the demonstration of a validated state of control to infer the adequacy of our contamination control efforts. 2.1.11.3

Process Simulations

An indirect means of assessing a facility’s aseptic processing performance is the process simulation (or media fill) test [38]. This test substitutes a growth medium for the product in the process from the point of sterilization through to closure of the product container. The expectation is that successful handling of the growth media through the operating steps provides assurance that product formulations handled in a similar fashion would also be successful [39]. Process simulations

TERMINAL STERILIZATION

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culminate in the incubation of the media-filled containers with success defined as a limited number of contaminated units in a larger number of filled units. The result is a contamination rate for the media fill, and not a direct indication of the level of sterility assurance afforded to aseptically processed materials using the same procedures. At the present time, the level of sterility provided to aseptically processed materials cannot be measured. The FDA and EMEA have harmonized their expectations relative to process simulation performance, but they have also asserted that the goal in every process simulation is zero contamination [1, 2]. This formalized expectation and recognition that patient safety should always be preeminent have resulted in substantial improvements in aseptic processing technology over the last 20 years.

2.1.12 TERMINAL STERILIZATION Terminal sterilization is a process by which product is sterilized in its final container. Terminal sterilization is the method of choice for products that are sufficiently stabile when subjected to a compatible lethal treatment. Because the process utilized is expected to be lethal to the microorganisms present, is highly reproducible, and generally readily validated, there is a clear preference for its use [1, 40, 41]. The predominant method for terminal sterilization is moist heat, and a substantial percentage of sterile products are processed in this manner. (Estimates range from 5 to 15% of all sterile products are terminally sterilized.) The sterilization often requires the attainment of a balance between sterility assurance and degradation of the material’s essential properties [42]. The overkill sterilization method is preferred for heat-resistant materials, and may be usable for terminal sterilization where the formulation can tolerate substantial heat input. The bioburden/biological indicator approach uses less heat input but requires increased control over the titer and resistance of the bioburden organisms present. The large-volume parenteral (LVP) industry sometimes uses dedicated nonaseptic filling systems for its containers prior to subjecting them to terminal treatments. These LVP systems may approach the aseptic designs described earlier, but they are not supported by the same levels of environmental monitoring nor process simulation. Application of terminal sterilization at small volume parenteral producers may be done after the product is aseptically filled, although this practice is usual only where the firm produces predominantly aseptically filled products and would not have a filling system dedicated to terminally sterilized formulations. Product that will be subject to terminal sterilization may be filled under clean conditions with reduced environmental monitoring and control. However, control of total particulate levels requires unidirectional airflow for critical filling or assembly processes. Terminal sterilization is most commonly accomplished by moist heat. Terminal sterilization by other means is certainly possible, and a very limited number of parenteral drugs are treated with dry heat or radiation after filling. There is growing interest in the use of radiation, including low-energy E-beam, as a terminal treatment suggesting more products will be processed in this manner. Although there are numerous advantages to terminal sterilization, there can be very good reasons for aseptically filling products that are stabile enough to be com-

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patible with a sterilization process. For example, multichamber containers that cannot withstand terminal sterilization may provide a very important safety benefit to the patient by reducing aseptic admixture or reconstitution in the clinic. These aseptic activities when conducted in clinics are generally not able to be done within anything like the controls required in industrial aseptic processing. It is often beneficial to discuss processing technology choices with regulatory authorities early in the development of a new product.

2.1.13

CONCLUSION

The manufacture of parenteral drugs by aseptic processing has long been considered a difficult technical challenge. These products require careful control and stringent attention to detail to assure their safety. Aseptic processing done with discipline and taking advantage of the numerous technical developments that have occurred over the years results in sterile products that can be administered with complete confidence. The wider adaptation of advanced aseptic processing will result in further evolutionary improvements in aseptic processing. The industry is at the beginning of an era in which human-scale aseptic processing will be completely replaced by separative technologies and process automation. Additionally, improved in-process controls are likely to be implemented making validation easier and easing the compliance burden.

APPENDIX Parenteral Drug Association, Bethesda, Maryland TM 1: Validation of Steam Sterilization Cycles, 1978 TR 3: Validation of Dry Heat Processes used for Sterilization & Depyrogenation, 1981 TR 7: Depyrogenation, 1985 TR 11: Sterilization of Parenterals by Gamma Irradiation, 1988 TR 13: Fundamentals of an Environmental Monitoring Program, 2001 TR 22: Process Simulation Testing for Aseptically Filled Products, 1996 TR 26: Sterilizing Filtration of Liquids, 1998 TR 28: Process Simulation Testing for Sterile Bulk Pharmaceutical Chemicals, 2006 TR 34: Design & Validation of Isolator Systems for the Manufacture & Testing of Health Care Products, 2001 TR 36: Current Practices in the validation of Aseptic Processing, 2002 TR 40: Sterilizing Filtration of Gases, 2005 International Society For Pharmaceutical Engineering, Tampa, Florida Baseline Guide, Vol. 3: Sterile Manufacturing Facilities, 1999 Baseline Guide, Vol. 4: Water and Steam Systems, 2001 Baseline Guide, Vol. 5: Commissioning and Qualification, 2001

REFERENCES

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REFERENCES 1. U.S. Food and Drug Administration (FDA) (2004), Guideline on sterile drug products produced by aseptic processing, FDA, Washington, DC. 2. European Union (EU) (2006), Annex 1—Sterile medicinal products—draft revision. 3. International Organization for Standardization (ISO), international standard 14644 1-3. 4. U.S. Food and Drug Administration (FDA) (2004), Pharmaceutical CGMPs for the twenty-first century—A risk-based approach, FDA, Washington, DC. 5. International Conference on Organization (ICH) (2005), Draft consensus guideline quality risk management Q9, draft. 6. Whyte, W., and Eaton, T. (2004), Microbiological contamination models for use in risk assessment during pharmaceutical production, Eur J Parenteral Pharm Sci, 9(1). 7. Whyte, W., and Eaton, T. (2004), Microbial risk assessment in pharmaceutical cleanrooms, Eur J Parenteral Pharm Sci, 9(1). 8. Agalloco, J., and Akers, J. (2006), Simplified risk analysis for aseptic processing: The Akers-Agalloco method, Pharm Technol, 30(7), 60–76. 9. International Organization for Standardization (ISO) (2004), Cleanrooms and associated controlled environments—Part 7: Separative devices (clean air hoods, gloveboxes, isolators and mini-environments), ISO 14644-7. 10. Agalloco, J. (2006), Thinking inside the box: The application of isolation technology for aseptic processing, Pharm Technol., p. S8–11. 11. Lysford, J., and Porter, M. (2003), Barrier isolators history and trends, Pharm Eng, 23(2), 58–64. 12. ISPE (2005), Restricted access barrier systems (RABS) for aseptic processing, ISPE definition, Aug. 16. 13. Wikol, M. (2004), GoreTM vial isolator, ISPE presentation, Feb. 12. 14. Py, D. (2004), Development challenges for intact sterile filling, PDA presentation, Mar. 9. 15. Thilly, J. (2004), CVFL technology from lab scale to industry, PDA presentation, Mar. 8. 16. ISPE (2001), Water and Steam Systems Baseline® guide. 17. ISPE (1999), Sterile Manufacturing Facilities Baseline® guide. 18. ISPE (2001), Water and Steam Systems Baseline® guide. 19. PDA (2006), Technical Monograph 1, Industrial moist heat sterilization in autoclaves, draft 17. 20. Perkins, J. (1969), Principles and Methods of Sterilization in Health Sciences, Charles Thomas, Springfield, IL. 21. Phillips, G. B., and Morrissey, R. F. (1993), Sterilization Technology: A Practical Guide for Manufacturers and Users of Health Care Products, Van Nostrand Reinhold, New York. 22. Agalloco, J. (1998), Sterilization in place technology and validation, in Agalloco, J., and Carleton, F. J., Eds., Validation of Pharmaceutical Processes: Sterile Products, Marcel Dekker, New York. 23. PDA (1981), Technical Report 3, Validation of dry heat processes used for sterilization and depyrogenation. 24. Case, L., and Heffernan, G. (1998), Dry heat sterilization and depyrogenation: Validation and monitoring, in Agalloco, J., and Carleton, F. J., Eds., Validation of Pharmaceutical Processes: Sterile Products, Marcel Dekker, New York. 25. Burgess, D., and Reich, R. (1993), Industrial ethylene oxide sterilization, in Phillips, G. B., and Morrissey, R. F. Eds., Sterilization Technology: A Practical Guide for Manufacturers and Users of Health Care Products, Van Nostrand Reinhold, New York.

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26. Sintim-Damao, K. (1993), Other gaseous sterilization methods, in Phillips, G. B., and Morrissey, R. F. Eds., Sterilization Technology: A Practical Guide for Manufacturers and Users of Health Care Products, Van Nostrand Reinhold, New Youk. 27. Meltzer, T., Agalloco, J., et al. (2001), Filter integrity testing in liquid applications; Revisited, Part 1, Pharm Technol, 25(10), and Part 2, Pharm Technol, 25(11). 28. PDA (1998), Technical Report 26, Sterilizing filtration of liquids. 29. PDA (2005), Technical Report 40, Sterilizing filtration of gases. 30. Agalloco, J., and Carleton, F. J., Eds. (1998), Validation of Pharmaceutical Processes: Sterile Products, Marcel Dekker, New York. 31. PDA (2001), Technical Report 13, Fundamentals of an environmental control program. 32. USP 〈1116〉 (2005), Microbiological control and monitoring environments used for the manufacture of healthcare products, Pharm Forum, 31(2), Mar.–Apr. 33. Agalloco, J. (1996), Qualification and validation of environmental control systems, PDA J Pharm Sci Technol, 50(5), 280–289. 34. PDA (2006), Technical Report 28, Process simulation testing for sterile bulk pharmaceutical chemicals. 35. Trappler, E. (1998), Validation of lyophilization, in Agalloco, J., and Carleton, F. J., Eds., Validation of Pharmaceutical Processes: Sterile Products, Marcel Dekker, New York. 36. Chapman, K. G. (1984), The PAR approach to process validation, Pharm Technol, 8(12), 22–36. 37. Food and Drug Administration (FDA) (2004), PAT guidance for industry—A framework for innovative pharmaceutical development, manufacturing, and quality assurance, FDA, Washington, DC. 38. PDA (1998), Technical Report 22, Process simulation testing for aseptically filled products. 39. Agalloco, J., and Akers, J. (2006), Aseptic processing for dosage form manufacture: Organization & validation, in Carleton, F. J., and Agalloco, J. P., Eds., Validation of Pharmaceutical Processes: Sterile Products, Marcel Dekker, New York. 40. Food and Drug Administration (FDA) (1991), Use of aseptic processing and terminal sterilization in the preparation of sterile pharmaceuticals, FR 56, 354–358. 41. PIC/S41. (1999), Decision trees for the selection of sterilisation methods (CPMP/QWP/054/98). 42. PDA (2006), Technical Monograph 1, Industrial moist heat sterilization in autoclaves, draft 17.

ADDITIONAL READINGS Akers, J. (2001), An overview of facilities for the control of microbial agents, in Block, S. S., Ed., Disinfection, Sterilization and Preservation, 5th ed, Lippincott, Williams and Wilkins, Philadelphia, pp. 1123–1138. Akers, J., and Agalloco, J. (1997), Sterility and sterility assurance, J Pharm Sci Technol 51, 72–77. Cole, J. C. (1990), Pharmaceutical Production Facilities—Design and Application, Ellis Norwood, Chicester. Institute of Environmental Science and Technology (IEST) (1995), Compendium of standards, practices, and similar documents relating to contamination control, CC009/ IESCC009.2, IEST, Mt. Prospect, IL.

ADDITIONAL READINGS

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Ljungvist, B., and Reinmueller, B. (1995), Ventilation and Airborne Contamination in Clean Rooms, Pharmacia A/B, Stockholm. Reinmuller, B. (2000), Microbiological risk assessment of airborne contaminants in clean zones, Bulletin No. 52, Royal Institute of Technology/Building Services and Engineering, Stockholm. United States Pharmacopoeia/National Formulary (2006), 29, Chapter 1116, Microbial evaluation of clean rooms, Rockville, Maryland, pp. 2969–2976.

SECTION 3 FACILITY

3.1 FROM PILOT PLANT TO MANUFACTURING: EFFECT OF SCALE-UP ON OPERATION OF JACKETED REACTORS B. Wayne Bequette Rensselaer Polytechnic Institute, Troy, New York


Motivation Background 3.1.2.1 Pharmaceutical Process Development 3.1.2.2 Batch Reactors 3.1.2.3 Reaction Calorimetry 3.1.3 Laboratory Vessels and Reaction Calorimeters 3.1.3.1 Material and Energy Balances 3.1.3.2 Estimating Fluid Properties and Heat Transfer Coefficients from Calorimeter Data 3.1.3.3 Estimating Heat Flows 3.1.3.4 Relating Heat Flows and Conversion 3.1.3.5 Semibatch Reactions 3.1.3.6 Rapid Scale-Up Relationships 3.1.3.7 Strategy under a Cooling System Failure 3.1.4 Heat Transfer in Process Vessels 3.1.4.1 Heat Transfer Relationships 3.1.4.2 Effect of Reactor Type, Jacket Heat Transfer Fluid, and Reactor Fluid Viscosity 3.1.4.3 Pilot- and Production-Scale Experiments 3.1.5 Dynamic Simulation Studies 3.1.6 Summary References


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140

EFFECT OF SCALE-UP ON OPERATION OF JACKETED REACTORS

3.1.1

MOTIVATION

There are many phases of process development between the discovery of an active pharmaceutical ingredient and the design, construction, and operation of a manufacturing process to produce a drug. A sequence of reactions and separations that is successful at the bench scale may lead to a process that is unsafe, is difficult to operate, or produces unsatisfactory product at the manufacturing scale. A manufacturing process typically has a large sequence of steps, involving several different unit operations (heat exchangers, reactors, separators, etc.), and a complete review of the design and scale-up of these unit operations would constitute a chemical engineering curriculum; thus, the focus of this chapter is the scale-up of jacketed batch chemical reactors from the laboratory to the pilot plant and manufacturing. These reaction vessels often serve many functions, including mixing, heating, cooling, distillation, and crystallization. Temperature control for laboratory reactors is typically easy because of high heat transfer area–reactor volume ratios, which do not require large driving forces (temperature differences) for heat transfer from the reactor to the jacket. Pilot- and full-scale reactors, however, often have a limited heat transfer capability. A process development engineer will usually have a choice of reactors when moving from the laboratory to the pilot plant. Kinetic and heat of reaction parameters obtained from the laboratory reactor, in conjunction with information on the heat transfer characteristics of each pilot plant vessel, can be used to select the proper pilot plant reactor. Similarly, when moving from the pilot plant to manufacturing, a process engineer will either choose an existing vessel or specify the design criteria for a new reactor. A necessary condition for operation with a specified reactor temperature profile is that the required jacket temperature is feasible. We have therefore chosen to focus on heat transfer–related issues in scale-up. Clearly there are other scale-up issues, such as mixing sensitive reactions. See Paul [1] for several examples of mixing scaleup in the pharmaceutical industry. In this chapter we discuss important issues as we move from laboratory to pilot plant and manufacturing. A review of batch process operation and pharmaceutical research is covered in Section 3.1.2, followed by laboratory vessels and reaction calorimetry in Section 3.1.3. In Section 3.1.4 heat transfer in process vessels is presented, including the effect of reactor type and heat transfer fluid on the vessel heat transfer capability. In Section 3.1.5 dynamic behavior based on simulation studies is discussed.

3.1.2 3.1.2.1

BACKGROUND Pharmaceutical Process Development

Anderson [2] presents a wide range of topics on pharmaceutical process development, including a number of different problems related to process scale-up, such as solvent and reagent selection, purification, and limitations to various operations. He notes that most reactors used for scale-up operations are selected for flexibility in running many different processes, especially for pilot plants and multiproduct manufacturing plants.

BACKGROUND

141

Pisano [3] discusses the management of process development projects in the pharmaceutical industry. Case studies are used to illustrate the effect of resource allocation decisions at different stages of a project. While there has been a focus on product development in the pharmaceutical industry, clearly process development plays an important role in getting a product to market and lowering the long-term product manufacturing costs. 3.1.2.2

Batch Reactors

Batch processes present challenging control problems due to the time-varying nature of operation. Chylla and Haase [4] present a detailed example of a batch reactor problem in the polymer products industry. This reactor has an overall heat transfer coefficient that decreases from batch to batch due to fouling of the heat transfer surface inside the reactor. Bonvin [5] discusses a number of important topics in batch processing, including safety, product quality, and scale-up. He notes that the frequent repetition of batch runs enables the results from previous runs to be used to optimize the operation of subsequent ones. LeLann et al. [6] discuss tendency modeling (using approximate stoichiometric and kinetic models for a reaction) and the use of model predictive control (linear and nonlinear) in batch reactor operation. Studies of a hybrid heating–cooling system on a 16-L pilot plant are presented. Various aspects of the effect of process scale-up on the safety of batch reactors have been discussed by Gygax [7], who presents methods to assess thermal runaway. Shukla and Pushpavanam [8] present parametric sensitivy and safety results for three exothermic systems modeled using pseudohomogenous rate expressions from the literature. Caygill et al. [9] identify the common factors that cause a reduction in performance on scale-up. They present results of a survey of pharmaceutical and fine chemicals companies indicating that problems with mixing and heat transfer are commonly experienced with large-scale reactors. 3.1.2.3

Reaction Calorimetry

The microanalytical methods of differential thermal analysis, differential scanning calorimetry, accelerating rate calorimetry, and thermomechanical analysis provide important information about chemical kinetics and thermodynamics but do not provide information about large-scale effects. Although a number of techniques are available for kinetics and heat-of-reaction analysis, a major advantage to heat flow calorimetry is that it better simulates the effects of real process conditions, such as degree of mixing or heat transfer coefficients. Regenass [10] reviews a number of uses for heat flow calorimetry, particularly process development. The hydrolysis of acetic anhydride and the isomerization of trimethyl phosphite are used to illustrate how the technique can be used for process development. Kaarlsen and Villadsen [11, 12] provide reviews of isothermal reaction calorimeters that have a sample volume of at least 0.1 L and are used to measure the rate of evolution of heat at a constant reaction temperature. Bourne et al. [13] show that the plant-scale heat transfer coefficient can be estimated rapidly and accurately from a few runs in a heat flow calorimeter. Landau et al. [14] use a heat flow calorimeter to investigate feasible pilot plant operating conditions for the production of a pharmaceutical intermediate. They

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determine kinetic and heat flow parameters using the calorimeter and further estimate heat transfer parameters for a pilot-scale reactor. Simulation studies are used to find the required jacket temperature for desired batch reactor temperature profiles. Semibatch operation is shown to be safer than normal batch operation. Landau [15] provides a detailed review of reaction calorimetry, including mathematical expressions for energy balances, and a number of application examples.

3.1.3

LABORATORY VESSELS AND REACTION CALORIMETERS

As reviewed in Section 3.1.2.3, reaction calorimeters can be used to better understand and characterize scale-related process phenomena, such as mixing and heat transfer. A heat flow calorimeter, the Mettler RC1e, is shown in Figure 1. A schematic of a similar calorimeter system is shown in Figure 2 [16]. A heat flow calorimeter can be used to estimate: • • •

Physical parameters (heat capacity) Reaction rate constants Heat transfer coefficients (overall, U or, or film, hi)

3.1.3.1

Material and Energy Balances

The overall energy balance for a process with no reaction has the form Energy accumulation = energy in heat transfer from jacket + energy in by calibration probe − energy lost by ambient heat transfer

FIGURE 1

Mettler RC1e heat flow calorimeter system (www.mt.com).

143

LABORATORY VESSELS AND REACTION CALORIMETERS Stirrer n

Condenser

Additional heater Qadd

TR pHR pR

Tw,in Tw,out and

mw

Tamb Tj,out F3

F2

Liquid surface

F1

Inert gas venting Circulation thermostat, heater, chiller

Oil jacket Scale

Scale

FIGURE 2

Scale

Tj,in

Outlet valve

Schematic of HEL SIMULAR reaction calorimeter. From ref. 16.

which is shown mathematically as (mc p )r

dT = −UA(T − Tj ) + qcal − kloss (T − Tamb ) dt

(1)

where (mcp)r is the reactor thermal capacitance, T is the reactor temperature, Tj is the jacket temperature, U is the overall heat transfer coefficient, A is the area for heat transfer, qcal is the heat flow from the calibration probe, and the final term accounts for heat loss from the reactor system. The thermal capacitance is composed of the fluid in the vessel as well as the inert components in contact with the fluid, including the vessel wall, agitator (stirrer), and sensors (e.g., thermocouple), as shown in the equation (mc p )r = V ρc p + mv c pv

(2)

where V is the volume of liquid, ρ is the liquid density, cp is the liquid heat capacity, mv is the mass of the vessel wall and other inerts, and cpv is the average heat capacity of the vessel wall and inerts. The inert contributions and heat transfer to the ambient can be found from extensive calibration studies. For small-scale reactors, such as reaction calorimeters, the thermal mass of the inerts can be significant. The thermal capacitance ratio, sometimes called the Lewis number, is given as φ=

(mc p )r mv c pv = 1+ V ρc p V ρc p

(3)

which can be on the order of 1.5–2 for a small-scale reactors and adiabatic calorimeters but is often 1.05–1.10 for small pilot plant reactors and less than 1.02 for manufacturing-scale reactors.

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3.1.3.2 Estimating Fluid Properties and Heat Transfer Coefficients from Calorimeter Data In a heat flow calorimeter, a feedback controller is used to maintain a constant desired reactor temperature by adjusting the jacket temperature. From (1), with a constant calibration probe heat flow, at steady state (dT/dt = 0), the overall heat transfer coefficient can be found from qcal − kloss (T − Tamb ) T − Tj

UA =

(4)

Also, the fluid heat capacity can be found by ramping up the reactor temperature and using (mc p )r =

−UA(T − Tj ) + qcal − kloss (T − Tamb ) dT/dt

(5)

and solving for cp from (2), assuming that the reactor inert component contributions are known from previous studies. An example calibration study is shown in Figure 3, where a constant heat flow is applied from 35 to 42 min, enabling the heat transfer coefficient to be estimated from the temperature difference using Equation (4). Then, the heat capacity is estimated from the temperature ramp applied between 5 and 20 min. It should be noted that the heat transfer coefficient and heat capacity of the fluid may vary with concentration and temperature. Typically, calibration experiments are performed before and after the reaction; then the heat transfer coefficient and heat capacity are assumed to vary linearly with conversion or batch time. For polymerization reactions in particular, the viscosity can increase tremendously with conversion, causing a substantial decrease in the heat transfer coeffi-

RC1 Calibration profiles 28 Determine UA Temperature, °C

26 24 22 20

reactor jacket

Determine cp 18

0

10

20

30 Time, min

40

50

60

FIGURE 3 Example reaction calorimetry study without reaction. The overall heat transfer coefficient area can be found during the steady-state temperature difference and known calibration probe heat flow, between 35 and 42 min. The heat capacity can then be found from the temperature ramp between 5 and 20 min.


145

cient. Reaction experiments can be run at several temperatures to find the functional relationship with temperature. Since the heat transfer area as a function of liquid volume is known, the overall heat transfer coefficient U can be calculated from (4). The overall heat transfer coefficient is calculated as 1 1 xg = + U hi kg

(6)

where the jacket side resistance is negligible. The glass vessel heat transfer resistance (xg/kg, thickness/thermal conductivity) can be used to find the reactor fluid heat transfer coefficient (hi). 3.1.3.3

Estimating Heat Flows

The reaction heat flow can be found by rearranging (1), with the calibration heat probe replaced by the reaction heat flow, to find qr = (mc p )r

dT + UA(T − Tj ) + kloss(T − Tamb ) dt

(7)

The total heat released during the reaction can be found by integrating (7), tf

Qtot = ∫0 qr dt

(8)

or, represented as a scaled (per-unit mass) total heat release, Qtot =

Qtot Qtot = Vρ m

(9)

The molar heat of reaction can be found from ΔH rxn =

−Qtot nrxn

(10)

where nrxn is the molar amount reacted. As a “first-pass” calculation, if it is assumed that the dominant heat transfer resistance is on the reactor side, then the overall heat transfer coefficient (U) from (4) can be used for scale-up. 3.1.3.4

Relating Heat Flows and Conversion

The reaction heat flows are directly related to the conversion of reactants [14]. Consider a first-order reaction of a limiting reactant, with the rate expression dC = −kC dt

(11)

146


where C is the molar concentration of the reactant. The heat flow is qr =

( dCdt ) ΔH

rxn

V = −kC ΔH rxn V

(12)

with an initial heat flow of qr 0 = −kC0 ΔH rxn V

(13)

dividing (12) by (13), we find the relationship between concentration and heat flow: C q = r C0 qr 0

(14)

For an isothermal reaction, the solution to (11) is C = e − kt C0

(15)

so, the heat flow for an isothermal reaction is qr = e − kt qr 0

(16)

Thus, the reaction rate constant k can be estimated from the reaction heat flow without making any concentration measurements. Assuming an Arrhenius rate expression k = A0 e − E/RT

(17)

the rate constant at several temperatures can be used to estimate the frequency factor (A0) and activation energy (E). (See ref. 14 for an example application.) 3.1.3.5

Semibatch Reactions

For extremely exothermic reactions it is necessary to slowly add the feed over time, that is, operate in a semibatch fashion. The heat flow for a semibatch reaction can be found from qr = UA(T − Tj ) + (mc p )r

dT f c pf (T − Tf ) +m dt

(18)

f is the mass flow rate of the feed stream. If the reactor temperature is where m maintained constant, this reduces to f c pf (T − Tf ) qr = UA(T − Tj ) + m

(19)


147

For reactions with essentially instantaneous kinetics, the reaction rate is limited by the feed addition rate. For other reactions, particularly if the reactor is operated at too low of a temperature, a reactant concentration can “build up,” eventually reaching an unsafe level that could lead to a rapid temperature rise and explosion. It is important for these reactions to monitor the heat flow to confirm that the reactant concentration is not increasing to unacceptable levels. 3.1.3.6

Rapid Scale-Up Relationships

Lacking knowledge of the larger scale reactor, it is tempting to simply assume that only the area for heat transfer varies upon scale-up. A natural parameter is the cooling time,1 defined as τ co =

(mc p )r V ρc p φ = UA UA

(20)

The heat transfer area varies with the square of the vessel diameter, and the volume varies with the cube of the vessel diameter. Thus the area–volume ratio (A/V) varies with volume as A 1 ~ V V 1/3

(21)

The inverse cooling time relationship for scale-up from volume V1 to V2 is

( )

⎡ UA ⎤ ⎡ UA ⎤ V1 ⎢⎣ V ρc φ ⎥⎦ = ⎢⎣ V ρc φ ⎥⎦ V 2 p p 2 1

1/3

(22)

The required reactor-jacket temperature difference on scale-up, with a constant Lewis number, is [T − Tj ]2 = [T − Tj ]1

( ) V2 V1

1/3

(23)

so the temperature difference can increase dramatically when a process is scaled up several orders of magnitude. Reactor-jacket temperature difference constraints can be particularly important for glass-lined vessels, where the limit is often 75 °C. 3.1.3.7

Safety under a Cooling System Failure

In the event of a cooling system failure it can be assumed that the reactor operates adiabatically. The adiabatic temperature rise can be found from

1

The notion of cooling time can be understood by writing (1) and assuming no calibration energy or heat loss. Then (1) becomes τco(dT/dt) = −(T − Tj). If a constant temperature difference T − Tj is applied, it will take τco time units for the reactor temperature to change by the temperature difference.

148


ΔTad =

Qtot (mc p )r

(24)

and the final temperature is Tfinal = Tinitial + ΔTad

(25)

As long as the final temperature is less than some critical “onset” temperature where a secondary decomposition reaction occurs, then the process can safely handle a cooling system failure. If a batch reactor temperature cannot be assured to remain less than the onset temperature after a cooling system failure, then a semibatch operation should be used. As noted in Section 3.1.3.5, it is necessary to assure that reactant concentration is not increasing above an onset concentration where a similar decomposition could occur with a cooling system failure.

3.1.4

HEAT TRANSFER IN PROCESS VESSELS

Based on initial heat flow calorimetry studies, a process development engineer must choose the appropriate reactor vessels for pilot plant studies. A pilot plant typically has vessels that range from 80 to 5000 L, some constructed of alloy and others that are glass lined. In addition some vessels may have half-pipe coils for heat transfer, while others have jackets with agitation nozzles. A process drawing for a typical glass-lined vessel is shown in Figure 4. In Sections 3.1.4.1 and 3.1.4.2 we review fundamental heat transfer relationships in order to predict overall heat transfer coefficients. In Section 3.1.4.3 we review experimental techniques to estimate heat transfer coefficients in process vessels. 3.1.4.1

Heat Transfer Relationships

Reactor-Side Coefficient The reactor-side heat transfer coefficient is calculated as hi = a

ki Re0i .67 Pr 0i .33 Di

(26)

where a is the agitation constant (0.33), ki is the fluid thermal conductivity, Rei is the Reynolds number, and Pri is the Prandtl number, Rei =

Pri =

2 Dag N ρi μi

(27)

μ i c pi ki

(28)


149

SRW 3525 drive E Lubricated dry mechanical seal

Drive nozzle face 10˝ 13˝ (3˝ Nozs.)

13 1/4˝ (4˝ Nozs.)

1/2˝ Cplg.

2˝ Cplgs. (Two)

5˝

54˝ O.D.

Optional side supports

48˝ I.D. C D

14 1/4˝ (6˝ Noz.)

Fin Battle w/RTD Temperature Sansor

A B

23˝ Span Cryo-Lock CBT

F

1 1/2˝ agit. nozs.

4 1/4˝ 1 1/2˝ Cplgs. 6˝

(4) 3/4˝ dia. holes equally spaced on a 10˝ BC

1/2˝ Cplg. 3˝ Noz.

3˝ (Offset)

18˝

3˝ Legs (four) 45˝ Leg circle 12˝

FIGURE 4 Typical 300- or 500-gal jacketed vessel (www.pfaudler.com).

and N is the agitator rotation rate. It should be noted that the film heat transfer coefficient varies inversely with the viscosity, that is,

hi ~

1 μ

0.33 i

(29)

Reactions where the viscosity increases substantially with conversion, such as some polymerization reactions, can be particularly difficult to control upon scale-up. Jacket-Side Coefficient Here the calculations are shown for a jacket equipped with agitation nozzles that greatly increase the jacket fluid velocity. The jacket “swirl velocity” vj is calculated (iteratively) from the nonlinear algebraic relationship [17] n (vn − v j ) = m

( )

4 fL ⎛ v2j ⎞ ⎜ ⎟ ρAf De ⎝ 2 ⎠

(30)

150


where m n the is the nozzle mass flow rate, vn is the nozzle velocity, the friction factor is 2 × 0.023 Re0.2

(31)

kj 0.027 Re0j .8 Prj0.33 De

(32)

f =

the jacket-side film coefficient is hj =

and the Reynolds and Prandtl numbers are Rej =

Prj = Overall Coefficient the resistances,

De vj ρj μj

(33)

μ j c pj kj

(34)

The overall heat transfer coefficient is found from the sum of

xg 1 1 1 x = + + m+ + ffi + ff j U hi hj km kg

(35)

which includes reactor film, jacket film, vessel metal, vessel glass, and fouling factors for both the reactor and jacket sides. 3.1.4.2 Effect of Reactor Type, Jacket Heat Transfer Fluid, and Reactor Fluid Viscosity Here we present examples of how the reactor type and heat transfer fluid affect the heat transfer coefficient. When the reactor fluid has a low viscosity, the dominant heat transfer resistance tends to be on the jacket side. When the reactor fluid has a high viscosity, however, the dominant resistance is typically on the reactor side. Parameter values for the studies are presented in Figures 5–7 and are given in the literature [18]. The overall heat transfer coefficient is much higher for an alloy reactor/half-pipe jacket than for a glass-lined carbon steel reactor/agitation nozzle jacket, as shown in Figure 5, where Syltherm is the heat transfer fluid. Syltherm has a significantly lower heat transfer coefficient than an ethylene glycol mixture, as shown in Figure 6, but is capable of operating over a wider range of temperatures. The reactor fluid viscosity has a tremendous effect on the overall heat transfer coefficient, as shown in Figure 7. This can be particularly important in polymerization reactions where viscosity increases with conversion.


151

Overall U, English units

80 Half pipe Jacket w/nozzles

70 60 50 40 30 20

–50

0

50 100 150 Jacket temperature °C

200

250

FIGURE 5 Overall heat transfer coefficient for 500-gal reactors. Comparison of alloy half pipe with glass-lined carbon steel (GLCS). Syltherm is the heat transfer fluid. (From ref. 18, with permission.)

60

Syltherm Glycol

55


50 45 40 35 30 25 20 15

–50

0


200

250

FIGURE 6 Overall heat transfer coefficient for 500-gal GLCS reactor. Comparison of Syltherm with Glycol. (From ref. 18, with permission.)

3.1.4.3

Pilot- and Production-Scale Experiments

The relationships shown in Section 3.1.3 are also pertinent to large-scale reactors. By using different solvents and volumes of solvent, pilot and production reactor heat transfer characteristics can be determined from a series of experiments. A primary limitation, compared to reaction calorimeter characterization, is that a calibration probe is rarely available. Thus, heat-up and cool-down studies, performed

152

EFFECT OF SCALE-UP ON OPERATION OF JACKETED REACTORS 500 gal. GLCS. 1 cp vs. 3000 cp 60


55 1 cP 3000 cP

50 45 40 35 30 25 20 15 10 –20

0

20


100

120

FIGURE 7 Overall heat transfer coefficient for 500-gal GLCS reactor with glycol heat transfer fluid. Comparison of effect of reactor-side viscosity.

100

temperature, °C

80 60 40 20 0 0

20

40

60

80 100 120 Time, min

140

160

180

FIGURE 8 Temperature profiles (jacket inlet, jacket outlet, and reactor) for a pilot plant reactor. (From ref. 19.)

by varying the jacket temperature and observing the changes in the reactor temperature (for solvents with known heat capacity), are used to characterize the reactor. The inverse cooling time, UA dT/dt = (mc p )r Tj − T

(36)

can be estimated from the temperature data collected from a heat-up/cool-down study. A characteristic example for a pilot-scale reactor is shown in Figure 8. The


153

28 27 mCp/UA, min

26 cool-down

25 24 23

heat-up

22 21 20 19

0

10

20

30

40

50

60

70

80

90

Jacket temperature, °C

FIGURE 9

Cooling time estimates based on data presented in Figure 8. (From ref. 19.)

resulting cooling time estimates are shown in Figure 9. Notice that the overall heat transfer coefficient is clearly a function of the jacket temperature. The reduced heat transfer at the lower jacket temperatures is due to the strong relationship between viscosity and temperature for the 40% glycol solution used in the jacket. The discontinuity in the cooling time estimate at around 45 °C may be due to two factors. One factor is the assumption of no heat loss from the vessel, which would tend to lower the UA estimates during the heat-up phase. Another factor is the assumption that the metal and glass inerts in the reactor are at the temperature of the reactor; in practice it might be a better assumption that the reactor wall in particular is at a temperature that is intermediate between the jacket and reactor temperatures. The fluid and inert thermal masses can be independently estimated by conducting experiments with a number of different solvent amounts. From the cooling time expression (mc p )r mv c pv V ρc p = + UA UA UA

(37)

writing this as a function of the reactor fluid volume, (mc p )r mv c pv ρc p = + ⋅V UA UA UA

(38)

and conducting experiments at a number of different fluid volumes or, equivalently, masses (Vρ), (mc p )r mv c pv c p = + ⋅Vρ UA UA UA

(39)

the linear regression can be used to find the slope and intercept and thus estimate the UA and mvcpv terms [19]. This approach is shown in Figure 10 for a jacket tem-

154

EFFECT OF SCALE-UP ON OPERATION OF JACKETED REACTORS 30

mCp/UA, min

28 26 24

22 20

18 200

350

250 300 Mass of water, kg

FIGURE 10 Linear regression to estimate thermal mass and UA. (From ref. 19.)

To cold HT fluid system

TC1 TC2

To hot HT fluid system

From cold HT fluid system

From hot HT fluid system

FIGURE 11 Characteristic pilot plant vessel control strategy. Slave (secondary) controller based on jacket outlet temperature is shown. The control valve is on the outlet stream to minimize temperature gradients (when switching from hot to cold fluids) that would be imposed if the valve was on the inlet. (From ref. 18, with permission.)

perature of 60 °C (based on a total of eight experiments at five different reactor fluid volumes).

3.1.5

DYNAMIC SIMULATION STUDIES

Older pilot plant and manufacturing processes often used steam for heating and water for cooling, with a switch-and-purge strategy between the two modes. Recent process designs have two heat transfer fluid systems (hot and cold heat transfer fluids) that are used for most of the heating and cooling needs. In addition, some vessels may have nitrogen coolers for cryogenic operation. A simplified schematic for a jacket heat transfer service is shown in Figure 11 [18]. Here, two separate heat transfer fluid headers are used, and the control valve is on the outlet stream to reduce the temperature shocks that might occur if a single

DYNAMIC SIMULATION STUDIES

155

control valve was on the inlet stream. Depending on the range of temperatures, either ethylene glycol or a proprietary fluid such as Syltherm is used. Depending on whether heating or cooling is needed, either the hot or cold process control valve is open. Similarly, on–off valves return fluid to the appropriate distribution system. Although the heat transfer fluid can be used over a wide range of temperatures, the film heat transfer coefficient is a strong function of temperature due to viscosity effects. The “cooling time” of a large reactor operating at a low temperature can be substantially longer than that of a small reactor operating at a high temperature due to this strong temperature effect. Simulation studies can be used to: • •

• •

Understand the effect of heat transfer fluid Understand possible performance limitations due to scale and operating conditions Test the effect of specified temperature gradient constraints Assist with controller design and selection of tuning parameters for system start-up

Various levels of models can be used to describe the behavior of pilot-scale jacketed batch reactors. For online reaction calorimetry and for rapid scale-up, a simple model characterizing the heat transfer from the reactor to the jacket can be used. Another level of modeling detail includes both the jacket and reactor dynamics. Finally, the complete set of equations simultaneously describing the integrated reactor/jacket and recirculating system dynamics can be used for feedback control system design and simulation. The complete model can more accurately assess the operability and safety of the pilot-scale system and can be used for more accurate process scale-up. In the simulation studies that follow, it is assumed that the reactor and jacket are well mixed, resulting in differential equations for the material and energy balances [18]. The reactor shell (including a glass lining, if used) and reactor internals (agitator and baffles) are at the same temperature as the reactor, so their “thermal mass” is included in the reactor energy balance. Similarly, the jacket shell is at the jacket temperature, with an associated thermal mass. The heat transfer area A is proportional to the reactor liquid level (between volumes associated with the minimum and maximum heat transfer area); also, the reactor shell thermal mass varies linearly with the liquid level. Heat transfer coefficients are calculated using the relationships presented in Section 3.1.4; see Garvin [20] or Dream [21] for detailed examples. Parameters, viscosity in particular, are a function of temperature. We focus on the effect of reactor size and material of construction on the expected dynamic behavior of the reactors. Details on the model development and simulation environment are presented elsewhere [18]. Figure 12 illustrates that a vessel can have significantly different dynamic behavior depending on whether it is being heated or cooled (for illustrative purposes, the freezing point of water is neglected in this simulation). The increase in reactor temperature results in a much faster response than a decrease for two reasons: (i) the jacket heat transfer fluid has a much higher viscosity (resulting in a lower overall heat transfer coefficient) at low temperatures and (ii) the fluid flow rate/jacket temperature gain is proportional to

156

EFFECT OF SCALE-UP ON OPERATION OF JACKETED REACTORS 100

Temperature, °C

80 60 40 20 0 –20 –40 0

20

40

60

80

100 120 140 160 180 200 Time, min

FIGURE 12 Comparison of responses for ±30 °C reactor temperature setpoint changes at t = 10 min; 500-gal GLCS filled with water (1925 kg).

100 Water Organic

90

Temperature, °C

80 70 60 50 40 30 20

0

20

40

60

80

100 120 140 160 180 200 Time, min

FIGURE 13 Comparison of temperature responses for 30 °C batch setpoint change; 500-gal GLCS, water (1925 L) vs. organic (1700 L).

the difference between the jacket temperature and make-up fluid temperature, which becomes small at low jacket temperatures. Notice that the initial response for the temperature increase is constrained by the ramp limit of 5 °C/min on the jacket temperature. The temperature response of an organic solvent is much faster than water because of the heat capacity difference, as shown in Figure 13. The previous plots were for simple heating/cooling applications (ref. 18 presents further studies for cryogenic and semibatch systems).

REFERENCES

3.1.6

157

SUMMARY

In this chapter we have presented an overview of scale-up considerations involved as one moves from bench-scale reaction calorimetry to larger scale pilot plant and production reactors. Our focus has been on heat transfer and single-phase processes, addressing primarily the problem that the heat transfer area per unit reactor volume decreases with scale. Clearly, there are many challenging problems associated with multiphase vessels, with evaporation/distillation and crystallization as obvious examples, but these topics are beyond the scope of this chapter.

REFERENCES 1. Paul, E. L. (1988), Design of reaction systems for specialty organic chemicals, Chem. Eng. Sci., 43(8), 1773–1782. 2. Anderson, N. G. (2000), Practical Process Research and Development, Academic, New York. 3. Pisano, G. P. (1997), The Development Factory, Harvard Business School, Boston. 4. Chylla, R. W., and Hasse, D. R. (1993), Temperature control of semi-batch polymerization reactors, Comp. Chem. Eng., 17(3), 257–264. 5. Bonvin, D. (1998), Optimal operation of batch reactors—A personal view, J. Proc. Cont., 8(5–6), 355–368. 6. LeLann, M. V., Cabassud, M., and Casamatta, G. (1999), Modeling, optimization and control of batch chemical reactors in fine chemical production, Annu. Rev. Control, 23, 25–34. 7. Gygax, R. W. (1990, Feb.), Scale-up principles for assessing thermal runaway risks, Chem. Eng. Prog., 86(2), 53–60. 8. Shukla, P. K., and Pushpavanam, S. (1994), Parametric sensitivity, runaway, and safety in batch reactors: Experiments and models, Ind. Eng. Chem. Res., 33(12), 3202–3208. 9. Caygill, G., Zanfir, M., and Gavrildis, A. (2006), Scalable reactor design for pharmaceuticals and fine chemicals production. 1: Potential scale-up obstacles, Org. Proc. Res. Dev., 10(3), 539–552. 10. Regenass, W. (1985), Calorimetric monitoring of industrial chemical processes, Thermochim. Acta, 95, 351–369. 11. Kaarlsen, L. G., and Villadsen, J. (1987), Isothermal reaction calorimeters—I. A literature review, Chem. Eng. Sci., 42(5), 1153–1164. 12. Kaarlsen, L. G., and Villadsen, J. (1987), Isothermal reaction calorimeters—II. Data treatment, Chem. Eng. Sci., 42(5), 1165–1173. 13. Bourne, J. R., Buerli, M., and Regenass, W. (1981), Heat transfer and power measurements in stirred tanks using heat flow calorimetry, Chem. Eng. Sci., 36, 347–354. 14. Landau, R. N., Blackmond, D. G., and Tung, H.-H. (1994), Calorimetric investigation of an exothermic reaction: Kinetic and heat flow modeling, Ind. Eng. Chem. Res., 33, 814–820. 15. Landau, R. N. (1996), Expanding the role of reaction calorimetry, Thermochim. Acta, 289, 101–126. 16. Obenndip, D. A., and Sharratt, P. N. (2006), Towards an information-rich process development. Part I: Interfacing experimentation with qualitatitive/semiquantitative modeling, Org. Proc. Res. Dev., 10(3), 430–440.

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17. Bolliger, D. H. (1982), Assessing heat transfer in process-vessel jackets, Chem. Eng., Sept. 20, 95–100. 18. Bequette, B. W., Holihan, S., and Bacher, S. (2004), Automation and control issues in the design of a pharmaceutical pilot plant, Control Eng. Practice, 12, 901–908. 19. Zima, A., Spencer, G., and Bequette, B. W. (1996), Model development for batch reactor calorimetry and control, Preprint, presented at the AIChE Annual Meeting, Chicago, IL, Nov. 1996. 20. Garvin, J. (1999), Understand the thermal design of jacketed vessels, Chem. Eng. Prog., 95(6), 61–68. 21. Dream, R. F. (1999), Heat transfer in agitated jacketed vessels, Chem. Eng., Jan., 90–96.

3.2 PACKAGING AND LABELING Maria Inês Rocha Miritello Santoro and Anil Kumar Singh University of São Paulo, São Paulo, Brazil


Introduction Packaging Materials 3.2.2.1 General Considerations 3.2.2.2 Glass as packaging material 3.2.2.3 Plastic as Packaging Material 3.2.2.4 Metal as Packaging Material 3.2.2.5 Applications: Some Examples 3.2.3 Quality Control of Packaging Material 3.2.3.1 General Considerations 3.2.3.2 Packaging Components 3.2.3.3 Inhalation Drug Products 3.2.3.4 Drug Products for Injection and Ophthalmic Drug Products 3.2.3.5 Liquid-Based Oral Products, Topical Drug Products, and Topical Delivery Systems 3.2.3.6 Solid Oral Dosage Forms and Powders for Reconstitution 3.2.4 Importance of Proper Packaging and Labeling 3.2.5 Regulatory Aspects 3.2.5.1 General Considerations 3.2.5.2 Food, Drug and Cosmetic Act 3.2.5.3 New Drugs 3.2.5.4 Labeling Requisites 3.2.5.5 Prescription Drugs 3.2.5.6 Drug Information Leaflet 3.2.5.7 Other Regulatory Federal Laws 3.2.5.8 Fair Packaging and Labeling Act 3.2.5.9 United States Pharmacopeia Center for the Advancement of Patient Safety 3.2.5.10 National Agency of Sanitary Vigilance (ANVISA, Brazil)


159

160

PACKAGING AND LABELING

3.2.5.11 International Committee on Harmonization (ICH) 3.2.5.12 European Union Regulatory Bodies References

3.2.1

INTRODUCTION

The packaging of a pharmaceutical product fulfils a variety of roles, such as product presentation, identification, convenience, and protection until administration or use. Selection of packaging requires a basic knowledge of packaging materials, the environmental conditions to which the product will be exposed, and the characteristics of the formulation. Several types of packaging are used to contain and protect the pharmaceutical preparations, such as the primary packaging around the product and secondary packaging such as a carton and subsequent transit cases [1]. The principal objective of the modern pharmaceutical industry is to manufacture pharmaceutical preparations presenting high quality, identity, purity, effectiveness, and innocuity in order to guarantee the satisfaction and safety of patients. The development of a new drug must involve the synthesis of a molecule, determination of its pharmacological activity, industrial-scale production, and its commercialization to guarantee quality of the final product. Packaging system development, including primary and secondary packaging components, is of critical importance. The material should be selected based on the characteristics of pharmaceutical product and dosage form. After the production phase, packaging must be planned according to regulatory requirements and its quality should be controlled according to the specifications. Commercially, the packaging material is used as a barrier to protect the pharmaceutical preparations against external factors that can degrade them and consequently decrease their effectiveness and increase toxic effects. Once the type of packaging material is decided based on such factors as size, shape, capacity, and physicochemical properties, all these data, including quality control tests, should be included in the specification of the products in order to assure the therapeutic effectiveness during its shelf life. Several types of materials are in use in the preparation of containers and closure systems: glass, plastics, metals, and combinations of these materials. However, care should be taken in the selection of appropriate material. These materials should not present any physical or chemical reactivity that could modify drug activity, quality, purity, or physical characteristics of the drug and pharmaceutical preparations. Any minor modification in the pharmacopeial specification is acceptable if it does not present a threat to patient’s health. The aim of this chapter is to discuss the importance of the packaging and labeling of pharmaceutical preparations. The role of packaging and labeling in the pharmaceutical industry has grown substantially over the past decade. The total packaging operation is part of any drug development program. Pharmaceutical products generally require a standard of packaging which is superior to that of most other products in order to support and comply with their main requirements, such as efficacy, integrity, purity, safety, and stability.

PACKAGING MATERIALS

161

For these reasons packaging technology should be based on the understanding of pharmaceutical products, characteristics of formulations, and dosage forms, including the physical and chemical properties of the drug substance. In the past, packaging concerns often arose only during the later steps of product development. Today, packaging is integrated with the development step and is among the earliest considerations of new pharmaceutical preparations being studied. Labels of products can vary from the simple to the extremely complex. But, even at the most basic level, product identification should meet regulatory requirements. More complex are the labels that make use of bar code technologies. New components such as microchips, biosensors, and deoxyribonucleic acid (DNA) arrays are making possible the development of new technologies leading to finished products individually packed that require specialized packaging materials and design expertise. The challenge now is to maintain low packaging cost, that is, always integrated into the cost of the product itself. Packaging in the post–World War II period benefited immensely from the commercialization of plastics, which were little known or used in prior years. Since then, the packaging industry has openly adopted plastics as a powerful new tool in the development of new packaging forms and functions. Quality control of a packaging component starts at the design stage. All aspects of package development that may give rise to quality problems must be identified and minimized by good design. Identifying and correcting mistakes in packaging will avoid product recall and rejection of pharmaceutical preparations [2, 3].

3.2.2 3.2.2.1

PACKAGING MATERIALS General Considerations

Packaging refers to all the operations, including filling and labeling, through which a bulk product should pass to become a finished product. Usually, sterile filling is not considered part of the packing process, although the bulk product is contained in a primary container. A packaging component means any single part of a container closure system. Typical components are containers (e.g., ampules, vials, bottles), container liners (e.g., tube liners), closures (e.g., screw caps, stoppers), closure liners, stopper overseals, container inner seals, administration ports [e.g., on large-volume parenterals (LVPs)], overwraps, administration accessories, and container labels [4]. A primary packaging component is one that is or may be in direct contact with the dosage form. A secondary packaging component is one that is not and will not be in direct contact with the dosage form [4]. A container closure system refers to the sum of packaging components that together contain and protect the dosage form. This includes primary packaging components and secondary packaging components, if the latter are intended to provide additional protection to the drug product. A packaging system is equivalent to a container closure system [4]. The role of packaging material on the overall perceived and actual stability of the dosage form is well established. Packaging plays an important role in quality maintenance, and the resistance of packaging materials to moisture and light can

162


significantly affect the stability of drugs and their dosage forms. It is crucial that stability testing of dosage forms in their final packaging be performed. The primary role of packaging, other than its esthetic one, is to protect the dosage forms from moisture and oxygen present in the atmosphere, light, and other types of exposure, especially if these factors affect the overall quality of the product on long-term storage [5]. The compliance packaging such as for fixed-dose combination pills and unit dosage form packaging is a therapy-related intervention and is designed to facilitate medication regimens and so potentially improve adherence. Compliance packaging can be defined as a prepackaged unit that provides one treatment cycle of the medication, to both the pharmacist and the patient, in a ready-to-use package. This innovation type of packaging is usually based on blister packaging that contain unit therapeutic dose for one time use. The separate dosage units and separate days are usually indicated on the dosage cards to help remind the patient when and how much of the medication to take, for example, blister packed oral dosage forms with drug information leaflets and contraceptive pills [6, 7]. The selection of packaging material for any pharmaceutical product is as important as proper pharmaceutical dosage form. To guarantee the safe and adequate delivery of drug product to the patient and improve patient compliance, the manufacturer should consider the following factors: 1. Compatibility and safety concerns raised by the route of administration of the drug product and the nature of the dosage form (e.g., solid or liquid based) 2. Kinds of protection the container closure system should provide to the dosage form (e.g., photosensitive, hygroscopic, easily oxidized drug products) 3. Potential effect of any treatment or handling that may be unique to the drug product in the packaging system 4. Patient compliance to the treatment and ease of drug administration 5. Safety, efficacy, and quality of drug product throughout its shelf-life The acquisition, handling, and quality control of primary and secondary packaging materials and of printed materials should be accomplished in the same way as that for the raw materials. The printed materials should be stocked in a reserved place so the possibility of unauthorized access is avoided. The labels and other rejected printed materials should be stored and transported with proper identification before being destroyed. There should be a destruction record of the printed materials. Each batch of printed material and packaging material should receive a specific reference number for identification. The identification affixed on the containers, on the equipment, in the facilities, and on the product containers should be clear, without ambiguity, and in a format approved by the company and contain the necessary data. Besides the text, differentiated colors indicating its condition could be used (e.g., in quarantine, approved, rejected, and cleaned). The packing materials should attend to the specifications, giving emphasis to the compatibility of the same with the pharmaceutical product that it contains. The material should be examined with relation to visible physical and critical defects as well as the required specifications.

PACKAGING MATERIALS

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Glass as Packaging Material

A packaging system found acceptable for one drug product may not be appropriate for another. Each application should contain enough information to show that each proposed container closure system and its components are suitable for the intended use. Nonsterile Products Solids Some topical drug products such as powders may be considered for marketing in glass bottles with appropriate dispenser. These topical drug products may be sterile and could be subject to microbial limits. The most common glass-packed solid oral dosage forms are oral powders and granules for reconstitution. A typical solid oral dosage form container closure system is a glass bottle (although plastic bottles are also used) with a screw-on or snap-off closure. A typical closure consists of a metal cap, often with a liner and frequently with an inner seal. The dry powders that are reconstituted in their marketed container need not be sterile; however, the possibility of an interaction between the packaging components and the reconstituting fluid can’t be discarded. Although the contact time will be relatively short when compared to the component/dosage form contact time for liquid-based oral dosage forms, it should still be taken into consideration when the compatibility and safety of the container closure system are being evaluated. Powders for oral administration that are reconstituted in their market container, however, have an additional possibility of interaction between the packaging components and the reconstituting fluid. Although the contact time will be relatively short when compared to the component/dosage form contact time for liquid-based oral dosage forms, it should still be taken into consideration when the compatibility and safety of the container closure system are being evaluated. Nonsolids For nonsterile products the preservative provides some protection, but continual microbial challenge will diminish the efficacy of the preservative, and spoilage or disease transmission may occur [8]. Antimicrobial preservatives such as phenylmercuric acetate are known to partition into rubbers during storage, thus reducing the formulation concentration below effective antimicrobial levels [9]. A complication of modern packaging is the need for the application of security seals to protect against deliberate adulteration and maintain consumer confidence. Sterile Products The sterile dosage forms share the common attributes that they are generally solutions, emulsions, or suspensions and are all required to be sterile. Injectable dosage forms represent one of the highest risk drug products (Table 1). Any contaminants present (as a result of contact with a packaging component or due to the packaging system’s failure to provide adequate protection) can be rapidly and completely introduced into the patient’s general circulation. Injectable drug products may be liquids in the form of solutions, emulsions, or suspensions or dry solids that are to be combined with an appropriate vehicle to yield a solution or suspension.

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Although ophthalmic drug products can be considered topical products, they have been grouped here with injectables because they are required to be sterile and the descriptive, suitability, and quality control information is typically the same as that for an injectable drug product. The potential effects of packaging component/dosage form interactions are numerous. Hemolytic effects may result from a decrease in tonicity and pyrogenic effects may result from the presence of impurities. The potency of the drug product or concentration of the antimicrobial preservatives may decrease due to adsorption or absorption. A cosolvent system essential to the solubilization of a poorly soluble drug can also serve as a potent extractant of plastic additives. A disposable syringe may be made of plastic, glass, rubber, and metal components, and such multicomponent construction provides a potential for interaction that is greater than when a container consists of a single material. Injectable drug products require protection from microbial contamination (loss of sterility or added bioburden) and may also need to be protected from light or exposure to gases (e.g., oxygen). Performance of a syringe is usually addressed by establishing the force to initiate and maintain plunger movement down the barrel and the capability of the syringe to deliver the labeled amount of the drug product. Solids For solids that must be dissolved or dispersed in an appropriate diluent before being injected, the diluent may be in the same container closure system (e. g., a two-part vial) or be part of the same market package (e.g., a kit containing a vial of diluent). Sterile powders or powders for injection may need to be protected from exposure to water vapor. For elastomeric components, data showing that a component meets the requirements of U.S. Pharmacopeia (USP) elastomeric closures for injections will typically be considered sufficient evidence of safety. Nonsolids The package must prevent the entry of organisms; for example, packaging of sterile products must be absolutely microorganism proof—hence the continued use of glass ampules. Liquid injections are classified as small-volume parenterals (SVPs), if they have a solution volume of 100 mL or less, or as LVPs, if the solution volume exceeds 100 mL [10]. Liquid-based injectables may need to be protected from solvent loss. An SVP may be packaged in a vial or an ampule. An LVP may be packaged in a vial, a glass bottle or, in some cases, as a disposable syringe. Packaging material for vials, and ampules are usually composed of type I or II glass. Stoppers and septa in cartridges, and vials are typically composed of elastomeric materials. Pharmaceuticals may interact with packaging and containers, resulting in the loss of drug substances by adsorption onto and absorption into container components and the incorporation of container components into pharmaceuticals. Diazepam in intravenous fluid containers and administration sets exhibited a loss during storage due to adsorption onto glass [11, 12]. Glass surfaces are also known to adsorb drug substances. Chloroquine solutions in glass containers decreased in concentration owing to adsorption of the drug onto the glass [13].

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Rubber closures are also known to absorb materials, including drugs. Absorption of preservatives such as chlorocresol into the rubber closures of injectable formulations has been studied extensively [13]. The water permeability of rubber closures used in injection vials is considered an important parameter in assessing the closures, but quantitative prediction of water permeability through rubber closures is difficult because the diffusion coefficient of water is dependent on relative humidity [14]. Liquid-based oral drug products are usually dispensed in glass bottles (sometimes in plastic), often with a screw cap with a liner, and possibly with a tamper-resistant seal or an overcap that is welded onto the bottle. The same cap liners and inner seals are sometimes used with solid oral dosage forms. A laminated material can be used to overwrap glass bottles for extra safety. The USP-grade glass packaging components are chemically resistant and can be considered sufficient evidence of safety and compatibility. In some cases (e.g., for some chelating agents), a glass packaging component may need to meet additional criteria to ensure the absence of significant interactions between the packaging component and the dosage form. Several ophthalmic preparations are commercialized in glass containers. Although the risk factors associated with ophthalmic preparations are generally considered to be lower than for injectables, any potential for causing harm to the eyes demands caution. A large-volume intraocular solution (for irrigation) may be packaged in a polyolefin (polyethylene and/or polypropylene) container. The liquid-based oral dosage forms may be marketed in multiple-unit bottles. The dosage form may be used as is or admixed first with a compatible diluent or dispersant. Liquid-based oral drug products in glass container must meet the requirements for USP containers. Glass containers are accepted as sufficient evidence of safety and compatibility. Performance is typically not a factor for liquid-based oral drug products but should be considered while treating pressurized liquid-based oral drug products (e.g., elixir spray). Topical dosage forms such as unpressurized sprays, lotions, ointments, solutions, and suspensions may be considered for marketing in glass bottles with appropriate dispenser. Some topical drug products, especially ophthalmic, are sterile or may be subject to microbial limits. In these cases, packaging material and handling should be done as those for injectables. 3.2.2.3

Plastic as Packaging Material

For plastic components, data from USP biological reactivity tests will typically be considered sufficient evidence of safety. Whenever possible, extraction studies should be performed using the drug product. If the extraction properties of the drug product vehicle may reasonably be expected to differ from that of water (e.g., due to high or low pH or to a solubilizing excipient), then drug product should be used as the extracting medium. If the drug substance significantly affects extraction characteristics, it may be necessary to perform the extractions using the drug product vehicle. If the total extract significantly exceeds the amount obtained from water extraction, then an extraction profile should be obtained. It may be advisable to obtain a quantitative extraction profile of an elastomeric or plastic packaging

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component and to compare this periodically to the profile from a new batch of the packaging component. Extractables should be identified whenever possible. Nonsterile Products Solids The most common solid oral dosage forms are capsules and tablets. A typical solid oral dosage forms container closure system is a plastic, usually highdensity polyethylene (HDPE), bottle with a screw-on or snap-off closure and a flexible packaging system such as a pouch or a blister package. A typical closure consists of a cap, often with a liner, frequently with an inner seal. If used, fillers, desiccants, and other absorbent materials are considered primary packaging components. A change in the selection of packing materials combined with a change in storage conditions or conditions during administration of the drug products may provoke stability problems. Many studies have been conducted on predicting the role of packaging in moisture adsorption by dosage forms. Adsorption of moisture by tablets contained in polypropylene films was successfully modeled from storage temperature and the difference in water vapor pressure between the inside and outside of the packaging [15]. Chemical and physical degradation of packaged dosage forms caused by moisture adsorption has been predicted from the moisture permeability of the packaging. For example, strength changes of lactose–corn starch tablets in strip packaging [16] and discoloration of sugar-coated tablets of ascorbic acid [17, 18] were predicted using the moisture permeability coefficient of the packaging. Typical flexible forms of packaging containing solid oral dosage forms are the blister package and the pouch. A blister package usually consists of a lidding material and a forming film. The lidding material is usually a laminate which includes a barrier layer (e.g., aluminum foil) with a print primer on one side and a sealing agent (e.g., a heat-sealing lacquer) on the other side. The sealing agent contacts the dosage form and the forming film. The forming film may be a single film, a coated film, or a laminate. A pouch typically consists of film or laminate which is sealed at the edges by heat or adhesive. Solid oral dosage forms generally need to be protected from the potential adverse effects of the following: 1. Water vapor (e.g., moisture may affect the decomposition rate of the active drug substance or the dissolution rate of the dosage form) 2. Incident light (e.g., in case of photosensitive products) 3. Reactive gases (e.g., oxygen could provoke oxidative reactions) Carefully selected packaging material may help protect drug products. For example, a blister or pouch and use of secondary packing may be used to protect pack photosensitive material, especially when a dark polymeric film with a covering lid made of aluminum is used for blister packing. Blister packaging using multilayer HDPE material and selection of an adequate sealing technique may help prevent moisture in the blister system. However, plastics and glass for packaging of solid oral dosage forms and for powders for reconstitution should meet the requirements

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of the USP container test. Incorporating oxygen adsorbents such as iron powder into packaging units can reduce the effect of oxygen. Protection from light can be achieved using primary packaging (packaging that is in direct contact with the dosage forms) and secondary packaging made of light-resistant materials. May be involved in the photolytic degradation kinetics. The velocity of the photochemical reaction may be affected not only by the light source, intensity, and wavelength of the light but also by the size, shape, composition, and color of the container. Great effort should be taken to stabilize a formulation in such a way that the shelf life becomes independent of the storage conditions. The photostability of drugs and excipients should be evaluated at the formulation development stage in order to assess the effects of packaging on the stability of the final product. Molsidomine tablet preparations in inadequate primary containers (blister) without secondary containers when exposed to irradiation may produce morpholine. These results illustrate the importance of packaging for the stability of molsidomine [19]. Three standard tests for water vapor permeation have been established by the USP for use with solid oral dosage forms. 1. Polyethylene containers (USP 〈661〉) [10] 2. Single-unit containers and unit-dose containers for capsules and tablets (USP 〈671〉) 3. Multiple-unit containers for capsules and tablets (USP 〈671〉) [10] The cotton and rayon used as fillers in solid oral dosage form containers may not meet pharmacopeial standards, but through appropriate tests and acceptance criteria for identification and moisture content, their adequacy should be shown. For example, rayon has been found to be a potential source of dissolution problems for gelatin capsules and gelatin-coated tablets. Desiccants are often used to eliminate moisture in packaging when the moisture resistance of the packaging is not sufficient to prevent exposure. The utility of desiccants has been assessed based on a sorption–desorption moisture transfer model [20]. Desiccants or other absorbent materials are primary packaging component. The components should differ in shape and/or size from the tablets or capsules with which they are packaged. Their composition should be provided and their inertness should be proved through appropriate tests, and acceptance criteria should be established. A topical powder product may be marketed in a sifter-top container made of flexible plastic tubes or as part of a sterile dressing (e.g., antibacterial product). The topical formulations in a collapsible tube can be constructed from low-density polyethylene (LDPE), with or without a laminated material. Normally, there is no product contact with the cap during storage. Thus usually there is no cap liner, especially in collapsible polypropylene screw caps. Normally separate applicator devices are made from LDPE. Product contact is possible if the applicator is part of the closure, and therefore an applicator’s compatibility with the drug product should be established, as appropriate (e.g., vaginal applicators). Nonsolids Typical liquid-based oral dosage forms are elixirs, emulsions, extracts, fluid extracts, solutions, gels, syrups, spirits, tinctures, aromatic waters, and suspen-

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sions. These products are usually nonsterile but typically need to be protected from solvent loss, microbial contamination, and sometimes exposure to light or reactive gases (e.g., oxygen). The presence of a liquid phase implies a significant potential for the transfer of materials from a packaging component into the dosage form. The higher viscosity of semisolid dosage forms and transdermal systems may cause the rate of migration of leachable substances into these dosage forms to be slower than for aqueous solutions. Due to extended contact, the amount of leachables in these drug products may depend more on a leachable material’s affinity for the liquid/semisolid phase than on the rate of migration. In addition to absorption onto and absorption into containers, transfer of container components into pharmaceuticals may affect the perceived stability/ quality of drug dosage forms. Adsorption of volatile components from rubber closures onto freeze-dried parenterals during both dosage form processing and storage brought about haze formation upon reconstitution [21–23]. Leaching of dioctyl phthalate, a plasticizer used especially in polyvingl chloride (PVC) plastics, into intravenous solutions containing surfactants was observed [24, 25]. Plastics contain additives to enhance polymer performance. PVC may contain phthalate diester plasticizer, which can leach into infusion fluids from packaging [26]. The liquid-based oral dosage forms may be marketed in multiple-unit bottles or in unit-dose or single-use pouches or cups. The dosage form may be used as is or admixed first with a compatible diluent or dispersant. A liquid-based oral drug pouch may be a single-layer plastic or a laminated material. The pouches may use an overwrap, which is usually a laminated material. For LDPE components, data from USP container tests are typically considered sufficient evidence of compatibility. The USP general chapters do not specifically address safety for polyethylene (HDPE or LDPE), polypropylene (PP), or laminate components. In such cases, an appropriate reference to the indirect food additive regulations [27] is typically considered sufficient. This reference is considered valid for liquidbased oral dosage forms which the patient will take only for a relatively short time. For liquid-based oral drug products which the patient will continue to take for an extended period, that is, months or years, and is expected to extract greater amounts of substances from plastic packaging components than from water (presence of cosolvents), additional extractable information may be needed to address safety issues. Topical dosage forms such as creams, emulsions, gels, lotions, ointments, pastes, and powders may be marketed in plastic materials. Topical dosage formulations are for local (not systemic) effect and are generally applied to the skin or oral mucosal surfaces. Some vaginal and rectal creams and nasal, otic, and ophthalmic solutions may be considered for topical drug products. A rigid bottle, a collapsible tube, or a flexible pouch made of plastic material may be used for liquid-based topical product. These preparations are marketed in a single- or multiple-unit container. For example, dissolved drug (or any substance, e.g., benzocaine) may diffuse in the suppository base and can, for instance, partition into polyethylene linings of the suppository wrap.

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Topical delivery systems are self-contained, discrete dosage forms that are designed to deliver drug for an extended period via intact skin or body surface, for example, transdermal, ocular, and intrauterine. These systems may be constructed of a plastic or polymeric material loaded with active ingredients or a coated metal. Each of these systems is generally marketed in a single-unit soft blister pack or a preformed tray with a preformed cover or overwrap. The compatibility and safety for topical delivery systems are addressed in the same manner as for topical drug products. Performance and quality control should be addressed for the rate-controlling membrane. Sterile Products Nonsolids An SVP may be packaged in a disposable cartridge, a disposable syringe, or a flexible bag made of polymeric plastic. Flexible bags are typically constructed with multilayered plastic (Table 2). An LVP may be packaged in a vial, a flexible bag, or, in some cases, a disposable syringe. Packaging material for cartridges, syringes, vials, and ampules are usually composed of polypropylene (Table 2). Stoppers and septa in cartridges and syringes are typically composed of elastomeric materials. An overwrap may be used with flexible bags to retard solvent loss and to protect the flexible packaging system from rough handling. Diazepam in intravenous fluid containers and administration sets exhibited a loss during storage due to adsorption onto and absorption into plastics [11, 12]. Absorption of clomethiazole edisylate and thiopental sodium into PVC infusion bags was observed [28]. The pH dependence of adsorption/absorption of acidic drug substances such as warfarin and thiopental and basic drug substances such as chlorpromazine and diltiazem indicates that only the un-ionized form of the drug substance is adsorbed onto or absorbed into PVC infusion bags [29]. The absorption was correlated to the octanol–water partition coefficients of the drugs, suggesting that prediction of absorption from partition data is possible [30, 31]. Polymers such as Nylon-6 (polycaprolactam) are known to adsorb drug substances such as benzocaine [32]. The ophthalmic drug products are usually solutions marketed in a LDPE bottle with a dropper built into the neck. A few solution products use a glass container due to stability concerns regarding plastic packaging components. 3.2.2.4

Metal as Packaging Material

Metal tubes constructed of a single material are the packaging material of choice for topical dosage forms and may be tested readily for stability with a product. Tubes with a coating, however, present additional problems. The inertness of coating material must be established through adequate tests and guarantee that it completely covers underlying material. The coating material must be resistant to creaking, leaking, leaching, and solvent erosion. For example, frequently used aluminum tubes have demonstrated reactivity with fatty alcohol emulsions, mercurial compounds, and preparations with pH below 6.5 and above 8.0. Nonreactive, epoxy linings have been found to make aluminum tubes resistant to attack [6].

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TABLE 1 Plastic Additives Type

Purpose

Examples

Lubricants

Improve processability

Stabilizers

Retard degradation

Plasticizers

Enhance flexibility, resiliency, melt flow Prevent oxidative degradation Minimize surface static charge Minimize coefficient of friction, especially polyolefins Color additives

Stearic acid paraffin waxes, polyethylene (PE) waxes Epoxy compounds, organotins, mixed metals Phthalates

Antioxidants Antistatic agents Slip agents

Dyes, pigments

Hindered phenolics (BHT), aromatic amines, thioesters, phosphites Quaternary ammonium compounds

Source: From ref. 6.

TABLE 2 Parenteral Drug Administration Devices Sterile Device Containers for blood products Disposable syringes Irrigating solution containers Intravenous infusion fluid containers Administration sets

Catheter

Plastic Material Polyvinyl chloride Polycarbonate, polyethylene, polypropylene Polyethylene, polypropylene, polyvinyl chloride Polyethylene, polypropylene, polyvinyl chloride Nylon (spike), polyvinyl chloride (tubing), polymethylmethacrylate (needle adapter), polypropylene (clamp) Teflon, polypropylene, thermoplastic elastomers


Some examples of plastic additives and parenteral drug administration devices used as packaging materials for sterile products can be seen in Tables 1 and 2. Ophthalmic ointments are marketed in a metal tube with an ophthalmic tip. Ophthalmic ointments that are reactive toward metal may be packaged in a tube lined with an epoxy or vinyl plastic coating. Metal containers, pressurized or not, may also be used for topical drug products. Topical dosage forms include aerosols, emulsions, gels, powders, and solutions and may be marketed in metallic flasks, pressurized or not. Topical dosage formulations are for local (not systemic) effect and are generally applied to the skin or oral mucosal surfaces. Some vaginal and rectal creams and nasal and otic spray drug products may be considered for marketing in metallic containers for topical use. A number of topical products marketed as a pressurized aerosol may be dispensed in a metallic bottle with a screw cap. Topical dosage forms in aluminum tubes usually include a liner. A tube liner is frequently a lacquer or shellac whose composition should be stated. A metallic pressurized packaging system for a liquid-

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based topical product may deter solvent loss and may provide protection from light when appropriate. The droplet size of topical aerosol sprays does not need to be carefully controlled, and the dose usually is not metered as in inhalers. The spray may be used to apply the drug to the skin (topical aerosol) or mouth (lingual aerosol) and the functionality of the sprayer should be addressed. The drug product has no contact with the cap and short-term contact with the nozzle. A topical aerosol may be sterile or may conform to acceptance criteria for microbial limits. However, the physical stability of aerosols can lead to changes in total drug delivered per dose and total number of doses that may be obtained from the container. 3.2.2.5 Applications: Some Examples Many research papers in the scientific literature present studies showing the importance of the effect of packaging materials on the stability of pharmaceutical and cosmetic preparations: 1. Santoro and co-workers [33] presented results of the stability of oral rehydration salts (ORSs) in different types of packaging materials. The objective of the research was to give guidance on the adequate choice of packaging material presenting the indispensable characteristics in order to protect ORS preparation. This pharmaceutical preparation is essential to children living in developing countries with tropical climate and its distribution is one of the programs of the World Health Organization (WHO) [34]. It has been proved in several research papers that water is the most important factor in the component’s degradation of ORSs. To proceed with the study, the pharmaceutical formulation was prepared by a pharmaceutical manufacture. The batch was packed in six types of packaging material. After storage of samples for 36 weeks maintained at ambient temperature, at ambient temperature and 76% relative humidity, and at 40°C with 80% relative humidity, analyses of water determination were made at different intervals of time. Water determination was performed by loss on drying at 50°C and Karl Fisher methods. The studied ORS preparation contained anhydrous glucose (20 g), sodium chloride (3.5 g), trisodium dehydrate citrate (2.9 g), and potassium chloride (1.5 g) According to the results, the packaging material that better protected the ORS preparation is the one constituted of polyester (18 g), aluminum (35%), and polyethylene (50 g). 2. The effect of packaging materials on the stability of sunscreen emulsions was also studied by Santoro and co-workers [35, 36]. The purpose of the research was to study the stability of an emulsion containing UVA, UVB, and infrared sunscreens after storage in different types of packaging materials (glass and plastic flasks, plastic and metallic tubes). The samples, emulsions containing benzophenone-3 (B-3), octyl methoxycinnamate (OM), and Phycocorail , were stored at 10, 25, 35, and 45°C and representative samples were analyzed after 2, 7, 30, 60, and 90 days. Stability studies were conducted by analyzing samples at predetermined intervals by highperformance liquid chromatography (HPLC) along with periodic rheological measurements. The proposed HPLC method enabled the separation and quantitative determination of B-3 and OM present in sunscreens. The method was successfully applied in

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the stability studies of the emulsions. The method is simple, precise, and accurate; there was no interference from formulation components. The sample emulsions stored at different temperatures presented similar rheological behavior, at least during the period of the study (three months). Most of the samples showed a pseudoplastic non-Newtonian thixotropic profile. There were no significant changes in the physical and chemical stability of emulsions stored in different packaging material. The studied glass and plastic packaging materials were found adequate for storing referred solar protector emulsions. 3. Sarbach and co-workers [48], studied the effect of plastics packaging materials on parenteral pharmaceutical products. Compatibility studies of these containers with different contents are required for drug registration. The authors demonstrated the migration phenomena which occurred between a trilaminated film and a parenteral solution of metronidazole at 0.5%. The main migration products found in the solution were e-caprolactam and a phthalic derivative. The authors also separated several unidentified compounds probably coming from the polyurethane adhesive. 4. Molsidomine is sensitive to light and shows a fast decomposition in solutions and in tablets. Thoma and co-workers [37] showed the importance of light-resistant packaging material for photolabile pharmaceuticals. They irradiated molsidomine preparations over a period of 72 h in a light cabinet according to storage at daylight for about 4–6 weeks. Losses of 23–90% in tablets and 43–60% in solutions were found. The photodegradation could be overcome by selection of suitable packaging materials, colorants or vanillin. The degradation product morpholine after dansylation was determined by HPLC and showed contents of 0.10–0.67 mg in tablets and 0.10–0.38 mg/mL in solution after irradiation. These examples, among many others described in the scientific literature, illustrate the importance of proper selection of packaging material for the stability and effectiveness of pharmaceutical dosage forms. 3.2.3 3.2.3.1

QUALITY CONTROL OF PACKAGING MATERIAL General Considerations

Several regulatory agencies as well as private agencies [Food and Drug Administration (FDA), British Pharmacopoeia, WHO, USP] [4, 10, 34, 38] have issued guidelines on the safety evaluation of materials and container closure systems. However, the ultimate proof of the safety and suitability of a container closure system and the packaging process is established by full shelf life stability studies. An important step in such evaluations is characterization of the packaging materials and the chemicals that can migrate or extract from container closure system components to the drug product. This extractable material belongs to diverse chemical classes that can migrate from polymeric materials, such as antioxidants, contaminants, lubricants, monomers, plasticizers, and preservatives. Such basic information is critical to understanding the biological safety and suitability of a container. Establishing the safety of container closure systems is of key importance to the medical and pharmaceutical industries (Table 3). It is no less important than the contents themselves. The FDA’s document “Guidance on Container Closure Systems for Packaging Human Drugs and Biologics” makes this point clear [4].

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TABLE 3 Examples of Packaging Concerns for Common Classes of Drug Products Degree of Concern Associated with Route of Administration

Likelihood of Packaging Component–Dosage Form Interaction High

Highest

Inhalation aerosols and solutions; injection; injectable suspensions

High

Ophthalmic solutions and suspensions; transdermal ointments and patches; nasal aerosols and sprays Topical solutions and suspensions; topical and lingual aerosols; oral solutions and suspensions

Low

Medium

Low

Sterile powders and powders for injections and inhalation powders

Topical powders; oral powders

Oral tablets and oral (hard and soft; gelatin) capsules

The FDA’s guidance document requires the evaluation of four attributes to establish suitability: protection, compatibility, safety, and performance/drug delivery. The document also provides a structured approach to ranking packaging concerns according to the route of drug administration and likelihood of packaging component–dosage form interaction. A container closure system acceptable for one drug product cannot be assumed to be appropriate for another. Each product should have sufficient information to establish that a container and its components are right for their intended use [4]. To establish suitability, all four attributes must be evaluated and be shown to pose no concern to the drug product or to product performance. Suitability refers to the tests used for the initial qualification of the container closure system with regard to its intended use. The guidance defines what tests must be done to evaluate each of the attributes of suitability. While the tests and methods described in Table 4 allow one to provide data that the container closure system is suitable for its intended use, an application must also describe the quality control (QC) measures that will be used to ensure consistency in the packaging components. The principal considerations for the QC measures are the physical characteristics and the chemical composition. By choosing two or three of the tests done in the initial suitability study, a QC program can be established that will ensure the consistency of the container closure system (Table 4). Protection A container closure system should provide the dosage form with adequate protection from factors (e.g., temperature, light) that can cause a degradation in the quality of that dosage form over its shelf life. Common causes of such degradation are exposure to light, loss of solvent, exposure to reactive gases (e.g., oxygen), absorption of water vapor, and microbial contamination. A container intended to provide protection from light or offered as a light-resistant container must meet the requirements of the USP 〈661〉 light transmission test. The procedure requires the use of a spectrophotometer, with the required sensitivity

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and accuracy, adapted for measuring the amount of light transmitted by the plastic material used for the container. The ability of a container closure system to protect against moisture can be ascertained by performing the USP 〈661〉 water vapor permeation test. The USP sets limits on the amount of moisture that can penetrate based upon the size and composition of the plastic components [HDPE, LDPE, or polyethylene terephthalate (PET)]. Evaluating the integrity of the container can be done in several ways. Two of the most common tests are dye penetration and microbial ingress. Container closure systems stored in a dye solution and exposed to pressure and vacuum cycles are examined for dye leakage into the container. The microbial ingress is similar in fashion but determines the microbial contamination of the contents when soaked in a media contaminated with bacteria. Other quantitative tests that can be run are vacuum/pressure decay, helium mass spectrometry, and gas detection. Compatibility Packaging components that are compatible with a dosage form will not interact sufficiently to cause unacceptable changes in the quality of either the dosage form or the packaging component. A leachability study designed to evaluate the amount and/or nature of any chemical migrating from the plastic material to the drug product should be considered. The study should evaluate substances that migrate into the drug product vehicle for the length of shelf life. The drug product should be evaluated at regular intervals, such as at one, three, or six months or one or two years, until the length of the shelf-life claim has been met. Analytical techniques such as liquid chromatography/mass spectrometry (LC/ MS) to evaluate nonvolatile organics, gas chromatography/mass spectrometry (GC/ MS) to evaluate semivolatile organics, and inductively coupled plasma (ICP) spectroscopy to detect and quantitate inorganic elements should be a part of this study. Unknown impurities and degradation products can be identified using liquid or gas chromatography along with MS. Information or substances identified from extractable chemical evaluation can be used to help prepare standards specific for the plastic container being studied during leachability studies. Development and validation of the selective analytical method should be thoroughly studied before its application in the detection of leachable chemicals in active drug substance and drug product. Organoleptic and chemical changes such as precipitates, discoloration, strange odor, and pH modification are signs of degradation of drug product. Changes in the physical characteristics of the container, such as brittleness, should be evaluated using thermal analysis and hardness testing. An infrared spectroscopic scan can fingerprint the materials and also provide proof of identity. Spectrophotometry and LC with ultraviolet detection can be used for the analysis of drug product stored at different stress conditions. These tests can be used for the quality control of drug product as well as for conducting stability studies on different products stored in the same container material. Safety Packaging components should be constructed of materials that will not leach harmful or undesirable amounts of substances to which a patient will be exposed when being treated with the drug product. This consideration is especially


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TABLE 4 Properties of Suitability Concerns and Interactions Attributes

Concerns and Interactions

Proposed Methods

Protection

Exposure to light, moisture, microbial ingress, and oxidation from presence of oxygen Leachable induced degradation, absorption or adsorption of drug, precipitation, change in pH, discoloration, brittleness of packaging materials No leached harmful or undesirable amounts of substances to expose patients treated with drug

USP〈661〉 light transmission and water vapor permeation, container integrity (microbial ingress, dye penetration, helium leak) Leachability study (migration of chemicals into drug product) using LC/MS, GC/MS, ICP/AA, pH, appearance of drug and container, thermal analysis (DSC, TGA), and infrared (IR)

Compatibility

Safety

Performance

Container closure system functionality, drug delivery

Extraction study (USP physicochemical tests–plastics), USP elastomeric closures for injections, toxicological evaluation, USP biological reactivity and complies with CFR, additives and purity Functionality (improved patient compliance or use), delivery (transfer dose in right amount or rate)

Abbreviations: DSC, differential scanning calorimetry; ICP, Inductively coupled plasma spectrometer; AA, Atomic absorption. Source: From ref. 39.

important for those packaging components which may be in direct contact with the dosage form, but it is also applicable to any component from which substances may migrate into the dosage form (e.g., an ink or adhesive). Determining the safety of a packaging component is not a simple process, and a standardized approach has not been established. However, an extraction study should be one of the first considerations. A good knowledge regarding possible extractable material could help analysts develop specific and selective methods to identify extractables from container closure components under various control extraction study conditions. Precise information on the synthesis of the polymer and descriptions of the monomers used in the polymerization, the solvents used in the synthesis, and the special additives that have been added during material production as well as knowledge of degradation products that may be released into the drug product are also important. Some potential extractable chemicals from packaging materials are water soluble, while others are soluble only in nonpolar environments. The USP includes physicochemical tests for plastics based on water extracts, while water, alcohol, and hexane extracts are required for polyethylene containers under controlled temperature and time parameters (70°C for 24 h for water and alcohol and 50°C for 24 h for hexane). The USP physicochemical tests for extractables should be a part of all suitability programs, regardless of the criticality of the drug dosage form. USP elastomeric closures for injections should also be a part of the extractables study to establish safety. These USP tests, which have evolved over many years, are relevant, sensitive, rapid, and inexpensive. They help establish material safety.

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The safety of material can be guaranteed by using appropriate analytical methods and instrumentation to identify and quantitate extracted chemicals. Liquid and gas chromatography and MS are powerful analytical tools that can separate and quantitate volatile and nonvolatile chemicals along with useful structural information. The mass spectrum or fragmentation pattern acquired for each molecule makes these excellent and effective tools for identifying unknown impurities or degradation products. Toxicological evaluation of identified and unidentified impurities from a container can help improve the safety index of drug products. The toxicological evaluation should take into consideration container closure system properties, drug product formulation, dosage form, route of administration, and dose regimen. A close correlation between chemical and toxicological information can provide better control on safety and compatibility of containers and closures. Performance The fourth attribute of the suitability of the container closure system, performance and drug delivery, refers to its ability to function in the manner for which it was designed. There are two major considerations when evaluating performance. The first consideration is functionality that may improve patient compliance, [e.g., a two-chamber vial or intravenous (IV) bag], or improve ease of use (e.g., a cap that contains a counter, a prefilled syringe). The second consideration is drug delivery, which is the ability of the packaging system to deliver the right amount or rate (e.g., a prefilled syringe, a transdermal patch, a metered tube, a dropper or spray bottle, a dry-powder inhaler, and a metered-dose inhaler). 3.2.3.2

Packaging Components

Quality control refers to the tests typically used and accepted to establish that, after the application is approved, the components and the container closure system continue to possess the characteristics established in the suitability studies. To ensure consistency, protection, compatibility, safety, and performance of the packaging components, it is necessary to define QC measures that will be used to ensure consistency in the packaging components. These controls are intended to limit unintended postapproval variations in the manufacturing procedures or materials of construction for a packaging component and to prevent adverse effects on the quality of a dosage form. The USP tests and studies for establishing suitability and QC of container closure system and for associated component materials are summarized in Table 5. Hydrolysis and oxidation are the two main routes of degradation for the majority of drugs. To gain more information, the drug could be subjected to a range of temperature and relative humidity conditions. In addition, photostability studies could be conducted by exposure to artificial or natural light conditions. Elevated temperature, humidity, and light stress the drug and induce rapid degradation. Harmonized guidelines are available for new drug substances and products and may provide useful information to characterize degradation processes and selection of appropriate packaging material. The primary packaging must physically protect the product from the mechanical stresses of warehousing, handling, and distribution. Mechanical stress may take a


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TABLE 5 U.S. Pharmacopeia General Tests and Assays Chapter 〈1〉 〈51〉 〈61〉 〈71〉 〈87〉 〈88〉 〈161〉 〈381〉 〈601〉 〈661〉

〈671〉 〈691〉 〈771〉 〈1041〉 〈1151〉

Topic Injections Antimicrobial preservatives—effectiveness Microbial limit tests Sterility tests Biological reactivity tests, in vitro Biological reactivity tests, in vivo Transfusion and infusion assemblies Elastomeric closures for injections, biological test procedures, physicochemical test procedures Aerosols Containers: light transmission; chemical resistance—glass containers; biological tests—plastics and other polymers; physicochemical tests—plastics; containers for ophthalmics—plastics; polyethylene containers; polyethylene terephthalate bottles and polyethylene; terephthalate G bottles; single-unit containers and unit-dose containers for nonsterile; solid and liquid dosage forms; customized patient medication packages Containers—permeation: multiple-unit containers for capsules and tablets; single-unit containers and unit-dose containers for capsules and tablets Cotton (or the monograph for purified rayon USP) Ophthalmic ointments Biologics Pharmaceutical dosage forms


variety of forms, from impact through vibration in transit and compression forces on stacking. The demands for mechanical protection will vary with product type: Glass ampules will require greater protection than plastic eye drop bottles, for example. Other protection is required from environmental factors such as moisture, temperature changes, light, gases, and biological agents such as microorganisms and, importantly, humans. The global market for medicinal products requires that the products are stable over a wide range of temperatures ranging from subzero in the polar region, 15°C in temperate zones, up to 32°C in the tropics. Along with this temperature variation, relative humidity can vary from below 50% to up to 90%, a feature that the packaging should be able to resist if necessary. The majority of packaging materials (including plastics) are to some degree permeable to moisture and the type of closure employed, such as screw fittings, may also permit ingress of moisture. The susceptibility of the product to moisture and its hygroscopicity will have to be considered and may require packaging with a desiccant or the use of specialized strip packs using low-permeability materials such as foil. Temperature fluctuations can lead to condensation of moisture on the product and, with liquids, formation of a condensate layer on top of the product. This latter problem is well known and can lead to microbiological spoilage as the condensate is preservative free.

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If the product is sensitive to photolysis, then opaque materials may be required. Most secondary packaging materials (e.g., cartons) do not transmit light, but in some cases specialized primary packaging designed to limit light transmission is employed. The package must also prevent the entry of organisms; for example, packaging of sterile products must be microorganism proof—hence the continued use of glass ampules. For nonsterile products the preservative provides some protection, but continual microbial challenge will diminish the efficacy of the preservative, and spoilage or disease transmission may occur. The packaging material must not interact with the product either to adsorb substances from the product or to leach chemicals into the product. Plastics contain additives to enhance polymer performance. PVC may contain phthalate diester plasticizer, which can leach into infusion fluids from packaging. Antimicrobial preservatives such as phenylmercuric acetate are known to partition into rubbers and plastics during storage, thus reducing the formulation concentration below effective antimicrobial levels. A complication of modern packaging is the need for the application of security seals to protect against deliberate adulteration and maintain consumer confidence. The active products used must also be stability tested in the proposed packaging material. The FDA guidance for industry suggests considering consistency in physical and chemical composition. Using a few simple tests, the quality of components and ultimately the container closure system can be monitored. Physical Characteristics The physical characteristics of interest include dimensional criteria (e.g., shape, neck finish, wall thickness, design tolerances), physical parameters critical to the consistent manufacture of a packaging component (e.g., unit weight), and performance characteristics (e.g., metering valve delivery volume or the ease of movement of syringe plungers). Unintended variations in dimensional parameters, if undetected, may affect package permeability, drug delivery performance, or the adequacy of the seal between the container and the closure. Variation in any physical parameter is considered important if it can affect the quality of a dosage form. Physical considerations such as water vapor transmission to evaluate seal integrity, thermal analysis such as DSC to monitor melting point and glass transitions of plastics, and IR scanning to prove identity should be part of an ongoing quality control monitoring program. Chemical Composition The chemical composition of the materials of construction may affect the safety of a packaging component. New materials may result in new substances being extracted into the dosage form or a change in the amount of known extractables. The chemical composition may also affect the compatibility, functional characteristics, or protective properties of packaging components by changing rheological or other physical properties (e.g., elasticity, resistance to solvents, or gas permeability). The chemical composition should also be evaluated by performing the simple but informative USP physicochemical tests using water, drug product vehicle, and alcohol extractions of plastic components. Specifications should be set for nonvola-


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tile residue (total extractables) during the initial suitability tests and then used to monitor the level of polar and nonpolar extractables as part of a quality control plan. A change in the composition of packaging raw material or a change in formulation is considered a change in the specifications. Due care must be taken to guarantee the safety, compatibility, and performance of a new dosage form in a new packaging system. The use of stability studies for monitoring the consistency of a container closure system in terms of compatibility with the dosage form and the degree of protection provided to the dosage form is essential. Except for inhalation drug products, for which batch-to-batch monitoring of the extraction profile for the polymeric and elastomeric components is routine, no general policy concerning the monitoring of a packaging system and components with regard to safety is available. Secondary packaging components are not intended to make contact with the dosage form. Examples are cartons, which are generally constructed of paper or plastic, and overwraps, fabricated from a single layer of plastic or from a laminate made of metal foil, plastic, and/or paper. In special cases, secondary packaging components provide some additional measure of protection to the drug product. In such cases it could be considered a potential source of contamination and the safety of the raw materials should be taken into consideration. 3.2.3.3

Inhalation Drug Products

Inhalation drug products include inhalation aerosols (metered-dose inhalers); inhalation solutions, suspensions, and sprays (administered via nebulizers); inhalation powders (dry-powder inhalers); and nasal sprays. The carboxymethylcellulose (CMC) and preclinical considerations for inhalation drug products are unique in that these drug products are intended for respiratory tract compromised patients. This is reflected in the level of concern given to the nature of the packaging components that may come in contact with the dosage form or the patient (Table 4). In October 1998, the FDA issued guidance for industry regarding container closure systems such as metered-dose inhaler (MDI) and dry-powder Inhaler (DPI) drug products. 3.2.3.4

Drug Products for Injection and Ophthalmic Drug Products

Injectable dosage forms are sterile and represent one of the highest risk drug products. Injectable drug products may be liquids in the form of solutions, emulsions, and suspensions or dry solids that are to be combined with an appropriate vehicle to yield a solution or suspension. Cartridges, syringes, vials, and ampules are usually composed of type I or II glass or polypropylene frequently used to deliver SVP and LVPs. Flexible bags are typically constructed with multilayered plastic. Stoppers and septa in cartridges, syringes, and vials are typically composed of elastomeric materials. An overwrap may be used with flexible bags to retard solvent loss and to protect the flexible packaging system from rough handling. Injectable drug products require protection from microbial contamination (loss of sterility or added bioburden) and may also need to be protected from light or

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exposure to gases (e.g., oxygen). Liquid-based injectables may need to be protected from solvent loss, while sterile powders or powders for injection may need to be protected from exposure to water vapor. For elastomeric components, data showing that a component meets the requirements of USP elastomeric closures for injections should typically be performed to assure safety. For plastic components, USP biological reactivity tests are recommended to assure evidence of safety. Whenever possible, the extraction studies described in USP should be performed using the drug product. Extractables should be identified whenever possible. For a glass packaging component, data from USP “Containers: Chemical resistance—Glass containers” will typically be considered sufficient evidence of safety and compatibility. In some cases (e.g., for some chelating agents), a glass packaging component may need to meet additional criteria to ensure the absence of significant interactions between the packaging component and the dosage form. The performance of a syringe is usually addressed by establishing the force to initiate and maintain plunger movement down the barrel and the capability of the syringe to deliver the labeled amount of the drug product. Ophthalmic drug products are usually solutions marketed in a LDPE bottle with a dropper built into the neck or ointments marketed in a metal tube lined with an epoxy or vinyl plastic coating with an ophthalmic tip. Since ophthalmic drug products are applied to the eye, compatibility and safety concerns should also address the container closure system’s potential to form substances which irritate the eye or introduce particulate matter into the product (USP 〈771〉, ophthalmic ointments). 3.2.3.5 Liquid-Based Oral Products, Topical Drug Products, and Topical Delivery Systems The presence of a liquid phase implies a significant potential for the transfer of materials from a packaging component into the dosage form. Liquid-Based Oral Drug Products Typical liquid-based oral dosage forms are elixirs, emulsions, extracts, fluid extracts, solutions, gels, syrups, spirits, tinctures, aromatic waters, and suspensions. These products are usually nonsterile but may be monitored for changes in bioburden or for the presence of specific microbes. A liquid-based oral drug product typically needs to be protected from solvent loss, microbial contamination, and sometimes exposure to light or reactive gases (e.g., oxygen). For glass components, data showing that a component meets the requirements of USP “Containers: Glass containers” are accepted as sufficient evidence of safety and compatibility. For LDPE components, data from USP container tests are typically considered sufficient evidence of compatibility. The USP general chapters do not specifically address safety for polyethylene (HDPE or LDPE), PP, or laminate components. A patient’s exposure to substances extracted from a plastic packaging component (e.g., HDPE, LDPE, PP, laminated components) into a liquid-based oral dosage form is expected to be comparable to a patient’s exposure to the same substances through the use of the same material when used to package food [27].

IMPORTANCE OF PROPER PACKAGING AND LABELING

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Topical Drug Products Topical dosage forms include aerosols, creams, emulsions, gels, lotions, ointments, pastes, powders, solutions, and suspensions. These dosage forms are generally intended for local (not systemic) effect and are generally applied to the skin or oral mucosal surfaces. Topical products also include some nasal and otic preparations as well as some ophthalmic drug products. Some topical drug products are sterile and should be subject to microbial limits. A rigid bottle or jar is usually made of glass or polypropylene with a screw cap. The same cap liners and inner seals are sometimes used as with solid oral dosage forms. A collapsible tube is usually constructed from metal or is metal lined from LDPE or from a laminated material. Topical Delivery Systems Topical delivery systems are self-contained, discrete dosage forms that are designed to deliver drug via intact skin or body surface, namely transdermal, ocular, and intrauterine. Each of these systems is generally marketed in a single-unit soft blister pack or a preformed tray with a preformed cover or overwrap. Compatibility and safety for topical delivery systems are addressed in the same manner as for topical drug products. Performance and quality control should be addressed for the rate-controlling membrane. Appropriate microbial limits should be established and justified for each delivery system. 3.2.3.6

Solid Oral Dosage Forms and Powders for Reconstitution

The most common solid oral dosage forms are capsules and tablets. A typical container closure system is a plastic (usually HDPE) or a glass bottle with a screw-on or snap-off closure and a flexible packaging system, such as a pouch or a blister package. If used, fillers, desiccants, and other absorbent materials are considered primary packaging components. Solid oral dosage forms generally need to be protected from the potential adverse effects of water vapor, light, and reactive gases. For example, the presence of moisture may affect the decomposition rate of the active drug substance or the dissolution rate of a dosage form. The potential adverse effects of water vapor can be determined with leak testing on a flexible package system (pouch or blister package). Three standard tests for water vapor permeation have been established by the USP, namely polyethylene containers (USP 〈661〉), single-unit containers and unit-dose containers for capsules and tablets (USP 〈671〉), and multiple-unit containers for capsules and tablets (USP 〈671〉).

3.2.4


The Poison Prevention Packaging Act (www.cpsc.gov/businfo/pppa.html) requires special packaging of most human oral prescription drugs, oral controlled drugs, certain normal prescription drugs, certain dietary supplements, and many over-thecounter (OTC) drug preparations in order to protect the public from personal injury or illness from misuse of these preparations.

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In many countries there are very strict regulations for packaging of many drug substances. Nevertheless, special packaging is not required for drugs dispensed within a hospital setting for inpatient administration. Manufacturers and packagers of bulk-packaged prescription drugs do not have to use special packaging if the drug will be repackaged by the pharmacist. Various types of child-resistant packages are covered in ASTM International standard D-3475. Medication errors linked to poor labeling and packaging can be controlled through the use of error potential analysis. The recognition that a drug name, label, or package may constitute a hazard to safety typically occurs after the drug has been approved for use and is being marketed. Calls for change almost always result from accumulating reports of serious injuries associated with the use of a drug. Numerous reports of medication errors are being reported, some of which have resulted in patient injury or death. In a number of these reports, a medication was mistakenly administered either because the drug container (bag, ampule, prefilled syringe and bottle) was similar in appearance to the intended medication’s container or because the packages had similar labeling. Obviously, the severity of such errors depends largely on the medication administered. The problem of medical errors associated with the naming, labeling, and packaging of pharmaceuticals is being very much discussed. Sound-alike and look-alike drug names and packages can lead pharmacists and nurses to unintended interchanges of drugs that can result in patient injury or death. Simplicity, standardization, differentiation, lack of duplication, and unambiguous communication are human factors that are relevant to the medication use process. These factors have often been ignored in drug naming, labeling, and packaging. The process for naming a marketable drug is always lengthy and complex and involves submission of a new entity and patent application, generic naming, brand naming, FDA—or other corresponding organization all over the world—review, and final approval. Drug companies seek the fastest possible approval and may believe that the incremental benefit of human factor evaluation is small. Very often, the drug companies are resistant to changing, for example, brand names. Although a variety of private-sector organizations in many countries have called for reforms in drug naming, labeling, and packaging standards, the problem remains. Drug names, labels, and packages are not selected and designed in accordance with human factor principles. FDA standards or other corresponding organizations in other countries do not require application of these principles, the drug industry has struggled with change, and private-sector initiatives have had only limited success. A number of factors can contribute to the mistaking of one medication for another. Failure to read the package label is one cause. Another if a medication is stored in the wrong location or if clinicians select the medication based solely on the appearance of its package. Also, confusion can occur between medications with names that look alike or sound alike or between premixed medications packaged in similar-looking containers. Another potential source for confusion with premixed medications is the presence of different concentrations of the same medication in a particular location (e.g., a package with 100 mg/mL concentration of a drug could be mistaken for one with 10 mg/mL concentration). Daily, physicians, nurses, and pharmacists base medical decisions on the information provided by a drug product’s labeling and packaging. Unfortunately, poor


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labeling and packaging have been linked all too often to medication errors. To help practitioners avoid errors, drug manufacturers should present information in a clear manner that can be grasped quickly and easily. To determine what presentation is most clear, manufacturers should invite and consider the input of physicians, nurses, and pharmacists, because they work with these products every day and are more likely than label and package designers to discover potential problems. Such input provides the basis for failure and effect analysis (FMEA), also known as error potential analysis or error prevention analysis. FMEA is a systematic process that can predict how and where systems might fail. Using FMEA, health care practitioners examine a product’s packaging or labeling in order to identify the ways in which it might fail. A number of steps to reduce confusion and improve the readability of a drug product’s label have already been determined through the use of FMEA. The first step is to reduce label clutter. Only essential information, such as the brand and generic names, strength or concentration, and warnings, should appear prominently on the front label. Numerous deaths have been prevented through the addition of a warning to concentrated vials of injectable potassium chloride, for example. Another step includes the use of typeface to enhance distinctive portions of look-alike drug names on look-alike packaging. Medication errors are also associated with poor product packaging design. Unfortunately, medication errors linked to poor labeling and packaging are sometimes used in the health care environment to justify the damage. Participation of an expertise from health care practitioners, during labeling and packaging design phase, might have prevented several errors. Whether for established drugs or new entities going through the approval process, the principles of safe practice in naming, labeling, and packaging are the same and must be very well controlled. Safety experts may differ about specific details, but there is little disagreement about the fundamental principles that should be incorporated into the drug approval process. Based on reports of errors associated with packaging and labeling, many recommendations have been proposed. Some of them are: 1. Avoid storing medications with similar packaging in the same location or in close proximity. 2. Follow the American Society of Health System Pharmacists (ASHP) guidelines or other legislation of a specific country for preventing hospital medication errors [40, 41]. The ASHP’s recommendations include the following: Fully document all medication prescription and deliveries and instruct staff that discrepancy or misunderstanding about prescription or patient information should be verified with the prescribing physician. Staff members should be told that all caregivers (regardless of level) have the duty to question the prescribing physician (regardless of the physician’s relative position in the hospital hierarchy) if they have concerns about a drug, dose, or patient. Periodically train staffs in practices that will help avoid medication errors. Ensure that the medication storage and distribution to hospital locations outside the pharmacy are supervised by hospital pharmacy staff only.

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3.

4.

5. 6.


Nonpharmacists should not be allowed to enter the pharmacy if it is closed. Perform failure mode and effects analysis. This is a technique used to identify all medication errors that could occur, determine how they occur, and estimate what their consequences would be. Steps then should be taken to prevent errors from occurring, when possible, and to minimize the effects of any errors that do occur. Report any information relating to medication errors to the Medication Errors Reporting Program operated by USP convention [10] and the Institute for Safe Medication Practice (ISMP) or other corresponding institutions in the different countries. The program shares information on medication errors with health care professionals to prevent similar errors from recurring. Hospitals should report incidents in which a device caused or helped cause a medication error. Urge suppliers to provide clear and unique labels and packages for their various individual medications.

Some other considerations relating to standards for drug names, labeling standards, and packaging standards are as follows: 1. Standard for Drug Names. The most critical issue in drug name selection is that one name should not be easily confused with another. This applies to both generic and brand names. A name must neither sound like that of another drug nor look like another drug name when it is written out by hand. From the industry’s standpoint, the challenge is to find a name that is easy to recollect and appropriate for the connotation desired, do not lead astray (safe), and not already a trade name. Nowadays, increasing sophisticated and effective methods are available for determining the likelihood of confusion by sound or sight. 2. Labeling Standards. To minimize the possibility of error, labels should be easy to read and avoid nonessential material. The name of the drug, and not the name of the manufacturer, should be the most prominent feature and should be in at least 12-point type. The use of color is very controversial; some believe that all colors should be prohibited to force personnel to read the labels. In the 1990s, a Washington State legislator proposed that every drug product entering the state must have a color-coded label. There was concern on the part of many that the state legislature would turn this idea into law. The prospect of having to color-code all the drugs entering a single state galvanized a response by industry, regulators, practitioners, and safe experts who agreed to revise pharmaceutical labeling. A Committee to Reduce Medication Errors was formed to study the problem. The effort eventually satisfied the color coders and the proposed legislation was dropped. The committee made several recommendations for standardizing and simplifying labels: 1. Eliminate unnecessary words from the label, such as “sterile,” “nonpyrogenic,” and “may be habit forming.” 2. Allow some abbreviations such as “HCl” and “Inj.” 3. Make label information consistent.

REGULATORY ASPECTS

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4. Require that vials containing medication that must be diluted bear the words “Concentrated, must be diluted” in a box on the label, that the vial have a black flip-top with those words on it, and that the ampules carry a black band. 3. Packaging Standards. While there is no evidence that trademark colors and logos on boxes pose a problem, the use of color on bottle tops and labels creates many difficulties. There are dozens of drugs whose names are quite different but whose packages look alike. This creates the potential for error when people “see” what they expect to see on the label. Standards need to be set for color on both caps and labels. Some believe that prohibiting all color would be safest—in effect, taking away a cue that could divert someone from reading the label. 3.2.5 3.2.5.1

REGULATORY ASPECTS General Considerations

Once the finished dosage form is made, the product should be packed into the primary container and labeled. Additional packaging and labeling are also included. Because of the many products and labeling materials, personnel in this area must be alert to prevent mix-ups. Controls and in-process checks should be carried out throughout the packaging/labeling operation to ensure proper labeling. Some examples of good manufacturing practices (GMP) requirements specific to packaging and labeling in different countries are as follows: In the United States the requirements should be written procedures designed to assure that correct labels, labeling, and packaging materials are used for drug products; such written procedures should be followed. These procedures should incorporate features such as prevention of mix-ups and crosscontamination by physical or spatial separation from operations on other drug products. In Canada, packaging operations are performed according to comprehensive and detailed written operating procedures or specifications, which include identification of equipment and packaging lines used to package the drug, adequate separation, and, if necessary, the dedication of packaging lines packaging different drugs and disposal procedures for unused printed packaging materials. Packaging orders are individually numbered. In the European Union, the requirements should be formally authorized in the “packaging instructions” for each product containing pack size and type. They are normally included in process controls with instructions for sampling and acceptance limits [42]. 3.2.5.2

Food, Drug and Cosmetic Act

About 100 years after its foundation, the Congress of the United States recognized that subjects related to safety and public health could not exclusively be state dependent and measures should be taken to protect the population in vital areas. Therefore, the federal government became interested in regulating products for consumption.

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In 1906, the Congress approved the Wiley Law to avoid the production, sale, or transport of food, medications, and alcoholic beverages that were inadequate or falsified, poisonous, or harmful. It was the first food and medication regulation adopted in interstate commerce. The Congress was given power to regulate commerce between foreign nations and several U.S. states. In 1912 a civil code law was enacted prohibiting any false affirmation of curing or therapeutic effect on medication labels. The current law was enacted on June 27, 1938, and regulates food, medications, medical devices, and diagnostic and cosmetic products. The law of 1938 stopped regulating the trade of alcoholic beverages. This law stated, among other recommendations, the following: 1. The label of each medication had to give the name of each active component and the quantity of some specific substances, active or not. 2. Cosmetics had to be inoffensive and be properly labeled and packaged. The 1938 law states that the label of a medication should contain adequate information regarding its use. However, in practice, it became evident that some pharmaceuticals and medications had to be administered by or under the orientation of a medical practitioner, due to the inability of a layman to diagnose a disease, choose an effective treatment, and recognize the cure or the symptoms. Several products were thus classified, but “the prescription concept of a medication” was introduced only after Alteration in the Law of Durham-Humphrey’s in 1951. Since then, a label had to carry the warring “Caution, the Federal Law prohibits dispensation without medical prescription.” The use of these medications had to be restricted to prescription by a practitioner and the packing or printed material inside had to contain adequate information so that the practitioner could prescribe them safely. Alterations in 1962 of the 1938 Law constituted an attempt to establish rigid controls on the research, production, divulging, promotion, sale, and use of medications as well as to assure its quality, efficiency, and effectiveness [43]. 3.2.5.3

New Drugs

Before starting clinical trials in humans, an authorization should be obtained from the FDA. This is known as a clinical trial authorization request for a new medication (AEM), on which it is necessary to establish the following: 1. The name that best describes the medication, including the chemical name and the structure of any new molecule 2. A complete list of medication components. 3. A quantitative composition of the medication. 4. The name and address of the vendor and an acquired description of the new drug 5. The methods, facilities, and controls used for the production, processing, and packing of a new medication 6. All available results available from preclinical and clinical trials 7. Copies of medication labels and the informative material that will be supplied to the researchers

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8. A description of the scientific training and the appropriate experience considered by the proponent to qualify a researcher as an adequate expert to investigate the medication 9. The names and “curricula vitae” of all researchers 10. An investigation layout planned for test accomplishment in humans Solicitations for release of new medications are generally very extensive, sometimes thousands of pages. The information has to be enough to justify the affirmations contained in the label of the proposed medication with respect to effectiveness, dosage, and safety. The exact composition of the content on the medication label is usually decided by consensus between the proponent and the FDA. The requisites for solicitation of new medications, whether by prescription or not, are identical. The instructions contained in the medication labels for use without prescription should demonstrate that the medication can be used safely without medical supervision. Once the medications are perfected, the publicity related to them has to be routinely presented to the FDA. The rules of 1985 also changed the requisites regarding addendums that are necessary when alterations are proposed in the medication or in its labeling, for example. In regulations promulgated by the FDA on February 12, 1972, a clinic should be called upon regarding the effectiveness of a medication. After that the information may be included in the label or in the drug informative leaflet with eligible sentence and defined by dark lines that contour it [43]. Other dispositions contained in the alterations to the 1962 law are as follows: 1. Immediate registration with the FDA before starting the production, repacking, or relabeling of medications and later annual registration, with inspections to be made at least once every two years. 2. Supportive inspections in the factory, particularly where prescription medications are produced. 3. The procedures used by the manufacturers should be in conformity with the good manufacturing practices, which permits the government to better inspect of all the operations. 4. The common name should be presented on the label. 5. The publicity of a prescription medication should present a brief summary mentioning the secondary effects, the contraindications, and the medication effectiveness. 6. All antibiotics are subject for certification procedures. 3.2.5.4

Labeling Requisites

According to a 1962 law, the main requisites for labeling are as described below. The labeling of over-the-counter medications is regulated by the Food, Drug and Cosmetic Act, which states:

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A medication should be considered falsified unless the label contains: 1. Indications of adequate use and 2. Adequate warnings regarding the pathological indications in those it should not be used or not for children use, when its use can be dangerous for health, of dosages, methods or interval of administration, or unsafe application, of mode and in necessary form for patients’ protection.

“Indications of use” were defined in the regulations as information with which even a layman can use the medication safely and for the purpose to which it is designated. The label of an over-the-counter medication must refer to the active substances, but it is not necessary to indicate its relative quantity, except where the ingredient leads to habituation. In this case the warning “Can lead to habituation” should appear on the label. A drug can be considered falsified if it does not provide, besides indications of adequate use, warnings against its use in some pathological conditions (or for children) in which the medication can constitute a health risk. Regulations have suggested warnings that can be used for most well-known dangerous substances. 3.2.5.5

Prescription Drugs

Specific requisites for labeling of ethical medications or of prescription medications are also found in the Food, Drug and Cosmetic Act. These need not to contain “adequate indications of use”; however, they must contain indications for the practitioner, inside or outside the package in which the medication is going to be dispensed, with adequate information for its use. This information may include indications, effects, dosages, route of administration, methods, frequency and duration of administration, important dangers, contraindications, secondary effects, and cautions “according to which the practitioners can prescribe the medication assuredly and for the desirable effects, including those for which it is proclaimed.” Regarding all medications, the act requires that the label present a precise affirmation on the weight of the content, measure or counting, as well as the name and manufacturer’s address, packer or distributor. The label of a prescription medication destined for oral administration has to contain the quantity or proportion of each active substance. If the medication is for parenteral administration, the quantity or proportion of all the excipients have also to be mentioned on the label, except for those that are added to adjust pH or make it isotonic, in which case only the name and its effect are needed. However, if the vehicle for injection is water, this does not need to be mentioned. If the medication is not to be administered by any of the routes mentioned above, for example, a pomade or a suppository, all excipients must to be mentioned, except for perfuming agents. Perfumes can be designated as such without the need to mention the specific components. Coloring agents can be assigned without being specified individually, unless this is required in a separate section for regulation of coloring agents, and inoffensive substances added exclusively for individual identification of each product need not be mentioned.

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The only warning that is necessary, “Attention: the Law prohibits the dispensing without prescription,” should be on the label of a prescription medication or in its secondary packing if the label is too little to contain it. 3.2.5.6

Drug Information Leaflet

The inclusion of a drug information leaflet is not compulsory whatever the medication. However, all medications, whether prescription or of over the counter, have to contain a label with adequate indications for use. If the medication label does not have enough space to contain all the information, the drug information leaflet has to be included with necessary information. The drug information leaflet and labels containing indication information must include the date when the text was last revised. To satisfy the act, the drug information leaflet usually included in the prescription medication packaging should contain “adequate information on usage, including indications, effects, dosages, methods, route, frequency and duration of administration. Any important dangers, contraindications, secondary effects and cautions, based on which the practitioner can prescribe the medication safely and for desirable effects, including those for which a clam is made.” To present the information in a uniform manner, the FDA issued labeling policies describing its format and the order and headings for the drug information leaflet description, action, indication, contraindications, alerts, cautions, adverse reactions, dosage and administration, overdose (when applicable), and as it is supplied. The drug information leaflet can contain the following optional information: Animal pharmacology and toxicology Clinical studies References Other specific cautions on medication have to appear in a visible manner at the beginning of the drug information leaflet so that practitioners, pharmacists, and patients can easily see them. According to GMP, an inspector should be cautious with several aspects of drug production, including the following: 1. Product containers and other components have to be tested and be considered adequate for their intended use only if they are not reactive, departure byproducts, or even have absorption capacity; so that they do not affect the safety, identity, potency, quality, or purity of the medication or its components. 2. Packing and labeling operations should be adequately controlled to (1) guarantee that only those medications that own quality standards and attain established specifications in their production and control be distributed, (2) avoid mix-ups during the filling operations, packing, and labeling, (3) assure that the labels and labeling used are correct for the medication, and (4) identify the finished product with a batch or a control number that allows determination of the batch production and control history. Application of the federal law on food, drug, and cosmetics is the FDA’s responsibility, which is a subdivision of the Department of Health and Human Services.

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The institution is managed by a Commissaries and is subdivided into several departments: Food safety and applied nutrition (CFSAN), Drug evaluation and research (CDER), Biologics evaluation and research (CBER), Devices and radiological health (CDRH), Veterinary medicine (CVM), Toxicological research (NCTR), Regulatory affairs (ORA) and the office of the commissioner (OC) [4, 44]. 3.2.5.7

Other Regulatory Federal Laws

There are other federal laws with which a pharmacist should be familiar. Perhaps the most important are laws on packing and labeling, operations that are regulated by the FDA and the Federal Communications Commission (FCC). The law on packing and labeling is targeted mostly to protecting the consumer. In the case of liquid the ingredients should be on the visible part of the package. The law presents specific requisites concerning the location and size of the type. Violation of this law can lead to apprehension by the FDA or a withdrawal order from the FCC. Many times a pharmacist involved in developing a product is called upon in the publicity of the medication. For this, he or she must understand the politics of the regulatory agency involved. The FCC, according to the Federal Law of Commerce, has jurisdiction over the announcement and promotion of all consumables, including medications and cosmetics. This law extends to all publicity and has to do with practices of fraudulent publicity and with promotion that is understood to be false and fraudulent. In general, the FCC controls the publicity of nonprescription drugs and cosmetics with respect to false or fraudulent affirmations, and the FDA is responsible for labeling of medications and for all publicity related to prescription medications. The principal objective of this is to avoid unnecessary duplication of procedures while enforcing the law. The agencies work closely together and the FCC relies strongly on the FDA due to its scientific knowledge. Any government has the right to approve laws for its citizens’ protection. This right constitutes the base on which laws regulate the drug substance, the drug product, and its production, distribution, and sale. It is common that these laws exist at a district level, state level, and national level and deal with falsification and adulteration, fraudulent publicity, and maintenance of appropriated sanitary conditions. Most U.S. states specify the purity requisites, labeling, and applicable packaging of a medication that are generally defined in identical language in federal law. Almost all states, prohibit the commercialization of a new medication until an authorization request for commercialization of a new medication has been submitted to the FDA and has been approved. Medication labeling requisites in each state are established, just as the local laws are defined, taking into consideration arguments and information, such as name and place of activity (production), content quantity, drug name, name of ingredients, quantity or proportion of some ingredients, usage indications, warning regarding dependence, caution against deterioration (degradation), warning about situations in which the use can be dangerous, and special requisites for labeling of official drugs [43]. 3.2.5.8

Fair Packaging and Labeling Act [44]

The FDA through Fair Packaging and Labeling Act regulates the labels on many consumer products, including health products. Title 15: Commerce and Trade Chapter 39: Fair Packaging and Labeling Program [44]

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Section 1451. Congressional Delegation of Policy Informed consumers are essential to the fair and efficient functioning of a free market economy. Packages and their labels should enable consumers to obtain accurate information as to the quantity of the contents and should facilitate value comparisons. Therefore, it is hereby declared to be the policy of the Congress to assist consumers and manufacturers in reaching these goals in the marketing of consumer goods [44]. Section 1452. Unfair and Deceptive Packaging and Labeling: Scope of Prohibition (a) Nonconforming Labels It shall be unlawful for any person engaged in the packaging or labeling of any consumer commodity (as defined in this chapter) for distribution in commerce, or for any person (other than a common carrier for hire, a contract carrier for hire, or a freight forwarder for hire) engaged in the distribution in commerce of any packaged or labeled consumer commodity, to distribute or to cause to be distributed in commerce any such commodity if such commodity is contained in a package, or if there is affixed to that commodity a label, which does not conform to the provisions of this chapter and of regulations promulgated under the authority of this chapter. (b) Exemptions The prohibition contained in subsection (a) of this section shall not apply to persons engaged in business as wholesale or retail distributors of consumer commodities except to the extent that such persons (1) are engaged in the packaging or labeling of such commodities, or (2) prescribe or specify by any means the manner in which such commodities are packaged or labeled. Section 1453. Requirements of Labeling; Placement, Form, and Contents of Statement of Quantity; Supplemental Statement of Quantity (a) Contents of Label No person subject to the prohibition contained in section 1452 of this title shall distribute or cause to be distributed in commerce any packaged consumer commodity unless in conformity with regulations which shall be established by the promulgating authority pursuant to section 1455 of this title which shall provide that: •

•

•

(1) The commodity shall bear a label specifying the identity of the commodity and the name and place of business of the manufacturer, packer, or distributor; (2) The net quantity of contents (in terms of weight or mass, measure, or numerical count) shall be separately and accurately stated in a uniform location upon the principal display panel of that label, using the most appropriate units of both the customary inch/pound system of measure, as provided in paragraph (3) of this subsection, and, except as provided in paragraph (3)(A)(ii) or paragraph (6) of this subsection, the SI metric system; (3) The separate label statement of net quantity of contents appearing upon or affixed to any package: • (A) • (i) if on a package labeled in terms of weight, shall be expressed in pounds, with any remainder in terms of ounces or common or decimal fractions of

192

•

•

•


the pound; or in the case of liquid measure, in the largest whole unit (quart, quarts and pint, or pints, as appropriate) with any remainder in terms of fluid ounces or common or decimal fractions of the pint or quart; • (ii) if on a random package, may be expressed in terms of pounds and decimal fractions of the pound carried out to not more than three decimal places and is not required to, but may, include a statement in terms of the SI metric system carried out to not more than three decimal places; • (iii) if on a package labeled in terms of linear measure, shall be expressed in terms of the largest whole unit (yards, yards and feet, or feet, as appropriate) with any remainder in terms of inches or common or decimal fractions of the foot or yard; • (iv) if on a package labeled in terms of measure of area, shall be expressed in terms of the largest whole square unit (square yards, square yards and square feet, or square feet, as appropriate) with any remainder in terms of square inches or common or decimal fractions of the square foot or square yard; • (B) shall appear in conspicuous and easily legible type in distinct contrast (by topography, layout, color, embossing, or molding) with other matter on the package; • (C) shall contain letters or numerals in a type size which shall be • (i) established in relationship to the area of the principal display panel of the package, and • (ii) uniform for all packages of substantially the same size; and • (D) shall be so placed that the lines of printed matter included in that statement are generally parallel to the base on which the package rests as it is designed to be displayed; and (4) The label of any package of a consumer commodity which bears a representation as to the number of servings of such commodity contained in such package shall bear a statement of the net quantity (in terms of weight or mass, measure, or numerical count) of each such serving. (5) For purposes of paragraph (3)(A)(ii) of this subsection the term “random package” means a package which is one of a lot, shipment, or delivery of packages of the same consumer commodity with varying weights or masses, that is, packages with no fixed weight or mass pattern. (6) The requirement of paragraph (2) that the statement of net quantity of contents include a statement in terms of the SI metric system shall not apply to foods that are packaged at the retail store level.

(b) Supplemental Statements No person subject to the prohibition contained in section 1452 of this title shall distribute or cause to be distributed in commerce any packaged consumer commodity if any qualifying words or phrases appear in conjunction with the separate statement of the net quantity of contents required by subsection (a) of this section, but nothing in this subsection or in paragraph (2) of subsection (a) of this section shall prohibit supplemental statements, at other places on the package, describing in nondeceptive terms the net quantity of contents: Provided, That such supplemental statements of net quantity of contents shall not

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include any term qualifying a unit of weight or mass, measure, or count that tends to exaggerate the amount of the commodity contained in the package. Section 1454. Rules and Regulations (a) Promulgating Authority The authority to promulgate regulations under this chapter is vested in (A) the Secretary of Health and Human Services (referred to hereinafter as the “Secretary”) with respect to any consumer commodity which is a food, drug, device, or cosmetic, as each such term is defined by section 321 of title 21; and (B) the Federal Trade Commission (referred to hereinafter as the “Commission”) with respect to any other consumer commodity. (b) Exemption of Commodities from Regulations If the promulgating authority specified in this section finds that, because of the nature, form, or quantity of a particular consumer commodity, or for other good and sufficient reasons, full compliance with all the requirements otherwise applicable under section 1453 of this title is impracticable or is not necessary for the adequate protection of consumers, the Secretary or the Commission (whichever the case may be) shall promulgate regulations exempting such commodity from those requirements to the extent and under such conditions as the promulgating authority determines to be consistent with section 1451 of this title: (c) Scope of Additional Regulations Whenever the promulgating authority determines that regulations containing prohibitions or requirements other than those prescribed by section 1453 of this title are necessary to prevent the deception of consumers or to facilitate value comparisons as to any consumer commodity, such authority shall promulgate with respect to that commodity regulations effective to: •

•

•

(1) establish and define standards for characterization of the size of a package enclosing any consumer commodity, which may be used to supplement the label statement of net quantity of contents of packages containing such commodity, but this paragraph shall not be construed as authorizing any limitation on the size, shape, weight or mass, dimensions, or number of packages which may be used to enclose any commodity; (2) regulate the placement upon any package containing any commodity, or upon any label affixed to such commodity, of any printed matter stating or representing by implication that such commodity is offered for retail sale at a price lower than the ordinary and customary retail sale price or that a retail sale price advantage is accorded to purchasers thereof by reason of the size of that package or the quantity of its contents; (3) require that the label on each package of a consumer commodity (other than one which is a food within the meaning of section 321(f) of title 21) bear (A) the common or usual name of such consumer commodity, if any, and (B) in case such consumer commodity consists of two or more ingredients, the common or usual name of each such ingredient listed in order of decreasing predominance, but nothing in this paragraph shall be deemed to require that any trade secret be divulged; or

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(4) prevent the nonfunctional-slack-fill of packages containing consumer commodities. For purposes of paragraph (4) of this subsection, a package shall be deemed to be nonfunctionally slack-filled if it is filled to substantially less than its capacity for reasons other than (A) protection of the contents of such package or (B) the requirements of machines used for enclosing the contents in such package.

(d) Development by Manufacturers, Packers, and Distributors of Voluntary Product Standards Whenever the Secretary of Commerce determines that there is undue proliferation of the weights or masses, measures, or quantities in which any consumer commodity or reasonably comparable consumer commodities are being distributed in packages for sale at retail and such undue proliferation impairs the reasonable ability of consumers to make value comparisons with respect to such consumer commodity or commodities, he shall request manufacturers, packers, and distributors of the commodity or commodities to participate in the development of a voluntary product standard for such commodity or commodities under the procedures for the development of voluntary products standards established by the Secretary pursuant to section 272 of this title. Such procedures shall provide adequate manufacturer, packer, distributor, and consumer representation. (e) Report and Recommendations to Congress upon Industry Failure to Develop or Abide by Voluntary Product Standards If (1) after one year after the date on which the Secretary of Commerce first makes the request of manufacturers, packers, and distributors to participate in the development of a voluntary product standard as provided in subsection (d) of this section, he determines that such a standard will not be published pursuant to the provisions of such subsection (d), or (2) if such a standard is published and the Secretary of Commerce determines that it has not been observed, he shall promptly report such determination to the Congress with a statement of the efforts that have been made under the voluntary standards program and his recommendation as to whether Congress should enact legislation providing regulatory authority to deal with the situation in question. Section 1455. Procedures for Promulgation of Regulations (a) Hearings by Secretary of Health and Human Services Regulations promulgated by the Secretary under section 1453 or 1454 of this title shall be promulgated, and shall be subject to judicial review, pursuant to the provisions of subsections (e), (f), and (g) of section 371 of title 21. Hearings authorized or required for the promulgation of any such regulations by the Secretary shall be conducted by the Secretary or by such officer or employees of the Department of Health and Human Services as he may designate for that purpose. (b) Judicial Review; Hearings by Federal Trade Commission Regulations promulgated by the Commission under section 1453 or 1454 of this title shall be promulgated, and shall be subject to judicial review, by proceedings taken in conformity with the provisions of subsections (e), (f), and (g) of section 371 of title 21 in the same manner, and with the same effect, as if such proceedings were taken by the Secretary pursuant to subsection (a) of this section. Hearings authorized or required

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for the promulgation of any such regulations by the Commission shall be conducted by the Commission or by such officer or employee of the Commission as the Commission may designate for that purpose. (c) Cooperation with Other Departments and Agencies In carrying into effect the provisions of this chapter, the Secretary and the Commission are authorized to cooperate with any department or agency of the United States, with any State, Commonwealth, or possession of the United States, and with any department, agency, or political subdivision of any such State, Commonwealth, or possession. (d) Returnable or Reusable Glass Containers for Beverages No regulation adopted under this chapter shall preclude the continued use of returnable or reusable glass containers for beverages in inventory or with the trade as of the effective date of this Act, nor shall any regulation under this chapter preclude the orderly disposal of packages in inventory or with the trade as of the effective date of such regulation. 3.2.5.9 United States Pharmacopeia Center for the Advancement of Patient Safety [45] For nearly 33 years, the USP has been reporting programs for health care professionals to share experiences and observations about the quality and safe use of medications. This year, the USP Center for the Advancement of Patient Safety publishes its sixth annual report to the nation on medication errors reported to MEDMARX (Table 6). It was observed that drug product packaging/labeling is one of the main courses of medication errors in hospitals. 3.2.5.10

National Agency of Sanitary Vigilance (ANVISA, Brazil)

ANVISA is a federal organization linked to Brazil’s Health Ministry, which has the incumbency of looking after medication quality and other health products aimed at patients’ safety. Several documents regarding GMP and quality control are easily accessed. The agency is also responsible for establishing enforcing the rules and can take corrective measures and punish the offenders [46]. Product stability and compatibility with the conditioning material are distinct, separate, and complementary concepts which should be applied to the pharmaceutical product before being made available for health care. TABLE 6 Selected Causes of Error Related to Equipment, Product Packaging/Labeling, and Communication in ICUs Cause of Error Label (the facility’s) design Similar packaging/labeling Packaging/container design Label (manufacturer’s) design Brand/generic names look-alike

N (Nonharmful + Harmful)

Percent Harmful

1,236

6,9

Source: MEDMARX Data Report: A Chartbook of 2000–2004 Findings from Intensive Care Units (ICUs) and Radiological Services.

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In the compatibility test between formulation and the conditioning material, several options of conditioning materials are evaluated to determine the most adequate for the product. The environmental conditions and periodicity analyses can be the same as those mentioned for the stability studies for the formulation. In this phase, the possible interactions between the product and the conditioning material which is in direct contact with the medication are verified. Phenomena such as absorption, migration, corrosion, and others that compromise integrity can be observed. Considering that these types of tests are generally destructive, it is necessary to define the number of samples to be tested. In ANVISA’s documents, different types of tests are established that should be carried out with different types of available materials and employed for conditioning medications and cosmetics (cellulose packagings, metallic, plastic, pressurized, etc.) [46].

3.2.5.11

International Committee on Harmonization (ICH)

In the document “Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients (APIs)” of the ICH Harmonized Tripartite Guideline, the following instructions are given for packaging and identification labeling of APIs and intermediates [47]. General •

•

•

There should be written procedures describing the receipt, identification, quarantine, sampling, examination and/or testing and release, and handling of packaging and labeling materials. Packaging and labeling materials should conform to established specifications. Those that do not comply with such specifications should be rejected to prevent their use in operations for which they are unsuitable. Records should be maintained for each shipment of labels and packaging materials showing receipt, examination, or testing, and whether accepted or reject.

Packaging Materials •

•

•

Containers should provide adequate protection against deterioration or contamination of the intermediate or API that may occur during transportation and recommended storage. Containers should be clean and, where indicated by the nature of the intermediate or API, sanitized to ensure that they are suitable for their intended use. These containers should not be reactive, addictive, or absorptive so that the quality of the intermediate or API complies with the specifications. If containers are reused, they should be cleaned in accordance with documented procedures and all previous labels should be removed or defaced.

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Label Issuance and Control • •

•

• •

•

•

Access to the label storage areas should be limited to authorized personnel. Procedures should be used to reconcile the quantities of labels issued, used, and returned and to evaluate discrepancies found between the number of containers labeled and the number of labels issued. Such discrepancies should be investigated and the investigation should be approved by the quality unit(s). All excess labels bearing batch numbers or other batch-related printing should be destroyed. Returned labels should be maintained and stored in a manner that prevents mix-ups and provides proper identification. Obsolete and outdated labels should be destroyed. Printing devices used to print labels for packaging operations should be controlled to ensure that all imprinting conforms to the print specified in the batch production record. Printed labels issued for a batch should be carefully examined for proper identity and conformity to specifications in the master production record. The results of this examination should be documented. A printed label representative of those used should be included in the batch production record.

Packaging and Labeling Operations •

•

•

•

•

•

There should be documented procedures designed to ensure that correct packaging materials and labels are used. Labeling operations should be designed to prevent mix-ups. There should be physical or spatial separation from operations involving other intermediates or APIs. Labels used on containers of intermediates or APIs should indicate the name or identifying code, the batch number of the product, and storage conditions, when such information is critical to assure the quality of intermediate API. If the intermediate or API is intended to be transferred outside the control of the manufacturer’s material management system, the name and address of the manufacturer, quantity of contents and special transport conditions, and any special legal requirements should also be included on the label. For intermediates or APIs with an expiry date, the expiry date should be indicated on the label and certificate of analysis. For intermediates or APIs with a retest date, the retest date should be indicated on the label and/or certificate of analysis. Packaging and labeling facilities should be inspected immediately before use to ensure that all materials not needed for the next packaging operation have been removed. This examination should be documented in the batch production records, the facility log, or other documentation system. Packaged and labeled intermediates or APIs should be examined to ensure that containers and packages in the batch have the correct label. This examination should be part of the packaging operation. Results of these examinations should be recorded in the batch production or control records.

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Intermediate or API containers that are transported outside of the manufacturer’s control should be sealed in a manner such that, if the seal is breached or missing, the recipient will be alerted to the possibility that the contents may have been altered.

3.2.5.12

European Union Regulatory Bodies

European regulatory requirements say little to date about container closure integrity of parenteral or sterile pharmaceutical products. Regulations provide for package integrity verification of parenteral vials to be supported by the performance of sterility tests as part of the stability program. More specific information is described in the European Union (EU) 1998 “Rules Governing Medical Products in the European Union, Pharmaceutical Legislation.” These GMP regulations require that the sealing or closure process be validated. Packages sealed by fusion (e.g., ampules) should be 100% integrity tested. Other packages should be sampled and checked appropriately. Packages sealed under vacuum should be checked for the presence of vacuum. While not as detailed as the FDA guidances, it is evident that the EU rules also require the verification of parenteral product package seal integrity. It is important to note that the EU rules specifically require 100% product testing for fusion-sealed packages, sampling and testing of all other packages, and vacuum verification for packages sealed under partial pressure [42]. The vacuum/pressure decay test is performed by placing the package in a tightly closed test chamber, a pressure or vacuum is applied inside the chamber, and then the rate of pressure/vacuum change in the chamber over time is monitored. The rate or extent of change is compared to that previously exhibited by a control, nonleaking package. Significantly greater change for a test package is indicative of a leak.

REFERENCES 1. Griffin, J. P. Ed. (2002), The Textbook of Pharmaceutical Medicine, 4th ed., BMJ Publishing, London. 2. Harburn, K. (1990), Quality Control of Packaging Materials in the Pharmaceutical Industry, Marcel Dekker, New York. 3. O’Brien, J. D. (1990), Medical Device Packaging Handbook, Marcel Dekker, New York. 4. U.S. Food and Drug Administration (FDA) (1999, May) Guidance on container closure systems for packaging human drugs and biologics, U.S. Department of Health and Human Services, FDA, Washington, DC. 5. Yoshioka, S. (2000), Stability of Drugs and Dosage Forms, Kluwer Academic Publishers: New York, NY, USA, p 272. 6. Banker, G. S., and Rhodes, C. T. (2002), Modern Pharmaceutics, 4th ed., rev. and expanded, Marcel Dekker, New York. 7. Connor, J., Rafter, N., and Rodgers, A. (2004), Do fixed-dose combination pills or unitof-use packaging improve adherence? A systematic review. Br. World Health Org., 82, 935–939. 8. Bloomfield, S. F. (1990), Microbial contamination: Spoilage and hazard, in Denyer, S., and Baird, R., Eds., Guide to Microbiological Control in Pharmaceuticals, Ellis Horwood, Chichester, England, pp 29–52.

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9. Aspinall, J. A., Duffy, T. D., Saunders, M. B., and Taylor, C. G. (1980), The effect of low density polyethylene containers on some hospital-manufactured eye drop formulations. 1. Sorption of phenyl mercuric acetate, J. Clin. Hosp. Pharm., 5, 21–29. 10. United States Pharmacopeia (2006), 29th ed., United States Pharmacopeial Convention, Rockville, MD. 11. Parker, W. A., and MacCara, M. E. (1980), Compatibility of diazepam with intravenous fluid containers and administration sets, Am. J. Hosp. Pharm., 37, 496–500. 12. Mizutani, T., Wagi, K., and Terai, Y. (1981), Estimation of diazepam adsorbed on glass surfaces and silicone-coated surfaces as models of surfaces of containers, Chem. Pharm. Bull., 29, 1182–1183. 13. Yahya, A. M., McElnay, J. C., and D’Arcy, P. F. (1985), Binding of chloroquine to glass, Int. J. Pharm., 25, 217–223. 14. Vromans, H., and Van Laarhoven, J. A. H. (1992), A study on water permeation through rubber closures of injection vials, Int. J. Pharm., 79, 301–308. 15. Matsuura, I., and Kawamata, M. (1978), Studies on the prediction of shelf life. III. Moisture sorption of pharmaceutical preparation under the shelf condition, Yakugaku Zusshi, 98, 986–996. 16. Nakabayashi, K., Tuchida, T., and Mima, H. (1980), Stability of packaged solid dosage forms. I. Shelf-life prediction of packaged tablets liable to moisture damage, Chem. Pharm. Bull., 28, 1090–1098. 17. Nakabayashi, K., Shimamoto, T., and Mima, H. (1980), Stability of packaged solid dosage forms. II. Shelf-life prediction for packaged sugar-coated tablets liable to moisture and heat damage, Chem. Pharm. Bull., 28, 1099–1106. 18. Nakabayashi, K., Shimamoto, T., and Mima, H. (1980), Stability of packaged solid dosage forms. III. Kinetic studies by differential analysis on the deterioration of sugar-coated tablets under the influence of moisture and heat, Chem. Pharm. Bull., 28, 1107–1111. 19. Tonnesen, H. H. (1996), Photostability of Drugs and Drug Formulations, CRC Press, London. 20. Kontny, M. J., Koppenol, S., and Graham, E. T. (1992), Use of the sorption–desorption moisture transfer model to assess the utility of a desiccant in a solid product, Int. J. Pharm., 84, 261–271. 21. Pikal, M. J., and Lang, J. E. (1978), Rubber closures as a source of haze in freeze dried parenterals: Test methodology for closure evaluation, J. Parenteral drug Assoc., 32, 162–173. 22. Jaehnke, R. W. O., Kreuter, J., and Ross, G. (1990), Interaction of rubber closures with powders for parenteral administration, J. Parenteral sci. Tech., 44, 282–288. 23. Jaehnke, R. W. O., Kreuter, J., and Ross, G. (1991), Content/container interactions: The phenomenon of haze formation on reconstitution of solids for parenteral use, Int. J. Pharm., 77, 4755. 24. Moorhatch, P., and Chiou, W. L. (1974), Interactions between drugs and plastic intravenous fluid bags. II: Leaching of chemicals from bags containing various solvent media, Am. J. Hosp. Pharm., 31, 149–152. 25. Venkataramanan, R., Burckart, G. J., Ptachcinski, R. J., Blaha, R., Logue, L. W., Bahnson, A. C., and Brady, G. J. E. (1986), Leaching of diethylhexyl phthalate from polyvinyl chloride bags into intravenous cyclosporine solution, Am. J. Hosp. Pharm., 43, 2800– 2802. 26. Boruchoff, S. A. (1987), Hypotension and cardiac arrest in rats after infusion of mono (2ethylhexyl) phthalate (MEHP), a contaminant of stored blood, N. Engl. J. Med., 316, 1218–1219.

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27. U.S. Food and Drug Administration, Code of Federal Regulations (CFR)—Title 21, Food and drugs, Chapters 174–186, available: http://www.access.gpo.gov/nara/cfr/index.html, accessed Mar. 11, 2005. 28. Kowaluk, E. A., Roberts, M. S., Blackburn, H. D., and Polack, A. E. (1981), Interactions between drugs and polyvinyl chloride infusion bags, Am. J. Hosp. Pharm., 38, 1308–1314. 29. Illum, L., and Bundgaard, H. (1982), Sorption of drugs by plastic infusion bags, Int. J. Pharm., 10, 339–351. 30. Illum, L., Bundgaard, H., and Davis, S. S. (1983), A constant partition model for examining the sorption of drugs by plastic infusion bags, Int. J. Pharm., 17, 183–192. 31. Atkinson, H. C., and Duffull, S. B. (1990), Prediction of drug loss from PVC infusion bags, J. Pharm. Pharmacol., 43, 374–376. 32. Richardson, N. E., and Meakin, B. J. (1974), The sorption of benzocaine from aqueous solution by nylon 6 powder, J. Pharm. Phamacol., 26, 166–174. 33. Santoro, M. I. R. M., Kedor-Hackmann, E. R. M., and Moudatsos, K. M. (1993), Estabilidade de sais de reidratação oral em diferentes tipos de embalagem. Bol. Sanit. Panam., 115, 310–315. 34. World Health Organization (WHO) (2003), The International Pharmacopoeia, Tests and General Requirements for Dosage Forms: Quality Specifications for Pharmaceutical Substances and Tablets, 3rd ed., Vol. 5, WHO, Geneva. 35. Santoro, M. I. R. M., Oliveira, D. A. G. C., Kedor-Hackmann, E. R. M., and Singh, A. K. (2004), Quantifying benzophenone-3 and octyl methoxycinnamate in sunscreen emulsions, Cosm. & Toil., 119, 77–82. 36. Santoro, M. I. R. M., Oliveira, D. A. G. C., Kedor-Hackmann, E. R., and Singh, A. K. (2005), The effect of packaging materials on the stability of sunscreen emulsions, Int. J. Pharm., 13, 197–203. 37. Thoma, K., and Kerker, R. (1992), Photoinstability of drugs. 6. Investigations on the photosansibility of molsidomine, Pharm. Ind., 54, 630–638. 38. British Pharmacopoeia (2002), Her Majesty’s Stationary Office, London, pp A144, 135– 136, 196, 671–673, 778–780, 976–978, 1145–1146. 39. Albert, D. E. (2004), Evaluating pharmaceutical container closure systems, Pharm. & Med. Packaging News, 3, 76–78. 40. ASHP Council on Professional Affairs (1993), ASHP Guidelines on preventing medication errors in hospital, Am. J. Hosp. Pharm., 50, 305–314. 41. ASHP Council on Professional Affairs (2001), ASHP guidelines on preventing medication errors in hospital, Am. J. Hosp. Pharm., 58, 3033–3041. 42. European Pharmacopoeia (2001), 4th ed., Council of Europe, Strasbourg. 43. Lachman, L., Lieberman, H. A., and Kanig, J. L. (2001), Teoria e prática na indústria farmacêutica, Fundação Calouste Gulbenkian, Lisboa. 44. U.S. Food and Drug Administration, Fair Packaging and Labeling Act. Title 15— Commerce and Trade, Chapter 39—Fair Packaging and Labeling Program, available: http://www.fda.gov/opacom/laws/fplact.htm accessed Mar. 11, 2005. 45. Santell, J. P., Hicks, R. W., and Cousins, D. D. (2005), MEDMARX Data Report: A Chartbook of 2000–2004 Findings from Intensive Care Units and Radiological Services, USP Center for Advancement of Patient Safety, Rockville, MD. 46. Agência Nacional de Vigilância Sanitária (ANVISA) (2004), Guia de Estabilidade de Produtos Cosméticos, ANVISA, Brasília. 47. International Organization on Harmonisation (2000), ICH harmonized tripartite guideline: Good manufacturing practice guide for active pharmaceutical ingredients, available: http://www.ICH.org, accessed June 23, 2005. 48. Sarbach, C., Yagoubi, N., Sauzieres, J., Renaux, C., Ferrier, D., and Postaire, E. (1996), Migration of impurities from a multi-layer plastics container into a parenteral infusion solution, Int. J. Pharm., 140, 169–174.

3.3 CLEAN-FACILITY DESIGN, CONSTRUCTION, AND MAINTENANCE ISSUES Raymond K. Schneider Clemson University, Clemson, South Carolina


3.3.3

3.3.4

3.3.5

Introduction Planning for Project Success 3.3.2.1 Needs Assessment 3.3.2.2 Front-End Planning 3.3.2.3 Preliminary Design 3.3.2.4 Procurement 3.3.2.5 Construction 3.3.2.6 Start-Up and Validation 3.3.2.7 Summary Design Options 3.3.3.1 Clean-Facility Scope 3.3.3.2 Design Parameters 3.3.3.3 Architectural Design Issues 3.3.3.4 Materials of Construction 3.3.3.5 HVAC System 3.3.3.6 Clean-Room Testing 3.3.3.7 Utilities Construction Phase: Clean Build Protocol 3.3.4.1 General 3.3.4.2 Level I Clean Construction 3.3.4.3 Level II Clean Construction Maintenance Appendix A: Guidelines for Construction Personnel and Work Tools in a Clean Room Appendix B: Cleaning the Clean Room Bibliography


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3.3.1

INTRODUCTION

While there are discrete steps in the design and construction of a pharmaceutical manufacturing plant project, those projects deemed successful incorporate certain practices that promote flow of the construction process toward completion on time and within budget. Proper front-end planning is not completed until it results in appropriate values for design parameters, “buy-in” at all levels of management, and clear direction for the design phase. Engineering the clean room in accordance with recognized industry practice would produce construction documents that facilitate clear procurement and construction planning as well as a focused, efficient, construction effort. A full return on the energy expended through the construction phase cannot be realized without a well-executed start-up and validation process that provides baseline data for effective ongoing operation and maintenance. The steps in the clean-room construction project include: Needs assessment Front-end planning Preliminary design Construction document development Procurement Construction Start-up and validation One of the truisms of the construction industry is that the greatest impact on the cost of a facility can be made at the earliest stages of the process. The construction process can be likened to a snowball rolling down a snow-covered hill. It grows and gains momentum, seemingly taking on a life of its own, until it can only be brought under control with a major effort. So too with manufacturing plant projects. Careful work during the first three stages will ensure that the project begins on a welldirected course and moves to a successful conclusion. Sometimes the special nature of pharmaceutical manufacturing plant projects clouds the fact that building such a plant is in fact a construction project. The facility engineering team of a small to medium company may be tempted to turn away from such projects due to the projects’ perceived uniqueness and leave the key decision making to others. In fact, it is the construction experience of that team that is most required to keep the project costs under control. The way to accomplish this is for the team to be involved in the process from its earliest stages. Let us review the steps in such a project and identify what should occur at each step and the potential for trouble.

3.3.2 3.3.2.1

PLANNING FOR PROJECT SUCCESS Needs Assessment

It is during this early stage that a requirement for a clean manufacturing facility is perceived. The need for the facility may be precipitated by a new product, an

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improved product, an improved manufacturing methodology, new or more stringent regulation requirements, or perhaps a change in marketing strategy. At this point a study should be undertaken to determine the benefits to be realized by the new facility as well as the costs to be incurred. Costs arise from not only construction but also ongoing operation and maintenance. These costs are affected by the plant location and the availability of a trained or trainable workforce. Does the day-to-day operation of the facility generally require that special attire be worn? Are special procedures, possibly more time consuming than those presently used, required? It is important that this study is complete and accurate in order to prevent any unrealistic expectations on the part of management and plant operations and to permit advanced planning for revised procedures once the facility is in use. The study should describe the goals of the project, its impact on present operations, budget restraints, tentative schedule, and path forward. It will serve as the basis for front-end planning and will provide the standard against which the success of the program is measured. 3.3.2.2

Front-End Planning

While the needs assessment study may be conducted by a limited number of people, the front-end planning process should be open to all. Plant facilities people will be bearing the brunt of the responsibility for bringing the facility online, on schedule, and within budget. Process people are responsible for ensuring that the facility will adequately house the process equipment and that the facility incorporates sufficient space, utilities, process flow considerations, and provision for flow of people and material to support the goals of the building program. Human resources people have to staff the facility, either out of the present employee pool or from the general local labor market. They must know the requirements of potential employees as well as the conditions under which they will be working. Procurement people will be purchasing furnishings and process equipment for the plant as well as overseeing the contracts let to the design and construction professionals. Operations people should have input regarding design parameters such as temperature, humidity, lighting, vibration, cleanliness class, and energy needs. Materials handling people should participate in order to understand the requirements for storing and transporting raw materials as well as retrieving, storing, and shipping finished goods from the plant. An integral part of the front-end planning team should be the design professionals charged with developing the plant design based on client input in such a way as to satisfy as many requirements developed in needs assessment as possible. This team may be assembled internally but frequently is drawn from specialty builders, architectural and engineering (A&E) firms, and design/build firms active in the pharmaceutical industry. The team of design professionals should have pharmaceutical experience on facilities comparable in size and complexity to that being planned as well as extensive experience in construction projects of all types. The design team may offer design only, design/build, procurement, construction management, or combinations of these services. This design team should be considered a resource during the front-end planning phase. It is the wise client who takes advantage of the experience of the design team, permitting them a large role as facilitators of the planning sessions.

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An appropriate design team will demonstrate expertise in contamination control philosophies, space planning, code compliance, and mechanical and electrical design and will be familiar with materials of construction currently being used in pharmaceutical projects. It is frequently helpful to include a member of the construction team in the front-end planning effort to advise on constructibility of the facility being planned. Unrealistic construction schedules will be avoided and field rework will be minimized if appropriate attention is paid to the construction phase early in the planning process. 3.3.2.3

Preliminary Design

Front-end planning typically utilizes the expertise of client process people to convey the requirements of the pharmaceutical facility to the design team. With this information in hand the design team begins the facility design incorporating process needs, code requirements, safety issues, material and personnel flow, work-in-process storage, utility needs, and so on, into a first-cut approach. Client representatives have an opportunity to review the effort and begin fine tuning the design to incorporate late-breaking process changes. The preliminary design is a target that helps both the design team and the client solidify design goals. Change is inexpensive, and therefore encouraged, at this stage and buy-in by all concerned is a major objective of this phase of the design effort. A budget based on the agreed-upon preliminary design should be developed to make sure that the overall project is on course. This will minimize surprises further along in the design/build process. Ideally the design will be “cast in stone” at the end of the preliminary phase. This permits the production work on the design documents to proceed unhindered. The more unknowns left at the end of the preliminary phase, the more difficult it will be to complete design documents in a timely fashion. Construction Document Development The construction documents should convey the intent of the design team and client to the construction team. A good set of construction documents should result in a tight spread of construction bids as there should be little room for varying interpretation on the part of the potential construction contractors. The drawings should have sufficient notes to convey the design intent without creating a cluttered appearance. The written specifications should be as brief as possible consistent with clarity. Complicated documents create the impression that a project may be more involved, and therefore more costly, than it should be. Cautious contractors may unnecessarily inflate their bid to cover perceived contingencies. Specifications that are too wordy may be difficult to follow and similarly result in higher prices as bidders make sure all bases are covered. No one likes surprises. The development of construction documents should be a straightforward process with little involvement by the client except to monitor the process and ensure that the original design intent is followed. While changes will always occur during this phase (“cast in stone” is a euphemism for “let’s keep the changes under control”), they are certainly less costly at this point than during the construction phase. It is desirable to minimize such changes. A continuous sequence of changes suggests that the preliminary design phase was not entered into seriously. It demonstrates a lack

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of preparedness on the part of the client and a lack of ability to communicate and draw out the client’s needs on the part of the design team. A sense of clarity of purpose slips away with ongoing change and the possibility for errors in construction documents, which eventually surface as costly construction changes, increases. 3.3.2.4

Procurement

A detailed scope of work describing the materials and services required is a vital part of the procurement process. There is no purpose to keeping the project bidders in the dark regarding what is required of them. The role of the procurement function is to obtain maximum value, that is, the best quality and schedule at the lowest price. The clearer the scope of work and construction documents, the better will be the chance of this happening. A low price is not a good value if the schedule slips by several months as a result. A marginal plant that does not maintain design conditions or meet production goals is a poor value even if it was delivered within schedule. The procurement process should qualify potential bidders by ensuring that similar pharmaceutical projects have been delivered on time, within budget, and on schedule. References should be checked. It is expected that references offered by a potential bidder would have good things to say about that bidder, but this is not a certainty and pointed questioning about personnel, schedule, quality, change orders, follow-up, and so on, can help develop a warm feeling or an uncertain feeling about potential bidders. If bids are in fact quite close, it is the quality of references that might suggest a particular bidder be given preference. There are a number of ways in which the project can be procured. Use of in-house engineering and construction expertise may work in special situations or on smaller projects. Typically problems arise when facilities departments, stretched to their limit with ongoing plant requirements, must lower the priority of the new facility to meet other commitments. Schedules may stretch out unacceptably. A number of specialty contractors have proven over the years to be adept at installing small turnkey facilities of limited complexity in a timely and economical fashion. If extensive engineering is required, if local code compliance becomes an issue, if complex process requirements must be met, or if the client requirements exceed the experience of the supplier there could be cause for concern. Design/build is a popular approach in that it suggests a single source of responsibility for all phases of the project. Frequently firms billing themselves as “design/ build” are strong in either design or build, but not both. The strong design firm can put the essentials on paper but the final price and schedule may suffer. The strong construction firm may lack the expertise to create an appropriate manufacturing environment, particularly where clean-room expertise is required. The project may be outstanding in all respects except performance. A good review of references is essential before selecting a design/build firm. Construction management has been increasingly used on larger projects. A good construction management firm will work closely with the client-selected design company to review constructability and adequacy of construction documents. It will assist to qualify bidders, maintain schedule, track costs, administer and oversee, and generally ensure that a team incorporating the strongest skills is assembled to complete the project. Pharmaceutical experience is essential.

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3.3.2.5


Construction

The construction process should proceed smoothly if the remarks presented above are followed. Cost can increase during this phase if changes must be implemented. While change is inevitable, a construction change procedure negotiated during the bidding phase and in place during construction will keep such change from getting out of control. The requirement for “building clean” has arisen in recent years as more stringent clean rooms have become more popular. Imposing a clean construction protocol on contractors can lengthen the schedule and increase cost. The protocol should be developed during the construction document phase and be an integral part of the bid documents. Once the decision is made to work clean, protocols developed should be followed by everyone on the jobsite associated with the clean areas. A poorly conceived and enforced protocol will be a costly and futile exercise. The tendency to build clean on every new or retrofit project should be carefully evaluated and a practical protocol should be developed consistent with the needs of the project. Client end users should be encouraged to observe construction as it progresses. They will be more intelligent about how their facility was built and therefore more attuned to maintaining the facility once it is completed and in operation. While suggestions should be welcomed as construction progresses, it is important that a chain of command be enforced. Any questions or suggestions or concerns should not be expressed to workers on the site but rather through project management channels. In this way good ideas can be implemented and bad ideas shelved without impacting the construction effort in a negative manner. Note the one exception to this practice is in regard to safety. Everyone on the site has safety responsibility. Any unsafe acts should be questioned and supervisors consulted immediately.

3.3.2.6

Start-Up and Validation

Subcontractors on the jobsite should be responsible for start-up as well as installation of equipment. Equipment manufacturers typically have personnel available to ensure appropriate start-up procedures are followed. If several trades are involved in the installation of a particular piece of equipment, then one trade should be assigned, by contract, as having coordinating responsibility for that piece of equipment. This will minimize “finger pointing” when equipment does not start or operate properly. This can be a sensitive issue and a construction manager can set the tone for cooperation in this area. An independent contractor responsible to the construction manager or owner should do testing and balancing (TAB) of mechanical systems. All start-up should be complete and initial valve or damper settings made (and recorded) by the subcontractor before testing and balancing begins. The TAB contractor should not have to repair equipment or troubleshoot inoperative equipment but rather only adjust and verify performance of equipment. A separate contractor should certify clean-room areas. This might be the TAB contractor if that firm is suitably qualified. There should be no question of equipment being operative at this stage of the project since start-up and testing and balancing are complete. Certification is the verification of facility compliance

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with clean-room specifications. If the facility design is well conceived and the construction team has installed a quality project, any certification test failure will most likely be corrected through fairly minor adjustments. Failure of the clean room to pass certification tests might require redesign but more frequently requires some equipment adjustment or perhaps a filter repair and then a retest. It is important that a clear understanding of responsibility be communicated before problems are encountered. Failure to plan for potential problems could result in extending the schedule and incurring unforeseen costs at a crucial point in the project. 3.3.2.7

Summary

Recognizing the step-by-step process involved in even the smallest pharmaceutical project can help focus attention in a manner that will result in a successful project. The formal schedule of a well-conceived project will include needs assessment, front-end planning, and preliminary design. It is important that project progress is measured against such a schedule and not just by the visual impact caused by bricks and mortar being installed.

3.3.3 3.3.3.1

DESIGN OPTIONS Clean-Facility Scope

The purpose of this section is to identify design and construction options for those parts of a pharmaceutical facility intended to house process equipment. These suggestions are intended to assure that the facilities, when used as designed, will meet the requirements of current good manufacturing practices (cGMPs). Air cleanliness within the facility may range from International Organization for Standardization (ISO) 5 (Class 100) through ISO 8 (Class 100,000). In addition, areas may be considered clean or labeled as “controlled environment” without having a cleanliness class assigned to the space. Note that throughout this chapter cleanliness class will be described using the designation presented in the new ISO 14644 (e.g., ISO 5, ISO 8) and parenthetically as presented in the currently obsolete (but widely understood and quoted) U.S. Federal Standard 209 (e.g., Class 100, Class 100,000). A cleanliness classification in accordance with the latest revision of ISO 14644 is generally inadequate by itself to describe a facility used for pharmaceutical processes. The presence of viable particles (living organisms) within the particle count achieved by applying methods described in the standard may affect the product within the facility. A measure of both viable and nonviable particles is required to provide sufficient information upon which to base a decision regarding the suitability of the clean room for its intended purpose. The options presented herein are intended to provide facilities that will effectively restrict both viable and nonviable particles from entering the clean areas, minimize contamination introduced by the facility itself, and continuously remove contaminants generated during normal operations. Measurement of total particle count in the clean room is described in ISO 14644. This count may be composed of viable, nonviable, or nonviable host particles with a viable traveler. There is no generally accepted relationship between total particle

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count and viable particle count. While maintaining appropriate particle counts is important in clean-room design and operation, a protocol designed to identify viable particles should be inherent in the certification/validation testing of a pharmaceutical clean room. No facility design can compensate for excessive contamination generated within it. In addition to effective facility design, the user must also institute a routine maintenance program as well as maintain personnel and operational disciplines that limit particles both entering and being generated within the facility. While this section identifies options for contamination control in facility design, any such options must be implemented in accordance with all appropriate government and regulatory building and safety codes. The design guideline is nonspecific as regards biological or chemical materials that may be used within the facility but generally addresses bulk pharmaceutical chemical plants (BPCs), secondary manufacturing chemical plants, bulk biopharmaceutical plants, and plants used for fill and finish operations. Good practice as well as any regulations governing biological and pharmaceutical processes conducted within the facility must be adhered to as required and could modify some of the suggestions contained herein. 3.3.3.2

Design Parameters

The design of the facility is based upon specification of certain design parameters. These in turn are used to calculate building system equipment capacities and aid in the selection of the appropriate types of equipment that are required. Design parameters that may be critical are discussed below. Cleanliness Classification The classification of the clean areas is determined by the using organization consistent with the level of nonviable and viable particulate contamination acceptable to the process conducted within the facility. This may be governed by regulatory agencies, client organizations, or company protocols. Target goals are set for nonviable particle count in accordance with the ISO. Viable particle target goals should be stated in colony-forming units (CFU) per square centimeter. In accordance with ISO 14644, particle goals will typically be identified for “at rest” and “operational” modes. In the absence of other guidance governing the cleanliness classification and acceptable levels of microbial contamination of the clean room, the values presented in Table 1 may be used. The room grades presented are from most critical (A) to least critical (E). The definition of criticality is left to the clean-room user organization. Other Design Parameters Facility design parameters that support the process within the clean room should be established by the user organization. Parameters such as temperature, humidity, lighting requirements, sound level, and/or vibration may be process driven or comfort driven and therefore are selected to accommodate specific process or comfort requirements as determined by the end user. Local Control Under some circumstances, cleanliness requirements can be achieved through the use of localized controls such as clean tents, glove boxes, minienvironments, or isolators. These provide unidirectional filtered airflow within

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DESIGN OPTIONS

TABLE 1 An Example of Cleanliness Classification Goals Particle Countse Room Grade Af Bg C D E

Cleanliness Classa

At Rest

M3.5 (100) M3.5 (100) M5.5 (10000) M6.5 (100000) Uncontrolled

3,500 3,500 350,000 3,500,000 N/A

Microbial Contamination

Operational

Air Sample (cfu/m3)

Settle Plates (cfu/4 h)b

Contact Plates (cfu/plate)c

Glove Print (cfu/glove)d

3,500 35,000 3,500,000 N/A N/A

HPMC. Bothe solid and liquid drug formulations could be delivered with this system [94–96]. A novel capsule was made from ethyl cellulose for the time-controlled release of drugs in the colon [97]. Initially the ethyl cellulose capsule was prepared using a gelatin capsule with ethyl cellulose, followed by dissolution of the gelatin in water. The thickness of the ethyl cellulose capsule body was varied and the effect of wall thickness on the release of drugs in the capsules was investigated. Ethyl cellulose capsules contained a large number of mechanically made micropores (400 μm) at the bottom. Also located in the bottom of the capsule body was a swellable layer consisting of L-HPC. Above the swellable layer was the drug reservoir, which contained a mixture of the model drug, fluorescein, and a bulking agent, such as lactose or starch. The capsule was thus capped and sealed with a concentrated ethyl cellulose solution. After administration of drug-containing capsule, water molecules penetrated the capsule through the micropores in the bottom of the capsule body. Hydration and swelling of HPC induced an increase in the internal osmotic pressure, which resulted in the “explosion” of the capsule and a burstlike drug release was observed. By altering the thickness of the capsule, the lag time of the drug release could be altered. A similar approach for the pulsatile release of drug was reported in which a hydrostatic pressure was generated inside the capsules.

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CONTROLLED-RELEASE DOSAGE FORMS

Systems with Capsular Structure Several single-unit pulsatile dosage forms with a capsular design have been developed. Most of them consist of an insoluble capsular body which contains the drug and a plug which is removed after a predetermined lag time because of swelling, erosion, or dissolution. The Pulsincap system consists of a water-insoluble capsule body filled with the drug formulation. The capsule half is closed at the open end with a swellable hydrogel plug. The dimension and the position of the plug can control the lag time prior to the release. In order to assure a rapid release of the drug content, effervescent agents or disintegrants can be included in the drug formulation, in particular with water-insoluble drugs. The system is coated with an enteric layer which dissolves upon reaching the higher pH region of the small intestine. This system comprises insoluble capsules and plugs. The plugs consist either of swellable materials, which are coated with insoluble but permeable polymers (e.g., polymethacrylates), or of erodible substances, which are compressed (e.g., HPMC, polyvinyl alcohol, polyethylene oxide) or prepared by congealing of melted polymers (saturated polyglycolated glycerides of glyceryl monooleate). The erosion of the plug can also be controlled enzymatically: A pectin plug can be degraded by pectinolytic enzymes being directly incorporated into the plug [98–100]. Linkwitz et al. [101] described the delivery of agents from osmotic systems based on the technology of an expandable orifice. The system is in the form of a capsule from which the drug is delivered by the capsule’s osmotic infusion of moisture from the body. The delivery orifice opens intermittently to achieve a pulsatile delivery effect. The orifice forms in the capsule wall, which is constructed of an elastic material, preferably elastomer (e.g., styrene–butadiene copolymer), which strectches under apressure dufferential caused by the pressure rise inside the capsule as the osmotic infusion progessses. The orifice is small enough that when the elastic wall is relaxed, the flow rate of drug through the orifice is substantially zero, but when the elastic wall is stretched due to the pressure differential across the wall exceeding a threshold, the orifice expands sufficiently to allow the release of the drug at a phsiologically required rate. This osmotically driven delivery device as an implant can used in the anal–rectal passageway, in the cervical canal, as an artificial gland, in the vagina, as ruminal bolus, and the like. A core-shelled cylindrical dosage form is available comprising a hydrophobic polycarbonate coating and a cylindrical core of alternating polyanhydride isolating layer and drug-loaded poly[ethyl glycinate) (benzyl amino acethydroxamate) phosphazene] (PEBP) layer for a programmable drug delivery system for single-dose vaccine and other related applications [102]. The pulsatile release of model compounds [fluorescein isothiocyanate (FITC)–dextran and myoglobin] whith a certain lag time (18–118 h) was achieved on the basis of the pH-sensitive degradation of PEBP and its cooperative interaction with polyanhydrides. In another experiment, Jiang and Zhu [103, 104] designed laminated devices comprising of polyanhydrides as isolating layers and pH-sensitive complexes of poly(sebacic anhydride)-bpolyethylene glycol (PSA-b-PEG) and poly(trimellitytylimdoglycine-co-sebacic anhydride)-b-polyethylene glycol [P(TMA-gly-co-SA)-b-PEG] as protein-loaded layers. The release of model proteins [bovine serum albumin (BSA) and myoglobin] showed a typical pulsatile fashion. The lag time prior to the release correlated with the hydrolytic druation of polyanhydrides, which varied from 30 to 165 h depending on polymer type and isolating layer thickness.

RELEASE OF DRUGS FROM CONTROLLED-RELEASE DOSAGE FORMS

5.1.10.2

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Stimuli-Induced Controlled-Release Systems

Several polymeric delivery systems undergo phase transitions and demonstrate marked swelling–deswelling change in reponse to environmental changes, including solvent composition ionic strength, temperature, electric fields, and light [105]. Responsive drug release from those systems results from the stimuli-induced changes in the gels or micelles, which maydeswell, swell, or erode in response to the respecive stimuli. The mechanisms of drug release include ejection of the drug from the gel as the fluid phase synerses out and drug diffusion along a concentration gradient. pH-Responsive Drug Release Dosage Forms pH-sensitive enteric coatings have been used routinely to deliver drug to the small intestine. These polymer coatings are insensitive to the acidic conditions of the stomach yet dissolve at the higher pH environment of the small intestine. This pH differential has also been attempted for colonic delivery purposes, although the polymers used for colonic targeting tend to have a threshold pH for dissolution that is higher than for those used in conventional enteric coating applications [104–106]. The synthesis and characterization of series of novel azo hydrogels for colontargeting drug delivery have been described. The colon specificity is achieved dure to the presence of pH-sensitive monomers and azo cross-linking agents in the hydrogel structures. Most commonly, copolymers of methacrylic acid and methylmethacrylate that dissolve at pH 6 (Eudragit L) and pH 7 (Eudragit S) have been extensively investigated [106, 107]. This approach is based on the assumption that gastrointestinal pH increases progressively from the small intestine to the colon. The pH in the distal small intestine is usually around 7.5, while the pH in the proximal colon is closer to 6. To overcome the premature release of drugs, a copolymer of methacrylic acid, methylmethacrylate, and ethyl acetate (Eudragit ES), which dissolves at a slower rate and at a higher threshold pH (7–7.5), has been developed [108]. The trn A series of in vitro dissolution studies with this polymer have highlighted clear benefits over the Eudragit S polymer for colon targeting. A gamma scintigraphy study comparing the in vivo performance of these various polymers revealed that Eudragit S (coating over the tablets) was superior to the older polymers in terms of retarding drug release in the small intestine, although, in some cases, the coated tablets did not break up at all. pH-sensitive delivery systems are commercially available for mesalazine (5-iminosalicylic cid) (Asacol and Salofalk) and budesonide (Budenofalk and Entocort) for the treatment of ulcerative colitis and Crohn’s disease, respectively [109]. Natural polysaccharides are being used for the development of solid dosage forms for pH-dependent delivery and for targeting the release of drugs in colon [110]. Various major approaches utilizing polysaccharides are fermentable coating of the drug core, embedding of the drug in biodegradable matrix, and formation of drug–saccharide conjugate (prodrugs). A large number of polysaccharides have already been studied for their potential in these types of delivery systems, such as chitosan, alginate, pectin, chondroitin sulfate, cyclodextrin, dextrans, guar gum, inulin, amylose, and locust bean gum [111].

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Nifedipine concentration (ng/mL)

10 9 8 Colon arrival time

7 6 5 4 3 2

Complete disintegration

1 0 0

3

6

9

12

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21

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Initial disintegration Time (h)

FIGURE 10 Nifedipine plasma concentration–time profile from pectin–galactomannancoated tablets. (Adapted from ref. 110 with permission of Elsevier Copyright 2002.)

A pectin-and-galactomannan coating was developed by Lee et al. [112]. It consists of a conventional tablet coated with pectin and galactomannan. The coating from aqueous solutions of pectin and galactomannan was shown to be strong, elastic, and insoluble in gastric fluid. Figure 10 shows the plasma concentration profile of nifedifine from pectin–galactomannan-coated tablets and associated in vivo transit and disintegration characteristics. The mean plasma concentration of nifedifine was negligible for more than 5 h postdose and then increased rapidly. CODES Technology CODES is a unique colon-specific drug delivery technology that was designed to avoid the inherent problems associated with pH- or timedependent systems [113, 114]. The design of CODES exploited the advantages of certain polysaccharides that are only degraded by microorganisms available in the colon [115]. This is coupled with a pH-sensitive polymer coating. Since the degradation of polysaccharides occurred only in the colon, this system exhibited the capability to achieve colon delivery consistently and reliably. As schematically presented in Figure 11, one typical configuration consists of a core table coated with three layers of polymer coatings. The first coating (next to the core tablet) is an acid-soluble polymer (e.g., Eudragit E) and an outer coating is enteric with a HPMC barrier layer in between the oppositely charged polymers. The polysaccharides, degradable by enteroorganisms, generate organic acid, including mannitol, maltose, lactulose, and fructooligosaccharides. During the transit through the GI tract, CODES remains intact in the stomach, but the enteric and barrier coatings disolve in the intestines. In vivo performance of CODES in beagle dogs was studied using acetaminophen as the model drug and lactulose as the matrix-forming excipient in the core tablet. Compared with enteric-coated tablet, the onset of acetaminophen release form CODES was delayed more than 3 h, as shown in Figure 12.


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Enter coating

In stomach

Acid-soluble polymer coating

In small intestime

Lactulose Microflora In colon Organic acid

FIGURE 11 Conceptual design of CODES technology. (Adapted from ref. 114 with permission of Elsevier Copyright 2002.)

APAP plasma concentration (ng/mL)

1400 1200 COOES Enteric-coated core

1000 800 600 400 200 0 0

2

4

6

8 10 Time (h)

12

14

16

FIGURE 12 Percentage of acetaminophen released from CODES and enteric-coated core tablets in beagle dogs. (Adapted from ref. 114 with permission of Elsevier Copyright 2002.)

Thermoresponsive Drug Release Dosage Forms Temperature is the most widely utilized triggering signal for a variety of modulated or pulsatile drug delivery systems. The use of temperature as a signal has been justified by the fact that the body temperature often deviates from the physiological temperature (37°C) in the presence of pathogens or pyrogens. This deviation sometimes can be a useful stimulus that

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achivates the release of therapeutic agents from various temperature-responsive drug delivery systems for diseases accompanying fever. The drug delivery systems that are responsive to temperature utilize various polymer properties, including the thermally reversible coil/globule transition of polymer molecules, swelling change of networks, glass transition, and crystalline melting. Thermoresponsive hydrogels have been investigated as possible drug delivery carriers for stimuli-responsive drug delivery systems [116–118]. The common characteristics of temperature-sensitive polymers are the presence of hydrophobic groups, such as methyl, ethyl, and propyl groups. Of the many temperature-sensitive polymers, poly(N-isopropylacrylamide) (PIPPAm) is probably the most extensively used. PIPPAm cross-linked gels have shown thermoresponsive, discontinuous swelling/deswelling phases—swelling, for example, at temperatures below 32°C while shrinking above this temperature. A sudden temperature increase above the transition temperature of these gels resulted in the formation of a dense, shrunken layer on the gel surface, which hindered water permeation from inside the gel into the environment. Drug release from the PIPPAm hydrogels at temperatures below 32°C was governed by diffusion, while above this temperature drug release was stopped completely, due to the “skin layer” formation on the gel surface (on–off drug release regulation). Swelling–deswelling kinetics of conventional cross-linked hydrogels are normally the reciprocal of the square of the gel dimension. This mobility of the crosslinked chains in the gel is affected by the surrounding chains and the swelling–deswelling phases of the gel are governed by the collective diffusions of the network chains. Thus, to accelerate structural changes of the gel in response to external stimuli, several approaches have been developed which form porous structure within the gel and decrease gel size. Kaneko et al. [119, 120] introduced a method to accelerate gel swelling–deswelling kinetics based on the molecular design of the gel structure by grafting the free mobile linear PIPPAm chains within the cross-linked PIPPAm hydrogels. These novel graft-type PIPPAm gels had the same transition temperature as conventional cross-linked PIPPAm gels and existed in the swollen state below the transition temperature, while above this temperature, they shrank. Adense skin layer formed on the conventional PIPPAm gels upon temperature change above the transition temperature, which limited the complete shrinkage of the gel. In contrast, the PIPPAm-grafted gels showed rapid deswelling kinetics without the formation of a skin layer on the gel surface. This is probably due to the rapid dehydration of the graft chains formed by hydrophobic aggregation on the three-dimensional cross-linked gel chains. The low-molecular-weight compounds released immediately from conventional PIPPAm gels after a temperature increase, after which the release was teminated due to the formation of a dense impermeable skin layer on the surface. In comparison, 65% of the drug was released in one burst from free PIPPAm-grafted hydrogels with a graft molecular weight (MW) of 9000 following the temperature increase. Graft-type gels with a molecular weight of 4000 showed oscillating drug release profiles. The release of high-molecular-weight compound (e.g., dextran, MW 9300) from PIPPAm grafttype gels was shown to burst after a temperature increase of 40°C. The difference in drug release profiles for two graft-type gels is probably due to the different strengths of aggregation forces between the formed hydrophobic cores within the graft-type gels. That is, the high-molecular-weight graft chains formed more hydro-


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phobic cores within the gels upon the temperature increase, which induced rapid gel deswelling. Temperature-sensitive hydrogels can also be placed inside a rigid capsule containing holes or apertures. The on–off release is achieved by the reversible volume change of temperature-sensitive hydrogels. Such a device is called a squeezing hydrogel device because the drug release is affected by the hydrogel dimension. In addition to temperature, hydrogels can be made to respond to other stimuli, such as pH. In this type of system, the drug release rate was found to be proportional to the rate of squeezing of the drug-loaded polymer. Clinical applications of thermosensitive hydrogels based on NIPAAm and its derivatives have limitations [121]. The monomers and cross-linkers used in the synthesis of the hydrogels are still not known to be biocompatible and biodegradable. The observation that acrylamide-based polymers activate platelets upon contact with blood, together with the unclear metabolism of poly(NIPAAm), requires extensive toxicity studies before clinical applications can merge. Recently some studies have been conducted on anocomposite hydrogels for photothermally modulated drug delivery. Gold nanoshells can be designed to absorb light strongly at desired wavelengths, in particular, in the near infrared between 800 and 1200 nm, where tissue is relatively transparent [122]. When optically absorbing gold nanoshells are embedded in a matrix material, illuminating them at their resonance wavelength causes the nanoshells to transfer heat to their local environment. This photothermal effect can be used to optically modulate drug release from a nonshell polymer composite drug delivery system. To accomplish photothermally modulated release, the matrix polymer material must be thermally responsive. The authors observed the pulsatile release of insulin and other proteins in response to near-infrared irradiation when gold nanoshells were embedded in NIPAAm-coacrylamide hydrogels [122]. Electroresponsive Release An electric field as an external stimulus has advantages, such as the availability of equipment, which allow precise control with regard to the magnitude of current, duration of electric pulses, interval between pulses, and so on. Electrically responsive delivery systems are prepared from polyelectrolytes (polymers which contain relatively high concentration of ionizable groups along the backbone chain) and are thus pH responsive as well as electroresponsive. Under the influence of an electric field, electroresponsive hydrogels generally deswell or bend, depending on the shape of the gel that lies parallel to the electrodes, whereas deswelling occurs when the hydrogel lies perpendicular to the electrodes. Synthetic (e.g., acrylate and methacrylae derivatives) as well as naturally occurring polymers (including hyaluronic acid, chondroitin sulfate, agarose, carbomer, xanthan gum, and calcium alginate separately or in combination) have been used. Complex multicomponent gels or interpenetrating networks have been prepared in order to enhance the gels or interpenetrating networks have been prepared in order to enhance the gel’s electroresponsiveness [123]. Electrically enhanced transdermal delivery provides the time-dependent delivery. Ionotophoresis, the electromigrational movement of charged molecules through the skin under a low-voltage and continuous driving force, has been reported for a number of bioactive compounds, such as leutinizaing hormone relesing hormone (LHRH), testosterone, and buserelin.

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Electronic Microelectromechanical System for Controlled Release Electronic microelectromechanical devices are manufactured using standard microfabrication techniques that are used to create silicon chips for computers, and they often have moving parts or components that enable some physical or analytical function to be performed by the device. Microfabrication techniques, the same processing techniques used to make microprocessors for computers and other microelectronic devices, have been used increasingly to produce microscale devices whose primary functions are mechanical, chemical and optical in nature. Such devices are commonly referred to as microelectromechanical systems (MEMS) and are found in ink-jet printers, automotive applications, and microtube engines in the aerospace industry. MEMS for biological applications are classified as either microfluidic devices or nonmicrofluidic devices. The ultimate goal of MEMS is to develop a microfabricated device with the ability to store and release multiple chemical substances on demand by a mechanism devoid of moving its parts [124, 125]. A wide variety of microreservoirs, micropumps, cantilevers, rotors, channels, valves, sensors, and other structures have been fabricated, typically from the materials that have been demonstrated to be biocompatible and can be sterilely fabricated and hermetically sealed. The digital capabilities of MEMS may allow greater temporal control over drug release compared to traditional polymer-based systems, while the batchprocessing techniques used in the microelectronics industry can lead to greater device uniformity and reproducibility than is currently available to the pharmaceutical industry. The use of MEMS for drug delivery necessitates the existence of drug depot or supply within or on the device. One straightforward approach to achieve this drug reservoir is the fabrication of silicon microparticles that contain an internal reservoir loaded with drug. These devices could be used for oral drug delivery, with release of the drug triggered by binding of a surface-functionalized molecule to cells in the digestive tract. The completely implantable minipump made by Minimed has a pulsatile, radiocontrolled injection rate through a catheter into the intraperitoneal region [126]. One study found that patients with the implantable pump did not differ from control subjects on any meansure of psychosocial function but that puump users monitored their blood glucose levels more frequently and had lower average blood glucose levels. Even though this type of device may improve patient’s mbility and reduce infections by eliminating transcutaneous catheters, they may still be hampered by their size, cost, ability to deliver only drugs in solution, and limited stability of some drugs in solution at 37°C. Ikemoto and Sharpe [127] have developed a stepmotor micropump for the injection of nanoliter volumes of d-amphetamine solution into discrete brain regions of freely moving rats that was well tolerated. This micropump delivered a reliable volume of 50 nL per infusion over an hour at a rate of one infusion per minute. Another development in MEMS technology is the microchip. The microchip consists of an array of reservoirs that extend through an electrolyte-impermeable substrate. The prototype microchip is made of silicon and contains a number of drug reservoirs. Each reservoir is sealed at one end by a thin gold membrane of material that serves as an anode in an electrochemical reaction and dissolves when an electric potential is applied to it in an electrolyte solution. The reservoirs are filled with any combination of drug or drug mixtures in any form (i.e., solid, liquid, or gel) through the opening opposite the anode membrane by ink-jet printing or microinjection and


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are then sealed with a waterproof material. A cathode is also required for the electrochemical reaction to take place, and the cathode is usually made of the same conductive material as the anode to simplify the fabrication procedure. The device is submerged in an electrolyte solution containing ions and upon electric stimulation forms a soluble complex with the anode in its ionic form. When release is desired, an electric potential is applied between an anode membrane and a cathode, and the gold membrane anode is dissolved within 10–20 s and allows the drug in the reservoir to be released. This electric potential causes oxidation of the anode material to form a soluble complex with the electrolytes which when dissolves allowing release of the drug. Complex release patterns (such as simultaneous constant and pulsatile release) can be achieved from the microchips. The microchip has the ability to control both release time and release rate. The rate of release from a reservoir is a function of the dissolution rate of the materials in the reservoir, the diffusion rate of these materials out of the reservoir, or both. Therefore, the release rate from an individual reservoir can be tailored to a particular application by proper selection of the materials placed inside the reservoir [e.g., pure drug(s), drugs with polymers] [124, 125]. A microchip with insulin-filled reservoirs could eventually provide a better alternative for the treatment of insulin-dependent diabetes mellitus (IDDM) [125]. Because the microchip is capable of being programmed as well as integrated with other electronic devices, it is supposable that the microchip could be incorporated into a closed-loop biofeedback system. An electronic apparatus that continuously measures the blood glucose levels could provide the stimulus to the microchip and result in release of insulin into the bloodstream. Although such a system could still not perfectly mimic an endogenous system of healthy person, it could practically meet the needs of IDDM patients. Pulsatile release of synthetic gonadotropin– releasing hormone (GnRH) can be achieved with a programmed microchip. A subcutaneous implanted microchip containing 1000 drug reservoirs would be adequate to administer a month’s worth of drug therapy. The implanted microchip would be a convenient means to achieve the desired pharmacotherapeutic outcome of ovulation without interfering with the patient’s daily activities or causing phlebitis. While microchip drug delivery would be the most technologically advanced delivery system, it has itself limited storage capacity for therapeutic drugs [125]. Because most applications of this technology require implantation within bodily tissues, the question arises, “What would be done when the chip runs out of drug?” Some sort of procedure would be required to retrieve the empty chip cartridge once it has emptied. Due to the limited quantity of drug that can be stored on one chip, this technology is only ideal for potent drugs. If a larger dose of a medication is required, the chip would not be adequate for dispensing larger quantities of drug. Magnetically Induced Release Magnetic carriers receive their magnetic response to a magnetic field from incorporated materials such as magnetite, iron, nickel, and cobalt. For biomedical applications, magnetic carriers must be water based, biocompatible, nontoxic, and nonimmunogenic. Earlier, Langer et al. [128] embedded magnetite or iron beads into a drug-filled polymer matrix and then showed that they could activate or increase the release of the drug from the polymer by moving a magnet over it or by applying an oscillating magnetic field. When the frequency of

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the applied field was increased from 5 to 11 Hz, the release of BSA from ethylenevinylacetate copolymer (EVAc) matrices slowed in a linear fashion. The rate of release could be modulated by altering the position, orientation, and magnetic strength of the embedded materials as well as by changing the amplitude of frequency of the magnetic field. The micromovement within the polymer produced microcracks in the matrix and thus made the influx of liquid, dissolution, and efflux of the drug. Done repeatedly, this would allow the pulsatile delivery of insulin. Another mechanistic approach based on magnetic attraction is the slowing down of oral drugs in the gastrointestinal system. This is possible by filling an additional magnetic component into capsules or tablets. The speed of travel through the stomach and intestines can then be slowed down at specific positions by an external magnet, thus changing the timing and/or extent of drug absorption into stomach or intestines. Slowing down the passage of magnetic liposomes with a magnet actually increased the blood levels of drug. Babincova et al. [129] developed magnetoliposomes for triggered release of drug. In their delivery systems, they entrapped dextran–megnetite and model drug 6-carboxyfluorescein in the liposomes and used laser to trigger the release of drug. The magnetite absorbs the laser light energy to heat the lipid bilayer above the gel–liquid crystal-phase transsition temperature Tc, which is 41°C for dipalmitoyl-phosphatidylcholine. Liposomes made from this lipid release their content as soon as the temperature reaches this level. They have also suggested that the absorption of laser energy by magnetite particles provides a means for localized heating and controlled release of liposome with a single laser pulse. This may have potential applications for selective drug delivery, especially to the eyes and skin. Even though the magnetic-modulated therapeutic approach is promising, it still needs very careful attention for a number of physical and magnetism-related properties. The magnetic force, which is defined by its field and field gradient, needs to be large and carefully shaped to activate the delivery system within the target area. The magnetic materials should be tissue stable and compatible. Chemically Induced Release Gluose-Responsive Insulin Release Device A decrease in or the absence of insulin secretion from pancreatic islets is the cause of diabetes mellitus. An effective glucoseresponsive insulin delivery system should be composed of a glucose-sensing component and an insulin-releasing component. The sensing component detects a change in the glucose level and produces a signal that affects the releasing component. The magnitude of the signal increases with increasing glucose concentration, and so does the rate of insulin release. Based on this principle, various polymer-based glucoseresponsive delivery systems have been designed, most of which are hydrogels that can alter their volume and degree of hydration in response to glucose concentration. Several systems have already been developed which are able to respond to glucose concentration changes, such as glucose oxidase (GOD), which catalyzes glucose oxidation [130]. Glucosylated insulin bound to concanavalin (Con) A was released through exchange with external glucose, due to the difference in their binding constants. This system needs direct injection of microcapsules into the peritoneal cavity of patients, which may cause undesirable side effects arising from the immune response to Con A if Con A was directly exposed to immune systems after breakage


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of microcapsules. Obaidat and Park [131] prepared a copolymer of acrylamide and allyl glucose. The side-chain glucose units in the copolymer were bound to Con A. These hydrogels showed a glucose-responsive, sol–gel phase transition dependent upon the external glucose concentration. The nonlinear dependence of this sol–gel phase transition with regard to the glucose concentration was due not only to the increased binding affinity of allyl glucose to Con A compared to native glucose but also to the cooperative interaction between glucose-containing copolymer and Con A. Kataoka et al. [132] developed glucose and thermoresponsive hydrogels using acrylamidophenylboronic acid and N-isopropylacrylamide (IPAAm). The obtained gels, containing 10 mol % phenylboronic acid moieties, showed a transition temperature of 22°C in the absence of glucose. Below this temperature, the gels existed in a swollen state. The introduction of glucose to the medium altered the transition temperature of the gels in such a way that the transition temperature increased with increasing glucose concentration to reach 36°C at 5 g/L glucose concentration. Boronic acid was in equilibrium between the undissociated and dissociated forms. With increasing glucose concentration, the equilibrium shifted to increase the amount of dissociated boronate groups and gels became more hydrophilic. Although all of the glucose-sensitive insulin delivery systems are elegant and highly promising, many improvements need to be made for them to become clinically useful. First of all, the response of these systems upon changes in the environment occurs too slowly. In clinical situations, these systems need to respond to ever-changing glucose concentrations at all times, requiring hydrogels that can respond reproducibility and with rapid-response onset times on a long-term basis. An additional constant is that all the components used in the systems must be biocompatible. Chemotactic Factor-Induced Controlled-Release Systems With physical or chemical stress such as injury and broken bones, an inflammation reaction takes place at the injured site. At the inflammatory sites, inflammation-responsive phagocytic cells such as macrophages and polymorphonuclear cells play a role in healing the injury. During inflammation, hydroxyl radicals (OH•) are produced from the cells. Yui and co-workers [133, 134] developed inflammatory-induced hydroxyl radicals and designed drug delivery system which responded to the hydroxyl radicals and degraded in a limited manner. They used hyaluronic acid (HA), a linear aminopolysaccharide composed of repeating disaccharide subunits of N-acetyl-dglucosamine and d-guluronic acid. In the body, HA is mainly degraded by hyaluronidase, or hydroxyl radicals. Degradation of HA via the enzyme is very low in a normal state of health. Degradation via hydroxyl radicals, however, is usually dominant and rapid when HA is injected at inflammatory sites. These authors prepared cross-linked HA with ethyleneglycol diglydylether or polyglycerol polygluycidalether. These HA gels degraded only when the hydroxyl radicals were generated through the reaction between the iron (Fe2+) ions and the hydrogen peroxide in vitro. Thus, a surface erosion type of degradation was achieved. When microspheres were incorporated in the HA hydrogels as a model drug, these microspheres were released when hydroxyl radicals induced HA gel degradation. Furthermore, degradation of HA in vivo tests showed that HA gels are degraded only when inflammation was induced by surgical incision. Control HA gels were stable over 100 days. It is possible to treat locally in inflammatory diseases such as rheumatoid arthritis using anti-inflammatory drug incorporated in HA gels [135].

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5.1.11


SUMMARY

As pharmaceutical scientists have increased their knowledge of pharmacokinetics and pharmacodynamics, it has become apparent that these factors can result in more efficacious drugs. The number of new drug entities appearing on the market yearly has declined and pharmaceutical manufacturers have shown a renewed interest in improving existing dosage forms and developing more sophisticated drug delivery systems, including those employing the principles of controlled drug release. Current research in this area involves numerous new and novel systems, many of which have strong therapeutic potential. In this chapter, we have tried to emphasize the importance of oral routes as well as others, such as ocular, transdermal, intrauterine, and vaginal. The various microencapsulation, nanoencapsulation, and liposome technologies and the release of drugs and bioactive compounds from such products have been described.

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5.2 PROGRESS IN DESIGN OF BIODEGRADABLE POLYMER-BASED MICROSPHERES FOR PARENTERAL CONTROLLED DELIVERY OF THERAPEUTIC PEPTIDE/PROTEIN Shunmugaperumal Tamilvanan* University of Antwerp, Antwerp, Belgium


Introduction Peptide/Protein-Loaded Microsphere Production Methods 5.2.2.1 Phase Separation (A Traditional Technique) 5.2.2.2 Double Emulsion (A Hydrous Technique) 5.2.2.3 Spray Drying (An Anhydrous Technique) 5.2.2.4 New Trends in Production Methods 5.2.3 Analytical Characterization of Peptide/Protein-loaded Microspheres 5.2.4 Immune System Interaction with Injectable Microspheres 5.2.5 Excipient Inclusion: Injectable Peptide/Protein-Loaded Microspheres 5.2.5.1 Solubility- and Stability-Increasing Excipients 5.2.5.2 Preservation-Imparting Excipients 5.2.6 Peptide/Protein Encapsulated into Biodegradable Microspheres: Case Study 5.2.6.1 Vaccines 5.2.6.2 Proteins 5.2.7 Conclusion References

*Current address: Department of Pharmaceutics, Arulmigu Kalasalingam College of Pharmacy, Anand Nagar, Krishnankoil, India Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad Copyright © 2008 John Wiley & Sons, Inc.

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5.2.1

INTRODUCTION

At the cellular level, deoxyribonucleic and ribonucleic acids (DNA and RNA, respectively) serve as an endogenious vehicle not only to store genetic information but also to transfer genetic information from one generation to their offsprings of all known living organisms. In addition, utilizing the rule of complementary base pairing, the DNA undergoes replication and transcription processes to produce respectively a new double-stranded DNA molecule and a complementary singlestranded RNA molecule. Following the translation process, peptide and protein are synthesized/constructed in ribosomal subunits through peptidic linkages between available 20 amino acids. The peptide and protein thus constructed perform a wide variety of functions and each cell contains several thousands of different proteins. Peptide- and protein-mediated, important physiological and biological processes of the human body include ligands/hormones for signaling, enzymes for biotransformation reactions, receptors for pharmacological response elucidation, antibodies in immune system interactions, transcription, and translation. Hence these molecules play a vital role to ensure proper development and functioning of entire organs of the human body. Webster’s New World Dictionary defines a drug as “any substances used as a medicine or as an ingredient in a medicine.” Indeed, peptides and recombinant proteins are highly potent, relatively macromolecular and promising therapeutic agents that emerged out from the significant development of biotechnic and biogenetic engineering technologies. Peptide and protein therapeutics include semisynthetic vaccines, monoclonal antibodies, growth factors, cytokines, soluble receptors, hormones, and enzymes. The advent of recombinant DNA technology allowed the possibility of the commercial production of proteins for pharmaceutical applications from the early 1980s and, in fact, manufacture of therapeutic proteins represented the first true industrial application of this technology [1]. During the 1980s the term biopharmaceutical became synonymous with therapeutic protein produced by recombinant DNA technology (or, in the case of a small number of therapeutic monoclonal antibodies, by hybridoma technology). Clinical evaluation of nucleic acid–based drugs used for the purposes of gene therapy and antisense technology commenced in the 1990s, and today the term biopharmaceutical also encompasses such (as-yet-experimental) drugs [2]. The first such recombinant therapeutic protein (insulin) was approved for general medical use only 24 years ago. Today there are in excess of 100 such products approved in some world regions at least, with 88 having received approval within the European Union (EU). This represents 36% of all new drug approvals since the introduction of the new centralized European drug approval system in 1995 [3]. Over the coming decade, therefore, in the region of a dozen new therapeutic proteins should, on average, gain regulatory approval each year. While EU figures are difficult to locate, the American Association of Pharmaceutical Researchers and Manufacturers (PhRMA) estimates that there are currently some 371 biotechnology medicines in development [4]. Out of these 371 biotechnology medicines, as estimated by PhRMA, more than 300 are protein based, with recombinant vaccines and monoclonal/engineered antibodies representing the two most promising categories. Incidentally, all 88 biopharmaceutical products currently approved within the EU are protein based. Of the proteins thus far approved, hormones and cytokines represent the largest product categories (23 and 18 products, respectively). Hormones approved include several recombinant insulins, displaying both native and modified amino acid

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sequences. In addition, several recombinant gonadotrophins [follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotrophins (hCG)] have been approved for the treatment of various forms of subfertility/infertility. Cytokines approved include a range of recombinant hematopoietic factors, including multiple erythropoietin-based products used for the treatment of anemia as well as a colony-stimulating factor aimed at treating neutropenia. Additional approved cytokines include a range of interferon-based products, mainly used to treat cancer and various viral infections, most notably hepatitis B and C, and a recombinant tumor necrosis factor (TNF) used as an adjuvant therapy in the treatment of some soft tissue cancers. Blood-related approved therapeutic proteins include a range of recombinant blood coagulation factors used to treat hemophilia, recombinant thrombolytics, and recombinant anticoagulants. Additional product categories include a range of subunit vaccines containing at least one recombinant component [mainly hepatitis B surface antigens (HBsAg)] and a variety of monoclonal antibody–based products indicated for the treatment/detection of various cancers or the prevention of organ transplant rejection. In summary, ailments that can be treated more effectively by this new class of therapeutic agents include cancers, autoimmune disease, memory impairment, mental disorders, hypertension, and certain cardiovascular and metabolic diseases [5, 6]. Poor absorption and easy degradation by endogenous proteolytic enzymes present in eye tissues, nasal mucosa, and gastro intestinal tract and low transdermal bioavailabilities due to relatively large size make the peptide/protein molecules to be administered only through parenteral routes either by multiple injections or infusion therapy in order to achieve desired therapeutic plasma levels for prolonged periods of time. Nevertheless, because of remarkably short half-lives within the in vivo arena, the therapeutic usuage of most of the peptide/protein is practically possible only through daily multiple injections under close medical supervision. Hence, the commercial success of peptides/proteins as therapeutic agents depends mainly on development of novel drug delivery systems which could potentially reduce the injection frequencies and thus eliminate the accompanying serious problem of patient compliance. Among the several technologies that have been suggested for reducing injection frequencies of therapeutic peptide/protein, microspheres prepared from biodegradable polymers are widely recognized for controlled drug delivery following parenteral administration. Polyester polymers such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and their copolymer poly(lactic acid-co-glycolic acid) (PLGA) are used routinely for the preparation of injectable microspheres after taking into consideration their well-known biocompatibility, controlled biodegradability, absorbability, and no toxicity of degradation products [7]. Furthermore, the PLGA types and related poly(hydroxyalkanonates) have a long history of medical and pharmaceutical use in fields as diverse as sutures, bone fixatives, artificial skins and cartilages, dental materials, materials for bone generation, drug delivery, and many others, as reviewed by Ueda and Tabata [8]. In conjunction with a long safety record of PLGA polymers, at least 12 different peptide/protein-loaded PLGA microsphere products are available in the market from nine different companies worldwide for the treatment of some life-threatening diseases (Table 1). In recent years, poly(ε-caprolactone) (PCL) has been investigated as an alternative to PLGA to make microspheres [9, 10]. A glimpse of ongoing research activities utilizing biodegradable polymer-based microspheres for various peptide/protein is shown in

396


TABLE 1 Currently Marketed Preparations (Injectable Microspheres) Containing Peptide/Protein Molecules Commercial Name

API

Polymer

Company

Lupron Depot

Leuprolide

PLGA or PLA

TAP

Enantone Depot

Leuprolide

PLGA or PLA

Takeda

Trenantone

Leuprolide

PLGA or PLA

Takeda

Enantone Gyn

Leuprolide

PLGA or PLA

Takeda

Sandostatin LAR Nutropin

Octreotide Somatropin

PLGA-glucose PLGA

Novartis Genentech

Trelstar Depot Decapeptyl SR Decapeptyl Suprecur MP Somatuline LA Parlodel LAR

Triptorelin Triptorelin Triptorelin Buserelin Lanreotide Bromocriptine

PLGA PLGA or PLA PLGA PLGA PLGA PLGA-Glu

Pfizer Ipsen-Beaufour Ferring Aventis Ipsen-Beaufour Novartis

Indication Prostate cancer, endometriosis Prostate cancer, endometriosis Prostate cancer, endometriosis Prostate cancer, endometriosis Acromegaly Growth deficiencies Prostate cancer Prostate cancer Prostate cancer Endometriosis Acromegaly Parkinsonism

Abbreviations: PLA: polylactide; PLGA: poly(lactide-co-glycolide); API: active pharmaceutical ingredient; PLGA-Glu: poly(d,l-lactide-co-glycolide-d-glucose).

Table 2 (incorporating refs. 12–35). However, overcoming the propensity for peptides/proteins to undergo degradation processes during incorporation into the biodegradable microspheres or after injection into the body awaiting release is one of the key hurdles in bringing microencapsulated systems for these drugs to market. This partially explains the limited and only a countable number of formulations available on the market. Furthermore, irrespective of the various microencapsulation techniques adopted to prepare peptide/protein-loaded microspheres, several transfer-required processes such as filtration, centrifugation, and vacuum or freeze drying are necessary to obtain a final product, and these processes might be obstacles when scaling up the manufacturing technique to produce sufficient quantities of sterile material for clinical trial and, ultimately, commercialization [11]. This chapter encompasses investigations made progressively on the design of injectable peptide/protein-loaded PLGA microspheres. It covers an update on the state of art of the manufacturing of peptide/protein-loaded microspheres through both conventional and newer microencapsulation techniques, different analytical methods used for microsphere characterization, immune system interaction with microspheres following parenteral administration, and potential application of microspheres having therapeutic peptides/proteins. Special emphasis is given particularly on various instability problems and investigated mechanistic ways to obviate the possible instability problems of peptide/protein drug during microsphere preparation as well as its release from the microspheres. It should be added that although the chapter focuses mainly on PLGA microspheres, many of the destabilization mechanisms and stabilization approaches described herein can be valid to some extent for other polymeric delivery systems, too.

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INTRODUCTION

TABLE 2 Injectable Peptide/Proteins/Vaccines Encapsulated in Biodegradable Microspheres Peptides, Protein, Vaccine

Technique

Polymer

Reference

Vaccine SPf 66 malaria vaccine Multivalent vaccines of Haemophilus influenzae type b (Hib), diphtheria toxoid (DT), tetanus toxoid (TT), pertussis toxin (PT) Rotavirus

Double emulsion Spray drying

PLGA PLGA

28 29

Double emulsion

PLG

30

Polypeptides and Proteins Insulin

Double emulsion Double emulsion

PLA polyethylene glycol (PEG) PLA

Recombinant human epidermal growth factor (rhEGF) Ribozyme Vapreotide (somatostatin analogue) Insulinlike growth factor-1 (IGF-1) Ornitide acetate leuteinizing hormone releasing hormone [(LHRH) antagonist] Vascular endothelial growth factor (VEGF) Human chorionic gonadotropin (hCG) Calcitonin FITC-bovine serum albumin (BSA) Levonorgestrel and ethinylestradiol Recombinant human bone morphogenetic protein Transforming growth factor beta Recombinant human erythropoietin (rhEPO) Protein-C Ovalbumin Human serum albumin Bovine serum albumin

13

Double emulsion Spray drying

PLA, PLGA PLGA

14 15

Double emulsion

PLGA-PEG

16

Dispersion/solvent extraction/evaporation

PLA, PLGA

17

Single emulsion

PLGA/PEG

18

Double emulsion

PLA, PLGA

19

Double emulsion Double emulsion

PLGA Poly(ε-caprolactone)

20 9

Double emulsion

Poly(ε-caprolactone)

10

Double emulsion

PLGA

21–24

Double emulsion

PLGA or PLGA-PEG LPLG-PEO-LPLG

25–27

Modified double emulsion Double emulsion Double emulsion Double emulsion Nonaqueous oil-in-oil (o/o) emulsion

PLA PLGA PLA PLG

12

31 32 33 34 35

Abbreviations: FITC: fluroscein isothiocyanate; PLG: poly(lactide-co-glycolide); LPLG-PEO-LPLG: copoly(l-lactic-co-glycolic acid-b-oxyethylene-b-l-lactic-co-glycolic adic); PEO: polyethylenenoxide.

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5.2.2 PEPTIDE/PROTEIN-LOADED MICROSPHERE PRODUCTION METHODS The development of delivery systems for therapeutic peptides/proteins depends on biophysical, biochemical, and physiological characteristics of these molecules, including molecular size, biological half-life, immunogenicity, conformational stability, dose requirement, site and rate of administration, pharmacokinetics, and pharmacodynamics [36]. Unlike conventional drug molecules, the unique conformational structure of peptidic/proteinic therapeutic agents poses a great challenge right from the beginning of the selection of suitable microencapsulation techniques to make microspheres. Table 3 lists the considerations to be taken before choosing a particular encapsulation technique. Apart from the traditional phase separation technique, other techniques suitable for peptide/protein-loaded microsphere production can be divided into two main categories: during microsphere preparation, those involved in utilizing a hydrous environment such as emulsion-based methods and those based on an anhydrous environment such as spray freeze drying, spray drying, freeze drying, grinding, jet milling, liquid-phase antisolvent precipitation, and supercritical CO2-based methods [37–40]. In the following sections, the various production techniques to make injectable peptide/protein-loaded microspheres are briefly introduced; however, a detailed discussion is beyond the scope of this chapter. 5.2.2.1

Phase Separation (A Traditional Technique)

Polymer phase separation or coacervation is an excellent technique for the encapsulation of water-soluble drugs including peptide/protein into a final microsphere product [41]. The peptide/protein molecule is dispersed in solid form into solution containing dichloromethane and PLGA. Silicone oil is added to this dispersion at a defined rate, reducing the solubility of polymer in its solvent. The polymer-rich liquid phase (coacervate) encapsulates the dispersed peptide/protein molecules and embryonic microspheres are subjected to hardening and washing using heptane. The process is quite sensitive to polymer properties, and residual solvent is also an important issue. Decapeptyl [triptorelin, a luteinizing hormone releasing hormone (LHRH) analogue] [42] and Somatuline LA (lanreotide, a somatostatin analogue) [43] are microsphere commercial products developed by this technique (Table 1). TABLE 3 Factors in Selection of Microencapsulation Method to Prepare Peptide/Protein-Loaded Microspheres Optimal peptide loading High microsphere yield Batch content uniformity Interbatch reproducibility Peptide stability during preparation and release Size uniformity Adjustable release profile Low burst release Flowability of final product Residual solvent and polymer monomer control Sterilization (both aseptic and terminal)

PEPTIDE/PROTEIN-LOADED MICROSPHERE PRODUCTION METHODS

5.2.2.2

399

Double Emulsion (A Hydrous Technique)

Oil-in-water (o/w) and water-in-oil-water (w/o/w) are the two hydrous techniques representing respectively the single- and double-emulsion formation during microsphere preparation. However, the w/o/w technique is most commonly employed [44]. In this process, peptides/proteins in aqueous solution are emulsified with nonmiscible organic solution of polymer to form a w/o emulsion. Dichloromethane serves as organic solvent and the o/w primary emulsion is formed using either highspeed homogenization or ultrasonication. This primary emulsion is then rapidly transferred to an excess of aqueous medium containing a stabilizer, usually polyvinyl alcohol. Again homogenization or intensive stirring is necessary to initially form a double emulsion of w/o/w. Subsequent removal (by evaporation) of organic solvent by heat, vacuum, or both results in phase separation of polymer and core to produce microspheres. Instead of solvent evaporation, solvent extraction with a large quantity of water with or without a stabilizer can also be undertaken to yield microspheres containing peptide/protein. Although the w/o/w microencapsulation technique seems to be conceptually simple to carry out, the particle formation process is quite complicated, and a host of process parameters influence the properties of peptide/ protein-loaded PLGA microspheres [45]. In spite of that, different peptides and proteins such as bovine serum albumin (BSA) or ovalbumin (OVA), insulin, recombinant human insulinlike growth factor-1 (rhIGF-1), recombinant human epidermal growth factor (rhEGF), human chorionic gonadotropin (hCG), protein C, recombinant human bone morphogenetic protein (rhBMP), and calcitonin, along with antigens and other therapeutically relevant proteins such as recombinant human erythropoietin (rhEPO), have been successfully encapsulated (see Table 2) by the w/o/w double-emulsion technique. Lupron Depot/Enantone Depot/Trenantone/ Enantone Gyn (all having leuprolide acetate, a LHRH analogue) are very popular commercial microsphere products produced by this technique [46, 47], available both in the EU and United States, for the treatment of either prostate cancer of man or infertility (endometriosis) of women (Table 1).

5.2.2.3

Spray Drying (An Anhydrous Technique)

Spray drying is a rapid, convenient technique which can be conducted under aseptic conditions. First, a polymer—prevalently PLGA is applied—is dissolved in a volatile organic solvent such as dichloromethane or acetone. The protein is suspended as a solid or emulsified as aqueous solution in this organic solution by homogenization. After that, the resulting dispersion is atomized through a (heated) nozzle into a heated airflow. The organic solvent evaporates, thereby forming microspheres with dimensions of typically 1–100 μm. The microspheres are collected in a cyclone separator. For the complete removal of the organic solvent, a vacuum drying or lyophilization step can follow downstream. The internal structure of the resulting polymeric microspheres depends on the solubility of the peptide/protein in the polymer before being spray dried leading to the formation of reservoir- or matrix-type products (see Figure 1). When the initial dispersion is solution, the final product obtained following spray drying is matrix or monolithic type, that is, polymer particles with a dissolved or dispersed nature of the active ingredient (defined as microspheres). Conversely, when the initial

400


Polymer

Polymer

Drug

Drug

(a)

(b)

FIGURE 1 Polymeric delivery systems: (a) reservoir systems (microcapsules); (b) matrix systems (microspheres).

dispersion is in suspension, the product obtained is reservoir type, that is, a distinct polymeric envelope/shell encirculating a liquid core of dissolved active ingredient (defined as microcapsules). Recombinant human erythropoietin [48] and bromocriptine mesylate, Parlodel Depot [49], are examples of microspheres (matrix type) obtained by the spray drying technique. 5.2.2.4

New Trends in Production Methods

Several issues such as reducing cost, reducing scale-up difficulties, improving protein stability, allowing for terminal sterilization, and eliminating the need for organic solvents during addition of the peptide/protein motivate the development of new methods to manufacture microspheres. Moreover conventional microencapsulation methods involve relatively harsh conditions that are not generally tolerated by peptide/protein molecules without stabilization. Therefore, new and improved processes shielding the peptide/protein from deleterious conditions have been proposed and evaluated. Modified Conventional Methods The w/o/w solvent evaporation or extraction is probably one of the most widely used methods for peptide and protein microencapsulation [44], despite its many drawbacks. Improvements and alternatives have therefore been proposed such as oil in water (o/w), *o/w (the asterisk including cosolvent) and oil in oil (o/o) [50]. Utilising a modified w/o/w method, the rhIGF-1 was encapsulated into PLGA microspheres after increasing the pH of the protein solution from 4.5 to 5.5–6.0, where rhIGF-1 formed a viscous gel [51]. High entrapment efficiency of fully bioactive protein was achieved, and 92–100% of pure, monomeric, and bioactive rhIGF-1 was released in vitro over 21 days. The lowering of the rhIGF-1 solubility at pH 5.5–6.0 probably restricted its conformational flexibility and changes upon exposure to the polymer solvent. Without pH adjustment, approximately 10–32% of rhIGF-1 was lost upon solvent exposure, due to degradation and aggregation. Elsewhere, a w/o1/o2 system was investigated for encapsulating different proteins and peptides, with the o1 and o2 phases consisting of acetonitrile/dichloromethane and liquid paraffin/Span 80, respectively [52]. The acetonitrile mediated the partial mixing of the w and o1 phases and subsequent protein/peptide precipitation, which was a


401

prerequisite for microencapsulation. The proteins BSA, tetanus toxoid (TT), and lysozyme did precipitate at low acetonitrile concentration, resulting in efficient microencapsulation (more than 90%), while a decapeptide and a linear gelatine did not precipitate so rapidly, resulting in poor entrapment. TT and lysozyme released during the burst phase (15%) maintained their bioactivity, although lack of further release suggested aggregation within the microspheres. Another approach consisted of dispersing the protein antigen in a mineral oil before encapsulation into PLGA microspheres by a o1/o2/w method [53]. The mineral oil (o1) was intended as a barrier to protect the antigen during emulsification with the polymer solution and from exposure to moisture during release. Over 92% of enzyme-linked imunosorbert assay (ELISA) reactive TT was released from the reservoir-type microspheres in a pulsatile pattern, proceeding with an initial burst and followed by a second release pulse between 14 and 35 or 35 and 63 days, depending on the polymer type used. The latter stage of release was ascribed to TT diffusion through the oily phase, once an appreciable loss of polymer mass had occurred. The authors claimed the mineral oil was the key to protecting the solid antigen during polymer erosion, where acidic degradants and moisture would otherwise have led to antigen inactivation. To improve solvent extraction, a novel method using a static micromixer was presented where a w1/o dispersion (aqueous BSA in organic PLGA solution) is fed into an array of microchannels and the extraction fluid (w2) into a second array of interdigitated channels [54]. The two fluids, transported separately through the channels, are discharged through an outlet slit where alternating fluid lamellae are formed with the w1/o fluid lamella disintegrating into microdroplets, which harden quickly to form microspheres. This process offers easy scale-up, methodological robustness, continuous production, and a simple setup, making it ideally suited for aseptic production, a strongly needed feature for microsphere vaccine formulations. ProLease Technology (Cryogenic Spray Drying) A variation of the conventional spray drying method is a cryogenic method which will described below. A novel low-temperature spraying technique (called ProLease technology) for preparing PLA and PLGA microspheres has been reported by Khan et al. [55] and the group at Alkermes [56, 57]. The method relies on the use of stabilizing and release controlling agents, low processing temperature, and nonaqueous microencapsulation. Typically, a protein powder is micronized, possibly with a stabilizer, by spray freeze drying and then suspended in an organic polymer solution. The suspension is atomized into a vessel containing liquid N2 underlaid by frozen ethanol (extraction solvent). The atomized droplets freeze in the liquid N2 and deposit on the surface of the frozen ethanol. As liquid N2 evaporates, the frozen ethanol liquefies (Tm approximately −110°C) so that the frozen polymeric droplets will transfer into the ethanol where the polymer solvent is extracted, yielding solid microspheres [58, 59]. To date, the ProLease system has been effectively applied to the encapsulation of zinc-complexed human growth hormone in PLGA microspheres, resulting in a onemonth effect after one single injection [37, 57, 60]. As a reference, the recombinant human growth hormone (rhGH) was unstable in contact with ethyl acetate or dichloromethane [61]. The only protein-containing PLGA microspheres, Nutropin Depot, is produced by this novel technique. However, this product containing rhGH

402


marketed initially in the United States in 1999 was pulled from the market voluntarily by the manufacturer in June 2004 because of high costs of production and commercialization (http://www.gene.com/gene/news/press-releases/, accessed May 25, 2006). ProLease technology was also used for encapsulating recombinant human vascular endothelial growth factor (rhVEGF) and rhIGF-1 [62, 63]. Both proteins were stabilized in aqueous solution, prior to spray freeze drying, and encapsulated (9– 20% w/w) into PLGA microspheres. The microspheres also contained ZnCO3 (3– 6% w/w) as release modifier. The resistance of rhIGF-1 to aggregation and oxidation, determined from in vitro release studies, hardly changed. Protein, released in an almost pulsatile fashion over 21 days, was composed of predominantly monomeric rhIGF-1 with only minor amounts (∼6%) of degradants forming toward day 21. Similarly, the integrity of rhVEGF dimer released over 21 days was good and its bioactivity remained largely unaffected, regardless of the extent of aggregation and degradation. In view of these studies, ProLease technology appears to have potential for sustaining antigen stability and release from microspheres. Techniques Using Supercritical Fluids Generally, the application of supercritical (SC) fluids for the encapsulation of peptides and proteins has been fueled by the recognition that the established methods implicate some drawbacks. The application of supercritical fluids, especially of supercritical carbon dioxide (CO2), can minimize or even eliminate the use of organic solvents and renders work at moderate temperatures possible [64]. The term supercritical defines the area above the critical point, which specifies the final point of the liquid–gas phase transition curve. Beyond that critical point, isobar/isotherm alterations of pressure or temperature alter the density of the critical phase but do not lead to a separation into two phases. A density change is directly associated with a change of the solvent power, and thus the method features a high variability. Usually CO2 is used as supercritical fluid due to its critical point (Tc = 31.1°C, Pc = 73.8 bars), which can be easily reached. That allows a moderate working temperature and leaves no toxic residues since it returns to the gas phase at ambient conditions. Two SC CO2-based processes have been reported for the preparation of drug-loaded polymeric microspheres: first, the rapid expansion from supercritical solutions (RESS) process, whereby a SC CO2 solution of an active agent and a polymeric carrier is rapidly expanded. This quickly transforms the SC CO2 into a liquid that is a much poorer solvent, thereby precipitating the active agent–carrier mixture as small particles [65]. Second is the aerosol solvent extraction system (ASES), also referred as the gas antisolvent spray precipitation (GAS) process [66]. Here, a solution of the active agent and the polymeric carrier is sprayed into a chamber loaded with SC CO2. The SC CO2 extracts the solvent from the spray droplets and induces coprecipitation of the active agent and the polymeric carrier in the form of small, solvent-free particles [67, 68]. However, the use of organic solvents cannot be avoided, which is to be deemed a major disadvantage of both techniques. In peptide/protein pharmaceuticals, the GAS process is predominantly applied for the preparation of microparticulate protein powders as an alternative to common drying processes. However, Winters et al. [69] reported an increase of β-sheet aggregates during the precipitation of lysozyme, trypsin, and insulin as a consequence of stress parameters such as organic solvent, pressure, and shear forces. One reason


403

why these methods were not credited as encapsulation techniques for protein within PLGA may be the tendency of several polymers to rapidly precipitate and agglomerate during the process [70]. ASES has been compared with conventional spray drying in terms of effects on the stability of the peptide tetracosactide [71]. Almost no intact peptide was recovered from spray-dried PLA particles, whereas the tetracosactide was well protected against oxidation during ASES (∼94% unmodified peptide). In general, the particle formation step seems to be less detrimental to proteins than the loading step. For example, emulsification in an aqueous phase or spray drying of rhEPO/PLGA emulsions was mild compared to the first emulsification step [72]. Also, variation of the particle formation step (spray drying or coacervation) had a minor impact on diphtheria toxoid (DTd) antigenicity when compared to other process variables [73]. A serious limitation of GAS, ASES, and RESS for producing microspheres is the need of polymer types that form discrete crystalline domains upon solidification, such as l-PLA [74, 75]. The advantages of these methods offer (e.g., over spray drying) are the low critical temperatures for processing (34°C) and the avoidance of oxygen exposure during atomization, with both parameters being potentially important to peptide/protein stability. Ultrasonic Atomization Ultrasonic atomization of w/o dispersions is presently under investigation for preparing especially protein antigen containing microspheres. In one setup, the atomized antigen/polymer dispersion was sprayed into a nonsolvent where the polymer solvent was extracted, resulting in microspheres [76]. A comparable technique was proposed where the antigen or polymer dispersion was atomized into a reduced pressure atmosphere and the preformed microspheres hardened in a collection liquid [77]. Similarly, PLGA solutions were also atomized by acoustical excitation and the atomized droplets transported by an annular stream of a nonsolvent phase [aqueous polyvinyl alcohol (PVA)] into a vessel containing aqueous PVA [78]. Solvent evaporation and microsphere hardening occurred in the vessel over several hours. The main advantages of these atomization techniques encompass the possibility of easy particle size control and scale-up, processing at ambient or reduced temperature, and the suitability for aseptic manufacturing in a small containment chamber such as an isolator. In Situ Formed Injectable Microspheres All the encapsulation techniques discussed so far rely on the preparation of solid microspheres. However, a method for preparing a stable dispersion of protein containing semisolid PLGA microglobules has been reported [79]. Here, a protein dissolved in PEG 400 was added to a solution of PLGA in triacetin or triethyl citrate. This mixture, stabilized by Tween 80, was added dropwise and under stirring to a solution of Miglyol 812 or soybean oil, containing Span 80, resulting in a stable dispersion of protein inside semisolid PLGA microglobules. The microglobules remained in an embryonic state until mixed with an aqueous medium, so that the water-miscible components were extracted and protein containing matrix-type microspheres formed. Myoglobin was encapsulated and found to remain physically unchanged (circular dichroism analysis) after the process and during storage of the microglobular dispersion (15 days, 4°C).

404


Preformed Porous Microspheres A new approach for attaining sustained release of protein is introduced involving a pore-closing process of preformed porous PLGA microspheres [80]. Highly porous biodegradable PLGA microspheres were fabricated by a single w/o emulsion solvent evaporation technique using Pluronic F127 as an extractable porogen. The rhGH was incorporated into porous microspheres by a simple solution dipping method. For its controlled release, porous microspheres containing rhGH were treated with water-miscible solvents in the aqueous phase for production of pore-closed microspheres. These microspheres showed sustained-release patterns over an extended period; however, the drug loading efficiency was extremely low. To overcome the drug loading problem, the pore-closing process was performed in an ethanol vapor phase using a fluidized-bed reactor. The resultant pore-closed microspheres exhibited high protein loading amount as well as sustained rhGH release profiles. Also, the released rhGH exhibited structural integrity after the treatment. Charged (Anionic and Cationic) PLGA Microspheres PLGA or any other type of microspheres can be readily decorated with positive or negative surface charges by simply preparing the particles by a w/o/w solvent evaporation/extraction process where the second water phase contains a cationic emulsion stabilizer [hexadecyltrimethylammonium bromide; poly(ethyleneimine); stearylamine] or an anionic emulsifier [sodium dioctyl-sulfosuccintate; sodium dodecyl sulfate (SDS)]. Such compounds attach tightly to PLGA surfaces during preparation and provide the necessary surface charge for ionic adsorption of counterions. It is known that a protein’s surface charge depends on its isoelectric point (pI) and the pH of the medium in which it is dispersed. The use of particles with ionic surface charge offers several advantages over classical microencapsulation, among which the mild conditions for loading are probably the most attractive. PLGA microspheres with surfaceadsorbed protein antigens and DNA have been highly efficient in inducing strong immune responses, as reviewed by Singh et al. [81] and Jilek et al. [82]. Nonetheless, it remains to be shown whether such particles are also suitable for eliciting longterm immunity after one or two injections. Jabbal-Gill et al. [83, 84] noted the tendency for microencapsulated protein antigens to distribute heavily at the surface of PLGA microspheres and developed polymeric lamellar substrate particles (PLSP) by precipitating a highly crystalline poly(l-lactic acid)/organic solvent solution with water, followed by removal of remaining organic solvent with nitrogen purge. The particles, which can be sterilized by gamma irradiation and stored as a suspension for several months without changes to antigen absorption [84], possessed a large lamellar surface area and highly negative zeta potential (∼−35 to −42 mV) and could adsorb significant amounts of antigen (up to 50 μg/mg microspheres) depending on pH, ionic strength, antigen–polymer ratio, and other factors. Release of protein antigen (TT) could be extended to over 1 month with minimal antigenic losses in released antigen, although most of the antigen was lost to the initial burst or to apparent irreversible adsorption (as indicated by the absence of reaching 100% release). Elevated antibody responses in mice were elicited using PLSP similar to one dose of aluminum adjuvant following subcutaneous administration of OVA at elevated doses (100 or 300 μg). Both immunoglobulin IgG1 and IgG2a antibody subtypes were of similar magnitude over 28 days in the PLSP/OVA groups, and cellular immunity was also observed following

ANALYTICAL CHARACTERIZATION

405

immunization with a 38-kDa protein antigen against tuberculosis [85]. Similarly, Kazzaz et al. [86] created anionic PLGA microparticles by substituting the standard nonionic emulsifier PVA with anionic SDS during microsphere preparation. In addition to eliciting elevated antibody responses in mice relative to the soluble antigen, the adsorbed antigen elicited a potent cytotoxic T-cell (CTL) response, similar to that observed after infection from virus expressing the p55 gag and polymerase proteins. Moreover, the CTLs were formed from the more challenging intramuscular route but not significantly by the soluble antigen, even at elevated doses. The SDS-PLGA particles could also be gamma irradiated before adsorption and were shown to effectively boost antigen in nonhuman primates [87].

5.2.3 ANALYTICAL CHARACTERIZATION OF PEPTIDE/ PROTEIN-LOADED MICROSPHERES An area requiring additional efforts is analytical characterization of peptides and proteins encapsulated in PLGA microspheres. The high complexity of the therapeutic peptides and proteins requires not only physicochemical methodologies but also immunochemical and biological techniques for the characterization and quality control of these substances. In general, the analytical methods can be broadly viewed from the following study perspectives: methods meant for microsphere product quality checking, methods used for peptide/protein stability identification inside the microspheres, and methods called for peptide integrity detection following liberation from the microspheres immediately upon placement in release medium either in vitro or in vivo. Therefore, in most cases, a combination of several analytical methods is necessary for a comprehensive characterization of the peptide/protein under investigation and for appropriate quality control of the product concerning identity, purity, and potency. However, some of the analytical methods have potentially appealing applications to interplay among the mentioned perspectives. In Table 4, a selection of widely used analytical methods is given, showing which technology is applicable for the testing of identity, purity, and potency of peptides and proteins. In addition, peptide/protein integrity evaluation is indeed likely to be affected by artefacts during the sample preparation before analysis and during the analysis itself. Therefore, artefacts might prevent the scientist from critically ascribing detected protein denaturation to manufacturing conditions [88]. In order to measure the extent of peptide/protein degradation within the carriers and during release, the encapsulated molecule has to be removed from the polymeric matrix. Moreover, for avoiding artefacts such as underestimation of drug content, recovery methods need to be tried by an empirical trial-and-error approach as each peptide/protein is different one from the other. Recovery methods so far reported include extraction-based method with the help of potentially deleterious organic solvents, hydrolysis of the polymer matrix with alkaline medium, dissolution of polymer matrix in an organic solvent, recovery of suspended insoluble protein by filtration [89], total protein quantification after complete digestion of carriers followed by amino acid analysis [90, 91], electrophoretic extraction of the protein using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) [92, 93], and direct dissolution of both the polymer and the protein drug in a single liquid

406


TABLE 4 Analytical Methods for Characterization and Quality Control of Pharmaceutical Peptides and Proteins Indicated Usage/Checking Methods

Identity

Purity

Potency

+

+

−

− − −

+ + +

− − −

+ + − + + + + +

+ + + + + + + +

− − − − − − − −

− + −

+ + +

− − −

− − +

− − −

+ + −

− −

− −

+ +

Physicochemical Chromatography Reversed-phase high-performance liquid chromatography (HPLC, RP-1) Ion exchange Affinity Size exclusion chromatography (SEC) Spectroscopy Infrared spectroscopy Raman spectroscopy Fluorescence spectroscopy Ultraviolet/visible (UV/VIS) spectroscopy NMR spectroscopy Mass spectrometry Circular dichroism (CD) Matrix-assisted light desorption ionization–time-of-flight (MALDI-TOF) mass spectrometry Electrophoresis Capillary electrophoresis SDS–polyacrylamide gel electrophoresis (PAGE) Isoelectric focusing Immunochemical Radioimmunoassay (RIA) ELISA Western blot Biological In vivo assays In vitro (cell culture) assays Abbreviation: SDS: sodium do decyl sulfate.

phase containing water-miscible organic solvents such as acetonitrile or dimethylsulfoxide (DMSO) [94, 95]. Following successful recovery of peptide/protein molecule from the microspheres, a simple spectrophotometric method does not always allow discrimination between the monomeric protein form and its aggregates. However, HPLC might separate these species and thus provides more accurate qualitative data [96]. But HPLC cannot quantify exclusively the amount of active protein antigen, as is the case with ELISA techniques [97]. Nowadays, Fourier transform infrared (FTIR) spectroscopy has become a popular, noninvasive method, as it is able to characterize the secondary structure of entrapped proteins [26, 95, 98–101]. Only recently, the integrity of their primary structure was evaluated, thanks to a new matrix-assisted laser

IMMUNE SYSTEM INTERACTION WITH INJECTABLE MICROSPHERES

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desorption ionization–time-of-flight (MALDI-TOF) mass spectrometry method [94, 102]. The method was shown to require little sample material and only a simple dissolution of the carrier was needed prior to the analysis. The MALDI-TOF allowed elucidation of a new degradation pathway, that is, peptide acylation within PLGA carriers resulting from a chemical interaction between peptide and degraded polymer [102]. Moreover, the method was also useful for quantification, and it should be underlined that no interference from PLGA was detected during the measurements. For all the reasons cited above, mass spectrometry should be considered one of the most promising methods for protein analysis inside polymeric carriers including microspheres. Using erythropoietin as an example, an exploratory and elaborative discussion was made on the analytical techniques used for the characterization and quality control of pharmaceutical peptides and proteins [103]. A similar discussion was also done on the analytical techniques critical to (as a part of) the quality assurance after process changes of the production of a therapeutic antibody [104]. With an increasing level of sophistication in the design of new protein antigens and adjuvants (including polymer controlled-release systems), efforts both in the United States and the EU are underway to respond with more appropriate regulations [105–107]. For example, the Committee for Proprietary Medicinal Products (CPMP), the primary scientific body in EU regulatory matters, is currently updating its “notes for guidance,” which guide/direct industry and regulatory authorities on content and evaluation of marketing authorization applications for vaccines [105]. Early drafts of these updates include more rigorous guidelines for new nonaluminum-based adjuvants, including antigen stability requirements (see Sesardic and Dobbelaer [105] for a discussion). Similar discussions ongoing in the United States have attempted to standardize requirements of controlled-release parenterals [106, 107], including specifics regarding in vitro release assays and the need to account for 80% or more of the encapsulating agent during the release period.

5.2.4 IMMUNE SYSTEM INTERACTION WITH INJECTABLE MICROSPHERES Since microspheres are capable of forming a drug depot, the encapsulated peptide or protein is being slowly released over days or months at the injection site. Interestingly, the size of microspheres plays an important role in immune response. Microspheres with sizes smaller than 10 μm can be directly taken up macrophages (and dendritic cells) through a phagocytosis mechanism while sizes greater than 10 μm need to undergo biodegradation before phagocytosis can occur [108]. It was shown that within a few days of intramuscular injection PLGA microspheres less than 10 μm are completely engulfed in a thin layer of connective tissue and thus evidenced infiltration by macrophages as a consequence of wound-healing response to injected particles [109]. It is feasible that the influx of these macrophages may cause degradation of the encapsulated protein and available protein released in the vicinity of the microspheres. Furthermore, it has been suggested that these macrophages are capable of producing proteolytic enzymes [110], which may result in the release and circulation of altered, inactive, or immunogenic forms of the encapsulated peptide or protein.

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On the other hand, degradation, protein antigen release, location, and antigen presentation of microspheres larger than 10 μm are expected to be different from smaller ones. Larger microspheres can provide an extracellular depot for secondary immune responses by way of B-cell stimulation [111–113]. In both cases, upon administration of microspheres, a foreign-body response occurs resulting in an acute initial inflammation despite the excellent tissue compatibility and biodegradability properties of polymers such as PLGA. This initial inflammation is followed by the infiltration of small foreign-body giant cells and neutrophils [114]. These immune cells could consume the released peptide or protein and produce an immune response. However, if released protein is recognized as a self-protein (e.g., homologus), the probability of an immune response by these cells is reduced. It is therefore always essential to release the protein in its native conformation. The release of aggregated or denatured protein from the microspheres may, in fact, result in an unwanted immune response [115]. It should be added that systematic studies to explore the effects of tissue response on the bioavailability of incorporated peptide or protein drug have not appeared extensively in the literature, with a few exceptions as described below. Using a light microscopic technique, bumps containing residual amounts of microspheres were observed at the injection site two weeks after administration of TT-encapsulated PLGA microspheres to mice and guinea pigs [116]. These bump formations may be due to chronic reactions, long-term immunogenicity, and immunological priming of mice and guinea pigs against the injected polymeric microspheres. The immunogenicity of microsphere-encapsulated vaccines can be varied to some extent by changing the physicochemical properties of the microspheres, for example, size, surface properties, and release kinetics of the antigen from the microspheres [111]. An interesting review by Jiang et al. [117] details the various reports on the relationship between in vitro protein antigen stability and immunogenicity, modulation of cell-mediated immune responses, and different formulation approaches to achieve the appropriate immune response with microencapsulated vaccine antigens. There has been some debate, arising from some animal experiments, that the antigenicity does not directly correlate with immunogenicity. However, the stability of protein antigens is considered to play a significant role in the quality and magnitude of immune response for the controlled-release single-dose vaccines as degraded or nonantigenic proteins may not be able to provide a continuous boost for generation of protective levels of high-affinity antibodies.

5.2.5 EXCIPIENT INCLUSION: INJECTABLE PEPTIDE/ PROTEIN-LOADED MICROSPHERES Peptide and protein molecules are highly prone to degradation mechanisms that can be divided into two classes: physical and chemical [118]. Whereas chemical degradation leads to the loss of the protein’s primary structure through oxidation, deamidation, peptide bond hydrolysis, isomerization, disulfide exchange, and covalent aggregation, physical degradation refers to the changes in the higher order structure (secondary and above) mainly by noncovalent aggregation and precipitation. In particular, aggregates formation during the encapsulation process must be avoided because these aggregates always represent loss of therapeutic efficacy and

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increased immunogenicity which can endanger the patient’s health [119, 120]. The following few examples indicate the fragility of peptide and protein molecules due to physical or chemical degradation: Aggregation of insulin has been well characterized and depends on unfolding of the insulin molecules [121]; aggregation of lyophilized formulations of BSA, β-galactoglobulin, and glucose oxidase are attributed to disulfide interchange [118]; deamidation contributes to reduction in catalytic activity of lysozyme [122] and ribonuclease at high temperatures [123]; and peptide bond hydrolysis results in loss of activity of lysozyme when heated to 90–100°C [122]. A recent introduction to this list is formaldehyde-mediated aggregation pathway (FMAP) unique to formaldehyde-treated protein antigens such as TT [117, 124, 125]. The formulator of injectable microspheres for peptide and protein faces multiple challenges: (i) to maximize physical and chemical stability, (ii) to prolong biological half-life, (iii) to increase absorption, (iv) to decrease antigenicity, and (v) to minimize metabolism. Thus, it is quite obvious that the fabrication of peptide- and protein-loaded microspheres requires several kinds of excipients for effective stabilization or immobilization of encapsulated therapeutic molecules. Excipients of choice are included specifically for controlling protein degradation in microspheres due to (a) external and internal environmental changes, (b) manipulating the initial burst release, (c) preventing protein adsorption onto delivery devices, and (d) neutralizing the causative acidic microclimate formation due to the acids liberated by the biodegradable lactic/glycolic-based polymers. Therefore, it is generally best to find conditions to stabilize the protein before other aspects of the formulation, such as controlled-release characteristics, are optimized. Typically, the appropriate excipients for the protein under investigation are experimentally selected among various substances by screening. This tedious experimental screening is partly necessary due to the present inability to predict protein stability after addition of such excipients. Moreover, since individual entrapped peptides and proteins differ in terms of physicochemical properties and chemical/therapeutic function, each species is expected to demonstrate a different degree of sensitivity to stress and react differently to the same stabilization strategy. For example, a sugar, amino acid, or antacid excipient may be required to stabilize protein, each of which can increase water uptake in the polymer matrix leading to an increase in release rate. In the scenario in which controlled-release conditions are optimized before such a stabilizer has been identified, it is likely that upon addition of the new stabilizer the release kinetics may change enough to require reformulation. Certainly, there is a sharp contrast between encapsulating a highly water soluble protein [126] or a poorly soluble zinc–protein complex [37, 57]. Switching between these two cases would be expected to alter the requirements in the formulation necessary to attain the controlled-release function (e.g., low versus high polymer matrix permeability for the protein, respectively) because protein solubility in water may be important for any diffusion component of release. The principal stresses causing instability of encapsulated peptide/proteins in PLGA microspheres are elaborated in a book chapter [127] and in a jounal publication [128]. This subject was again reexamined in a review based on new findings since the previous book chapter by the same author [129]. An interesting review from the same research group was published on the biodegradable PLGA microparticles for injectable delivery of vaccine antigens [117], where they focused on mechanistic approaches to improve the stability of PLGA-encapsulated protein antigens.

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BIODEGRADABLE POLYMER-BASED MICROSPHERES Excipients used

Solubilization

Moisture-induced instability prevention

FIGURE 2 problems.

Preservation

Stabilization

Microclimate pH-induced instability prevention

Flow chart of excipients used to prevent/minimize protein instability

Another review by Bilati et al. [130] also envisioned the strategic approaches for overcoming peptide and protein instability within biodegradable nano- and microparticles. The reader is also referred to related publications edited by Sanders and Hendren [131] and Senior and Radomsky [132] for information on excipients used in injectable peptide/protein-loaded formulations including microspheres. This section will not cover all excipients used in parenteral protein formulations because the aforementioned publications already do so. Rather this section highlights examples of synergestic and antagonistic interactions that have been reported mainly between the excipients and the peptide/protein drugs, especially before microsphere preparation, followed by a brief discussion of a major instability problem of proteins/peptides inside the microspheres. The published research paper is being organized according to major functions of parenteral excipients, namely, solubilization, stabilization, and preservation (see Figure 2) [133]. 5.2.5.1

Solubility- and Stability-Increasing Excipients

The traditional approach is to solubilize directly the peptide/protein in organic solvents. This can be achieved by different means. Cleland and Jones [61] assumed that native protein conformation could be maintained by precipitating the protein at its pI. The molecule is then free of charge and can be readily solubilized in organic solvents. Conversely, an alternative concept is based on the freeze drying of the protein at a pH away from its pI value before formulating it. It was thought that this strategy could increase protein solubility and stability in various polar and water-miscible organic solvents such as DMSO [134, 135]. It should be noted that a preformulation procedure consisting of using spray freeze drying with a suitable excipient was able to stabilize BSA before encapsulation by a nonaqueous method [35]. Using the dissolution approach, lysozyme was successfully formulated but incomplete lysozyme release from microspheres was observed and ascribed to aggregation [136]. Protein solubility can also be increased via an ion-pairing mechanism. The protein is modified by adding an oppositely charged surfactant that binds to the protein, so as to obtain a neutral hydrophobic entity and thus reduce direct contact between the protein and the organic solvent. Positively charged proteins and negatively charged surfactants should be employed, since cationic surfactants might have toxic side effects. This technique was shown to improve lysozyme conformational stability after a hydrophobic complex between lysozyme and oleic acid [137, 138]. A new interesting concept is to encapsulate an aggregated protein in a reversibly dissociable form in order to avoid the formation of irreversible aggregates during processing and to promote the sustained release of the native monomeric form. Growth hormone was successfully formulated with this approach [139].


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Cyclodextrins (CD) have emerged as very effective additive compounds for solubilizing hydrophobic drugs. In the parenteral dosage form area, modified cyclodextrins such as hydroxylpropyl-β-cyclodextrin and sulfobutylether-β-cyclodextrin have been reported to solubilize and stabilize many injectable drugs, including dexamethasone, estradiol, interleukin-2, and other proteins and peptides [140] without apparent compatibility problems [141]. In addition, CD-containing formulations (either 0.1 M sulfobutylether-β-cyclodextrin or 0.1 M hydroxylpropyl-βcyclodextrin) were shown to cause less damage to venous epithelial cells at the site of injection compared with formulations containing organic cosolvents [142]. When CD were coentrapped in the internal aqueous phase, erythropoietin (EPO) covalent aggregate formation was significantly reduced during microsphere preparation by the double-emulsion method [72] and lysozyme stability was improved [88]. Although the precise mechanism is unclear, interactions between amino acids and the hydrophobic inner cavity of CD may play a role [143]. However, CD showed no protecting effect on insulinlike growth factor-1 (IGF-1) [144] and hepatitis B core antigen (HBcAg) [145] and even promoted the loss of superoxide dismutase activity at high CD concentrations [146]. By contrast, carboxymethylcellulose (CMC) did not efficiently stabilize HBcAg and GH against dichloromethane-induced denaturation [61, 145]. Various types (α, β, and γ) of CD were examined for encapsulating TT in PLGA microspheres [147], with γ-hydroxypropyl-cyclodextrin effectively increasing TT encapsulation. However, CD also showed low efficiency in retaining spray-dried TT antigenicity, probably due to antigenic epitopes being buried inside the molecular CD core [147]. Surfactants have the ability to lower surface tension of protein solutions and prevent protein adsorption and/or aggregation at hydrophobic surfaces such as PLGA. Among them, nonionic surfactants are generally preferred as ionic surfactants might bind to groups in proteins and cause denaturation. Tween 20 was shown to greatly reduce the rate of formation of insoluble aggregates of recombinant human factor XIII caused by both freeze thawing and agitation stresses [148]. Maximum protection occurs at concentrations close to the critical micelle concentration of Tween 20, independent of initial protein concentration. In another report, Tween 20 at a 1% (w/v) concentration caused precipitation of a relatively hydrophobic protein (Humicola lanuginosa lipase) by inducing nonnative aggregates [149]. Similarly, nonionic surfactants such as Tween 20 or 80 were not good stabilizers for lysozyme and rhGH against the unfolding effect of the water–dichloromethane interface. It has been assumed that both the hydrophilic (PEG chains) and hydrophobic (fatty acid chain) parts of the polysorbate molecules were preferentially partitioned in the dichloromethane phase, leading to low protection efficacy [61, 98]. Exchange of Tween 20 for a less hydrophobic surfactant, PEG 3350, provided almost complete rhGH recovery irrespective of protein concentration. However, an opposing trend was seen with EPO encapsulation in PLGA microspheres [72]. Encapsulated protein aggregates increased (∼15%) with different PEG types codissolved in the w1 phase. Conversely, when three nonionic surfactants of different hydrophilic–lipophilic balances (HLBs) were coencapsulated with insulin by the w/o/w double-emulsion method, only Tween 20 was able to improve insulin stability within particles and to limit formation of high-molecular-weight products during the sustained-release period [150].

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Tween 80 is well known to protect proteins against surface-induced denaturation [151]. Tween 80 was demonstrated to reduce hemoglobin aggregation in solution by preventing the protein from reaching the air–liquid interface or the liquid–surface interfaces [152]. Polyoxyethylene surfactants such as Tween 80 can form peroxide impurities after long-term storage. Knepp et al. [153] concluded that Tween 80 and other nonionic polyether surfactants undergo oxidation during bulk material storage and subsequent use and the resultant alkyl hydroperoxides formed can contribute to the degradation of proteins. In such formulations, they further reported that thiols such as cysteine, glutathione, and thioglycerol were most effective in stabilizing protein formulations containing peroxide-forming nonionic surfactants. The Pluronics, also known as poloxamers (e.g., poloxamer 188, British Pharmacopoeia standard) are a well-studied series of commercially available, nonionic, triblock copolymers with a central block composed of the relatively hydrophobic poly(propylene oxide) flanked on both sides by blocks of the relatively hydrophilic poly(ethylene oxide) [154, 155]. The Pluronics possess an impressive safety profile and are approved selectively by the Food and Drug Administration (FDA) for pharmaceutical and medical applications, including parenteral administration [156]. The strong safety profile, commercial availability, ease of preparation, and wellstudied physical properties make the Pluronics particularly appealing for drug delivery purposes. They have been used in several patented protein formulations as stabilizers and sustained-release injectables in development as solubilizing and stabilizing agents [157]. However, poloxamers, like Tweens, can form peroxide impurities over time. Poloxamer 188 was successfully used when mixed with PLGA for prolonged release of active interferon-α (INF-α) [158], but such a formulation had no effect on BSA secondary structure compared to PLGA alone [35]. Poloxamer 188 was not effective in preventing nerve growth factor (NGF) aggregation during in vitro release from microspheres generated by spray drying [159]. Complex interactions between poloxamer, BSA, and PLGA were believed to have influenced BSA microencapsulation [160]. The gelling property of the amphiphilic poloxamer 407 was successfully employed for urease encapsulation. The protein was likely protected during the microsphere preparation by a hydrated gelled structure due to the hydrophilic polyoxyethylene chains [161]. EPO aggregates in PLGA microspheres decreased when poloxamer 407 was incorporated at a level of 10% (w/w) [72]. Interleukin-1α (IL-1α) was protected by phosphatidylcholine from damage during the double-emulsion process but underwent inactivation during microsphere incubation [162]. Sodium dodecyl sulfate significantly reduced insulin aggregation at the dichloromethane–water interface, whereas dodecyl maltoside did not, this surfactant being more efficient at air–water or solid–water interfaces [163]. It should be mentioned that surfactants are used along with sugars, proteins, and polymers effectively for solubilization and stabilization purposes of peptide/protein in microspheres. Bilati et al. [130] give an overview on various proteins and polymers that act as stabilizing excipients during the development of peptide/protein-loaded microspheres. 5.2.5.2

Preservation-Imparting Excipients

Prevention/Minimization of Moisture-Induced Instability Moisture- and microclimate acid pH–induced instability (typically the aggregation) of the peptide/protein


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encapsulated in PLGA microspheres has been monitored. Even several formulation strategies to inhibit these instability problems are being actively investigated. If the protein is expected to exist in the solid state within the PLGA polymer, the protein is remarkably prone to aggregation when formulated under conditions that allow moisture- and microclimate acid pH–induced instability. The two covalent aggregation mechanisms commonly described during exposure of the solid protein to moisture are the disulfide interchange/exchange [164] and the FMAP, which is operative for protein antigens that have been detoxified with formaldehyde exposure [124]. In the former pathway, the reaction is typically initiated by a thiolate ion on the protein or free thiolate ions that accompany β elimination of an intact disulfide [165]. Decreasing the concentration of the reactive species (e.g., lowering pH to favor the nonionized thiol, covalently blocking the thiol group, or oxidizing free thiols as they appear with divalent copper ion) has been shown to block this mechanism [165]. To inhibit the FMAP, strongly formaldehyde-interacting amino acids such as histidine and lysine [166] have been colyophilized with the formalinized protein antigen. On exposure to moisture, the amino acids appear to bind with the reactive Schiff base or equivalent electrophile [167] in the protein before a neighboring protein nucleophile can react to form an intermolecular cross-link [124]. Sorbitol has also been identified to inhibit the FMAP of TT at the maximal aggregating water content of the antigen, about 30 g H2O/g protein [168], although whether this is a humectant effect [169] or a possible covalent reaction with the highly reactive electrophile in the antigen has not been determined. Several techniques have been developed to successfully bypass the destabilizing stress either by altering the role of water in the solid or immobilizing the protein or, alternatively, by directly inhibiting the aggregation. Clearly, one of the most significant findings in the field of peptide/protein stability in polymers is the success of the immobilization strategy of Zn2+ precipitation, as performed with human growth hormone [37, 57, 170, 171]. The 2 : 1 mole ratio Zn–protein complex, which immobilizes the rhGH as a solid precipitate in a near-native state [99], has been shown to confer superior stability on the protein encapsulated in PLGA for a one-month release incubation. Since then, other proteins such as INF-α [172] and NGF [173] were also stabilized in PLGA microspheres by this approach. Another interesting approach originating in the patent literature is the precipitation of erythropoietin with salting-in salt, ammonium sulfate [174], which is a technique commonly used in protein processing. Other methods to alter the role of water in the reaction involve the addition of agents that alter the amount of water sorbed in the polymer and/or the activity of the water present. For example, both water-soluble salts (NaCl) and antacid excipients (Mg(OH)2) are known to dramatically increase the amount of water sorbed in PLGAs, with the former due to osmosis and the latter to a complex effect of neutralizing acidic degradation products and end groups of the polymer (which also involves an osmotic component) [126]. In contrast, for a given moisture content, humectants such as sorbitol, which dissolve in water bound to the protein, reduce the available free water necessary to mobilize the protein or perform other roles in deleterious reactions [169]. The alternative to bypassing the deleterious role of moisture is to inhibit the aggregation mechanism directly. Several ways to accomplish this have been reported, particularly in the solid state and in the absence of the polymer. Well-referenced and useful book chapters by Johnson [175] and Carpenter and Chang [176] are

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available to thoroughly focus on the importance of making a lyophilized powder before loading the peptide or protein into an injectable microspheres. It has been stated that, in comparison to protein solution, the protein in the solid state would be less susceptible to shear forces that occur during an emulsification procedure or denaturation at oil–water interfaces. However, special precautions should be taken during freeze drying because the drying process itself will expose the protein to destabilizing stresses. To circumvent this problem, cryo- and lyoprotectants and bulking agents are usually included along with a peptide or protein solution while it undergoes the drying stages of the lyophilization process. Cryo- and Lyoprotectants and Bulking Agents Various mechanisms are proposed to explain why excipients serve as cryo- or lyoprotectants. The most widely accepted mechanism to explain the action of cryoprotection is the preferential exclusion mechanism [177]. Excipients that will stabilize proteins against the effects of freezing do so by not associating with the surface of the protein. Such excipients actually increase the surface tension of water and induce preferential hydration of the protein. Examples of solutes that serve as cryoprotectants by this mechanism include amino acids, polyols, sugars, and polyethylene glycol. For lyoprotection, that is, stabilization of proteins during the drying stages of freeze drying and during storage in the dry state, two mechanisms are generally accepted. One is the water substitute hypothesis [178] and the other is the vitrification hypothesis [179]. Both are legitimate theories, but both also have exceptions; that is, neither fully explain the stabilization of proteins by excipients during dehydration and dry storage [180]. The water substitute hypothesis states that a good stabilizer is one that hydrogen bonds to the protein just as water would do where it presents and, therefore, serves as a water substitute. Sugars are good water substitutes. (It may at first appear contradictory that sugars can serve both as cryoprotectants because of being excluded from the surface of the protein and as lyoprotectants that hydrogen bond to the protein. However, keep in mind that the excluded solute concept involves a frozen aqueous system whereas the water substitute concept occurs in a dry system.) This is why many freeze-dried protein formulations contain sucrose or trehalose. Nevertheless, during a w/o/w procedure to prepare peptide/protein-loaded PLGA microspheres, sugars are often added to the inner aqueous phase. Trehalose was shown to partially improve the BSA secondarystructure protection within PLGA microspheres and to facilitate BSA monomer release [26]. Trehalose and mannitol had a significant effect on the recovery of soluble nonaggregated interferon-γ (INF-γ) and rhGH after emulsification and ultrasonication [61], whereas no or very little protecting effect on IGF-1 against these stress factors was observed [144]. No effect of trehalose, mannitol, and sucrose was observed against o/w interface-induced degradation of lysozyme, whereas lactose and lactulose significantly improved its structural stability and activity, mostly if these additives were also added to the second aqueous phase [88, 100]. Lysozyme and trypsin activity was not improved by addition of sucrose, which was unable to protect them from an emulsion-induced denaturation and from sonication [98, 181]. Mannitol and sucrose dissolved together in the inner aqueous phase had slight effect on NGF activity [182] and neither mannitol nor lactose improved HBcAg immunogenicity during dichloromethane/water emulsification [145]. Surprisingly, sucrose and trehalose even decreased urease bioactivity, showing the opposite effect to that


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expected [161]. Coencapsulation of maltose reduced α-chymotrypsin aggregation [183]. With respect to microspheres generated by spray drying, trehalose was effective in retaining TT antigenicity [147] and in preventing BSA secondary-structure degradation [35]. Trehalose protected efficiently NGF during the processing but did not prevent its aggregation during in vitro release [159]. The vitrification hypothesis states that excipients that remain amorphous (glass formers) form a glassy matrix with the protein with the matrix serving as a stabilizer. Acceptance of this hypothesis requires formulators to determine glass transition temperatures of formulations to be freeze dried and to develop freeze-dry cycles that maintain drying temperatures below the glass transition temperature. Reports are available to indicate that excipient stabilizers, which are capable of undergoing crystallization during storage, caused degradation (typically aggregation and loss of potency) of the protein [176, 184, 185]. Freeze-dried formulations typically contain one or more of the following bulking agents: mannitol, lactose, sucrose, trehalose, dextran 40, and povidone. These excipients may also serve as cryo- and/or lyoprotectants in protein formulations. Fakes et al. [186] studied these bulking agents for moisture sorption behavior before and after freeze drying. Moisture uptake certainly can affect drug stability in the freezedried state, particularly with peptides and proteins. When selecting a bulking agent, these properties, particularly the tendency for moisture uptake, must be considered by the formulation scientist in developing an optimally stable freeze-dried formulation. Several excipients can serve as stabilizers for proteins that are unstable during the drying phases of freeze drying and/or during long-term storage in the dry state. Typically, additives that will crystallize during lyophilization (e.g., mannitol) or will remain amorphous but unable to hydrogen bond to the dried protein (e.g., dextran) are not effective lyoprotectants for proteins. Excipients that will crystallize during freeze drying will also be relatively ineffective, as was shown with sucrose in H. lanuginosa lipase formulations [149]. However, these authors also reported that sucrose crystallization could be inhibited by decreasing the mass ratio of sucrose to protein and by minimizing the moisture content that serves to decrease the glass transition temperature during storage. The reverse can also be true for certain small molecules. For example, excipients (mannitol or sodium bicarbonate) that promoted the crystallization of cyclophosphamide during freeze drying stabilized the final product whereas excipients that did not allow crystallization (e.g., lactose) destabilized the final product [187]. Costantino et al. [188] studied the effects of a variety of parenteral excipients on stabilizing human growth hormone in the lyophilized state. Mannitol, sorbitol, methyl a-d-mannopyranoside, lactose, trehalose, and cellobiose all provided significant protection of the protein against aggregation, particularly at levels (131 : 1 excipient-to-protein molar ratio) to potentially satisfy water binding sites on the protein in the dried state. At higher excipient-to-protein ratios, mannitol and sorbitol crystallized and were not as effective in stabilizing the protein compared with low levels in which they remained in the amorphous, proteincontaining phase. Reducing sugars may not be as effective as other bulking agents, cryoprotectants, or lyoprotectants because they may potentially react with proteins via the Maillard reaction. For example, glucose will form covalent adducts with side-chain amino acids lysine and arginine of human relaxin [189]. In addition, a significant amount of serine cleavage from the C terminal of the B chain of relaxin was formed when

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glucose was used as the excipient. These reactions did not occur if mannitol and trehalose replaced glucose in the lyophilized formulation. Lactose will react with primary amines in the well-known Maillard-type condensation reaction to form brown-colored degradation products [190]. Thus, lactose is known to be incompatible with amine-containing compounds such as aminophylline, amphetamines, and amino acids/peptides. This reaction occurs more readily with amorphous lactose than crystalline lactose. Hydrophilic additives such as glucose are known to increase the porosity of microspheres, causing an increase in permeability to mass transport and a higher burst. However, a significant reduction in initial burst release of a highly watersoluble model peptide, octreotide acetate, from poly(d,l-lactide-co-glycolide) microspheres by the coencapsulation of a small amount of glucose (e.g., 0.2% w/w) was reported [191]. Using the double emulsion–solvent evaporation method of encapsulation, the effect of glucose on initial burst in an acetate buffer pH 4 was found to depend on polymer concentration, discontinuous phase/continuous phase ratio, and glucose content. Extensive characterization studies were performed on two microsphere batches, ±0.2% glucose, to elucidate the mechanism of this effect. However, no significant difference was observed with respect to specific surface area, porosity, internal and external morphology, and drug distribution. Continuous monitoring of the first 24-h release of octreotide acetate from these two batches disclosed that, even though their starting release rates were close, the microspheres plus glucose exhibited a much lower release rate between 0.2 and 24 h compared to those without glucose. The microspheres plus glucose showed a denser periphery and a reduced water uptake at the end of the 24-h release, indicating decreased permeability. However, this effect at times was offset as glucose content was further increased to 1%, causing an increase in surface area and porosity. In summary, these authors concluded that the effects of glucose on initial burst are determined by two factors: (1) increased initial burst due to increased osmotic pressure during encapsulation and drug release and (2) decreased initial burst due to decreased permeability of microspheres [191]. Mannitol is probably the most widely used bulking agent in lyophilized formulations because of its many positive properties with respect to crystallinity, high eutectic temperature, and matrix properties. However, some lots of mannitol can contain reducing sugar impurities that were implicated in the oxidative degradation of a peptide in a lyophilized formulation [192]. Mannitol at or above certain concentrations and volumes in glass vials is well known to cause vial breakage because of the unique crystallization properties of mannitol-ice during the primary drying states of freeze drying [193, 194]. Other Freeze-Dry Excipients High-molecular-weight carbohydrates such as dextran have higher glass transition temperatures than peptides/proteins. Therefore, when mixed with proteins, the overall glass transition temperature presumably can be increased with resultant increases in protein storage stability. Typically, carbohydrates (sucrose, trehalose, or dextran) alone do not result in appreciable increases in the storage stability of proteins. However, combinations of disaccharide and polymeric carbohydrates do tend to improve protein storage stability [195]. However, singular carbohydrates (sucrose or trehalose at 60 mM) were also just as effective in stabilizing a model recombinant humanized monoclonal antibody as combinations of sucrose and mannitol or trehalose and mannitol. Interestingly, with this


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model monoclonal antibody, mannitol alone at 60 mM provided less protection during storage than sucrose or trehalose alone. A specific sugar/protein molar ratio was sufficient to provide storage stability for this particular monoclonal antibody [196, 197]. Low-molecular-weight additives such as osmolytes (N,N-dimethylglycine, trehalose, and sucrose) or salts (sodium chloride, sodium phosphate, ammonium sulfate, and sodium citrate) were found to be highly effective in stabilizing keratinocyte growth factor, both against thermal denaturation and enhancing long-term storage stability [198]. Nevertheless, the stabilizing properties of osmolytes appear to be balanced between their binding to (deteriorating effect) and exclusion from (stabilizing effect) the peptide/protein surface. As binding or exclusion predominantly results from hydrophobic interactions, hydrogen bonding, and electrostatic interactions, the sum of the various interaction parameters are dissimilar for different proteins. Therefore, it becomes crucial to examine the individual nature of the additive toward each individual protein and to assess whether it will offer a stabilizing or destabilizing effect [199, 200]. Polyvinyl pyrrolidone (PVP) and glycine were found to stabilize lyophilized sodium prasterone sulfate whereas dextran 40 or mannitol did not [201]. PVP and glycine stabilized the pH of the reconstituted solution by neutralizing the acidic degradation product, sodium bisulfate, formed by the hydrolysis of prasterone sulfate. Dextran 40 or mannitol was ineffective because of no buffer capacity. Buffering agents, such as phosphate–citrate buffer and some neutral and basic amino acids (l-arginine, l-lysine, and l-histidine), also stabilized prasterone sulfate. l-Cysteine is an example of an amino acid that did not stabilize the drug, presumably because of its weak buffer capacity. Although the efficiency of proteinic additives for protein stabilization has been clearly demonstrated in several occasions even during encapsulation processes [31, 72, 98, 144], their use in pharmaceuticals is at present not desirable from a strictly regulatory point of view. Additionally, such agents might contribute to complicate all subsequent protein characterization within the formulation. Among these additives, albumins and gelatins are those mainly used for protection purposes. The protective effect of albumins against protein unfolding and aggregation has been extensively documented and is likely due to their surface-active properties (see Bilati et al. [130] for details). Prevention/Minimization of Microclimate pH-Induced Instability Evidence for acidification within degrading microspheres is investigated and local pH values between 1.5 and 4.7 are being reported [202–204]. Methods to measure microclimate pH in PLGA microspheres include (i) ensemble average measurements using electron paramagnetic resonance (EPR) [203, 204], nuclear magnetic resonance (NMR) [205], and potentiometry and (ii) direct visualization techniques such as confocal imaging of pH-sensitive dyes [206, 207]. In the EPR method, the constant of hyperfine splitting, 2aN, was used to determine an average pH inside PLGA microspheres. Because the experiments relied on the mobility of spin-labeled protein, with an increase of the microviscosity in the later hours of the experiments, the spectra of EPR was changed and the signal-to-noise ratio decreased to prevent the measurement of pH throughout the release period [203]. The potentiometric measurements can give rapid values of pH for thin polymer films, and the pH of the thin water film between the electrode and polymer mimics the microclimate pH of aqueous pores inside the polymer-based drug delivery system. However, it is difficult to mimic

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microclimate pH of a small-scale system, such as microspheres or nanospheres, which may have unique microstructures, excipient/drug distributions, and transport characteristics. Overall, the ensemble average measurements described above could give a general picture of microclimate pH at the macroscopic level. However, the microscopic level of the detection can only be achieved through direct visualization techniques, such as microscopic imaging. Shenderova et al. [207] first developed the confocal microscope imaging method to relate the microclimate pH with the fluorescent intensity. Because of the difficulty of controlling and predicting the fluorescein concentration in the aqueous pore inside the microsphere, the method was only semiquantitative. Fu et al. [206] improved the confocal microscopic imaging method by coencapsulating two dextran fluorescent dye (NERF and SNARF-1) conjugates inside microspheres and related the ratio of the two dye images with microclimate pH in order to eliminate the poorly controlled effects of dye concentration and pore distribution. However, both of the dyes emit in the green range (535 nm for NERF and 580 nm of SNARF), giving rise to poor resolution without a narrow-bandwidth detector. Because of the high noise-to-signal ratio from the ratio images, the prediction of pH is also expected to be semiquantitative. In order to overcome the aforementioned drawbacks in microclimate pH measurement, a new quantitative ratiometric method based on laser scanning confocal microscopic imaging was developed to create a pixel-by-pixel neutral range microclimate pH map inside PLGA microspheres [208]. This method was then applied to both acid-neutralized and nonneutralized PLGA microspheres during extended incubation in physiological buffer. In another study, the PLGA water-soluble acid distribution has been measured with prederivatization HPLC [209]. Ongoing acidification of the microsphere interior was shown to induce deamidation and covalent dimerization of nonreleased insulin [202]. Despite the evidence of acidification mentioned above, there is controversy on this subject. It has been pointed out that the sampling scheme has a significant impact on the degree of acidification; frequent replenishment of the release medium or the use of a dialysis bag can effectively prevent the acidification of the medium with subsequent reduced protein degradation [93, 210]. It is unsure, however, whether this also reflects the situation in vivo, in which the PLGA microspheres are often surrounded by a fibrous capsule that may reduce efflux of acidic degradation products from the PLGA matrix [93]. On the other hand, studies on rhGH-loaded PLGA microspheres showed a reasonable in vitro–in vivo correlation (IVIVC) only when a strong high-capacity buffer [200 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4] was used, which effectively minimized the pH drop [211]. As indicated by the prevention of acid-induced physical aggregation of BSA in an abstract [212], three principal ways have been identified thus far to avoid the formation of highly acidic microclimate regions in the PLGAs during protein release: (i)

Increasing the permeability of the polymer to facilitate escape of the watersoluble hydrolytic products of the PLGA polyester [125] (ii) Decreasing the degradation rate of the polyester [213] (iii) Coencapsulating additives to neutralize the weak acids formed by PLGA hydrolysis [126]

PEPTIDE/PROTEIN ENCAPSULATED INTO BIODEGRADABLE MICROSPHERES

419

In addition, two more ways that are likely to favor a lowering of microclimate pH are elevated initial acid content in the polymer [214] and low-frequency release media exchange [206]. The concept of controlling polymer permeability is difficult because attempts to increase permeability can spoil the controlled-release function of the polymer and cause the encapsulated protein to be released too rapidly. For example, Jiang and Schwendeman [213] increased the permeability of slow-degrading PLA (molecular weight (MW) 145 kDa) by blending in PEG (MW 10 or 30 kDa) at 0, 10, 20, and 30%. Insoluble BSA aggregation in the PLA microspheres containing 4.5–5% w/w BSA was found in 0 and 10% PEG after a one-month incubation, but not in those preparations containing 20 or 30% PEG. The structural integrity of BSA was also intact in the stabilized formulations. However, between 10 and 30% PEG, the release rate of BSA increased rapidly and by 30% PEG, 60% of the protein encapsulated was released in only three days [213]. In contrast, an abstract [212] implied that 5% BSA encapsulated in a more permeable PLA (MW 77 kDa), the BSA formed 2 g/day) should be evaluated for its safety [6]. Organoleptic properties such as color, taste, and odor are assessed qualitatively to set bulk drug specifications. If the drug has an unacceptable taste or odor, the chemistry group is advised to make a suitable salt form of the drug.

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6.2.2.1

ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS

Crystallinity and Polymorphism

The majority of the drugs exist in crystalline form and are characterized by their crystal habit and crystal lattice. The crystal habit describes the external morphology of the crystal, including shape and size, while the crystal lattice describes the internal arrangement of molecules in the crystal (Figures 1a and b). Drug molecules arrange in more than one way in a crystal, and this difference in the internal arrangement of crystals is known as polymorphism. The polymorphs have the same elemental composition but differ in their physical, chemical, thermodynamic, stability, and spectroscopic properties. A crystal lattice represents the space in which molecules arrange in different ways. Organic molecules arrange in one or more of the seven possible crystal systems: triclinic, monoclinic, orthorhombic, tetragonal, rhombohedral, hexagonal, and cubic [7]. Each crystal system is characterized by its threedimensional geometry and angles between the different crystal faces. The crystal lattice geometry is obtained using single-crystal X-ray diffractometry (XRD) and the details can be found elsewhere [7]. The difference in the crystal lattice of a drug arises as a result of the difference in packing of the molecules if the molecules are conformationally rigid (e.g., chlordiazepoxide) or due to the differences in conformation for flexible drug molecules (e.g., piroxicam). Although polymorphs differ in their internal crystal lattice, it may not be necessarily reflected in their external crystal habit (Figure 1b). In other words, a drug can exist in different crystal habits without any change in the internal crystal lattice (isomorphs). Crystal habit is mainly dependent on crystal growth conditions [8]. For example, Figure 1a shows two different crystal habits for a given crystal lattice. A prismatic crystal habit will result if the growth is equal in all directions, while plates are formed if the growth is slow in one direction. Alternatively, needle-shaped crystals (acicular) are formed when the growth is slow in two directions. Thus, the crystal habits can

(a)

(b)

(c)

FIGURE 1 Schematic of crystal habits, polymorphs and amorphous drug forms. (a) Two crystal habits are shown. The internal crystal lattice is the same while the external morphology is different. (b) In a crystal the molecules are arranged in a regular fashion. However, the arrangement may vary depending on how the molecules orient themselves in the internal crystal lattice. The internal crystal lattice is different in all the three polymorphic forms. The polymorphs may or may not differ in their external morphology. (c) Random arrangement of molecules in amorphous form.

PHYSICAL/BULK CHARACTERISTICS

937

be altered without any change in the internal crystal lattice by varying the crystallization conditions. The polarity of the crystallizing solvent mainly influences the crystal habit by preferentially adsorbing to one surface of the crystal face. Similarly, surfactants or additives are added to the crystallization medium to prevent or promote the growth of a specific crystal habit [8]. Crystal habits mainly differ in physicomechanical properties such as packing, flow property, compressibility, and tablettability. Acetaminophen crystallizes as polyhedral crystals when crystallized from water and as plates when crystallized from ethanol–water (Figure 2a). Both these crystal habits are isomorphic [9], that is, have the same internal crystal arrangement, since their melting points and heats of fusion were similar (melting point 178 °C and ΔHf = 177 kcal/mol). The polyhedral crystals have better flow and

(a) 4.0 3.5

ln [1/(1–D)]

3.0 2.5 2.0 1.5 1.0 0.5

Polyhedral crystals Thin platelike erystals

0.0 0 10 20 30 40 50 60 70 80 Compression pressure (MPa) (b)

FIGURE 2 Difference in crystal habit of acetaminophen and resultant difference in compressibility (a) Acetaminophen crystallizes as either platy crystals or polyhedral crystals depending on the solvent of crystallization. Both crystal habits have the same internal crystal lattice since they showed the same melting point. (b) Difference in compression behavior of two crystal habits. The x axis represents the compression pressure while the y axis represents the densification of the drug sample on compression. This plot is known as Heckel plot. The polyhedral crystal habit shows a higher densification implying better compressibility than plate like crystals. [From Garekani, H. A., Ford, J. L., Rubinstein, M. H., and Rajabi-Sahboomi, A. R., International Journal of Pharmaceutics, 187, 77–89, 1999. Reproduced with permission from Elsevier.)

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compression properties than platy crystals, which were brittle and fragmented during tableting (Figure 2b). Crystal habits are characterized using optical and electron microscopy, but their internal crystal lattice should be confirmed using DSC, XRD, and spectroscopic techniques. Polymorphs are generated by crystallizing the drug from various solvents. The solvents are usually those that are used in the synthesis and purification of the bulk drug but may also include solvents used in drug formulations [10]. By convention, polymorphs are named based on their order of discovery, such as forms I, II or A, B or α, β. In general form I is considered the most stable and least soluble form, while form II is considered the more soluble and least stable form. The least stable and more soluble polymorphic form is usually called the metastable form. They are not “unstable” but “metastable,” because the least stable form can remain stable provided the conditions are controlled to prevent its conversion to the more stable polymorphic form. Polymorphs are characterized by their solubility and stability differences with respect to temperature (Figure 3). Thermodynamically, polymorphs are classified as enantiotropic or monotropic depending on their thermal reversibility from one form to another [11]. Enantiotropic polymorphs are reversible polymorphs, where one form (form I) is more stable at higher temperature, while the other form (form II) is stable at lower temperature. They are characterized by a transition point (Ts) below the melting points of both forms (Figure 3a). The transition point represents the temperature at which one form converts to another. In the temperature–solubility curve, this is represented by the intersection of the solubility curves of both forms; that is, at the transition temperature, both polymorphs have the same solubility. As shown in Figure 3a, form II can convert to form I at a temperature above Ts, while form I can convert to form II at a temperature below Ts. On the other hand, monotropic polymorphs are not reversible but can only convert from the metastable form to the stable form. Here, Ts is higher than the melting point of both forms (Figure 3c). Both forms are stable in the entire temperature range below Ts. The different polymorphs are generated based on their solubility differences in a given solvent. According to Ostwald’s rule [12], the least stable or highly energetic form (form II, or metastable) will precipitate out first from a supersaturated solution followed by the stable or less energetic form (form I). Supersaturation is achieved by antisolvent addition or by altering the temperature. So, if the initial precipitate is separated rapidly, it would have predominantly the metastable form. Alternatively, the stable form can be melted and rapidly cooled to crystallize the metastable form. A stable or metastable polymorphic form is also used as a “seed” to preferentially grow and isolate the desired form during drug crystallization [13]. Several rules have been proposed to differentiate enantiotropic and monotropic polymorphs [11, 13]. A simple way to differentiate enantiotropic and monotropic polymorphs is the use of the heat–cool cycle in DSC [11]. As shown in Figure 3b, the enantiotropic polymorph is characterized by the appearance of solid–solid endothermic transition of form II to I followed by melting of form I. On cooling the melt of form I followed by reheating, the same thermogram is regenerated, proving the reversibility of the polymorphs. In monotropic polymorphs (Figure 3d), the thermogram is characterized by melting of metastable (form II) and recrystallization to form I followed by melting of form I. On cooling and reheating the sample, the transition and recrystallization peaks are not seen, indicating the irre-


939

Tm,I Solubility

Solubility

Tm,II I II

Tm,II II

Tm,I

I Ts

Ts

Temperature (°C) (a)

Temperature (°C) (c)

Endotherm

Heat flow

Cool cycle

Tm,I

Ts

First heat cycle

Heat flow

Endotherm Second heat cycle

Second heat cycle Cool cycle Tcrys Tm,II

Tm,I

Temperature (°C)

Temperature (°C)

(b)

(d)

First heat cycle

FIGURE 3 Difference between enantiotropic and monotropic polymorphs. (a) Solubility of enantiotropic polymorphs as function of temperature. The dotted line indicates the melting curve. Form I is less soluble below the transition temperature (Ts), while form II is more soluble above Ts. Form I has a higher melting point (Tm,I) than form II (Tm,II). Below Ts, form I is converted to form II and above Ts form II coverts to form I. (b) Thermogram generated from heat–cool–heat cycle in DSC. In the first heating cycle two endotherms are seen corresponding to conversion of form II to form I and melting of form I, respectively. On cooling both events show up as exotherms and on second heating cycle both endotherms reappear, indicating thermal reversibility of enantiotropic polymorphic pairs. (c). Solubility of monotropic pairs as a function of temperature. The Ts is above melting point of both forms. Forms I and II are stable in entire temperature range and their corresponding melting points are shown. (d) On heating in DSC, form II melts (Tm,II) followed by recrystallization (Tcrys) and subsequent melting of form I (Tm,I). In cooling cycle only melting of form I is seen as an exotherm and on reheating only one endotherm corresponding to form I is seen. This is typical of monotropic polymorphs which converts from form II to stable form I and not vice versa.

versible nature of these polymorphs. The heating rate in DSC is critical for characterizing the polymorphs, as a faster heating rate may not be able to identify the transition temperature, while a lower heating rate may lead to lower resolution of peaks. Therefore, it is a usual practice to generate DSC thermograms under different heating rates during polymorph characterization [11]. Also it is important to note that the sample preparation, particle size, and crucible type can affect the quality of the thermogram [5]. XRD is also another indispensable tool in identifying polymorphs. This is based on the differential scattering of X rays when passed

940


Intensity

100

50

10

20

30

30

40

50

30

40

50

2θ ( a)

Intensity

100

50

10

20

30

2θ ( b)

FIGURE 4 Schematic X-ray diffractograms of two polymorphic forms of a hypothetical drug. The x axis represents the detection angle and the y axis represents the intensity of the peak. As can be seen, there is a difference in the diffractograms due to the difference in the internal crystal lattice of polymorphs. The different internal arrangement in a crystal deflects the X ray at different angles.

through a powder sample. Typically, on passing through a powder sample, X rays will tend to get diffracted at various angles, and at some angle of detection, the X rays diffracted from the different planes of the crystal converge to form an amplified signal, which is detected by a photomultiplier tube. The angles at which the XRD peaks are obtained are characteristic for a polymorph (Figures 4a & b). The sample should be uniformly spread to get a good X-ray diffractogram, as an improper sample preparation may lead to variation in intensities due to the preferred orientation of a crystal in the XRD sample holder [14]. Other techniques, such as infrared (IR) spectroscopy and solid-state nuclear magnetic resonauce (NMR), are also used to characterize the polymorphs and are listed in Table 2.


TABLE 2

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Techniques to Characterize Different Crystalline Forms

Technique

Applications

Thermal analysis Differential scanning calorimetry Thermogravimetric analysis Hot-stage microscopy X-ray diffractometry Spectroscopy Infrared Near infrared Nuclear magnetic resonance

TABLE 3

Melting point, enthalpy of fusion, and crystallization; solid-state transformations Stoichiometry of solvates and hydrates; identifying vaporization and volatilization Visualization of solid-state transformations and desolvation events Identifying polymorphs; quantification of degree of crystallinity; crystal lattice geometry and solid-state transformations Characterization of polymorphs based on functional groups; characterization of hydrates and solvates In situ analysis of solid-state conversions; identification and quantification of polymorphs in dosage forms Useful to understand difference in molecular arrangement of polymorphs, hydrates, and solvates

Difference in Solubilities of Polymorphs

Drug Indomethacin Sulfathiazole Piroxicam

Melting Point (°C)

Solubility Ratioa

157, 163 177, 202 136, 154

1.1 1.7 1.3

a

Indicates ratio of solubility of low-melting polymorphic form to solubility of high-melting polymorphic form of drug.

Polymorphism has significant implications in the solubility, bioavailability stability, processing, packaging, and storage of solid drug substances [15–17]. The metstable polymorphic form may be used to improve the solubility of drug substances. Many drugs are known to exhibit polymorphism, particularly, steroids, barbiturates, anti-inflammatory drugs, and sulfonamides, which have a high probability of exhibiting polymorphism [15]. The existing knowledge on drug polymorphism is a good starting point for a preformulation scientist to anticipate polymorphs based on the drug chemistry. In some cases, polymorphism may provide an opportunity to improve the solubility of a drug. For example, form II of chloramphenicol palmitate has a higher dissolution rate resulting in significantly higher plasma concentration than form I when administered orally [15]. However, in many cases [16] the difference in solubility may not be significant enough to cause differences in oral bioavailability (Table 3). Although the polymorphs differ in their dissolution rates, it should be realized that once the drug goes into solution, they do not differ in their properties. If a drug’s absorption is limited by its poor membrane permeability, then the difference in solubility of polymorphs may not impact its bioavailability. Similarly, if the drug dissolution is rapid in comparison to the gastrointestinal (GI) transit time, then the difference in polymorph solubility will not influence its bioavailability [16].

942


More than the presence of metastable polymorph, it is the conversion of the metastable to the stable form during processing, storage, or use that is of great concern to the pharmaceutical scientist [17]. The unpredictability in “conditions” that result in the generation and conversion of one polymorphic form to another mainly aggravates such a situation [18]. This is exemplified by ritonavir, which is a classical case of appearance of a “new polymorphic form” after the drug was marketed [19]. Ritonavir, an anti-retroviral, drug was introduced in the market as a polymorphic form I in soft gelatin capsule in 1996. Two years later, a new polymorphic form II appeared in the formulation due to some unknown reasons causing the drug to be less soluble. The drug manufacturer withdrew the product due to failure of the batches in dissolution tests. After extensive investigation and reformulation, the drug was reintroduced in the market in 1999. Nonetheless, in spite of their unpredictability, a good preformulation team will be able to anticipate the different polymorphs during drug development. It should not be an impossible task given the recent advancements in high-throughput generation and characterization of polymorphs [20]. From an innovator’s perspective, the identification and thorough characterization of multiple polymorphs during drug discovery can extend the patent life and delay the market entry of generic manufacturers. On the other hand, it is also an opportunity for the generic manufacturers to generate new polymorphs with better solubility and stability for gaining market entry. The patent dispute on ranitidine polymorphs is a good example in this regard [21]. Two polymorphic forms of ranitidine were patented by the innovator company and the generic manufacturers had to find an appropriate method to manufacture the desired polymorph without the accompanying impurity of the other polymorph. This provided an edge for the innovator to extend the drug’s market exclusivity for a little longer than they would otherwise have had. If a pure polymorph cannot be generated, the extent of polymorphic impurity should be quantified and ensured from batch to batch. The preformulation scientist closely works with the synthetic chemist in setting specifications for polymorphs. 6.2.2.2

Hydrates/Solvates

In addition to drug molecules, solvent molecules also get incorporated in the crystal lattice, resulting in altered physicochemical properties. When the solvent is water, they are known as hydrates, while if it is any other solvent, they are known as solvates. They are also known as pseudopolymorphs or solvatomorphs. Hydrates are important in this regard as one-third of all marketed drugs are hydrates [13]. Depending on how the water is arranged inside the crystals, they are classified as isolated hydrates, channel hydrates, and ion-associated hydrates [13]. In isolated hydrates, the water molecules are separated from each other by the intervening drug molecules in the crystal lattice (e.g., cephadrine dihydrate). Channel hydrates result when water molecules are linked to one another forming a channel (e.g., theophylline monohydrate). The water molecules may be present either stoichometrically or nonstoichometrically within the crystal lattice. Ion-associated hydrates are typically seen when the water is metal ion coordinated (e.g., nedocromil zinc). Nonstoichometric channel hydrates are problematic due to the presence of diffusible water, which can easily move in and out of the crystal lattices [13, 22]. Hydrates or solvates are formed by crystallizing the drug in the presence of water or solvents. The hydrate formation is dictated by water activity in a given solvent

Heat flow

Percent weight loss


943

(a)

(b)

Tdehyd

Tm

Temperature (°C)

FIGURE 5 Characterization of hydrate. (a) TGA thermogram of monohydrate. The thermogram shows weight loss as a function of temperature. The step in the thermogram shows weight loss due to dehydration of a hydrate. (b) DSC thermogram showing endotherm at corresponding temperature (Tdehyd). The second endotherm indicates the melting point of the hydrate (Tm).

[22]. Hydrates are characterized using gravimetric methods such as thermogravimetric analysis (TGA) or by Karl Fischer titrometry [23]. In TGA, the loss of water/solvate on heating a sample is recorded as a thermogram (Figure 5). The mass change due to dehydration is seen as a step loss in the TGA thermogram. Based on the weight of the initial sample and its elemental composition, the number of water molecules can be calculated. The TGA curve in combination with a DSC thermogram helps to differentiate hydrates from other thermal transitions. In Figure 5, the endotherm in the DSC thermogram corresponds to water loss as indicated by the TGA curve. The TGA can be coupled to an IR or mass spectrometer to characterize solvates. Thermal microscopy is a useful qualitative tool to visualize the release of water from the drug crystals as a function of temperature [23]. Hydrate formation and dehydration significantly influences the processing and storage of drug products [17]. Hydrates may take up further water or dehydrate to lose water. Dehydration of hydrates leads to several possibilities [24], as shown in Figure 6. Hydrates on dehydration can form isomorphic desolvates retaining the same crystal lattice as the hydrate but without the water. Alternatively, hydrates can lose water and become anhydrous crystals. They can also lose water, forming amorphates with the loss of crystal lattice. Higher hydrates can lose water to form lower hydrates, for example, pentahydrate converting to di- or monohydrate. The hydrates also can exhibit polymorphism or on dehydration can convert to a different polymorphic form [13, 17]. Such solid-state transformations are possible during processing, such as granulation, tableting, and storage [17]. In general, hydrates are less soluble than anhydrous forms while solvates are more soluble than ansolvates in water. Ampicillin trihydrate is a classical example which shows lower solubility and lower plasma concentration than anhydrous ampicillin [15]. Preformulation studies

944

ROLE OF PREFORMULATION IN DEVELOPMENT OF SOLID DOSAGE FORMS Polymorphs Isomorphic desolvate

Anhydrous

Hydrate

Lower hydrate

Amorphous

FIGURE 6 Various possibilities that arise from dehydration of hydrate. A hydrate can dehydrate reversibly into various solid-state forms. It can dehydrate to form an anhydrous form of the drug or to a lower hydrate. Hydrate can also dehydrate to form an isomorphic desolvate where the crystal lattice is retained except for the absence of water. The crystal structure may also collapse on dehydration to form an amorphous form. Hydrates on dehydration can also result in different polymorphs.

provide valuable inputs to the formulator in selecting a suitable form of the drug. For example, ampicillin is hygroscopic and hence can be used in suspension dosage forms, while ampicillin trihydrate, which is non-hygroscopic, is used in solid dosage forms. 6.2.2.3 Amorphate Unlike a crystalline drug, an amorphous form of the drug does not have a regular crystal lattice arrangement and the molecules are arranged in random order (Figure 1c). Glass is a typical amorphous substance and so amorphous drugs are also known as glasses [25]. Amorphous form is prepared through milling, rapid cooling of a melt, rapid precipitation using an antisolvent, rapid dehydration of a hydrate, spray drying, or freeze drying [26]. Some of the above methods may also unintentionally produce an amorphous form during processing of the crystalline form of the drug [17]. For instance, milling during dosage form manufacture may produce an amorphous form unintentionally, as in the case of indomethacin. The amorphous form does not show a melting point but is characterized by a glass transition temperature (Tg). This temperature indicates the conversion of the amorphous form from a rigid glassy state to a more mobile rubbery state. Above Tg the amorphous form will tend to recrystallize and convert to the crystalline form, which then undergoes melting, as shown in Figure 7a. The Tg for an amorphous drug can vary depending on the storage conditions and thermal history of the sample and is sensitive to moisture, pressure, and temperature [26]. The Tg is seen only as a slight shift in the baseline due to a change in the specific heat capacity of the sample and is influenced by the heating rate in DSC [25]. In XRD, the amorphous form shows a shallow peak or halo, as opposed to sharp and intense peaks for a crystalline drug compound (Figure 7b). The main advantage of amorphous form of the drug substance state is its significantly higher solubility than the crystalline form of the drug, primarily due to the excess surface energy [16, 27]. Therefore, conversion of a crystalline drug into an amorphous form is one of the strategies to increase drug solubility. Table 4 compares


945

Heat flow

Endotherm

Tg

Tcrys

Tm

Temperature (°C)

(a)

Intensity

100

50

10

20

30

30

40

50

2θ (b)

FIGURE 7 Characterization of amorphous form. (a) DSC thermogram of amorphous substance. Thermogram is characterized by a glass transition temperature (Tg) above which the amorphous form is mobile and recrystallizes (Tcrys) into a crystalline form which finally melts (Tm). (b) Amorphous form that does not show any peaks in XRD as it does not have regular arrangement of molecules. Shallow peaks are indicative of an amorphous drug substance.

the solubility of amorphous and crystalline forms of a few drugs. However, the biggest challenge lies in the stabilization of the amorphous form to prevent it from converting to the less soluble crystalline form during storage and use. It should be noted that they can take up moisture to convert to a crystalline form or to a hydrate, resulting in decreased drug solubility. The moisture uptake can also lead to chemical degradation [26]. The stabilization of drug amorphates is usually accomplished by increasing the Tg using polymers and thus restricting their molecular mobility and

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TABLE 4 Comparative Solubilities of Amorphous and Crystalline Forms of Drugs Drug Carbamazepine Griseofulvin Glibenclamide

Solubility Ratioa 1.5–1.7 38–441 14

a

Indicates ratio of solubility of amorphous form to solubility of crystalline form of drug.

chemical reactivity [26]. Particle size reduction, which is a very common processing step in dosage form manufacturing, can result in varying degrees of amorphous and crystalline forms in the drug. In such cases it is essential to quantify the degree of crystallinity using various analytical techniques such as DSC and XRD [28]. 6.2.2.4

Hygroscopicity

Some solid drug substances have a tendency to absorb moisture from the atmosphere leading to physical and/or chemical instabilities. Hygroscopicity is the rate and extent of moisture adsorbed/absorbed by a solid substance. Solid drug substances may vary in their behavior to moisture and are classified as deliquescent, efflorescent, and effervescent. Deliquescent materials such as hydrochloride salts absorb moisture and become a liquid. Effervescent substances (e.g., a mixture of citric/tartaric acid and sodium bicarbonate) absorb moisture and release carbon dioxide. On the other hand, efflorescent substances such as hydrates may lose moisture depending on the relative humidity (RH). Therefore, it is important to study the moisture absorption behavior of drugs to choose the processing and storage conditions for the drug. Hygroscopicity is measured by exposing the drug sample to various RH in a dessicator. The RH is maintained at a constant level by using salt solutions of varying concentrations (e.g., KNO3, KCl) and the humidity is expressed with respect to the humidity of a saturated salt solution. Moisture sorption and desorption curves are generated to study the moisture uptake. The moisture absorption profile is generated by noting the increase in mass on exposure to varying RH and the desorption profile is generated by recording the change in weight with decreasing RH [23, 29]. This can be measured using a dynamic vapor sorption instrument. A typical sorption/desorption profile is shown in Figure 8 for an anhydrous and hydrate form of a hypothetical drug. As can been, for a hydrate the sorption and desorption profile is superimposable and is hence non-hygroscopic. On the other hand, the anhydrous form of the drug is hygroscopic and shows hysteresis on the sorption and desorption profile, indicating significant moisture uptake. Such profiles give useful clues to the preformulation scientist. Significant hysteresis is indicative of hydrate formation and can be used as a guide to evaluate the potential of hydrate formation. Further the profile also helps to differentiate hygroscopic and non-hygroscopic salts [29]. The profile also gives information on processing and storage conditions that can overcome solid-state transformations. For example, in Figure 8, it is seen that the anhydrous form does not take up moisture if RH is below 80% and it retains the moisture

947

Change in mass (%)


20

40

60

80

Relative humidity (%)

FIGURE 8 Sorption and desorption profile of hydrate and anhydrous form of hypothetical drug. The solid lines represent the profile for an anhydrous form of the drug, while the broken lines represent the profile for a hydrate form of the drug. Anhydrous form of the drug does not take up moisture until it reaches 80% RH, and on reducing the RH, it does not lose moisture until it reaches RH of 20%. The hysteresis is indicative of hygroscopicity and significant moisture uptake. The hydrate form of the drug does not show hysteresis but both the sorption and desorption curve superimpose on each other indicating that it is non-hygroscopic.

until the RH is reduced below 20%. The study can also be used to extract kinetic and temperature information on moisture uptake by drug substances. The studies conducted during preformulation testing should be representative of the anticipated processing and storage conditions of the drug. 6.2.2.5

Particle Characteristics

Drug particle characteristics such as size, shape, and surface area impact the drug’s processability and product performance. Particle size is the most influential among these as the other two properties can be related to it. When sufficient drug is available, the preformulation scientist characterizes the particle size and size distribution to set specifications for formulation and future drug lots. Table 5 lists the various methods used to measure particle size. The methods differ in their principle of operation and also in the range of particle sizes they can measure [30]. Usually, the gross particle morphology is characterized using a simple optical microscope and if required is further characterized using a scanning electron microscope. Lightscattering methods are commonly used to measure particle size due to their low sample requirements and ease of measurement. The instrument readout is in the form of a graph where the particle size is plotted against the percent frequency of particles in different particular size ranges. The results are used to set particle size specifications and understand polydispersity or multimodal particle size distribution in powders. Especially if the drug is potent, a narrow size distribution is desired to ensure drug homogeneity during formulation. The surface area of a powder bed is determined using the Brunauer, Emmett, and Teller (BET) method. In this method,

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TABLE 5

Methods for Particle Size Analysis

Method Sieve

Microscopy

Sedimentation

Light scattering

Principle Sieve analysis utilizes a series or stack, or nest of electro brass or stainless steel sieves that have smaller mesh at the bottom followed by meshes that become progressively coarser toward the top of the series. Useful for measuring particles in size range 10–50,000 μm Analysis is carried out on two-dimensional images (projected diameter) of particles which are assumed to be randomly oriented in three dimensions. Can measure particles in the size range 1–1000 μm. Electron microscopy is useful for analysis of particles in submicrometer range (0.01–100 μm). It also gives information on surface morphology and shape of the powder. Size analysis is based on sedimentation of particles as a function of their size due to gravitational pull or by using a centrifugal force. Can measure particles in the size range 0.01–100 μm. This is based on the principle of light scattering of particles as a function of their hydrodynamic radius. Commonly used, as it requires a small sample size and is a rapid method for particle size measurement. It can be used to measure particles varying in size from 0.001 to 100 μm.

N2 gas is passed through a powder bed and the surface area is calculated based on the volume of gas coming out in the absence and presence of the powder [31]. Particle size reduction is an initial step in the development of any dosage form. The data generated during preformulation testing guides the formulator in deciding about size reduction. Particles with size >100 μm generally require size reduction and particles in the size range of 10–40 μm are generally acceptable for solid dosage forms [32]. As mentioned in the earlier section, particle size reduction may lead to partial or complete amorphization of a powder, and this factor should be taken into consideration. Also worth mentioning are the other solid-state transformations that may take place during milling, such as the conversion of one polymorphic form to another or the desolvation of a hydrate [17]. It is important to maintain the particle size distribution within a narrow range to avoid powder stratification and avoid flowability issues during capsule filling or tablet compression. Particle size reduction increases drug solubility due to the enormous increase in surface area with decreasing particle size. Griseofulvin is often widely quoted in the literature in this regard, where the bioavailability of this water-insoluble drug is increased 10 times on reducing the particle size [32]. On the other hand, particle size reduction may be counterproductive for some drugs such as nitrofurantoin. Particle size reduction of nitrofurantoin causes rapid drug absorption with an associated increase in its adverse effects. In contrast, the slowly dissolving macrocrystals of nitrofurantoin do not cause adverse effects [32]. Excessive particle size reduction (40

Extremely poor

—

Carr’s index (%)

(c)

FIGURE 9 Measurement techniques for powder flow. (a) Angle of repose is determined by pouring a powder through a funnel and noting the angle that the powder heap makes with the horizontal surface. (b) Carr’s index is measured by pouring a known weight of the powder into a graduated cylinder and tapping it on a hard surface until the powder is consolidated. (c) Carr’s index is measured as percent, while angle of repose is measured in degrees, and both methods show good correlation. Powder flow is classified based on either of the measurement methods.

available for preformulation testing, either method may be used to estimate the powder flow property. Poor flowing powders may require glidants to improve their flow property. Compressibility is studied by compressing the drug in a hydraulic or IR press, and such experiments give early warnings to the formulator about capping and lamination issues in tablets. Altogether, these preformulation tests give valuable information to the formulator in deciding excipients and processes.

6.2.3

SOLUBILITY CHARACTERISTICS

Drug solubility is one of the physicochemical parameters that receives lots of attention during preformulation testing. In the initial stages, solubility studies are usually kinetically determined, where the drug is placed in contact with the solvents and then the solubility is assessed using turbidometric methods almost instantaneously.


951

Solvent required for 1 gm of solid

Practically insoluble >1000 mL Very slightly soluble 100–1000 mL Slightly soluble 100–300 mL Sparingly soluble 30–100 mL Soluble 10–30 mL Freely soluble 1–10 mL Very soluble pKa,counterion. In atropine sulfate, atropine has a pKa of 9.9 and sulfuric acid has a pKa of −3. It is important to mention that salts do not alter the intrinsic pKa of the drug but increase drug solubility by keeping pH on the ionization side of the drug’s pKa (Figures 11a and b). The salt formation is a futile exercise if the pKa of a drug is 10 and other solubilization strategies have to be pursued [33]. Inorganic ions such as hydrochloride and sodium are the most frequently encountered species in pharmaceutical salts, primarily because of the ease of salt formation and their low molecular weights. They provide significant increase in drug solubility and at the same time strong acids/bases may also be hygroscopic, posing problems during processing and storage. In those cases, salts are formed using weaker organic anions or cations such as mesylate, besylate, and choline. Table 7 lists some of the commonly used inorganic and organic counterions used in pharmaceutical salts. The pH provided by the salts significantly influences the drug dissolution and subsequent drug absorption from the GIT. Though salts increase drug ionization and aqueous drug solubility, it is the un-ionized form of the drug that is absorbed through the membrane. According to the well-known pH partition hypothesis [33], a weakly TABLE 6

Factors to Consider during Salt Selection

Relative pKa of the drug and the counterion Common-ion effects Crystal habit and crystallinity Polymorphic conversions Hygroscopicity Chemical stability Manufacturability Toxicity

Percent Ionization


pKa of drug

955

pKa of counterion

pH

Percent Ionization

(a)

pKa of counterion

pKa of drug

pH

(b)

FIGURE 11 Relative pKa of drug and salt-forming counterion. (a) For a weak acid, the pKa of the salt-forming counterion should be higher than the drug pKa to keep the pH on the ionization side. (b) For a weak base, the salt-forming counterion should have a pKa less than the drug’s pKa to keep the pH in the ionization side.

TABLE 7 Various Counterions Used to Form Drug Salts Chemical class Inorganic Organic

Salt-Forming Counterions Hydrochloride, hydrobromide, sulfate, nitrate, phosphate, sodium, potassium, calcium, and zinc Triethanolamine, ethanolamine, lactic acid, maleic acid, citric acid, acetic acid, choline, ethanesulfonic acid, oleate, and stearate

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acidic drug is primarily absorbed from the stomach (pH 1–3), where it remains in un-ionized form. A basic drug is expected to be absorbed from the intestines (pH 6.5–8), where it would be in the un-ionized form. But in many cases, the absorption of drugs cannot be satisfactorily explained by this theory. This is understandable if one considers the fact that drug dissolution from a salt is mainly influenced by the surface pH of the dissolving drug (microenvironment) rather than the bulk pH of the GI fluids. For instance, the surface pH of weakly acidic drug is 1.5 times higher than the bulk pH in the stomach, and therefore the dissolution of the salt of a weak acid will be 100 times faster than the free-acid form of the drug in stomach [40]. Similarly, it is the free base rather than its salt form which will dissolve faster in the gastric pH. So it is possible to modulate the drug solubility of a pharmaceutical salt irrespective of its location in the GIT. Considering the fact that most of the drugs are absorbed from the intestine (due to large surface area), it is desirable to select a salt that is not completely ionized or unionized at the intestinal pH to have optimal dissolution and absorption. An important phenomenon that is often overlooked during the salt selection process is the suppression of salt ionization in GI fluids due to the common-ion effect. This is particularly important with inorganic salts, where salt ionization can be suppressed by ions such as chloride and sodium which are abundant in GI fluids. A hydrochloride salt will ionize in solution, as shown in the Equation (12), but in gastric fluid, the presence of chloride ions suppresses the drug ionization [as shown by the thicker arrow in the reverse direction in Equation (13)] to maintain an equilibrium between the ionized and un-ionized form of the drug: DH + Cl − DH + + Cl −

(12)

DH + Cl − → DH + + Cl −

(13)

Hence it is important to study this effect during preformulation by testing the solubility of the salt in the presence and absence of sodium chloride. Although salts do not alter the pharmacological activity of the drug, safety is an important consideration in the selection of salts. From this perspective, salts are treated as a new molecule by the FDA. The safety of the salt is evaluated with respect to its route of administration and dose of the drug [37]. 6.2.3.2

Partition Coefficient

In simple terms, the partition coefficient represents the relative solubility of a drug in a hydrophobic and a hydrophilic solvent. The hydrophilic solvent is usually water or buffer (pH 7.4), while the hydrophobic phase is usually n-octanol. The partition coefficient (Ko/w) is defined as the ratio of concentration of the drug in the organic phase (Co) to drug concentration in the aqueous phase (Cw): Ko/w =

Co Cw

(14)

The choice of n-octanol is based on its ability to mimic the lipophilicity of the biological membranes [33] and further its solubility parameter (δ = 10.24; solubility


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parameter is a measure of internal cohesive energy) falls within the solubility parameter range of most drugs (8–12.4). The partition coefficient is determined by dissolving the drug in one of the phases and shaking both the phases together in a flask for 30 min to achieve equilibration. Then the drug concentration is determined from one of the phases, usually the aqueous phase, and the drug concentration in the oil phase is determined by subtracting the drug concentration in the aqueous phase from the total drug that was added. This value when expressed in logarithmic form is known as log P. The phase volume of the two phases is 1 : 1 but, if the drug is less soluble in the aqueous phase, the ratio (water–octanol) is changed to 1 : 10 or 1 : 20 to have a measurable drug concentration in the aqueous phase [33]. It is important to saturate the phases with respect to the other solvent before starting the experiment to rule out the influence of solvent partitioning on drug distribution between the two phases. Another important factor is the influence of drug ionization on the partition coefficient and this is particularly important when a buffer is used instead of water. The partition coefficient determined from Equation (14) is an apparent value rather than a true partition coefficient for ionic drugs. However, the true partition coefficient can be calculated from the apparent partition coefficient if the drug’s pKa and the pH of the drug solution are known [39], as shown in the equations

( (

1 ⎧log P − log app ⎪ 1 + 10( pH − pKa ) log P = ⎨ 1 ⎪log Papp − log ⎩ 1 + 10( pKa − pH )

) )

for weak acid

(15)

for weak base

(16)

During the initial stages of drug screening, the partition coefficient is calculated based on the chemical structure (C log P). This is done by assigning values to different fragments in the chemical structure [41]. The calculated values are only estimates, but they are useful to rank order a homologous series of compounds based on their lipophilicity for further lead optimization. Given the fact that drugs have to cross many biological membranes before reaching the site of action, the log P value has a significant influence on drug absorption, drug pharmacokinetics, and pharmacology. This is exemplified from the numerous structure–activity and structure–property relationships using log P [41]. The log P is important in assessing the oral absorption potential of a drug. If a drug has a low log P (5), it will be trapped in the lipophilic membrane. A log P of 1–5 is usually considered optimal for oral drug absorption [3]. For an ionic drug, the un-ionized form of the drug will be more lipophilic than the ionized form of the drug. Therefore, at any given pH in vivo, the relative proportion of ionized versus un-ionized form of the drug dictates drug dissolution and absorption through the membrane. 6.2.3.3

Drug Dissolution

Dissolution is the rate at which a solid drug goes into solution and is a critical determinant in the absorption of drugs from solid dosage forms. A drug has to go into solution before it can be absorbed. In vitro dissolution studies are a valuable

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tool for determining the influence of different solid-state properties on drug dissolution and vis-à-vis predict in vivo drug dissolution and absorption. It is used to screen drugs that show dissolution rate–limited absorption. The factors that influence drug dissolution rate (dc/dt) can be understood from the well-established Noyes–Whitney equation [42] dC kSCs = dt V

(17)

where k is the dissolution rate constant (cm/s), Cs is the saturated solubility of the drug, S is surface area of solid exposed to the solvent, and V is the volume of the dissolution medium. The dissolution rate constant is a function of the diffusion coefficient D of the drug through a stagnant aqueous layer adjacent to the dissolving surface and thickness h. Powder or particulate dissolution is carried out in a dissolution vessel in a specific volume of dissolution fluid (900 mL) which is stirred and maintained at 37 °C. Several configurations are available to study the dissolution of various dosage forms (Figures 12a and 12b). Various simulated physiological media are used to understand the in vivo behavior of drug and dosage forms [43]. The usual dissolution media include water, 0.1 N HCl, and pH 7.4 buffers. It is important to maintain sink conditions in the dissolution medium by keeping the drug concentration at 10% of saturated solubility of drug (to mimic in vivo). For poorly water soluble drugs, surfactants are often added to the dissolution medium for this purpose. During preformulation testing, particulate dissolution studies reveal the influence

Tablet

Tablet

Tablet

(a)

(b)

(c)

FIGURE 12 Various drug dissolution methodologies. (a) In the paddle method, the tablet is placed in the dissolution vessel containing dissolution medium and the paddle is rotated at defined rpm, while the dissolution vessel is maintained at body temperature. (b) In the basket method, the tablet is kept inside a meshed basket and rotated. (c) For IDR studies, the tablet is kept inside a die cavity and only one face of the tablet is exposed to the dissolution medium.


959

of particle size, crystal habit, and wettability of a drug substance. A formulator, on the other hand, routinely uses dissolution testing as a quality control tool in the design of dosage forms. Various models have been developed to describe the release kinetics of conventional and modified release dosage forms [44]. Since powder dissolution is influenced by changing surface area, it is not useful for delineating the effects of polymorphs, hydrates, and pharmaceutical salts. Instead, the intrinsic dissolution rate (IDR) is used. The IDR studies are conducted at a constant surface area and hence the dissolution rate observed is only a function of the intrinsic solubility of the drug. The Noyes–Whitney equation is modified for IDR, where the surface area is kept constant, and Equation (17) reduces to dC = k1Cs dt

(18)

where k1 is the intrinsic dissolution rate constant and dC/dt is the intrinsic dissolution rate (mg·cm2/s). For IDR studies, the drug (500 mg) is compressed in a hydraulic press (at 500 mPa) to a 13-mm disc. This disc is then loaded onto a holder which exposes only one surface of the disc to the dissolution medium (Figure 12c). The IDR is obtained by dividing the slope of the plot between the amount of drug dissolved and time by the area of solid exposed to the dissolution medium. The IDR predicts the influence of drug solubility on in vivo drug dissolution and absorption. A drug which has an IDR of 1 mg·cm2/s will not generally show dissolution rate– limited absorption in vivo. However, if the IDR falls between 0.1 and 1 mg·cm2/s, then further studies may be required to make a decision. Drugs with IDR < 0.1 mg·cm2/s show dissolution rate–limited absorption in vivo, necessitating drug solubilization strategies [45]. With respect to pharmaceutical salts, IDR is used to understand the influence of surface pH on drug dissolution and absorption. The common-ion effect can be studied by including 0.1–0.15 M NaCl in the dissolution medium. Also IDR is useful to understand the difference in solubility of polymorph and amorphous forms. However, in some cases, the compression force used in making the IDR disk may by itself induce solid-state transformations [45]. DSC, IR, and XRD must be used to identify the drug’s solid state before and after compression as well as at the end of the dissolution studies. A well-designed dissolution study is used as an early warning for drug molecules that would pose absorption problems in vivo. 6.2.3.4 Absorption Potential The ultimate goal of any drug development program is to develop an orally absorbable compound. Solubility and permeability are the two most critical parameters that dictate oral absorbability of a molecule. All other parameters are directly or indirectly related to these two physicochemical properties. As can be seen from Figure 13, there are several barriers that a drug needs to overcome before reaching the systemic circulation. The oral absorption of a drug is mainly limited by drug dissolution and/or by the drug permeation across the GI membrane. Considering their importance, drug solubility and permeability are screened very early in the drug discovery process. Solubility studies are typically run in a high-throughput format using a turbidometric method as discussed earlier. Based on this primary

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GI transit time GI membrane

Drug in solution

Dosage form

Solubility and dissolution

Systemic circulation

Mucous layer

FPM

Drug stability Permeability

FIGURE 13 Barriers to drug absorption. Drug from the dosage form should be soluble and dissolve in GI fluids before it can be absorbed. Drug dissolution is one of the major ratelimiting steps in drug absorption. Drug absorption may be affected if the drug is unstable in GI fluids. Furthermore, drug absorption will also depend on how long the drug resides in a particular region of the GIT. The drug has to diffuse through the highly viscous mucous layer before getting absorbed through the membrane. Membrane permeability is one of major the rate-limiting steps in absorption. After absorption the drug may be subject to first-pass metabolism (FPM) in the liver before reaching the systemic circulation. Dark arrows indicate that solubility and permeability are the most influential factors.

screen, detailed solubility and dissolution studies are carried out as the compound moves through different development phases (Figure 14a). A usual target for drug solubility during lead selection is 1 mg/mL for avoiding dissolution rate–limited absorption [46]. Drug permeability is commonly screened in the early discovery phase using CaCo-2 cell lines, which is a human colon carcinoma cell line. Using a 96-well format, the cell lines are used as a primary screen to rank order compounds based on permeability [4]. If designed properly, the cell culture studies can be used to understand the drug absorption mechanisms [47]. To avoid permeability-limited absorption in vivo, a drug should have a permeability coefficient of 2 × 10−4–4 × 10−4 cm/s [48]. Inputs from the drug metabolism team (based on liver microsomal studies) can give clues on the drug’s susceptibility to first-pass metabolism. Following cell culture studies, a select group of compounds are studied using isolated rat intestine and segmental absorption studies to understand the drug absorption mechanism and the site of drug absorption in the GIT (Figure 15b and 15a). This is further substantiated using in situ perfusion experiments in rats [49]. The details of the studies are depicted in Figure 15b. Some of these compounds are studied in whole animals of which a few may make it to clinical trials in humans.


Dissolution studies

Human studies

pH solubility profile

Whole-animal studies

Equilibrium solubility

Isolated rat intestine and In Situ perfusion in rats

Kinetic solubility

CaCo-2 cells

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Drug discovery and development (a)

(b)

FIGURE 14 Different methods to determine solubility and permeability during various stages of drug discovery and development. (a) In the initial stages the drugs are screened using kinetic solubility studies, which are later followed by equilibrium solubility studies, pH solubility profile, and dissolution studies during development phase. (b) Drug permeability is initially screened using CaCo-2 cell lines followed by rat intestinal studies. This is followed by pharmacokinetic studies in animals and finally the potential drug molecules are tested in humans in clinical trials.

Thus, the preformulation team in coordination with other discovery teams gets useful estimates on in vivo drug absorption. In addition to drug solubility and permeability, it is also important to consider the anticipated dose while assessing the absorption potential. A useful tool to optimize the drug’s physicochemical properties is the maximum absorbed dose (MAD) model [46]. The model predicts the dose that would be absorbed based on the drug’s solubility (Cs; solubility in intestinal pH of 6.5), absorption rate constant (ka; usually obtained from rat permeability studies), physiological factors such as gastric transit time (Ti; approximated as 4.5 h), and intestinal fluid volume (Vint; approximated as 250 mL): MAD = Cs kaVintTi

(19)

Using this model, the required solubility or permeability can be estimated for a given dose of the drug. For example, a drug with an anticipated human dose of 1 mg/kg (70 mg for a normal 70-kg subject) will require a solubility of 0.05 mg/mL provided the drug shows good permeability. Similarly, the absorption rate required to achieve the same dose for a drug with good solubility (1 mg/mL) is 0.001 min−1. The MAD model is helpful in guiding development teams on optimizing drug solubility and/or permeability. A further refinement of this model led to the evolution of the biopharmaceutics classification system (BCS), which classifies the drugs into four classes depending on their solubility and permeability (Figure 16). The BCS is applicable only to the oral route of administration, and according to this classification, a drug is considered to be highly soluble if the highest dose of the drug is soluble in a glass of water (250 mL) covering the entire pH range in GIT from 1 to 7.5, and a drug is considered to be highly permeable if the drug has more than 90% oral bioavailability [48]. The model has been developed based on the solubility and permeability characteristics

Drug solution


Drug solution

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Passive transport Carriermediated transport

pH 7.4 buffer

Passive transport

pH 7.4 buffer (a)

Rat intestine

Drug in

Drug out (b)

FIGURE 15 Intestinal permeability studies. (a) Everted rat intestine is used to study the mechanism of drug absorption. Isolated rat intestine is filled with drug solution and tied at both ends and the drug permeation into the external medium is measured (left). In another set of experiment, the rat intestine is everted with the internal mucosal membrane facing outside (right) and the serosal side facing inward. If the drug is passively absorbed, then there would not be any differences in permeability in these two experiments. If the drug is transported by carriers, then drug permeation would be seen only in the first experiment, as the carriers are present only on the mucosal side. (b) In situ intestinal perfusion studies are conducted in an anesthetized animal and the drug solution is pumped through a tube and drug coming out on the other side of the intestine is measured. The drug is also measured by sampling from the jugular vein in the animal. This is useful to measure the dynamic drug absorption into systemic circulation.

of marketed drugs. However, BCS is bound to become an important biopharmaceutical tool in lead optimization in drug discovery setting and also at the same time serving as a useful guide to develop new formulations in a generic setting. From a regulatory perspective, BCS provides a scientific basis to grant biowaivers. As per BCS, the dissolution test can serve as a surrogate tool for costly and time-intensive bioequivalence studies for generic drugs which are highly soluble. This requires the establishment of a good in vitro–in vivo correlation. At present, the FDA grants biowaivers to generic manufacturers of immediate-release dosage forms for drugs in class I (highly soluble and highly permeable) provided they can prove the “dissolution equivalence” of their product to that of the innovator’s drug product [50].


Class I

e.g., Ibuprofen, griseofulvin

e.g., Metronidazole, propranolol

Class IV

Class III

e.g., Atenolol, cimetidine

e.g., Amhotericin B, taxol

Low

Permeability

High

Class II

963

Low

Solubility

High

FIGURE 16 Biopharmaceutics classification system. The drugs are classified based on drug solubility and drug permeability. A drug is considered to be highly soluble if the highest dose of the drug is soluble in 250 mL of water varying in pH from 1 to 7.8 (GIT pH range). A drug is considered highly permeable if more than 90% of the drug is bioavailable by oral route. Class I drugs are highly soluble and highly permeable, class II drugs are poorly soluble but highly permeable, class III drugs are highly soluble but poorly permeable, and class IV drugs have poor solubility and permeability.

The dissolution equivalence is tested using the statistical dissolution model, termed f2 or similarity factor, and is described by the equation

( ) ∑ (R + T ) ⎤⎦⎥

1 ⎧ f2 = 50 log ⎨ ⎡1 + ⎢ ⎣ N ⎩

t

t =1− n

t

2

−0.5

⎫ × 100 ⎬ ⎭

(20)

where N is the number of dosage form units (12 units are tested), t is dissolution time points from 1 to n, Rt is the percent drug dissolved for the reference drug product, and Tt is the percent drug dissolved for the test product. Two dissolution profiles are considered similar, if f2 ≥ 50 and if the coefficient of variation does not exceed 20% at early dissolution time points (usually 10 min) and 10% at other time points in the pH range 1–7.5. However, if ≥85% of drug is dissolved in ≤15 min, then no comparison is required and the dissolution is based on a single time point determination [50]. The BCS paradigm can be used to develop strategies for enhancing drug solubility and/or permeability (Tables 8 and 9). Solubility enhancement may involve only

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TABLE 8

Solubility Enhancement Methods

Technique

Principle

Salt formation Particle size reduction Change of form Cosolvents

Complexation

Surfactants

Disperse systems

TABLE 9

Permeability Enhancement Methods

Method Transcellular transport

Paracellular transport

Carriermediated transport Blocking efflux pump

Increases drug solubility by keeping the pH at which the drug is ionized. Increase in surface area increases drug solubility. Particle size is reduced to nanodimensions (nanosuspensions) for increasing drug solubility. Crystalline drugs are converted to amorphous forms which show higher solubility than crystalline forms of the drug. Various water miscible cosolvents are used to increase the water solubility of drug. The cosolvents are selected based on the polarity of the drug. Common cosolvents include glycerol, ethanol, and propylene glycol. Drug is entrapped or complexed with excipients that can mask the lipophilic groups of the drug and enhance drug solubility in water. Cyclodextrins are commonly used to entrap hydrophobic drugs in the core, while the hydrophilic groups on the periphery help to solubilize the drug. Surfactants are characterized by the presence of lipophilic and hydrophilic groups and form spherical structures known as micelles in water at a certain concentration. The hydrophobic drug is entrapped in the hydrophobic core of the micelle while the hydrophilic groups on the periphery help to solubilize the drug. The hydrophobic drug is dissolved in an organic solvent and in addition may also contain an emulsifier. On contact with the intestinal fluids, the drug is emulsified (microemulsions) by bile salts and is absorbed through the intestine.

Mechanism Sorption promoters can be used to enhance the transcellular transport in the intestine, including bile salts and fatty acid esters. They tend to fluidize the lipid bilayer and enhance drug permeation across the membrane. Enhancement is achieved by modulating the tight junctions between the cells. Chelating agents such as ethylenediamine tetraacetic acid can chelate calcium ions and transiently open the tight junctions for drug transport. Nutrient transport carriers are utilized for drug transport. Prodrugs are designed to meet the structural requirements for carrier-mediated transport. P-glycoprotein is a major efflux mechanism that pumps out drug from the intestinal cells back into the intestinal fluid. Several drugs and food substances are known to inhibit p-glycoprotein and enhance drug permeation.

a physical intervention, as opposed to molecular modification for permeability enhancement. Generally [46], the permeability range of drugs varies by only 50-fold (0.001–0.5 min−1) in comparison to drug solubility, which varies by six orders of magnitude (0.1 μg/mL–100 mg/mL). Hence, the formulator has greater flexibility in altering the drug’s solubility in comparison to altering the drug’s permeability. This

STABILITY CHARACTERISTICS

Solubility/permeability

Early drug discovery

Maximum absorbable dose

Exploratory studies

BCS classification

Drug development

Ora ldosage form development

Clinical development

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FIGURE 17 Flow chart for determining absorption potential of a drug during drug discovery and development. Solubility and permeability studies from preclinical phase are used to calculate the maximum absorbable dose and, when correlated to BCS, this can provide information on its biopharmaceutics class. This is useful to estimate if the drug absorption would be dissolution and/or permeability limited for developing appropriate drug delivery strategies.

is evident form the fact [51] that the majority of the marketed drugs are highly soluble (>55% in classes I and III). Sometimes, enhancing the permeability by altering the drug’s chemical structure may be counterproductive. This because of the associated increase in molecular weight that attenuates the permeability enhancement gained with structural modification. On the other hand, optimization of drug solubility may be more fruitful if the poor permeability is overcome by increasing drug solubility to provide a high drug concentration at the absorption site. However, this may be a difficult strategy if the dose is very high. The preformulation team should use the “appropriate tools” at every stage of the drug discovery and development process to guide or alert other development teams about drug solubility and permeability issues (Figure 17).

6.2.4


Drug stability is an essential component of preformulation testing. Establishing the stability of the drug under a variety of conditions is an expensive and timeconsuming process. It cannot be overemphasized that the availability of a stabilityindicating assay is the key to stability studies. The preformulation scientist works closely with the analytical method development team in developing a stabilityindicating assay. During the early stages, a foolproof stability-indicating assay may

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not be available. This is understandable considering the fact that the initial drug lots are not pure and the purity is improved as the molecule progresses to subsequent development stages. Therefore, the intention of the preformulation scientist is not to generate a thorough kinetic rate profile of the drug but to broadly define the conditions under which the drug would be stable. Only relevant stability data are generated during various phases for developing preclinical and clinical formulations [52]. The stability data are evaluated and conveyed to the formulation development team upfront if stabilization and additional packaging requirements are needed. Often the chemical structure of the drug can give clues on the drug’s degradation pathway and its stability characteristics [53]. Table 10 lists some of the functional groups that are susceptible to common degradation pathways. Stability test conditions are chosen keeping in mind the environment that the drug will encounter during drug development, processing, storage, and use (Table 11). One parameter is studied at a time while keeping all other parameters constant. Apart from classical stability studies, such as hydrolysis, oxidation, and photolysis, the preformulation scientist has to determine the stability of drugs in unconventional matrices such as animal feed used for toxicological studies [34]. The stability of a drug in the animal feed is complicated by the feed composition, including enzymes and vitamins among others. The moisture content in the feed may also vary with storage temperature. In such cases, it is appropriate to study the stability of the drug under the storage conditions encountered in the toxicological laboratory. Sensitive techniques such as liquid chromatography/mass spectrometry (LC/MS) are used to evaluate drug stability in such complex mixtures. In general, most of the drugs undergo first-order degradation, while some drugs may follow zero-order kinetics and only a few drugs undergo second-order degradation [34]. The first- and zero-order reactions are readily differentiated by studying drug stability at two different initial drug concentrations. First-order kinetics will depend on initial drug concentration, while a zero-order reaction will be TABLE 10

Groups Susceptible to Common Degradation Pathways

Degradation Pathway Hydrolysis Oxidation Photolysis

TABLE 11 Parameters Temperature Moisture pH Oxygen Light

Functional Groups Esters, lactones, amides, lactams, oximes, imides, and malonic ureas Amines, sulfides, disulfides, sulfoxides, phenol anions, thiols, nitriles, and catechols Aromatic hydrocarbons, aromatic heterocyclics, aldehydes, and ketones

Stability Testing Conditions Conditions 5, 25, 30, 37, 40, and 60 °C 30, 45, 60, 75, and 90% RH 1, 3, 5, 7, and 9 at room and body temperature Sparging with 40% oxygen or adding 100–200 ppm of hydrogen peroxide 1.2 million lux hours of exposure to visible light and 200 h/m2 exposure to UV light (360–400 nm)


967

independent of initial drug concentration. Accelerated stability studies are conducted to expedite the degradative reactions, where temperature is the commonly used accelerant. The influence of temperature on drug stability kinetics is described by the Arrhenius equation: k = Ae − Ea /RT

(21)

where k is a reaction rate constant, A is a frequency factor, Ea is activation energy, R is the gas constant, and T is absolute temperature. According to the Arrhenius equation, every 10 °C rise in temperature increases the reaction rate by two- to fivefold [31]. The usual temperatures selected for early stability studies include 5, 25, 37, 40, and 60 °C to cover the temperatures encountered in processing, use, and storage of the drug product. Using the Arrhenius equation, the rate constant from higher temperatures can be extrapolated to determine the stability at room temperature [31]. The slope of the plot of the reciprocal of temperature and the rate constant gives the activation energy. The activation energy usually varies between 15 and 60 kcal/mol with a mean value of 19.8 kcal/mol [33]. A break in the line is usually indicative of change in the activation energy due either to change in the reaction order or the mechanism of degradation at higher temperature. In such cases, it becomes imperative to conduct detailed studies to understand the drug degradation mechanism. Some of the reactions seen at higher temperature may not be representative of the reactions at room temperature. Hence, short-term high-temperature studies should be supplemented with long-term real-time stability testing at room temperature. Additionally, the drug is also exposed to moisture, oxygen, and UV light (250–360 nm). The conditions used for stress studies may vary depending on the drug type and the drug development stage [54]. The stability studies in this chapter are discussed with respect to a solid dosage form which includes solid-state stability, limited liquid state stability, and drug–excipient compatibility. 6.2.4.1

Solid-State Stability

In general, solid-state reactions are slow, complex, and at times difficult to quantify. They may manifest as either physical and/or chemical instabilities. Physical instabilities include solid–solid transformations, desolvation of hydrates, and change in color [34]. On the other hand, chemical instabilities may involve a change in drug content as a result of hydrolysis, oxidation, or light-induced degradations [32]. The influence of temperature is studied by exposing the solid drug to increasing temperatures and also exposing the drug to various relative humidities at room temperature for two to eight weeks (Table 11). If substantial change is observed at higher temperatures, the drug samples stored at 5 °C are analyzed. If no degradation is seen at higher temperature, then none can be expected at room temperature. The results from higher temperature should be carefully interpreted. For instance, a hydrate may lose moisture at higher temperatures and make a drug unstable which otherwise would be stable at room temperature. Similarly, chlortetracycline hydrochloride converts from the β form to the α form at above 65% relative humidity, in contrast to ω1ω2ω3. To further illustrate the simultaneous effects of the factors on the release rate of drug, a three-dimensional response surface plot based on Equation (7) was generated, as shown in Figure 11. As can be clearly seen from this figure, the release rate can be significantly varied just by tailoring the CA–acetone and CA–water ratios without changing the coating material. In addition, Figure 11 shows that a slight maximum in release rate is observed as the ratio of composition of acetone to water increases. In order to validate the predictive capability of the empirical expression, two formulations with compositions given in Table 3 were selected randomly from the

CONCLUSION

1119

0.40

Release rate (mg/min)

0.30 0.20 0.09 –0.01

ω3 (0.25) ω2 (0.70)

ω1 (0.05)

ω1 (0.25) ω3 (0.25)

ω2 (0.90)

Weight fraction

FIGURE 11 Three-dimensional response surface plot of release rate as function of composition of coating solution.

TABLE 3 Composition of Coating Solution Randomly Selected for Testing Predictive Capability of Empirical Expression Compositon (wt %) Cellulose Acetate 7.5 10

Acetone

Water

Experimental Release Rate (mg/min)

82.5 82.5

10 7.5

0.09 0.027

Predicted Release Rate (mg/min)

R2

0.125 0.029

0.9816 0.9858

experimental design region. Experimental release rates estimated from the slope of the release profiles and corresponding rates predicted from Equation (7) are also listed in Table 3. According to the results, the maximum absolute percentage difference between the experimental and predicted release rates is 3.5%. This value lies within the residuals obtained in deriving Equation (7). Based on this comparison, it is fair to conclude that the empirical expression derived in this study can be used as a tool to predict the release rate of theophylline for any composition within the experimental design region.

6.5.7

CONCLUSION

This chapter has considered the controlled release of drugs from tablet coating systems. These systems are still the preferred route of drug administration due to

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their advantages of ease of administration, maintaining zero-order release rates, and better patient compliance; thus, they hold the major market share in the pharmaceutical industry along with the other formulations of oral drug delivery. In tablet coated systems, diffusion of drug through the coating is usually the rate-limiting step and the desired drug release rates are achieved by properly selecting the coating material and adjusting the morphology of the coating using suitable manufacturing methods. Coating characteristics such as glass transition temperature, crystalline content, and degree of cross-linking significantly determine the release rate of the drug. For a selected coating material, the structure of the coating plays a critical role in achieving useful release rates. The morpholology of the tablet coatings can be varied by incorporating plasticizer or water-soluble additives in the coating solution, by blending the polymers, or by applying multilayer composite coatings. Another approach is to apply asymmetric- and porous-type coatings. The morphological characteristics of these types of coatings, such as fraction of dense top layer and porous sublayer and size and shape of the pores, can be varied by optimizing the composition of the coating solution, evaporation conditions (temperature, relative humidity, and velocity of air), and the type of solvent or nonsolvent used in the coating solution. Asymmetric-type coatings can be used to facilitate osmotic delivery of drugs with low solubilities since high water fluxes can be achieved. These coatings allow us to control the release kinetics without altering the coating material or significantly varying the coating thickness. Currently, considerable research efforts have been directed toward developing protein drug delivery systems due to discovery of numerous protein and peptide therapeutics. The delivery of protein drugs is usually limited to parenteral administration and frequent injections are required due to their short half-lives in the blood. In this regard, development of oral delivery systems is necessary for patient compliance and convenience. The challenge in the design of the oral delivery systems is that they should protect the incorporated drugs from chemical and enzymatic degradation until the drug reaches the delivery site. The protein drug should not be influenced by pH or bacteria and enzymes along the gastrointestinal (GI) tract and should be delivered at the desired site with a desired efficiency. To achieve sitespecific delivery for protein- and peptide-based drugs, one of the strategies that has often been investigated is to coat the drug core with polymers that can respond to the stimuli of local environments such as pH and enzymes. For water-soluble protein drugs, such as insulin, an additional protective coating is usually required to isolate the drug from the surrounding water. Composite tablet coating materials which combine the enzymatic susceptibility and protective properties of polymers can be another solution for this problem. A systematic comparison study among various polymer combinations is required to select the appropriate coating materials for specific drugs. Not only the selection of the coating material but also the manufacturing technique for the preparation of tablet coatings plays a critical role and still remains one of the most challenging subjects in the controlled drug delivery area. Proteins are very sensitive to conditions that can occur during the coating process. Mechanical stresses during the preparation, exposure to a hydrophobic organic solvent, intermediate moisture level during hydration, and interaction between protein and polymer can easily inactivate the protein-based drug. Therefore, more research focused on both optimization of coating materials and manufacturing methods for encapsulating the protein and peptide drugs is necessary.

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107. Lai, J.-Y., Lin, F.-C., Wang, C.-C., and Wang, D.-M. (1996), Effect of nonsolvent additives on the porosity and morphology of asymmetric TPX membranes, J. Membr. Sci., 118, 49–61. 108. Broadhead, K. W., and Tresco, P. A. (1998), Effects of fabrication conditions on the structure and function of membranes formed from poly(acrylonitrile-vinylchloride), J. Membr. Sci., 147, 235–245. 109. Won, J., Park, H. C., Kim, U. Y., Kang, S., Yoo, S. H., and Jho, J. Y. (1999), The effect of dope solution characteristics on the membrane morphology and gas transport properties: PES/-BL/NMP system, J. Membr. Sci., 162, 247–255. 110. Young, T-H., Lin, D.-J., Gau, J.-J., Chuang, W.-Y., and Cheng, L.-P. (1999), Morphology of crystalline Nylon-610 membranes prepared by the immersion–precipitation process: Competition between crystallization and liquid–liquid phase separation, Polymer, 40, 5011–5021. 111. Chuang, W.-Y., Young, T.-H., Chiu, W.-Y., and Lin, C.-Y. (2000), The effect of polymeric additives on the structure and permeability of poly(vinyl alcohol) asymmetricmembranes, Polymer, 41, 5633–5641. 112. Won, J., Lee, H. J., and Kang, Y. S. (2000), The effect of dope solution characteristics on the membrane morphology and gas transport properties. Part 2: PES/BL system, J. Membr. Sci., 176, 11–19. 113. Fan, S.-C., Wang, Y.-C., Li, C.-L., Lee, K.-R., Liaw, D.-J., Huang, H.-P., and Lai, J.-Y. (2002), Effect of coagulation media on membrane formation and vapor permeation performance of novel aromatic polyamide membrane, J. Membr. Sci., 204, 67–79. 114. Mohamed, N. A., and Al-Dossary, A. O. H. (2003), Structure–property relationships for novel wholly aromatic polyamide-hydrazides containing various proportions of paraphenylene and meta-phenylene units. Part III: Preparation and properties of semipermeable membranes for water desalination by reverse osmosis separation performance, Eur. Polym. J., 39, 1653–1667. 115. Pinnau, I., and Koros, W. (1991), Structures and gas separation property asymmetric polysulfone membranes made by dry, wet, and dry/wet phase-inversion, J. Appl. Polym. Sci., 43, 1491–1502. 116. Sharpe, I. D., Ismail, A. F., and Shilton, S. J. (1999), A study of extrusion shear and forced convection residence time in the spinning of polysulfone hollow fiber membranes for gas separation, Sep. Purif. Technol., 17, 101–109. 117. Ismail, A. F., Ng, B. C., and Abdul Rahman, W. A. W. (2003), Effects of shear rate and forced convection residence time on asymmetric polysulfone membranes structure and gas separation performance, Sep. Purif. Technol., 33, 255–272. 118. Matsuyama, H., Teramoto, M., and Uesaka, T. (1997), Membrane formation and structure development by dry cast process, J. Membr. Sci., 135, 271–288. 119. Jansen, J. C., Macchione, M., and Drioli, E. (2005), High flux asymmetric gas separation membranes of modified poly(ether ether ketone) prepared by the dry phase inversion technique, J. Membr. Sci., 255, 167–180. 120. Jansen, J. C., Buonomenna, M. G., Figoli, A., and Drioli, E. (2006), Asymmetric membranes of modified poly(ether ether ketone) with an ultra-thin skin for gas and vapour separations, J. Membr. Sci., 272, 188–197. 121. Kleinbaum, D. G., Kupper, L. L., and Muller, K. E., (1987), Applied Regression Analysis and Other Multivariable Methods, 2nd ed., Duxbury, Belmont, pp. 658–659.

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APPENDIX The in vitro release profiles of the model drug theophylline are shown below in Figures A1 through A10.

FIGURE A1 Release of theohylline from tablet coatings made with 5% CA dissolved in 90% acetone and 5% water.


APPENDIX

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1130




APPENDIX

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1132



FIGURE A10 Release of theohylline from tablet coatings made with 12.5% CA dissolved in 77.5% acetone and 10% water.

6.6 TABLET COMPRESSION Helton M. M. Santos and João J. M. S. Sousa University of Coimbra, Coimbra, Portugal

Contents 6.6.1 6.6.2 6.6.3 6.6.4 6.6.5 6.6.6 6.6.7 6.6.8 6.6.9 6.6.10

Introduction Theory of Particle Compaction Compactibility Tablet Compression Equipment for Tablet Compression Tablet Press Tooling Tablet Engraving Tablet Shape and Profile Tablet Bisect Problems during Tablet Manufacturing 6.6.10.1 Capping and Lamination 6.6.10.2 Picking and Sticking 6.6.10.3 Mottling 6.6.10.4 Weight and Hardness Variation References

6.6.1

INTRODUCTION

Tablets are the most important pharmaceutical dosage from and their design has always been of great interest to pharmaceutical engineering. Since tablets are made by a process of die compaction, although commonly called tablet compression, many investigations have been involved in the task to describe the mechanisms involved in this process. Nevertheless, some considerations should be taken regarding the definitions of stages involved in tablet compression. Compression is one of two Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad Copyright © 2008 John Wiley & Sons, Inc.

1133

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TABLET COMPRESSION

stages involved in the compaction of a two-phase system due to the application of an external force. It is defined as the reduction in the bulk volume of a material as a result of gaseous phase [1, 2]. The second stage is consolidation, which is described as an increase in the mechanical strength of the material resulting in particleto-particle interaction [1, 2]. It is suggested that four mechanisms are basically involved in the process of compression of particles: deformation, densification, fragmentation, and attrition. The process of compression is briefly described as follows: small solid particles are filled in a die cavity and a compression force is applied to it by means of punches and then the formed monolithic dosage form is ejected. The shape of the tablet is dictated by the shape of the die while the distance between the punch tips at the point of maximum compression governs the tablet thickness, and the punch tip configuration determines the profile of the tablet. The compression cycle in a conventional rotary tablet press will be described in detail in this chapter and is illustrated in Figure 1. The physical and mechanical properties of tablets, such as density and mechanical strength, are significantly affected by the process. Since tablet compression relies on the ability of particulates to be compacted, the need to control the critical properties of the materials with respect to readiness or ability to compact is an important issue to the formulator. In order to compress a powder or granulation product into a tablet of specific hardness, a defined compression force must be applied. As pointed out by Shlieout et al. [3], by compressing a constant mass of powder, any variation in the applied force causes a change in the measured force. In addition, the substance itself plays an important role, that is, if it is of good compressibility, then the force needed for compression would be low. It is well known that this compressibility will depend on powder characteristics such as crystal habit and thermodynamic behavior. The structure and strength of tablets are often discussed in terms of the relationship between the properties of the particulate material and the properties of the formed tablet. The properties of a powder that control its evolution into a tablet

(a) Creating thecavity Fill cam

(e) Precompression Solid formation

FIGURE 1

(b) Filling the cavity Suction gravity feeder

(c) Metering thecavity Weight adjustment ramp and head scraper

(f) Compressing Solid formation

(g) Removing solid Ejection cam

(d) Containing the fill Tail over die Pull down after weightcontrol Punch-holding device

Compression cycle. (Courtesy of Thomas Engineering.)

THEORY OF PARTICLE COMPACTION

1135

during compression, which will also relate to the fracture toughness and the tensile strength of the tablets, are the compression mechanics of the particles and their dimensions. Generally, all materials have the ability to store some elastic strain; however, its extent will greatly vary for different materials and will depend upon the intrinsic nature of the material. There are many instances where a brittle material, or its surface, reduces significantly its cohesion or adhesion compared to that of a ductile material [4].

6.6.2 THEORY OF PARTICLE COMPACTION Basically, the process of tablet compression starts with the rearrangement of particles within the die cavity and initial elimination of voids. As tablet formulation is a multicomponent system, its ability to form a good compact is dictated by the compressibility and compactibility characteristics of each component. Compressibility of a powder is defined as its ability to decrease in volume under pressure, and compactibility is the ability of the powdered material to be compressed into a tablet of specific tensile strength [1, 2]. One emerging approach to understand the mechanism of powder consolidation and compression is known as percolation theory. In a simple way, the process of compaction can be considered a combination of site and bond percolation phenomena [5]. Percolation theory is based on the formation of clusters and the existence of a site or bond percolation phenomenon. It is possible to apply percolation theory if a system can be sufficiently well described by a lattice in which the spaces are occupied at random or all sites are already occupied and bonds between neighboring sites are formed at random. The transitional repacking stage is driven by the particle size distribution and shape. This will determine the bulk density as the powder or granulation product is delivered into the die cavity. In this phase, the punch and particle movements occur at low pressure. The particles flow with respect to each other, with the finer particles entering the void between the larger particles, and thus the bulk density of the granulation is increased. Various techniques have been utilized to determine the degree of the two consolidation mechanisms in pharmaceutical solids (initial packing of the particles and elimination of void spaces), namely the rate dependency technique. By applying this technique, stress relaxation data based on the Maxwell model of viscoelastic behavior indicate virtually no rate dependency for elastic or brittle materials. There is also an increase in the calculated yield pressure with an increase in punch velocity for viscoplastic materials such as maize starch and polymeric materials. This is attributed to the reduction of time necessary for the plastic deformation process to occur [6]. For brittle materials such as magnesium and calcium carbonates there is no observed change in the yield pressure with increasing punch velocity [6]. When a force is applied to a material, deformation occurs. When this deformation completely disappears after cessation of the external force, further deformation occurs. Deformations that do not completely recover after release of the stress are known as plastic deformations. The force required to initiate a plastic deformation is known as the yield stress. When the particles are so closely packed that no further filling of the voids can occur, a further increase of the compressional force causes deformation at the points of contact. Both plastic and elastic deformation may occur,

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TABLET COMPRESSION

although one type will predominate for a given material. The ability of materials to be compressed relies on their deformation behavior. The known extreme cases are as follows. For elastic bodies, the force applied to consolidation will be fully given back (action equals reaction). This is expressed as a completely elastic deformation. For plastic bodies, the force applied will be saved as energy in the body and will express no elastic deformation at all. During tablet building, these two processes never occur alone but occur only in combination, as mentioned before. As the external force is increased, the stresses within the particles become great enough and cracks may occur. Fragmentation furthers densification with the infiltration of the smaller fragments into the void spaces being responsible for increasing the number of particles and formation of new and clean surfaces that are potential bonding areas. The mechanisms of fragmentation and plastic deformation are not independent since both phenomena modify particle size distribution, and larger particles do not act as small particles with respect to plastic deformation [7]. The bonding of particles is governed by different mechanisms. The three most considered theories are mechanical theory, intermolecular theory, and liquid surface film theory. The first theory assumes that under pressure the individual particles experience elastic, plastic, or brittle deformation and that the edges of the particles intermesh, forming a mechanical bonding. According to Parrot [2], intermolecular theory states that under pressure the molecules at the points of true contact are close enough so that van der Waals forces interact to consolidate the particles. Liquid surface film theory relies on the presence of a thin liquid film, which may be the consequence of fusion or solution, at the surface of the particle, induced by the energy of compression. Even tough the applied force is not high, it is locally transmitted to small areas of true contact so that a very high pressure will exist at the contact surfaces. This high pressure plays an important role in the melting point and solubility of the material and proves to be essential to bonding. It follows that after releasing the pressure, solidification of the fused material would form solid bridges between the particles. An important consideration has been proposed by Zuurman et al. [8] to explain the action of some excipients during this phase. One of these excipients is magnesium stearate, which is widely used as a lubricant in order to prevent tablets from sticking to the die and punches and minimize wear of tooling. It has been proven that magnesium stearate can have an adverse effect on bonding between particles. The decrease of tablet strength is always considered to be the result of reduction of interparticle bonding due to the addition of a lubricant. The production of tablets with the desired characteristics depends on the stresses induced by elastic rebound and the associated deformation processes during decompression and ejection. Ideally, if only elastic deformation occurred, with the sudden removal of axial pressure the particles would return to their original form, breaking any bonds that may have been under pressure. Finally, as the lower punch rises and pushes the tablet upward, there is a continued residual die wall pressure and considerable energy may be expanded due to the die wall friction. As the tablet is removed form the die, the lateral pressure is relieved, and the tablet undergoes elastic recovery with an increase in the volume of the portion removed from the die. The compression cycle profiles may be used to characterize the consolidation mechanisms of powders as they help to characterize the extent of pressure distribution within the powder bed as well as the formed tablet. The compression behavior

THEORY OF PARTICLE COMPACTION

1137

of powder mixtures is usually characterized using the well-known Heckel equation [9, 10], which describes the relationship between the porosity of a compact and the applied pressure and is based on the assumption that the densification of the bulk powder in the die follows first-order kinetics: ln

1 = kP + A 1 − ρr

(1)

where ρr is the relative density of the compact at pressure σ, P is the applied pressure, and K and A are constants. The constants A and k are determined from the intercept and slope, respectively, of the extrapolated linear region of a plot of ln(1/1 − ρr) versus σ (compaction pressure). The Heckel constant k is related to the reciprocal of the mean yield pressure, which is the minimum pressure required to cause deformation of the material undergoing compression. The intercept obtained from the slope of the upper portion of the curve is a reflection of the densification after consolidation. A large value of k indicates the onset of plastic deformation at relatively low pressure. Thus, K appears to be a material constant. The correlation between k and the mean yield pressure Py gives Equation (2). The constant A is related to the densification during die filling and particle rearrangement prior to bonding [11]: k=

1 Py

(2)

A high ρr value indicates that there will be a high volume reduction of the product due to particle rearrangement. The constant A has been shown to be equal to the reciprocal of the mean yield pressure required to induce plastic deformation. A larger value for A (low yield pressure) indicates the onset of plastic deformation at relatively low pressure, a sign that the material is more compressible. The Heckel plot allows an interpretation of the mechanism of bonding. A nonlinear plot with small value for its slope (a small Heckel constant) indicates that the material undergoes fragmentation during compression. When the plot is linear, it indicates that the material undergoes plastic deformation during compression. In addition to the Heckel approach, other techniques may be applied to the characterization of powder compression. One of these approaches was proposed by Cooper and Eaton [12]:

( )

( )

− k2 V0 − V −k1 + a2 exp = a1 exp V0 − Vs P P

(3)

where V is the volume of the compact at pressure P (m3), V0 is the volume of compact at zero pressure (m3), Vs is the void-free solid material volume (m3), a1, a2, k1, and k2 are the Cooper–Eaton constants. The Kawakita equation [13] describes the relationship between volume reduction and applied pressure according to Equation (4), where P is the applied pressure, V0 is the initial bulk volume, V is the volume at pressure P, a and b are the constants characteristic of the powder under compression, and C is the degree of volume reduction [Equation (5)]:

1138

TABLET COMPRESSION

P P 1 = + C a ab C=

V0 − V V0

(4)

(5)

In the Kawakita equation the particle density is not introduced in the calculations since the model operates on the relative change in volume, which gives the same result whether the relative or the absolute volume is used. The problem in the calculation of this equation is to find the correct initial volume V0. 6.6.3

COMPACTIBILITY

Compactibility of a powdered mixture is defined as the ability of the material to be compressed into a tablet of a specified strength without changing its composition. Investigations have demonstrated that binary mixes of identical composition could have different organizations, depending on the surface energy and particle size of the fraction used. Actually, it has been demonstrated that it is possible to control the organization of binary mixes by modifying the particle sizes of the fractions blended if they have the appropriate surface energies [6]. Generally, only powders that form hard compacts under an applied pressure without exhibiting any tendency to cap or chip can be considered as readily compactible. The compactibility of pharmaceutical powders can be characterized by its tensile strength and indentation hardness, which can be used to determine three dimensionless parameters: strain index, bonding index, and brittle fracture index. To calculate the work of compaction during tableting, it is necessary to have accurate values of force and punch displacement. Differences in the dynamics of powder densification between eccentric and rotary machines were pointed out by Palmieri et al. [14] after compression of microcrystalline cellulose, lactose monohydrate, and dicalcium phosphate dehydrate at different compression pressures. The effect of the longer dwell time of the rotary machine press on the porosity reduction after the maximum pressure is reached is more noticeable in a ductile material such as microcrystalline cellulose. It has been shown that Heckel parameters obtained in the rotary press are in some cases different from those recovered in the eccentric machine because of the longer dwell time, machine deflection, and punch tilting occurring in the rotary press, although theoretically they could better describe the material densification in a high-speed production rotary machine. Williams and McGinity [15] studied and compared the compaction properties of microcrystalline cellulose from six different sources using tableting indices. It was demonstrated that storage of compacts at elevated humidity conditions prior to determining the tableting indices decreased the magnitude of the tensile strength, dynamic indentation hardness, and bonding index. Based on the differences in physicomechanical properties observed for the tableting indices, the authors stated that microcrystalline cellulose products from different sources are not directly interchangeable and showed that the tensile strength, indentation hardness, bonding index, and brittle fracture index for compacts composed of microcrystalline cellu-

COMPACTIBILITY

1139

lose in combination with either talc or magnesium stearate generally decreased as the amount of the lubricant was increased over the concentration range of 0–9%. Similar results were observed for admixtures of sodium sulfathiazole in combination with either talc or magnesium stearate. It was also demonstrated that the tensile strength, indentation hardness, and bonding index increased, and the brittle fracture index decreased as the percent of microcrystalline cellulose was increased in a mixture with sodium sulfathiazole. The results of a study conducted by Muller and Augsburger [16] suggest that the pressure–volume relationship determined during powder bed compression is affected by the instantaneous punch speed profile of the displacement–time waveform for all materials studied, even though they deform by different mechanisms. It appears that the instantaneous punch speed profile of the particular displacement–time waveform is a confounding factor of Heckel analysis. Moisute acts as a plasticizer and influences the mechanical properties of powdered materials for tablet compression. In the case of microcrystalline cellulose, at moisture levels above 5% the material exhibits significant changes consistent with a transition from the glassy state to the rubbery state [17]. The possible influence of moisture on the compaction behavior of powders was also analyzed by Gupta et al. [18]. This work evaluates the effect of variation in the ambient moisture on the compaction behavior of microcrystalline cellulose powder. The work conducted by Gustafsson et al. [19] evaluated the particle properties and solid-state characteristics of two different brands of microcrystalline cellulose (Avicel PH101 and a brand obtained from the alga Cladophora sp.) and related the compaction behavior to the properties of the tablets. The difference in fibril dimension and, thereby, the fibril surface area of the two celluloses were shown to be the primary factor in determining their properties and behavior. The compaction properties of pharmaceutical formulations can be studied experimentally using a variety of techniques, ranging from instrumented production presses to compaction simulators, and methods of analysis. The results are usually plotted as porosity–axial stress functions, which is of interest to compare different materials. However, there are some drawbacks on this type of evaluation. As mentioned by Cunningham et al. [20], this approach is deficient once it considers only the average stress along the direction of compaction, ignoring radial stress transmission and friction. There have been some attempts to overcome the analysis of compaction problems, mostly by introducing numerical modeling approaches. The modeling approaches often used in compaction analysis are (a) phenomenological continuum models, (b) micromechanically based continuum models, and (c) discrete-element models. The parameters that should be analyzed when tableting is under development are as follows: 1. Understanding the formulation and compositional effects on the compaction process, including axial loading and unloading along with ejection 2. Determination of the stress distributions within the powder compact, including residual stresses 3. Optimization of the tablet tooling design

1140

TABLET COMPRESSION

4. Estimation of the density distributions within a tablet that may influence dissolution or mechanical properties 5. Estimation of the compaction force necessary to obtain tablets having given properties 6. Taking into account the effect of the tablet material on the stress distribution on tooling to aid tool design 7. Assessment of the origin of defect or crack formation 8. Optimization of more complex compaction operations such as bilayer and trilayer tablets or compression-coated tablets The demonstration of the validity of the continuum-based modelling approach to tablet compaction requires familiarity with fundamental concepts of applied mechanics. Under the theory of such a mechanism, powder compaction can be viewed as a forming event during which large irrecoverable deformation takes place as the state of the material changes from loose packing to near full density. Moreover, it is important to define the three components of the elastoplastic constitutive models which arose from the growing theory of plasticity, that is the deformation of materials such as powder within a die: 1. Yield criterion, which defines the transition of elastic to plastic deformation 2. Plastic flow potential, which dictates the relative amounts of each component of plastic flow 3. Evolution of microstructure, which in turn defines the resistance to further deformation It is also known that the compression process can be described using static and dynamic models. In the case of static models, time is not considered, although it is a very important factor in the deformation process. The viscoelastic reactions are time dependent, especially for the plastic flow. Recently Picker [21] proposed a three-dimensional (3D) model to help explaining the densification and deformation mechanism experienced by differently deforming materials during compression. According to the author, a single description of the processes during tableting is possible, and thus densification and deformation properties can be clearly distinguished with a single model. This issue has been investigated over the last years, and a comprehensive approach has been developed for the analysis of compaction using continuum mechanics principles. This approach is based on the following components: 1. 2. 3. 4. 5.

Equilibrium equations (balance of forces transmitted through the material) Continuity equation (conservation of mass) Geometry of problem Constitutive behavior of powder (stress–strain behavior) Boundary conditions, including loading (e.g., displacement and velocity) and friction between tooling and powder 6. Initial conditions (e.g., initial relative density of powder)

TABLET COMPRESSION

1141

Due to the significant nonlinearity in material properties and contact stresses, a typical powder compaction problem cannot be solved analytically without major simplifications, and thus a numerical approach is required. The tableting properties of materials also depend on their deformation behavior. It is apparent that the tablet tensile strength is a strong function of the plastic work required for its formation but not a function of the elastic work recovered. Consequently, it is likely that strong and ductile interparticle functions, whose formation dissipated a significant plastic work, result in strong and tough compacts [4]. It should be mentioned that the material parameters do change with compaction and the use of constant material values, which is often applied, is not necessarily appropriate given the evolving microstructure of the deforming powder. The experimental characterization and accompanying analysis allow these material properties to be evaluated within the comprehensive framework of continuum mechanics, which can be useful in analyzing and predicting the effects of constitutive behavior, friction, geometry, loading schedule, and initial condition, for example, initial relative density and powder fill configuration. Ruegger and Celik [22] investigated the effect of punch speed on the compaction properties of pharmaceutical powders with one particular objective: to separate out differences between the effect of the compression and decompression events. Tablets were prepared using an integrated compaction research system. The loading and unloading speeds were varied independently of one another. In general, when the compression speed was equal to the decompression speed, the tablet crushing strength was observed to decrease as the punch velocity increased. When the compression speed was greater than or less than the decompression speed, the results varied, depending on the material undergoing compaction. The authors also stated that the reduction of the unloading speed had a similar effect on the directcompression ibuprofen; however, even greater improvement in the crushing strength was observed when the loading speed was reduced. As a major conclusion, it was demonstrated that the strength of tablets can be improved and some tableting problems such as capping can be minimized or prevented by modifying the rates of loading/unloading. It is important to notice that, in the case of interacting materials, the compatibility of a binary mix will depend mostly on the compatibility of the percolating material [23]. Accordingly, several industrial applications can be made over these findings. In the development phase, it is possible to modify the formulation of interacting systems to increase the drug content without losing the compatibility of the mix, whereas in the production phase, it is possible to increase the compatibility of a poorly compatible active ingredient by sieving or preferably by milling an excipient with good compression qualities without changing the composition of the mixture.

6.6.4 TABLET COMPRESSION The process of tablet compression is divided into three stages: filling, compression, and ejection of the tablet (see Figure 1). During the first stage of a compression cycle the lower punch falls within the die, creating a cavity which will contain the powder or granulation product that flows

1142

TABLET COMPRESSION

from a hopper. The fill volume is determined by the depth to which the lower punch descends in the die. At this moment the particles of the powder or granulation product flow with respect to each other, thus resulting in a close packing arrangement and the physical characteristics of the material (particle size, particle size distribution, density, shape, and individual particle surface properties) associated with process parameters such as flow rate and compression rate, and the relationship between the die cavity and the particle diameter will define the number of potential bonding points between the particles. The packing characteristic of the product to be compressed is greatly affected by the shape of the particles. Since the product to be compressed comprises components of different nature, the voidage of a closely packed system is considerably changed. When the upper punch goes down, its tip penetrates the die, confining the powder or granulation product, letting the particle rearrangement stage to continue and initiating the compression stage as the compression force is applied. As a result, forces resulting from the compression force are transmitted through the interparticulate points of contact created in the previous stage. The porosity of the powder bed is gradually decreased, the particles are forced into intimate proximity to each other, and stress is developed at the interparticulate points of contact. Once the particles have formed contacts, they will deform plastically under the applied load. Deformation of the particles will be characterized by elastic, plastic, fragmentation, or a combination of these phenomena, which will depend on the rate and magnitude of the external applied load, the duration of locally induced stress, and the physical properties of the product under compression. When the particles are in sufficiently close proximity, they are bonded. Particles bond as a result of mechanical interlocking, which is described as entanglement of the particles, phase transition at the points of contact, and intermolecular forces, namely the van der Waals force, hydrogen bonding, and ionic bonding. After formation of the tablet by application of a compression force follows the decompression stage, where the compression force is removed and the upper punch leaves the die. Then, the formed tablet undergoes a sudden elastic expansion followed by a viscoelastic recovery during ejection when the lower punch moves upward.

6.6.5

EQUIPMENT FOR TABLET COMPRESSION

The equipment employed for tablet compression is generally categorized according to the number of compression stations and dislocation mode. Therefore, eccentric model presses have only one compression station (one die and one pair of punches, upper and lower) while rotary models have multiple compression stations (each station with one die and one pair of punches, upper and lower). The basic difference between the two types of compression equipment is that for eccentric models the compression force applied during compression is due to the upper punch whereas for rotary models it is mainly applied by the lower punch. A rotary tablet press machine (Figure 2) comprises a housing in which the compression set and subsets (upper and lower roller assemblies) are mounted, the turret head, the upper cams, the weight control assembly and the lower cams, the hopper,

EQUIPMENT FOR TABLET COMPRESSION

(a)

1143

(b)

FIGURE 2 Rotary tablet press machine: (a) left-side view; (b) black-side view. (1) Cabinet, (2) compression, (3) turret, (4) gear, (5) weight control assembly and lower cams, (6) plate cams, (7) guarding, (8) hopper system, (9) feed frame assembly, (10) take-off chute, (11) aspiration assembly, (12) electrical system.

the feeder assembly, the take-off chute, the aspiration assembly, the gear box and the electrical unit, and the lubrication system. The compression zone is located on the back side of the equipment and employs a maximum load force limited by the type of tooling being used. It is of paramount importance to note that, if a load force is applied over the indicated limit, the press unit will not function properly, resulting in premature wear or possible damage to the tooling. The compression set comprises the hopper and feeder system, the die table, the upper and lower compression rollers, the upper and lower turrets, the excess-material scraper, the tablet stripper, the recirculation channel, and the aspiration system. The hopper is usually made of stainless steel and has the shape of a funnel to contain and deliver the product to be compressed. It may be provided with a window for the observation of the product level and may also be provided with low-level sensors that signal an alarm, shut off the engine, or activate the feeding mechanism to deliver the product when it falls below this level. The feeder system usually consists of three sections (in the case of force feeders) and is ideal for press performance at high speed. The first section of a force feeder system is where the hopper is connected and is responsible for the flow of the product from the hopper to the next sections. The second section is where the die cavities are filled to their maximum capacities, and the third section is where the weight control adjustment takes place. These sections contain paddle systems which prevent packing of the product. The

1144

TABLET COMPRESSION

speed of the paddles is adjustable and should be synchronized with the die table in order to prevent tablet weight variation. Better adjustment of the paddle speed could be achieved when keeping the lowest standard deviation of the compression force. The feeder system height above the die table is usually kept between 0.05 and 0.10 mm. When the product to be compressed is of very fine particles, this height should be kept at 0.025 mm. Presses are commonly equipped with a powder aspiration system which is connected to a vacuum source in order to remove excess powder from the die table. This assembly is essential for a high-speed press working for extended periods of time. Special attention must be taken when the powder product comprises an active ingredient of fine particle size. In this case, aspiration should be minimal in order to prevent loss of the active ingredient. The compression subsets comprise the upper roller assembly and the lower roller assembly. The upper roller assembly is located on the roof plate of the press and utilizes an adjustment system for the regulation of the insertion depth. The lower roller assembly is located on the underside of the die table and utilizes a device for the regulation of the tablet edge thickness. The turret head is fixed to the main shaft of the gear box. It is manufactured in two pieces (upper and lower) which guarantee the alignment between punches and dies. The gear box is mounted on the lower section under the die table and is responsible for transmitting the draft movement of the motor toward the turret head. The upper cams are responsible for guiding the upper punches around the circumference of the turret head. It comprises the filling stage track, which guides the upper punches in an up position during its passage over the feeder system; the upper lowering cam, which guides the upper punches down in order to keep their tips covering the cavities (precompression position) and directs the upper punches to their compression stage; the upper compression roller, which guides the upper punches to their compression position; and the upper filling cam, which guides the upper punches back to the filling track. The weight control assembly, which comprises the weight adjustment cam, is located in the lower section of the press and is regulated by an adjustment system. The lower cams are also located in the lower section of the press and comprise the preweight control (or fill cam), the weight adjustment cam, the lower lowering cam, and the ejection cam. The preweight control guides the lower punches to the full-fill position. The weight adjustment cam guides the lower punches up to the desired fill position. The lower lowering cam guides the lower punches to the precompression position. The ejection cam guides the lower punches and the formed tablets to the discharge position. It is recommended to operate the weight adjustment cam in the approximate center of the fill cam and because of this the fill cam is removable and available in different sizes having a range of approximately 10 mm with an increment range of 4 mm. The choice of the adequate fill cam for the operation of a tablet press with a particular product should be based on the density of the product. According to Figure 3, the fill cam can be adequately chosen when taking into account the density of the material to be compressed and thus the material column height in the die cavity. Rotary tablet presses could be designed to be single, double, or triple sided. A single-sided press comprises one hopper, one set of compression rolls, and one takeoff chute unit whereas double- and triple-sided presses comprise two and three each

EQUIPMENT FOR TABLET COMPRESSION Product column height (mm)

1145

Fill cam (mm) 6

10 8

14 12

18

22

16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

FIGURE 3

Fill cam depth as function of product column height.

of these units, respectively. Irrespective of the design of the rotary tablet press, the compression cycle is described as follows (Figures 1 and 4). The powder or granulation product contained in the hopper flows to the feeder which spread the product through a large area over the die table in order to provide enough time to fill the die cavity. The die cavity is created when the fill cam guides the lower punches to the full-fill position and enters the feeder area. Note that the die cavity is filled with an excess of the product at this stage of the cycle. Right after, the weight adjustment ramp and head guide the lower punches to the desired fill position. The excess of the product is removed by the scraper and is pushed back by the excess product stripper when entering the recirculation channel. At this stage the lower punches are guided to the first and second lower compression rols (precompression and main compression rolls, respectively) while the upper punches are guided by the upper lowering compression roll to the precompression position and to the compression position by the main compression roll. As the upper punch penetrates the die cavity until a predefined height, the main compression roll applies the compression load over the lower punch, compressing the product in the die. Soon after compression, the upper lifting cam allows the upper punch to leave the die cavity. Simultaneously, due to the ejection cam, the lower punch is pulled,

1146

TABLET COMPRESSION

Upper lifting cam

Upper lowering cam Main compression rolls

Precompressionon rolls

Scraper

Feeder

Tail over die Pull down after weight control cam

Ejection cam

Main compression rolls

Precompressionon rolls

Fill cam

Weight adjustment ramp and head

FIGURE 4 Compression cycle on rotary tablet press. (Courtesy of Thomas Engineering.)

ejecting the formed tablet to the die table. The ejected tablets are then stopped by a scraper and allowed to escape through a chute and collected. At this time the fill cam geometry makes the lower punch go down and a new compression cycle begins.

6.6.6 TABLET PRESS TOOLING Punches and dies are essential tools in the tableting process and therefore are critical to the quality of the tablets produced. Both tools are designed for long life under normal conditions of working, but, in spite of this, they are not proof against careless handing. It is important for those working with a tablet press to be familiar with the terminology used in the industry concerning the punches and dies. Table 1 describes the commonly used terms related to press tooling. Some of the press tooling parts can be identified in Figure 5. When considering a tableting operation, it is important not only to select the appropriate press tooling in terms of dimensional data but also to consider the material of which tools are made. Performance of the press tooling will in part be a function of the material selected for its manufacture. Usually the material and hardness of the compression tooling are left to the manufacturer’s discretion. There are various types of steel available for the manufacture of press tooling. It is important to recognize the individual characteristics of the steel regarding its composition and the percentage of each constituent element. Usually only a small amount of alloying element is added to steels (usually less than 5%) for the purpose of improving hardness and strength corrosion resistance, stability at high or low temperatures, and control of grain size. Some of these elements are as follows: 1. Carbon Principal hardening element. As the carbon content increases, its hardness increases. Increases the tensile strength of the steel.

TABLET PRESS TOOLING

1147

TABLE 1 Tooling Terminology Band Bakelite tip relief Barrel/shank Barrel-to-stem radius Barrel chamfer Barrel flutes Cup depth Die

Die bore Die chamfer Die groove Die height/ overall length Die lock Die outside diameter Die taper Head Head/dwell flat Inside head angle Key Keying angle Land Neck Outside head flat angle/radius Overall length Tip face/cup Tip length Tip relief

Tooling set Tooling station Relief/undercut Stem Working length

Area between opposing cup profiles formed by die wall Undercut groove between lower punch tip straight and relief; assures sharp corner to assist in scraping product adhering to die wall Surface controlled by turret punch guides to ensure alignment with die Provides smooth transition from tip length to barrel Chamfers at ends of punch barrel, eliminates outside corners Vertical slots machined into punch barrel to reduce bearing surface and assist in removing product in punch guides Depth of cup from highest point of tip edge to lowest point of cavity Component used in conjunction with upper and lower punches; accepts product for ocmpaction and is responsible for tablet’s perimeter size and configuration Cavity where tablet is made, shape and size determine the tablet Entry of die bore Groove around periphery of die to allow die to be fixed in press Overall height of die Mechanism used to lock die in position after it is installed in die table Outside diameter of die, compatible with die pockets in press Gradual increase in die bore from point of compaction to mouth of bore, assists ejection End of punch which guides it through press cam track Flat area of head that receives full force of compression rolls at time that tablet is being formed Area of contact with lower cam and upper cam Prevents rotational movement of punches ensuring alignment to shaped and multihole dies Relationship of punch key to tablet shape; position will be influenced by tablet shape, take-off angle, and turret rotation Area between edge of punch cup and outside diameter of punch tip Relieved area between head barrel which provides clearance for die Contact area with press cams and initial contact with pressure rolls Total punch length as measured from head flat to end of tip Portion of punch tip that determines contour of tablet face including tablet embossing Straight portion of stem effective inside die bore Portion of punch stem which is undercut or made smaller than punch tip straight; most common for lower punches in order to reduce friction from punch tip and die wall Complete set of punches and dies to accommodate all stations in tablet press Upper punch, lower punch, and die which accommodate one station in tablet press Mechanical clearance between stem and die bore, sharp edge between tip straight and undercut areas acts to clean die Area of punch opposite head which begins at end of barrel and extends to tip Length of punch from bottom of cup to head flat; together, upper and lower working lengths control tablet thickness and weight; also known as overall length, bottom of cup (OLBC)

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TABLET COMPRESSION

FIGURE 5 Identification of common parts of press tooling (upper and lower punches and die) according to ISO 18084, 2005: (1) upper punch, (2) lower punch, (3) die, (4) key, (5) land, (6) stem, (7) barrel-to-stem chafer, (8) cup depth, (9) tip face, (10) blended land, (11) face, (12) bore, (13) die grove, (14) protection radius or shoulder, (15) chamfer or radius, (16) outer diameter, (17) tip straight, (18) relief, (19) barrel-to-stem radius, (20) working length of tip, (21) overall length, (22) barrel, (23) working length, (24) barrel-to-neck radius, (25) neck-tohead radius, (26) inside head angle, (27) neck, (28) head, (29) head outer diameter, (30) outside head angle, (31) head flat, (32) key orientation angle, (33) upper punch face key position, (34) barrel diameter.

2. Manganese Increases ductility and hardenability of the steel. Also increases the rate of carbon penetration during carbonizing and imparts excellent wear resistance. 3. Nickel Improves the toughness and impact resistance of the steel and mildly increases its hardness. 4. Chromium Increases the hardness of the steel and improves its wear or abrasion resistance. It helps to limit grain size. If added in amounts greater than 5%, it can impart corrosion and wear resistances. 5. Molybdenum Improves hardenability and increases tensile strength of the steel. 6. Vanadium Produces a fine grain size and improves fatigue strength of the steel, just like molybdenum.


1149

7. Tungsten Is used in tool steels to maintain hardness at elevated temperatures. 8. Copper Increases corrosion resistance; nevertheless its content has to be controlled, otherwise the surface quality and hot-working behavior are compromised. The carbon steels comprise alloying elements not exceeding the defined limits of 1% carbon, 0.6% cooper, 1.65% manganese, 0.4% phosphorus, 0.6% silicon, and 0.05% sulfur. For the alloy steels, the limits exceed those for the carbon steels and may also include elements not found in carbon steels. The alloy steels have a specific designation according to the American Iron and Steel Institute (AISI). Such designation is a four-digit number where the first digit stands for the class of the alloy (e.g., 1, carbon; 2, nickel-chromium; 3, molybdenum; 4, chromium), the second digit designates the subclass of the alloy, and the last two digits designate the amount of carbon in 0.01%. The stainless steels comprise at least 10% chromium with or without the addition of any other alloying element. The tool steels are carbon steel alloys with an excess fo carbides which impart hardness and wear resistance. According to the AISI, tool steels are grouped as water hardening (W), shock resisting (S), cold-work oil hardening (O), cold-work medium-alloy air hardening (A), cold-work high-alloy high chromium (D), low alloy (L), carbon tungsten (F), low-carbon mold steels (P1–P19), other mold steels (P20–P39), chromium-based hot work (H1–H19), tungsten-based hot work (H20–H29), molybdenum-based hot work (H40–H59), high-speed tungsten based (T), and high-speed molybdenum based (M). The appropriate steel for press tooling should be selected based upon the toughness and wear resistance required by the application, and therefore it is mandatory to have satisfactory knowledge regarding the abrasiveness, corrosiveness, and lubricity of the product intended for compression as well as the desired dimensions of the punch tip. The toughness of the steel regards its ability to resist shock and its wear resistance regards the ability to resist physical damage or erosion due to product contact. For a clear understanding, the Thomas Engineering Press Tooling Manual [24] states the following: Punches manufactured from high carbon/high chromium steel may exhibit improved wear resistance characteristics, however under extreme compression force, the cup may crack due to the brittle nature of the steel. Steels with lower carbon and chromium levels will act conversely. While these steels may be useful in some applications, the majority will require a more moderate balance of toughness and wear resistance. Steel selection for dies is not as critical. In most cases, high wear resistance steel is preferred. The bulk of pharmaceutical tablet press punches are manufactured from S1, S5, S7, or 408 (11% chromium, 8% nickel) tool steel. The S series steels provide a good combination of shock and wear resistance and have a proven record of performance in tableting operations. At one time, 408 or 3% nickel steel was the industry standard because of its superior shock resistance toughness. The S grades however, which have only a slight loss in ductibility by comparison, offer much improved wear characteristics and have all but replaced 408 as the preferred general purpose punch steel. A2, D2 and D3 are high carbon/high chromium steel used for their excellent wear resistance. Among all the steels commonly used for press tooling, D3 has the highest wear resistance. However,

1150

TABLET COMPRESSION

its low toughness rating typically limits its use to dies only. D2 rates slightly lower in abrasion resistance than D3 but its increased toughness makes it suitable for punch use, provided the cup design is not too fragile. A2 is a compromise between the general purpose S grades and D2 in both toughness and wear. It can be used for punches as well as dies. Tungsten carbide, while not actually a steel, is extremely wear resistant and is commonly used to line dies. Punch tips can be manufactured from tungsten carbide; however, the cost of tooling is quite high and restricted to applications where tip fracture due to high compression forces is not likely. Ceramic materials such as partially stabilized zirconia can also be used as die liners. Ceramics offer high wear and corrosion resistance and lower tablet ejection forces than either steel or carbide due to their low coefficient of friction. S1, S7 and 408 provide some protection against mildly corrosive materials. More severe corrosion problems however, demand the use of stainless steel (440C) tooling. From the standpoint of wear, 440C falls between the S and D grades of tool steel. Its low toughness rating (comparable to D3) requires a strong cup design if tip fracture problems are to be avoided. One measure of tool steel quality is the rate of inclusions. Inclusion are unwanted impurities or voids and are present to some degree in all steels. After heat treatment, inclusions give rise to localized areas of stress concentration where microscopic cracks can later develop. Remelting of the steel at the foundry will further reduce a tool steel’s level of impurities; therefore improving the quality of the steel and subsequently its performance in the tooling environment. In cases where punch tip fracture is a problem, tooling suppliers may recommend a remelted or premium grade of particular steel as a means of eliminating the problem.

Concerning the configuration of compression tooling, the most commonly used are the so-called B (19 mm, or 3/4 in.) and D (21 mm, or 1 in.) tooling types. Additionally, these two types are classified into three specifications: the North American TSM (Tableting Specification Manual) [25], the European Union (EU) standard, and the Japan Norm (JN). The North American TSM is used in the United States and is the only standard officially supported by the governing body and published by the American Pharmacists Association. The EU standard and the JN are generally used in Europe and the Far East, respectively. In spite of the existence of these standard specifications, there are tablet press manufacturers that use their own configurations for tooling which have the disadvantage of being restricted to a specific tablet press. Figures 6 and 7 depict the three standard configurations of compression tooling. In addition, the International Organization for Standardization (ISO) standard 18084:2005 [26] comprises specifications of the main dimensions, including tolerances and characteristics of punches and dies. Regarding the importance of compression tooling to the performance of the tablet press and the quality of the tablets, it is of paramount importance that punches and dies are handled with care. The first criterion is the identification of the tooling; that is, punches and dies should be identified according to the standard and be designated by “upper punch without key,” “upper punch with key,” “lower punch with key,” “lower punch without key,” or “die,” the reference of the standard (e.g., TSM, EU, JN, ISO), and the punch or die diameter. Punches and dies should also have a marking that includes at least the manufacturer’s identification, the number of the punch in the series, and/or the identification number. Upon


1151

FIGURE 6 Tooling standards configurations.

receipt, after manufacturing, and prior to inspection, the punches and dies should be carefully and thoroughly cleaned and dried. Then, tooling should be lightly oiled, packed, and stored in a dry, cool place. Damage to the punches and dies should be avoided. Therefore, they should not be transported from place to place without protective package. During transportation, installation, and removal of tooling from the tablet press, cleaning, inspection, and storage, care must be taken to avoid hitting the tips of the punches. The visual and dimensional inspection of punches and dies should be carried out periodically. Visual inspection should be performed each time punches and dies are installed in and removed from the tablet press. Under normal conditions, slight wear is to be expected. When abnormal or excess wear is detected, the cause should be immediately investigated, inspecting the cams or components which touch the affected area of the tool. The importance of visual inspection resides on the fact that it may ensure the optimum life of the punches and dies, performance of the tablet press, and consistency and appearance of the tablets. In addition to the visual inspection, it is also recommended that dimensional inspection be performed at specific intervals throughout the life span of the punches and dies. The dimensional inspection not only ensures the consistency of hardness, weight, and thickness of the tablets, but also proves to be critical in diagnosing potential and real problem areas with regard to the tableting process and press. A typical schedule for the dimensional inspection may be as follows: 50%, 75%, 85%, 90%, and 95% of the historical or projected life cycle of the punches and dies. Therefore, the history or data base should be maintained for each set of tooling. Nevertheless, there is no general agreement on what dimensions of the punches and dies should be included in a

1152

TABLET COMPRESSION

Standard TSM (B-type punches)

TSM domed (B-type punches)

Standard TSM (D-type punches)

TSM domed (D- type punches)

EU standard

Japan norm

FIGURE 7 Tooling head configurations.

dimensional inspection. Some believe that a 100% inspection should be carried out while others defend that only critical dimensions (e.g., working length, cup depth, and overall length) should be inspected, believing that measuring any other dimension is either unnecessary, since it rarely if ever changes and therefore is not worthy of the time and expense of measuring, or cannot be properly measured with current equipment and is better served by a visual inspection. What is important when inspecting compression tooling is that the dimensional values are consistent within the set and tolerances and within specifications. Before proceeding with inspection, the measuring instruments should be calibrated to be certain that the dimensional values obtained are accurate and true.

TABLET SHAPE AND PROFILE

1153

6.6.7 TABLE ENGRAVING Engraving is the most common method for tablet surface marking identification. The engraving method could be embossed (letters or symbols are raised on the tablet surface and cut into the punch tip face) or debossed (letters or symbols are cut into the tablet surface and raised on the punch tip face). For engraving on the tablet surface some specifications should be considered: stroke width, angle of engraving, radius, depth, spacing, and engraving area. Generally, for uncoated tablet application, a stroke width between 15 and 20% of the letter height having an engraving angle of 30 ° is recommended. The radius should be between 50.8 μm (0.002 in.) and a value derived from dividing the stroke width by 2 times the cosine of the engraving angle. It is important to note that radii smaller than 50.8 μm or exceeding the maximum value are difficult to machine since it may decrease depth and definition of engraving. The depth is a function of the engraving angles, stroke, and radius for a given tablet size and, as a general rule, the depth should not exceed 50% of the stroke width, or no less than 88.9 μm (0.0035 in.). Spacing between letters or symbols should be a minimum of one stroke width. The available engraving area is based upon letter distortion due to the curvature or radius of the cup and thus, as a general rule, letter distortion is defined by the ratio of the outside depth of the engraving to the specified depth. Generally, distortion is present when this ratio exceeds 1 : 3. When engraving is considered for fillm-coated tablets, the recommended stroke width should be the same as recommended for uncoated tablets. The recommended engraving angle should be 35 °. However, engraving angles up to 40 ° can be used in extreme applications to allow coating solution flow. For stroke widths of 203.2 μm (0.008 in.) or less, a 30 ° angle is recommended to maintain minimum engraving depth. The recommended radius should be between 76.2 and 152.4 μm and a value derived by dividing the stroke width by 2 times the cosine of the engraving angle. It is important to note that radii will be determined by the flowability of the coating solution and coating process. As a general rule, stroke depth should be at least 177.8 μm. However, shallower depths can be used, provided that the film coating process is properly developed taking this factor into account. The spacing between the letters and symbols and the available engraving area considerations are the same as for uncoated tablets. The following equations can be used to determine stroke width and engraving radii: S = 0.15H ↔ 0.20 H Rmin = 50.8 μm

Rmax =

S 2 cos θ

(6) (7)

6.6.8 TABLET SHAPE AND PROFILE The more popular standard geometric shapes of tablets are the round and the caplet shapes. Other tablet shapes include the oval, elliptical, square, diamond, rectangular, and polygonal. The shape of tablets plays an important role in terms of aesthetics, process (printing, film coating, packaging, and shipping), and acceptability by the consumer (identification, help with swallowing).

1154

TABLET COMPRESSION

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

FIGURE 8 Common tablet shapes and profiles: (A) standard convex, (B) compound cup, (C) convex beveled, (D) flat faced plain, (E) flat faced bevel edged, (F) flat faced radius edged, (G) lozenge, (H) modified ball, (I) core rod with hole in center, (J) capsule, (K) modified capsule, (L) oval, (M) bullet, (N) arrow head, (O) triangle, (P) arc triangle, (Q) square, (R) pillow or arc square, (S) rectangle, (T) modified rectangle, (U) diamond, (V) pentagon, (W) hexagon, (X) octagon, (Y) almond.

In terms of design, the profile of a tablet also plays an important role in the aesthetics, packaging, orientation for printing processes, and handling. The profile of a tablet is important in the film coating process and even in helping with the swallowing. Applying a bisect score onto the tablet surface enables the tablet to be easily divided into smaller dosage amounts. Nowadays it is common for tooling suppliers to use software to provide 2D and 3D technical drawings of tablets and tooling. Such software may provide accurate details of tablets and tooling using only tablet dimensions as input and therefore enables fast evaluation by the manufacturing department prior to ordering prototypes. Figure 8 illustrates some tablet shapes and profiles. Flat-face, bevelled-edge tablets have many advantages due to their flatness, which provides the most compact tablet weight per volume weight, uniform hardness since the compression force is exerted evenly on the cup face, and engraving with no distortion. This tablet profile proves to be ideal for small tablets, especially when engraved, although the engraving area may be limited by the 381 μm (0.015 in.) radius on the bevel. On the other hand, compression tooling displays an inherent weakness in the punch cup design at the point where the bevel edge meets the cup flat. Attention must be paid since these types of tablets cannot be coated as they will stick together, or twin.

TABLET BISECT

1155

Shallow and standard concave tablets have the great advantage of displaying a maximum allowable area available for engraving without distortion as a result of the moderate curvature of the cup profiles and the absence of a bevel. The shallow and standard cup configurations are the strongest profiles per punch tip diameter. In addition, such profiles allow consistent distribution of the compression force over the cup face due to the slight curves involved in the cup, thus contributing to the production of tablets of uniform hardness. Nevertheless, caution must be taken concerning the cup depth since when it approaches the cup edge it may be less than the depth of the engraving. The major disadvantage of these profiles may be due to the angle of the cup profile to the tablet sidewall, which may lead to chipping at the tablet edge during film coating or handling. Caplet-shaped tablets are easier to swallow, aesthetically pleasing, and chipping at the tablet edge generally does not occur during film coating or handling due to the angle of the cup profile to the tablet sidewall. Nevertheless, the increased curvature of the cup reduces compression force by approximately 50% compared to the shallow and standard concave profiles. Distortion of engraving may also be a problem because of the more extreme curvature. During film coating caplet tablets have the potential of sticking together, or twinning, as the tablet sidewalls are parallel. This problem could be alleviated by applying a 76.2 mm (0.003 in.) drop (15.24– 20.32 cm, or 6–8 in. radius) to the sidewalls. The concave oval profile displays a maximum allowable area for engraving and a uniform distribution of the compression force over the cup face. Such an advantage is a consequence of the mild curvature of the cup profile and absence of a bevel. Structurally, concave oval tablets are the strongest of the non-round-shaped tablets. However, due to the angle of the cup profile to the tablet sidewall, chipping at the tablet edge may occur during film coating or handling. The compound cup profile could be used to provide round or oval tablets. This profile provides a good tablet weight per volume but simultaneously presents a weak cup edge, thus being the weakest of all cup configurations. Because of this, the maximum compression force is limited to the minor cup radius on the round shapes and the minor cup radius on the minor side for the oval shapes. In addition, the available engraving area is limited to the blending point of the two radii.

6.6.9 TABLET BISECT Usually known as score or break line, the tablet bisect has the purpose of easily breaking the tablet in predetermined small dosages. According to the TSM, the bisect types range from the most functional (the pressure sensitive, or type G) to the least functional (partial, or type H). Each bisect type has its own characteristic, as can be seen in Figure 9. Generally, the bisect is placed on the upper punch, especially when its depth exceed 40% of the cup depth, in order to avoid problems during ejection of tablets. Nevertheless, the bisect can be placed on the lower punch either when the upper punch is supposed to contain embossed characters or printing that makes difficult the existence of the bisect or when its depth does not exceed 40% of the cup depth. When it is desired to apply a bisect to the upper tablet’s surface but there is interference of engraving or printing, then a modified bisect design should be considered.

1156 Top view

TABLET COMPRESSION Profile (end) view

Profile (side) view

50% max of band thickness A

B

Break radius

C

95% of cup depth D

E Break radius

F Cup radius

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

Bisect radius

75% of cup depth

FIGURE 9 Tablet bisect for concave tooling (according to TSM): (A, B, C) pressuresensitive (type G), (D, E, F) cut through (type D) or European style, (G, H, I) decreasing (type C), (J, K, L) standard protruding (type A), (M, N, O) standard (type E), (P, Q, R) short (type B), (S, T, U) partial (type H).

When considering applying a bisect to a tablet’s surface, careful attention should be taken with respect to the tablet’s cup depth, band thickness, and hardness. Considering these aspects, the specifications for the bisect size are determined taking into account the tablet’s size, engraving or printing, and desired bisect design. The TSM acknowledges two different configurations of bisect for concave tablets: protruding and cut flush. The protruding configuration follows the curvature of a radiused cup and extends past the tip edge of the punch. The cut flush configuration

PROBLEMS DURING TABLET MANUFACTURING

1157

is the most popular bisect configuration since one may experience problems with the protruding configuration. This is explained by the fact that the protruding bisect may run into the tip edge of the lower punch if they become too close during the compression cycle of the press. Among the bisect styles acknowledged by the TSM, the cut-through, also known as the European style, can only be applied on radiused cup designed tablets. Other styles are the standard, the short, and the partial bisects. Compared to the standard style, the cut-through style is said to have the advantage of letting patients better break the tablet into smaller subunits. On the other hand, because the cut-through is wider at the center, it decreases the available tablet surface area for engraving or printing.

6.6.10


Due to either formulation or equipment, some problems can arise during the tablet compression process, such as capping and lamination, picking and sticking, mottling, double printing, weigh variation, and hardness variation. It is the early detection and accurate diagnosis of any of these flaws that can avoid tablet compression process failure and consequently improve its reliability, safety, reduce process downtime and the overall operating cost. Often, some of the above-mentioned problems are not detected during the development of a particular tablet formulation, only appearing during scale-up as the processing speed is increased. Some of the problems experienced during tableting can be solved by shifting the formulation or alleviated by altering the tableting conditions. 6.6.10.1

Capping and Lamination

Capping and lamination are common problems that can be experienced during tableting. Capping is defined as the splitting of one or both lids of a tablet from its body. Lamination is a precursor to capping since it involves the occurrence of layers in a compact parallel to the punch face. Sometimes capping is noticed not during the process but during physical testing, such as friability and hardness. An incipient theory proposed by Train [27] related lamination to radial elastic recovery of the compacted material during ejection. A once-accepted theory formulated that capping and lamination are the result of air entrapped in the tablet under pressure which tries to escape during ejection [28]. This theory is no longer widely accepted. Disagreement arises from the fact that some formulations cap or laminate even at low press speeds. Today, it is believed that the entrapped air may be related to capping but does not affect lamination. A widely accepted theory for lamination presented by Long [29] and reformulated by Ritter and Sucker [30] attributes capping to the residual die wall pressure. This pressure is said to cause internal shear stresses in the tablet causing the propagation of cracks, which results in lamination or capping. The propagation of cracks can be prevented by plastic relaxation of shear stresses. Therefore, materials having sufficient plasticity may not be susceptible to lamination. Some properties of the powder mixture, such as moisture content, type and amount of the binder, and

1158

TABLET COMPRESSION

particle size, are important formulation variables that could be assessed in order to impart plasticity, thus diminishing capping and lamination tendencies. Normally, drugs such as paracetamol, mannitol, ibuprofen, phenazone, and mefenamic acid have poor compression properties and produce tablets that are weak and frequently exhibit capping. Materials that deform elastically or exhibit time dependence are more susceptible to capping and lamination and/or strength reduction, especially as tableting rate is increased. The effect of punch velocity is most marked when transferring a material from an eccentric to a rotary press or when scaling up to larger production size tablet presses. In addition to the possible causes of capping and lamination discussed previously, one should also consider the possibility that shape of the tooling and tooling defects are sources of capping. In such cases the problem can simply be alleviated by repairing or altering press tooling. Usually the process of capping can be evidenced as an increase in tablet height within a few seconds after tablets are ejected from the die. A technique generally applied to characterize and prevent the capping and lamination of a material intended to be compacted is using the brittle fracture index (BFI). The BFI was designed by Hiestand et al. [31] and measures the ability of a material to relieve stress by plastic deformation around a defect. It is obtained by applying Equation (8) and compares the tensile strength of a tablet with a hole in its center (T0), which acts as a built-in stress concentrator defect, with the tensile strength of a similar tablet without a hole (T), both at the same relative density:

( )

T BFI = 0.5 ⎡ 0 − 1⎤ ⎢⎣ T ⎥⎦

(8)

It is said that a material showing a moderate to high BFI value (>0.5) is prone to laminate and cap during the process. A low value of BFI is desirable to minimize lamination and capping during tablet production. Indentation hardness is another measure which finds wide application in the pharmaceutical industry for the assessment of capping and lamination tendency. The indentation hardness measurement employs an indentation hardness tester and is defined as the hardness of a material determined by either the size of an indentation made by an indenting tool under a fixed load or the load necessary to produce penetration of the indenter to a predefined depth. An instrumented indentation hardness tester can be employed for that purpose since it has the ability to measure the intender penetration (H) under the applied force (F) throughout the testing cycle and is therefore capable of measuring both plastic and elastic deformation of the material under test. Another technique for the assessment of capping and lamination tendency which has been increasingly employed in the research-and-development phase of tablet manufacturing is acoustic emission. This technique relies on the fact that an abrupt change in stress within a material to be compacted generates the release of a transient strain energy designated as acoustic emission which results in a mechanical wave that propagates within and on the surface of a structure [5, 32]. Thus, this technique can discriminate between capped and noncapped tablets based on comparing the measured level of acoustic emission energy against a decision threshold.


1159

If it is desirable to overcome capping and lamination during the tableting process, the use of ultrasound-assisted presses could be a reliable solution. However, use this technique is still very recent since reports in the scientific literature extend only over the last decade [33]. In general, when capping and lamination are possible problems during tablet manufacture, an option could be the slower removal of force during decompression. This could be useful since capping tendency increases with increasing rates of decompression. However, better improvements could be achieved if the compression and decompression events are treated separately. By determining the effect of reducing either the loading or unloading speeds on the individual materials, it could be possible to increase crushing strength and eliminate or minimize the incidence of capping and lamination to greater extents. Thus, there is the need for a machine that is capable of customizing compaction profiles so that each formulation can be manufactured under an optimum set of conditions. 6.6.10.2

Picking and Sticking

Picking refers to adherence of powder to the punch surface. It is more problematic when the punch surfaces are engraved with logos or letters such as B, A, or O in order to produce debossed tablets. Sticking occurs when powder tends to adhere to the die leading to the development of an additional pressure to surpass friction between the formed compact and the die wall. As a result, the produced tablets show a rough surface at their edges. Furthermore, sticking can cause picking or damage the press punches by blocking the free movement of the lower punches leading to an increase of compaction pressure. Various approaches can be used to solve picking and sticking problems during tablet manufacturing, namely optimization of press tooling, process parameters, and formulation. Generally, it is important to find the optimal combination of formulation and process parameters, particularly when market image tablets are to be produced. In relation to formulation adjustment, an antisticking agent (talc is commonly used for this purpose) can be added to the powdered formulation in order to eliminate picking and sticking during manufacturing. Colloidal silicon dioxide may be the right choice when picking is evident since this excipient can impart smoothness to the punch surfaces. However, when adding colloidal silicon dioxide to the powder formulation, it would be necessary to add an extra lubricant in order to avoid sticking and facilitate ejection of tablets from the dies. In addition to the need for an extra excipient in the powder formulation, press tooling may need to be adjusted to improve tableting. For the production of market image tablets, logo or letters on the punches should be as big as possible. Additionally, punch tips may be plated with chrome in order to give a smooth and nonadherent surface. When a lubricant such as stearic acid or propylene glycol or any other raw material of low melting point is present in the powder formulation, the heat generated during tableting may cause softening of these ingredients, thus leading to sticking. To overcome this problem, it may be needed to refrigerate the powder load to be tableted or to equip the press machine with a cooling unit.

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6.6.10.3

TABLET COMPRESSION

Mottling

Mottling is defined as an uneven coloration of tablets or nonuniformity of color over the tablet surface. One of the possible causes of mottling may be the difference in color between the active principle and excipients, but sometimes it may be the result of degradation of the active ingredient which imparts spot zones over the surface of the tablets. Nonetheless, when colored compressed tablets are needed for aesthetic reasons, the foremost cause of mottling is dye migration to the periphery of granules during the drying process [34]. To overcome this problem, one should consider changing the solvent used for wet granulation or the binder agent, using a low drying temperature, or decreasing the particle size of the excipient. Another way to overcome mottling was demonstrated by Zagrafi and Mattocks [35] and suggests the inclusion of an adsorbent agent such as wheat or potato starch to the formulation. The adsorbent agent is said to adsorb the dye, retarding its migration then decreasing mottling.

6.6.10.4 Weight and Hardness Variation Weight and hardness variation are common problems experienced when tableting. Tablet weight is mainly affected by factors such as powder variation, tablet press condition and tooling, and flow of powder on the tablet press. Inconsistent powder or granulate density and particle size distribution are common sources of weight variation during tablet compression. Problems related to the density of the powder or granulate are often associated with overfilling of the die and recirculation of the product on the tablet press. A variation of particle size distribution of the powder or granulate can be the result of segregation due to transfer or static electricity. It might also vary because the product cannot withstand the handling and mechanical stress it undergoes before reaching the tablet press. Weight variation can arise as a result of a poorly prepared or operated tablet press. To solve this problem, one should inspect the press performance. Attention must be taken when dealing with a new die table on a load tablet press. In such a case, operation of the tablet press must regard the up-and-down motion of the punches within 76.2 μm of the setting without neglecting the conditions of the pressure rolls and cams. Inspection of the critical dimensions of tablet press tools is recommended. At least three dimensions of the upper and lower punches should be inspected: the working length, the cup depth, and the overall length. The working length is the key factor affecting tablet weight. Therefore, the length of each punch must be correct and identical. The cup depth and the overall length are not critical with regard to controlling tablet weight. Therefore comprehensive inspection and evaluation of the press tooling are essential to minimize deviation of tablet thickness, weight, and hardness. During the course of a compression operation it is also important to not neglect the level of the product in the hopper. Head pressure is a critical factor related to the amount of product in the hopper. The more product present in the hopper, the greater the head pressure, and vice versa. Therefore, when the head pressure varies, so does the weight of the tablets. So, in order to maintain a constant head pressure,

REFERENCES

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thus reducing a potential variation of weight, compression should be conducted within a narrow range of the powder or granulate product in the hopper. The fill cam is another factor that can have a profound effect on tablet weight. The choice of an adequate fill cam regarding some characteristics of the powder or granulate product allows the die cavity to be properly overfilled. Usually, in order to maintain consistent tablet weight during compression, it is recommended to overfill the cam by 10–30% of its volume. Basically, any tablet press part that is ultimately related to the powder product flow can have a mild or profound impact on weight control. It is important to remember that the scraper blade tends to become worn by die table rotation and powder product abrasion. Therefore, periodic inspection of its condition and replacement are recommended. Nevertheless, the scraper blade proper condition is important but also its adjustment since if it is not set up correctly, powder product may accumulate on the die table, leading to problems with weight control. Tablet hardness variation is intimately related to weight variation and, accordingly, to the influence of compression variables such as dwell time, tablet thickness, and working length of the punches. Thus, to solve a hardness variation, consistency of the tablet weight must be checked first. If the predefined weight is achieved but hardness is out of limits, then precompression and compression forces should be adjusted while keeping tablet thickness within target limits. Although dwell time might be a source of hardness variation, adjustment of this parameter may be detrimental to the whole process since the compression rate is slowed. Occasionally, when the tablet weight target is kept within limits but hardness varies, the problem may be due to the formulation. As mentioned previously, the correct use of punches and dies is of paramount importance and periodic inspection is mandatory in order to ensure the compression process has not been compromised. So, when it becomes hard to achieve tablet hardness, it is recommended to first verify tablet weight and thickness consistency and then try to adjust the precompression. The choice to increase the tablet weight even if it is within limits or to reduce the tablet press speed is not convenient and should be used only when there are no more options.

REFERENCES 1. Çelik, M. (1994), Compaction of multiparticulate oral dosage forms, in Ghebre-Sellassier, I., Ed., Multiparticulate Oral Drug Delivery, Marcel Dekker, New York, pp. 181–216. 2. Parrot, E. (1990), Compression, in Lieberman, H. L., and Schwartz, J. B., Eds., Pharmaceutical Dosage Form, 2nd ed., Marcel Dekker, New York, pp. 201–244. 3. Shlieout, G., Wiese, M., and Zessin, G. (1999), A new method to evaluate the consolidation behavior of pharmaceutical materials by using the Fraser-Suzuki function, Drug. Dev. Ind. Pharm., 25(1), 29–36. 4. Mohammed, H., Briscoe, B. J., and Pitt, K. G. (2006), A study on the coherence of compacted binary composites of microcrystalline cellulose and paracetamol, Eur. J. Pharm. Biopharm., 63(1), 19–25. 5. Leuenberger, H., and Leu, R. (1992), Formation of a tablet: A site and bond percolation phenomenon, J. Pharm. Sci., 81(10), 976–982. 6. Khossravi, D., and Morehead, W. T. (1997), Consolidation mechanisms of pharmaceutical solids: A multi-compression cycle approach, Pharm. Res., 14(8), 1039–1045.

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7. Masteau, J. C., and Thomas, G. (1999), Modelling to understand porosity and specific surface area changes during tabletting, Powder Technol., 101(3), 240–248. 8. Zuurman, K., Van der Voort Maarschalk, K., and Bolhuis, G. K. (1999), Effect of magnesium stearate on bonding and porosity expansion of tablets produced from materials with different consolidation properties, Int. J. Pharm., 179(1), 107–115. 9. Heckle, R. W. (1961), An analysis of powder compaction phenomena, Trans. Metall. Soc. AIME, 221, 1001–1008. 10. Heckle, R. W. (1961), Density-pressure relationship in powder compaction, Trans. Metall. Soc. AIME, 221, 671–675. 11. Shivanand, P., and Sprockel, O. L. (1992), Compaction behavior of cellulose polymers, Powder Technol., (69), 177–184. 12. Cooper, A. R., and Eaton, L. E. (1962), Compaction behavior of several ceramic powders, J. Am. Ceram. Soc., 45(3), 97–101. 13. Kawakita, K., and Ludde, K. H. (1970), Some considerations on powder compression equations, Powder Technol., 4, 61–68. 14. Palmieri, G. F., Joiris, E., Bonacucina, G., Cespi, M., and Mercuri, A. (2005), Differences between eccentric and rotary tablet machines in the evaluation of powder densification behaviour, Int. J. Pharm., 298(1), 164–175. 15. Williams, R. O.3rd, and McGinity, J. W. (1989), Compaction properties of microcrystalline cellulose and sodium sulfathiazole in combination with talc or magnesium stearate, J. Pharm. Sci., 78(12), 1025–1034. 16. Muller, F. X., and Augsburger, L. L. (1994), The role of the displacement-time waveform in the determination of Heckel behaviour under dynamic conditions in a compaction simulator and a fully-instrumented rotary tablet machine, J. Pharm. Pharmacol., 46(6), 468–475. 17. Amidon, G. E., and Houghton, M. E. (1995), The effect of moisture on the mechanical and powder flow properties of microcrystalline cellulose, Pharm. Res., 12(6), 923–929. 18. Gupta, A., Peck, G. E., Miller, R. W., and Morris, K. R. (2005), Influence of ambient moisture on the compaction behavior of microcrystalline cellulose powder undergoing uniaxial compression and roller-compaction: A comparative study using near-infrared spectroscopy, J. Pharm. Sci., 94(10), 2301–2313. 19. Gustafsson, C., Lennholm, H., Iversen, T., and Nystrom, C. (2003), Evaluation of surface and bulk characteristics of cellulose I powders in relation to compaction behavior and tablet properties, Drug. Dev. Ind. Pharm., 29(10), 1095–1107. 20. Cunningham, J. C., Sinka, I. C., and Zavaliangos, A. (2004), Analysis of tablet compaction. I. Characterization of mechanical behavior of powder and powder/tooling friction, J. Pharm. Sci., 93(8), 2022–2039. 21. Picker, K. M. (2004), The 3D model: Explaining densification and deformation mechanisms by using 3D parameter plots, Drug. Dev. Ind. Pharm., 30(4), 413–425. 22. Ruegger, C. E., and Celik, M. (2000), The effect of compression and decompression speed on the mechanical strength of compacts, Pharm. Dev. Technol., 5(4), 485–494. 23. Barra, J., Falson-Rieg, F., and Doelker, E. (1999), Influence of the organization of binary mixes on their compactibility, Pharm. Res., 16(9), 1449–1455. 24. Press Tooling Manual (2003), Thomas Engineering Inc. Hoffman Estates, IL, USA. pp. 1–38. 25. Tableting Specification Manual, 7th ed., American Pharmacists Association, p. 130. 26. International Organization for Standardization (ISO), 18084:2005, 1st ed, ISO, Geneva, pp. 1–13.

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27. Train, D. (1956), An investigation into the compaction of powders, J. Pharm. Pharmacol., 8(10), 745–761. 28. Burlinson, H. (1968), Tablets and Tabletting, Heinemann, London. 29. Long, W. M. (1960), Radial pressures in powder compaction, Powder Metall., 6, 73–86. 30. Ritter, A., and Sucker, H. B. (1980), Studies of variables that effect tablet capping, Pharm. Tech., (3), 57–65, 128. 31. Hiestand, E. N., Bane, J. M., Jr., and Strzelinski, E. P. (1971), Impact test for hardness of compressed powder compacts, J. Pharm. Sci., 60(5), 758–763. 32. Joe Au, Y. H., Eissa, S., and Jones, B. E. (2004), Receiver operating characteristic analysis for the selection of threshold values for detection of capping in powder compression, Ultrasonics, 42(1–9), 149–153. 33. Rodriguez, L., Cini, M., Cavallari, N., Passerini, N., Saettone, M. F., Monti, D., and Caputo, O. (1995), Ultrasound-assisted compaction of pharmaceutical materials, Farm Vestn., (46), 241–242. 34. Armstrong, N. A., and Palfrey, L. P. (1989), The effect of machine speed on the consolidation of four directly compressible tablet diluents, J. Pharm. Pharmacol., 41(3), 149–151. 35. Zografi, G., and Mattocks, A. M. (1963), Adsorption of certified dyes by starch, J. Pharm. Sci., 52(Nov.), 1103–1105.

6.7 EFFECTS OF GRINDING IN PHARMACEUTICAL TABLET PRODUCTION Gavin Andrews, David Jones, Hui Zhai, Osama Abu Diak, and Gavin Walker Queen’s University Belfast, Belfast, Northern Ireland


6.7.3

6.7.4

6.7.1

Introduction Milling Equipment 6.7.2.1 Ball Mill 6.7.2.2 Fluid Energy Mill 6.7.2.3 Hammer Mill 6.7.2.4 Cutting Mill Powder Characterization Techniques 6.7.3.1 Powder Sampling 6.7.3.2 Particle Density and Voidage 6.7.3.3 Particle Surface Area 6.7.3.4 Particle Shape Effect of Particle Size Reduction on Tableting Processes 6.7.4.1 Wet Granulation Processes 6.7.4.2 Mixing Processes 6.7.4.3 Flowability of Pharmaceutical Powders 6.7.4.4 Compression Processes References

INTRODUCTION

The importance of size reduction in relation to pharmaceutical active agents and excipients is well known, and the aim of this chapter is to identify methods for particle size reduction, discuss how particle size and shape are characterized, and


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EFFECTS OF GRINDING IN PHARMACEUTICAL TABLET PRODUCTION

recognize the importance of controlling particle characteristics to ensure the success of pharmaceutical powder processing and the manufacture of elegant pharmaceutical products. An initial overview of the implications of size reduction within pharmaceutics and the importance of comminution in relation to variability of active pharmaceutical ingredient (API) surface area, efficacy, and ultimately dosing regimen required to maintain optimum therapeutic effects will be addressed. This will encompass examples from a diverse range of dosage forms, including oral, parenteral, and topical systems. The effects of particle size on the essential characteristics of powders intended for compression (tablets, capsules) such as fluidity and compressibility will be addressed. The need for uniformity of size and the effects of particle size distribution on the homogeneity of mixing/blending and in essence on the uniformity of APIs within the final manufactured dosage form will be highlighted.

6.7.2

MILLING EQUIPMENT

There are many factors that must be taken into consideration in choosing milling equipment. Some of these factors are related to required product specifications such as particle size distribution, but additionally, physical and chemical properties of the material such as particle shape and moisture content must also be taken into consideration. Furthermore, other factors that are related to production requirements (mill capacity and the required production rate) must be carefully balanced to ensure the correct choice of milling equipment. 6.7.2.1

Ball Mill

A ball mill consists of a hollow cylinder mounted such that it can be rotated on its horizontal longitudinal axis (Figure 1). The length of the ball mill is slightly greater than its diameter. A ball mill reduces particle size by subjecting particles to impact and attrition forces generated by moving steel balls or pebbles (grinding medium) that typically occupy 30–50% of the total volume of the mill. It is common for a ball mill to contain balls of different diameters that aid size reduction. Generally, larger diameter balls have a higher tendency to act upon coarse feed materials

(a)

FIGURE 1

(b)

(c)

Ball mill in operation showing correct cascade action.

MILLING EQUIPMENT

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whereas smaller diameter balls facilitate the formation of fine product by reducing void spaces between the balls. The most important factors governing the performance of the mill and the achievement of the desired particle size are as follows: 1. Amount of material required for subsequent testing (sample volume) 2. Speed of rotation of ball mill A high volume of powder feed produces a cushioning effect whereas small sample volumes cause a loss of efficiency and abrasive wear of the mill parts. The amount of material to be milled in a ball mill may be expressed as a material-to-void ratio (ratio of the volume of material to that of the void in the ball charge). As the amount of material is increased, the efficiency of a ball mill is increased until the void space in the bulk volume of ball charge is filled; then, the efficiency of milling is decreased by further addition of material. Rotational speed is the most significant factor controlling the particle size specification. The optimum speed of rotation is dependent on mill diameter. At low angular velocities the balls move with the drum until the force due to gravity exceeds the frictional force of the bed on the drum, and the balls then slide back to the base of the drum. This sequence is repeated, producing very little relative movement of balls so that size reduction is minimal. At high angular velocities the balls are thrown out onto the mill wall by centrifugal force and no size reduction occurs. At about two-thirds of the critical angular velocity where centrifuging occurs, a cascading action is produced. Balls are lifted on the rising side of the drum until their dynamic angle of repose is exceeded. At this point they fall or roll back to the base of the drum in a cascade across the diameter of the mill. By this means, the maximum size reduction occurs by impact of the particles with the balls and by attrition. The critical speed of a ball mill is the speed at which the balls just begin to centrifuge with the mill. Thus, at the critical speed, the centrifugal force is equal to the weight of the ball. At and above the critical speed, no significant size reduction occurs. The critical speed nc is given by the equation nc =

76.6 D

where D is the diameter of the mill. A larger mill reaches its critical speed at a slower revolution rate than a smaller mill. Ball mills are operated at from 60 to 85% of the critical speed. Over this range, the output increases with the speed; however, the lower speeds are for finer grinding. An empiric rule for the optimum speed of a ball mill is n = 57 − 40 log D where n is the speed in revolutions per minute and D is the inside diameter of the mill in feet.

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In practice, the calculated speed should be used initially in the process and modified as required. The use of a ball mill is advantageous in that it may be used for both wet and dry milling and additionally can be successfully employed in batch and continuous operation. Also, the installation, operation, and labor costs involved in ball milling are extremely low in comparison to other techniques, which makes this technique economically favorable. 6.7.2.2

Fluid Energy Mill

Fluid energy milling acts by particle impaction and attrition that are generated by a fluid, usually air (Figure 2). Fluid energy mills can reduce the particle size to approximately 1–20 μm. A fluid energy mill consists of a hollow toroid that has a diameter of 20–200 μm, depending on the height of the loop, which may be up to 2 m. Fluid is injected as a high-pressure jet through nozzles at the bottom of the loop with the high-velocity air, giving rise to zones of turbulence into which solid particles are fed. The high kinetic energy of the air causes the particles to impact with other particles with sufficient momentum for fracture to occur. Turbulence ensures that the high levels of particle–particle collision produce substantial size reduction by impact and attrition. The design of fluid energy mills provides an internal classification system according to their particle size in which the finer and lighter particles are discharged and the heavier, oversized particles, under the effect of centrifugal force, are retained until reduced to a significantly smaller size.

Centrifuging action throws coarser particles outward

Classifier removes fine particles and fluid Solids inlet

Zone of turbulence

Fluid inlet jets

FIGURE 2

Fluid energy mill.

MILLING EQUIPMENT

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Hammers

Feed

Screen Product

FIGURE 3

6.7.2.3

Hammer mill.

Hammer Mill

The main mechanism of size reduction produced by a hammer mill is by impaction that is generated from a series of four or more hammers hinged on a central shaft and enclosed within a rigid metal case (Figure 3). During milling the hammers swing out radially from the rotating central shaft. The angular velocity of the hammers produce strain rates up to 80 s−1, which are so high that most particles undergo brittle fracture. As size reduction continues, the inertia of particles hitting the hammers reduces markedly and subsequent fracture is less probable, so that hammer mills tend to produce powders with narrow particle size distributions. Particle retention within the mill is achieved using a screen, which allows only sufficiently milled particles (defined particle size) to pass through. Particles passing through a given mesh can be much finer than the mesh apertures, as particles are carried around the mill by the hammers and approach the mesh tangentially. For this reason, square, rectangular, or herringbone slots are often used. According to the purpose of the operation, the hammers may be square faced or tapered to a cutting edge or have a stepped form. The particle size achieved may be controlled variation in the speed of the hammers and additionally by careful selection of the size and type of screen. During the operation of a hammer mill the speed of rotation is critical such that below a critical impact speed the rotor turns so slowly that a blending action rather than milling is obtained. Such operating conditions result in significant rises in temperature. Moreover, at very high speeds, there is the probability of insufficient time between successive passes of the hammers for a significant mass of material to fall from the grinding zone. The hammer mill is particularly useful in achieving particles in the approximate size range of 20–40 μm and additionally in producing a particle size distribution that is extremely narrow. The equipment offers ease of use and high levels of flexibilty (speed and screen may be rapidly changed allowing rapid variation in achievable particle size), is easy to clean, and can be operated as a closed system, thus avoiding operator exposure to potent dusts and potential explosion hazards.

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Stationary knives

Rotating knives Screen

Product

FIGURE 4 Cutter mill.

6.7.2.4

Cutting Mill

Particle size reduction using a cutting mill involves successive cutting or shearing a sample using a series of knives attached to a horizontal rotor (Figure 4). This rotary motion pushes the sample against a series of stationary knives that are attached to the mill casing. Size reduction occurs by fracture of particles between the two sets of knives, which have a clearance of approximately a few millimetres. As with a hammer mill a screen is fitted at the base of the mill casing and acts to retain material until a sufficient degree of size reduction has occurred.

6.7.3 6.7.3.1

POWDER CHARACTERIZATION TECHNIQUES Powder Sampling

Powdered materials are used in a wide range of industries, no more so than in the pharmaceutical industry wherein powders are used for the manufacture of a wide range of dosage forms, the two most common being tablets and hard gelatin capsules. Orally administered solid dosage forms are the preferred and most patient convenient, primarily because of the ease of administration and the convenience of handling. Pharmaceutically, orally administered solid dosage forms are generally more favorable because of increased stability in comparison to their liquid counterparts (suspensions, syrups) and the increased control they offer in manipulating drug dissolution in vivo to suit end-use requirements. Solid dosage forms administered via the oral route are an intricate blend of pharmaceutical excipients (diluents,

POWDER CHARACTERIZATION TECHNIQUES

1171

binders, disintegrants, glidants, lubricants, and flavors) and APIs. In order to successfully manufacture acceptable pharmaceutical products, these materials must be adequately mixed and/or granulated to ensure that the resultant agglomerates possess the required fluidity and compressibility and, in addition, avoid demixing during postgranulation processes. Moreover, the final characteristics of tablets or capsules such as drug dissolution rate, disintegration time, porosity, friability and hardness are significantly influenced by the properties of the powder blends used during their manufacture. During product manufacture large volumes of powder blends are fed through production equipment/processes, and it is essential to be able to accurately determine, define, and control powder properties to ensure reproducible manufacture and product performance. Therefore the characterization of the physicochemical properties of powder blends is extremely important. It is well accepted that there are inherent difficulties in characterizing the entire mass of a bulk powder blend or process stream, so it is essential to remove and analyze discrete samples. Sampling is a useful technique that allows an appropriate aliquot to be withdrawn from the bulk so as to collect a manageable amount of powder which is representative of the batch [3], in other words, every particle should have an equal chance of being selected [4]. However, there are many circumstances that may result in the selection of nonrepresentative samples and hence the definition of powder characteristics that are not a true estimation of the entire bulk powder. Typically, powder masses with an extremely wide particle size distribution or diverse physical properties are highly likely to be heterogeneous, which may result in high levels of variability and samples that do not represent bulk mass. Moreover, powder characteristics may change because of the attrition and segregation during transfer that can make sampling extremely difficult. It is well accepted that two types of sampling errors are possible when removing small masses of powder from bulk [5]. 1. Segregation errors, which are due to segregation within the bulk and can be minimized by suitable mixing and the use of a large number of incremental samples to form a larger test sample. 2. Statistical errors, which arise because the quantitative distribution in samples of a given magnitude is not constant but is subject to random fluctuations. Consequently, it is an example of a sampling error that cannot be prevented but can be estimated and indeed reduced by increasing the sample size. Therefore, sampling procedures are of the greatest importance in order to reduce the effect of nonuniform size segregation and nonrandom homogeneity of a system to achieve statistically meaningful sampling results. Careful attention and faithful observance must be demonstrated and it is extremely important that sampling occurs when the powders are in motion [6] and samples are withdrawn from the whole stream for equal periods of time, rather than part of the stream for all of the time [3].

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TABLE 1 Stationary Bulk Sampling Sampling Devices Low volume powder sampler (Figure 5a)

Pneumatic lance sampler (Figure 5c)

Scoop sampler

Thief/spear probe sampler (Figure 5b)

Procedure of Sampling In operation the sampler is inserted into the product to be sampled. At a specific sampling depth the operator pushes down on the T bar, which opens the sampling chamber. When released the springloaded T bar will close the sampling chamber. A gentle flow of air out of the nozzle allows the probe to move through the powder bed. At the site, the air is slowly reversed to draw up a sample, which is collected against a porous plate at the end of the probe [7]. A single swipe of the scoop completely across the powder bulk collects the sample. Each collection should use opposite directions.

One or more cavities are stamped in a hollow cylinder enclosed by an outer rotating sleeve. The thief is inserted into sample with the cavities closed, once opened the sample fills the hole. The cavities are closed and the thief is withdrawn. It must be ensured that samples are withdrawn from different locations

Application and Characteristics Used for small quantity of sample powders. The sampler has a sampling chamber volume approximately equal to 2 mL.

Minimizes powder disturbance and therefore is better than a sample thief, but bias still cannot be avoided [8].

Suitable only for materials that are homogeneous within the limits set by the quantity of material taken by the scoop. It may be used for nonfree-flowing or damp materials where instrumental methods are inappropriate [9]. Thief samplers belong to two main classes, side sampler (has one or more cavities along the probe) and end sampler (has a single cavity at the end of the probe), which are the most common used for stored non-flowing material [10].

There are a number of sampling techniques for particle sampling, which can be classified in many different ways. Here, particle sampling techniques are divided into three parts: stationary bulk sampling (Table 1 and Figure 5), flowing stream sampling (Table 2 and Figure 6), and subsampling (Table 3 and Figure 7). The sampling devices, procedures and application overview of the common used techniques in corresponding fields are shown as follow.


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Press

(a)

Side sampler

End sampler (b) Porous plate

Fluidizing jets

Airflow (c)

FIGURE 5 Stationary bulk sampling: (a) low-volume powder sampler; (b) thief/spear probe sampler; (c) pneumatic lance sampler [7].

6.7.3.3

Particle Density and Voidage

Particle density may be defined as the total mass of the particle divided by its total volume; however, depending upon the different definitions of the total volume (or the different ways to measure the particle volume), there are various definitions of particle density in existence (see Table 4). In order to get clear understanding of the subtle differences between the definitions of various particle density types, an illustration can be formed as shown in Figure 8. Particle Density Methods Density is defined as the ratio of mass to volume, so the density determination can be separated into two steps: measurement of mass and measurement of volume. Determining the mass of an object is rather straightforward; however, it is much more difficult to directly determine the volume of a solid. The volume of a solid object with a regular geometric shape may be calculated

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TABLE 2 Flowing Stream Sampling Sampling Devices Auger sampler (Figure 6a) [Line sampler for stream]

Constant-volume sampler (Figure 6b) [Point sampler for stream]

Diverter sampler (Figure 6c) [Crosssectional sampler for stream]

Full stream sampler (Figure 6d) [Crosssectional sampler for stream]

Procedure of Sampling A pipe with a slot is placed inside the process stream, permitting easy capture of powder through the process stream cross section when rotated. Samples are subsequently then delivered into a separate container by gravitational forces. [6] Sampling occurs when the stream falls down through a pipe and a constant-volume container is inserted or withdrawn from the stream system.

The whole stream is diverted by opening a sliding cover or pivoting an external flap in the bottom of a gravity-flow chutes or pipes or screw conveyors [12]. The samples could be removed to a low-angle laser light-scattering instrument then returned to the process stream [6]. Samples are withdrawn from conveyors, carried out only on the return stroke.

Application and Characteristics While this is often used for stream sampling, it is difficult to collect a representative sample when stream is heterogeneous [10].

Designed to extract a constant volume of homogeneous granular material for subsequent chemical analyses and is not suitable for withdrawing samples for physical analyses [11]. The process could be automated and highly suitable method for online particle size measurement. [7]

Extremely useful for dusty materials provided the trough extends the whole length of the stream and does not overfill [6].

mathematically; however, in most conditions, the shape of a particle is often irregular, especially in powder technology, which makes it extremely difficult to measure geometrically. Therefore, various methods have been developed to determine the volume of particles and powders. The two most in use in both laboratory and industrial settings are liquid and gas displacement methods. The different values of particle density can also be expressed in a dimensionless form, as “relative density” (or specific gravity), which is the ratio of the density of the particle to the density of water. The discussion that follows will give an overview of the common methods used in particle density measurement.

POWDER CHARACTERIZATION TECHNIQUES Process stream

Process stream

Powder flow

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Sample Sampling position (a)

Discharge position (b)

Stream

Normal position Step 1

Sampling stroke Step 2

Discharging sample

Stream Step 3

To analyzer (c)

Samples (d)

FIGURE 6 Flowing stream sampling: (a) auger sampler [6]; (b) constant-volume sampler [6]; (c) diverter sampler; (d) full-steam sampler.

Measurement of Particle Density 1. Liquid Pycnometry Method There are several British standards that deal with liquid pycnometry applied to specific materials [18–23]. A pycnometer bottle is weighted empty (M1), and then full of liquid (M2). Following these two initial measurements, two subsequent measurements are made: a sample of powder approximately one-third of maximum container volume (M3) and the bottle filled to capacity containing the sample and water (M4). Great care is required in the final step to ensure that the liquid is fully wetted and all the air removed. Variations in recorded weight also arise depending on how much liquid escapes when the ground glass stopper is inserted in the liquid-filled container. It is extremely important that the liquid used in this procedure does not solubilize or react with the solid particles. Moreover, the solid particles must not absorb the selected fluid. 2. Gas Pycnometry Method Principally this method is similar to liquid pycnometry in that volume determination is achieved by detecting the pressure or volume change associated with the displacement of a gas (rather than liquid) by a solid object. Given that this method is largely dependent upon the diffusivity of the gas, helium is often used since it has a low molecular weight and a small atomic radius, allowing high diffusivity into small pores. Sample volumes are often displayed on a

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TABLE 3 Subsampling Sampling Devices Coning and quartering (Figure 7a)

Oscillating hopper sample divider (Figure 7c) Revolving sample splitter (Figure 7f)

Riffle/chute splitter (Figure 7e)

Spinning riffler (Figure 7d) Table sampler (Figure 7b)

Procedure of Sampling

Application and Characteristics

A cross-shaped cutter is used to separate the sample heap (which is first flattened at the top) into four equal parts. The segments are drawn apart and two opposite quadrants are combined together. This procedure is repeated at least 4 times until a small enough sample has been generated. Hopper (paddle) oscillates and powder falls into two collectors placed under the hopper (paddle). The revolving feeder distributes the sample material equally (in time) over a number of radial chutes, assuming constant rotational speed [14].

The first choice for non-freeflowing powders and nonflowing powders. Prone to operator bias as fine particles remain in the center of the cone and should never be used with free-flowing powders [13].

The sample is introduced to a rectangular area, divided by parallel chutes leading to two separate receptacles [14]. a steady stream of powder is run into a rotating basket of containers [8]. In a sampling table, powder flows down from the top of an inclined plane, holes and prisms splitting the powder. The powder that reaches the bottom of the plane is the sample.

Used for small quantity of samples. Sample size can be controlled by monitoring time over each collector [7]. Very easy to perform and several versions are available that are suitable for free-flowing powders, dusty powders, and cohesive powders. Handling quantities can vary from 40 L to a few grams. Well-accepted method for sample reduction that is highly suitable for free-flowing powders. Used to produce samples with a minimum volume of 5 mL. Useful in subsampling large samples [15]. Suitable for freeflowing materials [13]. Used for sample reduction with the advantages of low price and lack of moving parts.

digital counter on the testing equipment [24]; however, such volumes are easily calculated using the pressure change and the ideal gas law, PV = nRT. The true density of the particle can be measured using this method if the particles have no closed pores, while the apparent particle density can be measured if there are any closed pores. Additionally, if open pores are filled with wax, envelope volumes may


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Repeat

Sample Discard (a)

(b)

(c)

(d)

Container 1

A

A

A

Container 1 Container 2 (e)

(f)

FIGURE 7 Subsampling: (a) coning and quartering; (b) table sampler [6]; (c) oscillating hopper (paddle) sample divider [6]; (d) spinning riffler (BSI); (e) riffle/chute splitter (BSI); (f) revolving sample splitter [14].

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TABLE 4 Definitions of Density Terms Volumes in Definition Density Types Absolute powder density

Apparent particle density

Apparent powder density

Bulk density

Effective particle density Envelope density

Skeletal density

Tap density True density

Density Definitions Mass of powder per unit of absolute volume, which is defined as the solid matter after exclusion of all the spaces (pores and voids) (BSI) Mass of particles divided by its apparent particle volume, which is defined as the total volume of the particle, excluding open pores but including closed pores (BSI) Mass of powder divided by its apparent powder volume, which is defined as the total volume of solid matter, including open pores and closed pores and interstices (BSI) Mass of the particles divided by the volume they occupy, which includes the space between the particles (ASTM) Mass of a particle divided by its volume, including open pores and closed pores (BSI) Ratio of the mass of a particle to the sum of the volumes of the solid in each piece and voids within each piece, which is, within close-fitting imaginary envelopes, completely surrounding each piece (ASTM) Ratio of the mass of discrete pieces of solid material to the sum of the volumes of the solid material in the pieces and closed pores within the pieces (ASTM) Apparent powder density obtained under stated conditions of tapping (BSI) Mass of a particle divided by its volume, excluding open pores and closed pores (BSI)

ClosedPore Volume

OpenPore Volume

Interparticle Void Volume

Solid Material Volume

Note: BSI = British Standards Institute [16], ASTM = American Society for Testing and Material [17]. , included; , excluded.


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Bulk volume

Envelope/effective Apparent/skeletal True/absolute volume volume volume Eliminating Eliminating Eliminating interparticle open pores, closed pores void cracks volume volume volume

Porous particles in container

Bulk density

Envelope/effective density

Apparent/skeletal density

True/absolute density

FIGURE 8 Various density types. The density value increases from bulk density to true density while the volume value decreases from bulk volume to true volume.

be determined and the difference between envelope and apparent volume can yield the open-pore volume, which is sometimes used as a measure of porosity. 3. Hydrostatic Weighing Method The volume of a solid sample is determined by comparing the mass of the sample in air with the mass of sample immersed in a liquid with a known density. The volume of sample may be calculated using the difference between the two measured mass values divided by the density of the liquid. This method can be used to determine the bulk or apparent volume. It is extremely important that the suspending liquid does not interact with the powder under investigation. 4. Float–Sink or Suspension Method This method involves placing a solid sample into a liquid with known and adjustable density. The density of liquid is incrementally adjusted until the sample begins to sink–float (ASTM C729-75 [25]), or is suspended at neutral density in the liquid (ASTM C693-93 [26]). At the point of equilibrium the density of the sample is equal to the density of the liquid. 5. Bed Pressure Drop Method This technique is based on making measurements of bed pressure drop as a function of gas velocity at two voidages, when gas is passed through the bed of powder in the laminar flow regime [24]. During measurement pressure changes for at least four velocities must be measured. The effective particle density ρp can be calculated using the equation s1 ⎛ ρb1 ⎞ ⎛ ρp − ρb2 ⎞ = ⎜ ⎟ s2 ⎝ ρb2 ⎠ ⎝ ρp − ρb1 ⎠

3

where s is the gradient of pressure drop with gas velocity, ρb is the bulk density, ρp is the particle effective density. 6. Sand Displacement Method The sand displacement method is another useful way of measuring the envelope density of a particle using fine sand as the displacement media. Sand is mixed with a known amount of particles, then the density of the sample particles can be determined from the difference of the bulk density between sand alone and that with samples. 7. Mercury Porosimetry Method Mercury is a nonwetting liquid that must be forced to enter a pore by application of external pressure. Consequently it is an extremely useful and convenient liquid for measuring the density of powders and/or particles. This method can measure the apparent and true density of one sample by

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applying different pressures. At atmospheric pressure, mercury will resist entering pores smaller than about 6 μm in diameter, but at pressures of approximately 60,000 psi (414 MPa) mercury will be forced to enter pores with diameters as small as 0.003 μm [27]. Measurement of Bulk Density Bulk density is very important in determining the size of containers used for handling, shipping, and defining storage conditions for pharmaceutical powders and granules. It is a property that is also pertinent in defining the size of hoppers and receivers for milling equipment and for sizing blending equipment in the scale-up to pilot and to commercial production [28]. The concept of bulk density is the mass of particles divided by the bulk volume, which includes not only the envelope volume of particles but also the spaces between particles, so it should not be confused with particle density [24]. The most convenient method to measure bulk density is to fill the particles into a known volume container (usually cylindrical), level the surface, and weigh the particles in the container. The bulk density is calculated by the mass of the particles divided by the volume that can be read from the scale of the measuring cylinder. In order to minimize experimental errors, the container should be ideally at least 1 L in volume, and the ratio of length and diameter should be about 2 : 1. Also it is recommended to leave the sample for approximately 10 min to achieve an equilibrium volume (density) value before making readings. Given that the bulk volume associated with the particle mass is a mixture of air and solid material, the bulk density value is highly dependent on sample history prior to measurement. Calculation of the tapped density can then be achieved by tapping the bulk powder a specified number of times (to overcome cohesive forces and remove entrapped air) to determine the tapped volume of the powder. The tapped and bulk density values can be used to define the flowability and compressibility of a powder using Carr’s index and the Hausner ratio. 6.7.3.4

Particle Surface Area

Surface area is one of the most important characteristics in particle technology. Particles with a different surface area will express different physical properties that will subsequently affect many applications and ultimately final dosage form properties. Similar to particle density, there are various definitions relating to particle surface area [16]: 1. Adsorption surface area: the surface area calculated from an adsorption method. 2. BET surface area: the surface area calculated from the Brunauer, Emmett, and Teller theory of multilayer adsorption of a gas on a solid surface. 3. Calculated surface area: the surface area of a powder calculated from its particle size distribution. 4. Effective permeability mass-specific surface: the effective volume-specific surface divided by the effective solid density, determined by permeametry.


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5. Effective permeability volume-specific surface: the effective surface area divided by the effective solid volume, determined by permeametry. 6. Permeability surface area: the surface area of a powder calculated from the permeability of a powder bed under stated conditions. 7. Specific surface area (Sw): the surface area of a unit mass of material determined under stated conditions, where Sw is usually expressed in centimeters squared per gram or meters squared per gram and can be used for quality control purposes [28]. Particle Surface Area Determination Methods From the standard definitions of particle surface area, it can be seen that various determination methods are used for surface area measurement, such as adsorption (including Langmuir’s equation for monolayer adsorption and the BET equation for multilayer adsorption), particle size distribution, and permeability methods. The different methods are rarely in agreement because the value obtained depends upon the procedures used and also on the assumptions made in the theory relating the surface area to the phenomena measured. The most common methods used for measuring particle surface area are described below. 1. Gas Adsorption Method Gas adsorption methods measure the surface area of particles/powders through measurement of the amount of gas adsorbed onto the sample surface. The methods can measure both external and internal surfaces (including open pores in the particles) and can yield physically meaningful average particle sizes with nonporous materials [24]. The amount of gas adsorbed depends upon the nature of the solid (adsorbent) and the pressure at which adsorption takes place. The amount of gas (adsorbate) adsorbed can be found by determining the increase in weight of the solid (gravimetric method) or the amount of gas removed from the system due to adsorption by application of the gas laws (volumetric method [6]). The adsorption used in this method is physical adsorption, which is a relatively weak interaction between samples and gases and therefore can be removed by evacuation. In this method, a graph of the number of moles of gas adsorbed per gram of solid, at constant temperature, against the equilibrium gas pressure is called an adsorption isotherm. A point must be chosen on this isotherm corresponding to the completion of the adsorbed monolayer in order to calculate Sw [29]. 2. Permeametry Method This method is based on the fact that the flow rate of a fluid through a bed of particles depends on the pore space, the pressure drop across the bed, the fluid viscosity, dimensional factors such as the area of the bed, and specific surface area (Sw). The determination of permeability can be made either under continuous steady-state flow (constant flow rate) or under variable-flow (constant-volume) conditions. All of the permeability methods are based on the Kozeny–Carman equation, which is used to calculate a surface area of a packed powder bed from its permeability. The Kozeny–Carman equation is expressed as [16] Aε 3 Δp ⎞ Sk = ⎛⎜ ⎝ K (1 − ε)2 Lηq ⎟⎠

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where Sk = effective permeability volume-specific surface of powder assuming only viscous flow occurs in determination (Kozeny–Carman term) A = cross-sectional area of bed of powder perpendicular to direction of flow of air ε = porosity of bed of powder Δp = pressure difference across bed of powder K = Kozeny constant L = linear dimension of bed of powder parallel to direction of flow of air (commonly known as height of powder bed) η = viscosity of air at its temperature at time of determination q = rate of flow of incompressible fluid through bed of powder The specific surface area calculated here only involves the walls of the pores of the bed and excludes the pores within the particles. Therefore, the surface area measured in this method can be much smaller than the total surface area measured by gas adsorption methods [24]. 3. Particle Size Distribution Method The surface area of particles can be determined using particle size and particle shape values. The “equivalent spherical diameter” is used in this technique and many attempts to measure the surface area using this method have led to values that are significantly less than the true value (large deviations arising from inability to define particle shape due to surface irregularities and porosity). Surface area values calculated from particle size distribution methods will in effect establish the lower limit of surface area due to the implicit assumptions of sphericity or other regular geometric shapes and by ignoring the highly irregular nature of real surfaces [30]. Besides the three methods introduced above, there are many other methods of surface area determination: Any surface-dependent phenomenon can be used for such measurement [24]. Some available methods (mercury porosimetry, adsorption from solution, adsorption of dyes, chemisorption, density methods, and secondary ion mass spectroscopy) are explained in more detail elsewhere [6, 30, 31, 32].

6.7.3.5

Particle Shape

Particle behavior is a function of particle size, density, surface area, and shape. These interact in a complex manner to give the total particle behavior pattern [28]. The shape of a particle is probably the most difficult characteristic to be determined because there is such diversity in relation to particle shape. However, particle shape is a fundamental factor in powder characterization that will influence important properties such as bulk density, permeability, flowability, coatablility, particle packing arrangements, attrition, and cohesion [33–36]. Consequently it is pertinent to the successful manipulation of pharmaceutical powders that an accurate definition of particle shape is obtained prior to powder processing. A number of methods have been proposed for particle shape analysis, including shape coefficients, shape factors, verbal descriptions, curvature signatures, moment invariants, solid shape descriptors, and mathematical functions (Fourier series


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expansions or fractal dimensions); these are beyond the scope of this chapter but have been adequately described in other texts [37]. In the most simplistic means of defining particle shape, measurements may be classified as either macroscopic or microscopic methods. Macroscopic methods typically determine particle shape using shape coefficients or shape factors, which are often calculated from characteristic properties of the particle such as volume, surface area, and mean particle diameter. Microscopic methods define particle texture using fractals or Fourier transforms. Additionally electron microscopy and X-ray diffraction analysis have proved useful for shape analysis of fine particles. Particle Shape Measurement 1. Shape Coefficients and Shape Factors There are various types of shape factors, the majority based on statistical considerations. In essence this translates to the use of shape factors that do refer not to the shape of an individual particle but rather to the average shape of all the particles in a mass of powder. However, a method developed by Hausner [38] that uses three factors—elongation factor, bulkiness factor, and surface factor—may be used to characterize the shape of individual particles (Table 5). 2. Determining Particle Shape by Fourier Analysis Fourier transforms have been previously used to determine particle shape and the rollability of individual particles from the coefficients of the resulting series [39]. Moreover, fast Fourier transforms have been successfully used to determine coefficients and a particle “signature” by plotting ln An versus ln n, where An is the nth Fourier coefficient and n is the frequency [29, 40, 41]. In brief, Fourier method consists of finding the centre of gravity of a particle and its perimeter, from which a polar coordinate system is set up. Amplitude spectra of a finite Fourier series in closed form are used as shape descriptors of each particle [42]. Several research papers have focused on the characterization of individual particle shape using Fourier grain analysis or morphological analysis [43–44]. The method has also been extended to the measurement of particle shapes in a blend [45] and to relate particle attrition rate in a milling operation to particle shape [46]. 3. Determining Particle Shape by Electron Microscopy Electron microscopy has been used for the examination of fine powder dispersions and will provide information on particle shape perpendicular to the viewing direction. Standard shadowing procedures may be useful in obtaining information on shape in the third dimension. Scanning electron microscopy can give direct and valuable information on the shape of large particles [47]. 4. Determining Particle Shape by X-Ray Diffraction Broadening The broadening of X-ray diffraction lines is primarily a measure of the departure from singlecrystal perfection and regularity in a material and can therefore be used to characterize particle shape. This is the only method that gives the size of the primary crystallites, irrespective of how they are aggregated or sintered, and is of great value for determining the properties of fine powders [48, 49]. 5. Other Methods for Particle Shape Determination Gotoh and Finney [50] proposed a mathematical method for expressing a single, three-dimensional body

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TABLE 5 Shape Coefficients and Shape Factors Coefficients and Factors

Symbols

Volume shape coefficient

αv

Surface shape coefficient

Volume–surface shape coefficient

Shape factor

Sphericity shape factor

Circularity shape factor

Definitions and Equations αv =

V d3

αs

where V = average particle volume d = mean particle diameter S αs = 2 d

αvs

where S = average particle surface d = mean particle diameter αs α vs = αv

α0

Ψw

where αv = volume shape coefficient αs = surface shape coefficient αo = α vm n where αo = shape factor for equidimensional particle and thus represents part of αv which is due to geometric shape only αv = volume shape coefficient m = flakiness ratio, or breadth/thickness n = elongation ratio, or length/breadth Sphericity = (surface area of sphere having same volume as particle) / (surface area of actual particle) Circularity = (perimeter of particle outline)2 / 4π(cross-sectional or projection area of particle outline)

Source: From refs. 6 and 42.

by sectioning it as an equivalent ellipsoid with the same volume, surface area, and average projected area as the original body. Moreover, wedge-shaped photodetectors to measure forward light-scattering intensity have also been explored for determination of crystal shape [51]. More recently a technique referred to as time of transition (TOT) that was first introduced in 1988 has also been used for the analysis of particle size and shape [52, 53].

6.7.4 EFFECT OF PARTICLE SIZE REDUCTION ON TABLETING PROCESSES Particle size plays a critical role in the efficacy of a drug product. It can impact not only bioavailability but also the efficiency and success of production process and ultimately the properties of the final dosage form.

EFFECT OF PARTICLE SIZE REDUCTION ON TABLETING PROCESSES

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6.7.4.1 Wet Granulation Processes The particle size of an active pharmaceutical ingredient can have significant effect on the processing behavior of a formulation, such as granule growth during wet granulation and hence the resulting granule characteristics. The particle size of the starting material can affect the strength and deformability of moist granules and hence their behavior during the wet granulation process. The effect of particle size on granule growth is a function of several interacting factors, the balance of which largely depends on the nature of the material and the experimental conditions. Differences in granule structure and porosity, resulting from changes in starting material particle size, can also affect other characteristics (e.g., compressibility) of the granulation. Badawy et al. [57] studied the effect of DPC 963 (a nonnucleoside reverse transcriptase inhibitor) particle size on the granule growth, porosity, and compressibility of granules manufactured by a high-shear wet granulation process. It was found that DPC 963 granule growth in the high-shear granulator and the resulting granule compressibility and porosity were sensitive to relatively small changes in drug substance particle size. Decreasing the particle size resulted in more pronounced granule growth and enhanced the porosity and compressibility of the granulation. Higher pore volume for the granulation manufactured using the active ingredient with a smaller particle size may be the reason for its higher compressibility. The high granulation porosity resulted in an increased fragmentation propensity and volume reduction behavior of the granulation that led to increased compressibility. The more porous granulation has higher tendency to densify upon application of the compression force, resulting in closer packing of the particles.

6.7.4.2

Mixing Processes

Mixing may be defined as a unit operation that aims to treat two or more components, initially in an unmixed or partially mixed state, so that each unit of the components lies as nearly as possible in contact with a unit of each of the other components [2]. Whenever a product contains more than one component, mixing will be required in the manufacturing process in order to ensure an even distribution of the active component(s). It is well accepted that mixing solid ingredients is usually more efficient and uniform if the active ingredient and excipients are approximately the same size, which ultimately provides a greater uniformity of dose [1]. Particle size and particle size distribution are important in the powder-mixing process since they largely determine the magnitude of forces, gravitational and inertial, that can cause interparticulate movement relative to surface forces, which resist such motion. As a consequence of high interparticulate forces, as compared with the gravitational forces, powders of less than 100 μm mean particle diameter sizes are not free flowing. Powders that have high cohesive forces due to interaction of their surfaces can be expected to be more resistant to intimate mixing than those whose surfaces do not interact strongly [2]. In moving from one location to another, relative to neighboring particles, a particle must surmount a certain potential energy barrier that arises from forces resisting movement. This effect is a function of both particle size and shape and is most

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pronounced when high packing densities occur. Ideal mixing may be achieved when all the particles of the powder mix have similar size, shape, and density characteristics whereas segregation (demixing) may occur when powder blends are not composed of monosized near-spherical particles but contain particles that differ in size, shape, and density. Segregation is more likely to occur if the powder bed is subjected to vibration. The main reason for segregation in powder blends is the difference in the particle size of the components of the particles contained within the blend. Due to the high diffusivity of small particles, such materials move through the voids between larger particles and so migrate to the lower regions of the powder mix. Moreover, during mixing operations, extremely fine particles have a high tendency to be forced upward by turbulent air currents as the powder blend tumbles and subsequently become isolated from the mixing process through continuous suspension above the blend. When mixing is stopped, these particles will sediment and form a layer on top of the coarser particles. It is important to control the particle size distribution of pharmaceutical granules or powder blends because a wide size distribution can lead to a situation with a high probability of segregation. If this occurs within the hoppers of tablet machines, nonuniform products may be manufactured due principally to large weight variations. Tablet dies are filled by volume rather than weight, and consequently, the establishment of different regions within a hopper containing granules of different sizes (and hence bulk density) will contain a different mass of granules. This will lead to an unacceptable distribution of the active pharmaceutical content within the batch of finished product, even though the drug is evenly distributed by weight throughout the granules. 6.7.4.3

Flowability of Pharmaceutical Powders

Due to the relatively small particle size, irregular shape, and unique surface characteristics, many pharmaceutical powders have a high tendency to be extremely cohesive. This high level of cohesion results in “sticky” powders that have poor flowability, commonly resulting in large mass variability within the final product owing to unpredictable and variable filling of tablet dies. Powders with different particle sizes have different flow and packing properties, which significantly alter the volume of powder expelled from manufacturing equipment during, for example, encapsulation or tablet compression. In order to avoid such problems, the particle sizes of the active pharmaceutical ingredient and other powder excipients should be defined and controlled during formulation so that problems during production are avoided. Most notably, powder flowability is of critical importance in the successful production of acceptable pharmaceutical dosage forms. High levels of flowability within pharmaceutical powders is not just important in the final stages of manufacture but is essential for many industrial processes, particularly mass transport. Poor or uneven powder flow can result in excess entrapped air within powders, which may induce capping or lamination in specific high-speed tableting equipment. Moreover, uneven powder flow that is a direct result of the presence of excess fines within a powder blend will also promote increased particle–die wall friction, lubrica-

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tion problems, and very importantly increased dust contamination hazards to operating personnel. Although particle size is a significant factor controlling the flowability of pharmaceutical powders or granules, other factors must be considered. The presence of molecular forces between particle/granule surfaces increases the probability for cohesion and adhesion between solid particles. Cohesion may be defined as the attractive forces between like surfaces, such as component particles of a bulk solid, whereas adhesion may be defined as the attractive force between two unlike surfaces, for example, between a particle and a tablet punch. It is extremely important to appreciate that cohesive forces acting between particles in a powder bed are attributed mainly to short-range nonspecific van der Waals forces that are significantly altered as particle size and relative humidity change. Cohesion and adhesion are phenomena that occur at the surface of a solid and hence particles with an extremely large surface area will have greater attractive forces than those with a smaller surface area. Consequently particle surface area will have a dramatic effect on the flowability of pharmaceutical powders. Typically, fine particles with very high surface-to-mass ratios will be more cohesive than larger particles, which are influenced more by gravitational forces. Particles larger than 250 μm are usually relatively free flowing, but as the size falls below 100 μm, powders become cohesive and flow problems are likely to occur. Powders having a particle size less than 10 μm are usually extremely cohesive and resist flow under gravity. Although it has been previously stated that particles with similar particle sizes are desirable for pharmaceutical processes, a bulk powder mass with a narrow particle size distribution accompanied with dissimilar particle shapes can produce a bulk mass with inherently different flow properties, owing principally to differences in interparticle contact area. 6.7.4.4

Compression Processes

In general, the strength of a compressed powder depends on the inherent ability of the powder to reduce in volume during compression and the amount of interparticulate attraction in the final compact. The decrease in compact volume with increasing compression load is attributed normally to particle rearrangement, elastic deformation, plastic deformation, and particle fragmentation. Pharmaceutical materials normally consolidate by more than one of these mechanisms [58, 59]. Unmodified paracetamol crystals exhibit poor compressibility during compaction, resulting in weak and unacceptable tablets with a high tendency to cap [60]. Moreover the incidence of capping and lamination during production, following ejection of tablets from the die, depended on the plastic and elastic behaviors of the excipients used [61]. It has been suggested that materials undergoing plastic deformation, in contrast to elastic deformation, display enhanced bond formation and produce strong tablets. The effect of particle size on the compression properties of paracetamol oral dosage forms has been previously reported [62]. Heckel analyses plots indicated that the predominant mechanism of compaction of paracetamol was fragmentation with larger particle fractions experiencing more fragmentation than the smaller particles. Furthermore, Heckel analysis also indicated that, for a given applied

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pressure, the larger particles of paracetamol produced denser compacts than the smaller particles. The results of elastic–plastic energy ratios indicated that the majority of energy involved during compaction of paracetamol was utilized as elastic energy. This suggested a massive elastic deformation of paracetamol particles under pressure, resulting in weak and capped tablets. It was found that larger particles exhibited less elastic recovery and elastic energy compared to smaller particles. This was attributed to increased fragmentation of larger particles, resulting in increased bonding between particles due to the formation of more new, fresh, and clean particle surfaces.

REFERENCES 1. Lachman, L., Lieberman, H. A., and Kanig, J. L. (1986), The Theory and Practice of Industrial Pharmacy Textbook, 3rd ed., Leo & Febiger, Philadelphia. 2. Aulton, M. E. (2002), Pharmaceutics: The Science of Dosage Form Design Textbook, 2nd ed., Churchill Livingstone, London. 3. Svarovsky, L. (1990), Characterization of powders, in Rhodes, M. J., Ed., Principles of Powder Technology, Wiley, Chichester, pp. 35–69. 4. Hawkins, A. E. (1990), Characterizing the single particle, in Rhodes, M. J., Ed., Principles of Powder Technology, Wiley, Chichester, pp. 9–34. 5. Sommer, K. (1981), Sampling error on particle analysis, Aufbereit Tech., 22(2), 96–105. 6. Allen, T. (1997), Particle Size Measurement, 5th ed., Vol. 1, Chapman & Hall, London, pp. 1–62. 7. Venable, H. J., and Wells, J. I. (2002) Powder sampling, Drug Dev. Ind. Pharm., 28(2), 107–117. 8. Kaye, B. H. (1997), Powder Mixing, Chapman & Hall, London, pp. 77–95. 9. British Standard, BS 3406-1 (1986), Methods for Determination of Particle Size Distribution—Part 1: Guide to Powder Sampling, British Standard Institution, London. 10. Bicking, C. A. (1964), Sampling, in Standen, A. Ed. Kirk–OthmerEncyclopedia of Chemical Technology, 2nd ed., Interscience Publishers, New York, pp. 744–762. 11. Hulley, B. J. (1970), Sampling and Sample Conditioning in On-line Fertilizer Analysis, Chem. Eng., 77, 410–413. 12. Clarke, J. R. P., and Carr–Brion, K. G. (1996), Sampling systems for process analysers 2nd ed., The Bath Press, Avon, pp. 148–180. 13. Allen, T. (1964), Sampling and size analysis, Silic. Ind. 29(12), 509–515. 14. Petersen, L. (2004), Representative mass reduction in sampling—A critical survey of techniques and hardware, Chemometr. Intell. Lab. Syst., 74, 95–114. 15. Crosby, N. T., and Patel, I. (1995), General Principles of Good Sampling Practice, Royal Society of Chemistry, Cambridge. 16. British Standard BS 2955 (1993), Glossary of Terms Relating to Particle Technology, British Standards Institution, London. 17. ASTM (1994), Compilation of ASTM Standard Definitions, 8th ed., American Society for Testing and Materials, Philadelphia, PA. 18. British Standard BS 812 (1995), Testing Aggregates, Part 2: Methods of Determination of Density, British Standards Institution, London.

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19. British Standard 1016 (1980), Methods for the Analysis and Testing of Coal and Coke, Part 13: Test Special to Coke, British Standards Institution, London. 20. British Standard BS 1377 (1975), Methods of Tests for Soils for Civil Engineering Purposes, British Standards Institution, London. 21. British Standard BS 3483 (1974), Methods for Testing Pigments for Paints, Part B8: Determination of Density Relative to Water at 4°C, British Standards Institution, London. 22. British Standard BS 4550 (1987), Methods for Testing Cement, Part 3.2: Density Test, British Standards Institution, London. 23. British Standard BS 7755 (1998), Soil Quality, Part 5: Physical Method, Section 5.3: Determination of Particle Density, British Standards Institution, London. 24. Svarovsky, L. (1987), Powder Testing Guide, Methods of Measuring the Physical Properties of Bulk Powders, Published on behalf of the British Materials Handing Board by Elsevier Applied Science, London, pp. 3–33, 79–95. 25. ASTM C729-75 e1 (1995), Standard Test Method for Density of Glass by the Sink-Float Comparator, American Society for Testing and Materials, Philadelphia, PA. 26. ASTM C693-93 (1998), Standard Test Method for Density of Plastics by the Density-Gradient Technique, American Society for Testing and Materials, Philadelphia, PA. 27. Webb, P. (2001), Volume and Density Determinations for Particle Technologists, Micromeritics Instrument Corp, Georgia. 28. Lieberman, H., and Lachman, L. (1981), Pharmaceutical Dosage Forms Tablets, Vol. 2, Marcel Dekker, New York, pp. 112–150, 202–222. 29. Beddow, J. K., and Meloy, T. (1980), Testing and Characterization of Powders and Fine Particles, Heyden & Son, London, pp. 63–64. 30. Lowell, S., and Shields, J. E. (1984), Powder Surface Area and Porosity, 2nd ed., Chapman & Hall, London. 31. Beddow, J. K. (2000), Particle Characterization in Technology, Vol. I, CRC Press, Boca Raton, FL, 3–20. 32. Brunauer, S., Deming, L. S., Deming, W. S., and Teller, E. (1940), Adsorption of gases in multimolecular layers, J. Am. Chem. Soc., 62, 1723–1732. 33. Fonner, D. E., Banker, G. S., and Swarbrick, J. (1966), Micromeritics of granular pharmaceutical solids. 1. Physical properties of particles prepared by 5 different granulation methods. J. Pharm. Sci., 55, 181. 34. Ridgway, K., and Rupp, R. (1969), Effect of particle shape on particle properties, J. Pharm. Pharmacol., 21, 30–39. 35. Shotton, E., and Obiorah, B. A. (1975), Effect of physical properties on compression characteristics, J. Pharm. Sci., 64(7), 1213–1216. 36. Heyd, A., and Dhabbar, D., Particle shape effect on caking of coarse granulated antacid suspensions, Drug Cosmet. Ind. 125, 42–45. 37. Hawkins, A. E. (1993), The Shape of Powder-Particle Outline, Wiley, New York. 38. Hausner, H. H. (1967), Characterization of the Powder Particle Shape in Particle Size Analysis, Society for Analytical Chemistry, London. 39. Schwarcz, H. P., and Shane, K. C. (1969), Measurement of particle shape by Fourier analysis, Sedimentology, 13(3–4), 213–231. 40. Meloy, T. P. (1969), Screening, AIME, Washington, DC. 41. Meloy, T. P. (1977), Fast Fourier transforms applied to shape analysis of particle silhouettes to obtain morphological data, Powder Technology, 17, 27–35.

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42. Ehrlich, R., and Full, W. E. (1984), Fourier shape analysis—a multivariate pattern recognition approach, in Beddow, J. K., Ed., Particle Characterization in Technology, Vol. II, Morphological Analysis, CRC Press, Boca Raton, FL. 43. Meloy, T. P., Clark, N. N., Durney, T. E., and Pitchumani, B. (1985), Measuring the particle shape mix in a powder with the cascadograph, Chemical Engineering Science, 40(7), pp. 1077–1084. 44. Alderliesten, M. (1991), Mean particle diameters. part II: standardization of nomenclature. Part. Part. Syst. Charact., 8, 237–241. 45. Fairbridge, C., Ng, S. H., and Palmer, A. D. (1986), Fractal analysis of gas adsorption on syncrude coke, Fuel, 65, 1759–1762. 46. Shibata, T., and Yamaguchi, K. (1990), paper presented at the Second World Congress Particle Technology, Sept., Part 1, Kyoto, Japan. 47. Johari, O., and Bhattacharyya, S. (1969), The application of scanning electron microscopy for the characterization of powders, Power Technol., 2, 335. 48. Hillard, J. E., Cohen, J. B., and Paulson, W. M. (1970), Optimum Procedures for determining ultra fine grain sizes, in Burke, J. J., Reed, N. L., and Weiss, V., Eds., Ultrafine Grain Ceramics, Syracuse University Press, Syracuse, New York, pp. 73. 49. Oel, H. J. (1969), Crystal growth in ceramic powders, Gray, T. J., and Frechette, V. D., Eds., Kinetics of Reactions in Ionic Systems, Plenum, New York, p. 249. 50. Gotoh, K., and Finney, J. L. (1975), Representation of the size and shape of a single particle, Powder Tech., 12, 125–130. 51. Heffel, C., Heitzmann, D., Kramer, H., and Scarlett, B. (1995), paper presented at the 6th European Symp. Particle Size Characterization, Partec 95, Nurenberg, Germany. 52. Karasikov, N., Krauss, M., and Barazani, G. (1988), in Lloyd, P. J., Ed., Particle Size Analysis, Wiley, New York. 53. Manohar, B., and Sridhar, B. S. (2001), Size and shape characterization of conventionally and cryogenically ground turmeric (Curcuma domestica) particles, Powder Technol., 120, 292–297. 54. Abrahamsen, A. R., and Geldart, D. (1980), Behaviour of gas-fluidized beds of fine powders, Part I. Homogeneous expansion, Powder Technol., 26, 35–46. 55. Shibata, T., Tsuji, T., Uemaki, O., and Yamaguchi, K. (1994), Am. Inst. Chem. Eng. Part 1, 59, 95–100. 56. Realpe, A., and Velázquez, C. (2005), Powder Technol., 169, 108–113. 57. Badawy, S. I., Lee, T. J., and Menning, M. M. (2000), Effect of drug substance particle size on the characteristics of granulation manufactured in a high-shear mixer, AAPS PharmSciTech, 1(4). 58. De Boer, A. H., Bolhuis, G. K., and Lerk, C. F. (1978), Bonding characteristics by scanning electron microscopy of powder mixed with magnesium stearate, Powder Technol., 20, 75–82. 59. Duberg, M., and Nystrom, C. (1986), Studies on direct compression of tablets. 17. Porosity pressure curves for the characterization of volume reduction—Mechanisms in powder compression, Powder Technol., 46, 67–75. 60. Krycer, I., Pope, D. G., and Hersey, J. A. (1982), The prediction of paracetamol capping tendencies, J. Pharm. Pharmacol, 34, 802–804. 61. Malamataris, S., Bin-Baie, S., and Pilpel, N. (1984), Plasto-elasticity and tableting of paracetamol, Avicel and other powders, J. Pharm. Pharmacol. 36, 616–617. 62. Garekani, H. A., Ford, J. L., Rubinstein, M. H., and Rajabi-Siahboomi, A. R. (2001), Effect of compression force, compression speed, and particle size on the compression properties of paracetamol, Drug Dev. Ind. Pharm., 27(9), 935–942.

6.8 ORAL EXTENDED-RELEASE FORMULATIONS Anette Larsson,1 Susanna Abrahmsén-Alami,2 and Anne Juppo3 1

Chalmers University of Technology, Göteborg, Sweden AstraZeneca R&D Lund, Lund, Sweden 3 University of Helsinki, Helsinki, Finland 2

Contents 6.8.1

6.8.2

6.8.3

6.8.4

6.8.5 6.8.6

Introduction 6.8.1.1 Background 6.8.1.2 Biopharmaceutical Aspects on Oral ER Formulations 6.8.1.3 Influence of Drug Properties 6.8.1.4 Principles for Extended Drug Release Insoluble Matrix Tablets 6.8.2.1 Principles of Formulation and Release Mechanisms 6.8.2.2 Manufacturing of Insoluble Matrix Tablets Membrane-Coated Oral Extended Release 6.8.3.1 Principles of Formulation and Release Mechanisms 6.8.3.2 Manufacturing of Oral Membrane-Coated Systems Hydrophilic Matrix Tablets 6.8.4.1 Principles of Formulation and Release Mechanisms 6.8.4.2 Manufacturing of Hydrophilic Matrix Tablets Comparison and Summary of Different Technologies Other Oral ER Formulations References

6.8.1 6.8.1.1

INTRODUCTION Background

In order to achieve therapeutic effect, a drug needs to reach the right place in the body at the right time. For some drugs, this may be achieved by simple solutions or solid dosage forms with an instant drug release while, for others, one has to modify Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad Copyright © 2008 John Wiley & Sons, Inc.

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1192

ORAL EXTENDED-RELEASE FORMULATIONS

the drug release. To understand the literature within the area of modified drug release, it is important to be aware of the standard terms used for dosage forms within this field. Malinowski and Marroum have summarized these terms in the book Encyclopedia of Controlled Drug Delivery [1]. The authors state that modified-release (MR) formulations refer to “dosage forms for which the drug release characteristics of time course and/or location are chosen to accomplish therapeutic or convenience objectives not offered by conventional dosage forms.” One group of MR formulations is the delayed-release dosage form, which does not release the drug immediately after administration. One example of delayed-release formulation is the enteric-coated formulations. Another subgroup of MR formulations is the extended-release (ER) dosage forms, which are the focus of the present chapter. According to a definition from the U.S. Pharmacopeia (USP), ER formulations can be referred to as dosage forms that allow at least a twofold reduction in the dosing frequency compared to conventional dosage forms [2, 3]. The interest in oral ER formulations has dramatically increased in recent years. This can be seen in Figure 1, where the bars in the diagram correspond to the number of publications found in a search in the database SciFinder Scholar 2006 [4] that include the words oral extended release. This increase in publications confirms that there are many ongoing activities in this field. The expression oral extended release occurs in the database for the first time in 1954, when Yamanaka et al. utilized the slow dissolution rate of various salts of a drug (pyrimidine penicillin) to extend the period of time when the drug had a clinical effect [5]. In 1959 Robinson and Suedres made a formulation of sulfamethylthiadiazole together with hydrogenated castor oil, which was suspended in an aqueous vehicle, creating a formulation with extended drug release [6]. Later, in the late 1950s and early 1960s Sjögren and Fryklöf compressed active substances (e.g., pentobarbitone sodium and theophyl2000 1800

Number of publications

1600 1400 1200 1000 800 600 400 200 0 2001– 1996– 1991– 1986– 1981– 1976– 1971– 1966– 1961– >1960 2005 2000 1995 1990 1985 1980 1975 1970 1965 Five-year periods

FIGURE 1 Number of publications containing the words “oral extended release” identified in the database SciFinder Scholar 2006 for the five-year period 1960–2005 [4].

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INTRODUCTION

line) together with polyvinyl chloride (PVC) and obtained extended drug release from these insoluble matrix tablets [7]. In an early publication they showed that increased dose loadings or addition of channeling agents increased the drug release rate. It is also interesting to note that the most popular ways to prepare oral ER formulations today were already mentioned in an early review from the early 1970s [8]. 6.8.1.2

Biopharmaceutical Aspects on Oral ER Formulations

The clinical effect of low-molecular-weight substances is often related to the concentration of the drug in the blood plasma. Classical blood plasma profiles for both immediate-release (IR) and ER formulations are shown in Figure 2. It is well known that a drug only has a clinical effect when the concentration in the blood plasma is above the minimum effective concentration (MEC). If the concentration of the active substance is above the maximum safe concentration (MSC), the side effects will be unacceptable. The interval between the MEC and MSC is called the therapeutic window or therapeutic range, and the time when the concentration is above the MEC is called the “duration” of the drug. One aim of ER formulations is to increase the time the substance is above its MEC by continuous release of the drug from the formulation. Under optimal conditions the rate-limiting step in the drug absorption process of an ER formulation is its release rate, which then can be directly related to the concentration of the drug in the blood plasma. When the drug release rate from an ER formulation is constant, the blood plasma concentration will be constant under ideal conditions, whereas ER formulations with time-

30 MSC

25 20

Blood plasma concetration

Blood plasma concentration n

30

Therapeutic range

15 10

MEC

Duration 5 0

MSC

25 20 15 10

MEC

5

Conventional IR Ideal ER ER

0 0

5

10 15 Time (h) (a)

20

25

0

5

10

15

20

25

Time (h) (b)

FIGURE 2 (a) Schematic picture of blood plasma concentration profile after administration of a drug to an individual, including the MSC, MEC, therapeutic range, and duration. (b) Repeated administration of IR formulation (four times daily) of a drug with short pharmacokinetic half-time and administration once daily of an ideal ER formulation with constant drug release (broken line) or ER formulation with nonconstant drug release rate (dotted line).

1194


dependent drug release rate may give rise to time variations in the concentration of the drug in the blood plasma (Figure 2). The ER concept might offer several advantages, such as reduction in administration frequency, reduction of side effects, less irritation in the gastrointestinal tract, and improved patient compliance. Speers and Bonnano have also mentioned some economic aspects of ER formulations, such as the possibility to patent line extensions and to reduce manufacturing costs since fewer units are required to obtain the same effect [9]. On the other hand, ER formulations may have several drawbacks, for example, large variations in effect between patients due to varying physiological factors within the patient group, limited transit time for the ER formulation, drug stability problems during the gastrointestinal passage, and more severe complications such as dose dumping. In order to obtain a clinical effect by an orally administered drug, it is, for example, required that the drug is (i) dissolved and released from its formulation, (ii) transported over the mucosal barrier, and (iii) has passed from the lumen to the systemic blood circulation without being metabolized by, for example, the lumen or the liver. The drug dissolution rate and the rate of absorption of the dissolved active substance as well as the relation between these processes are important, in particular the dissolution process since the absorption of undissolved drug particles can be disregarded. The ER formulations can be a single-unit, monolithic system or multiple-unit systems containing many individual units with extended release. Multiple-unit systems consist of many small pellets and are normally produced by extrusion and spheronization or coating on inert spheres [2, 10]. The composition and ER mechanism can vary for multiple-unit systems, and some examples are membrane-coated reservoir systems and polymer- or lipid-based matrix systems, where the matrices can be made of both soluble and insoluble carriers [11–16]. From a biopharmaceutical point of view, the multiple-unit systems have many advantages, for example, a more consistent gastrointestinal transit compared to larger monolithic systems [17]. The gastrointestinal transit times for monolithic and multiple-unit systems were compared in a study by Abrahamsson et al. [17]. It was found that the gastric emptying time for the small multiple units was considerably shorter than that of larger monolithic systems (on average 3.6 and 9.6 h, respectively). The transit times through the small intestine were approximately equal, whereas the transit time in the colon for the multiple units was longer compared to the monolithic system, which was explained by different influence of the motility on the different systems. Another advantage with multiple-unit compared to monolithic systems is that the effects of dose dumping become less severe [2]. A breakage and instant drug release from one pellet will have considerably lower effect than breakage of one monolithic system. There may also be development and manufacturing advantages and disadvantages with multiple-unit systems compared to monolithic systems. The dose for multiple-unit systems may be easier to adjust since one can readily increase the number of pellets in the formulation and thus increase the dose. One can adjust the drug release profiles for multiple-unit systems by mixing pellets with different release profiles. The multiple-unit systems offer the possibility to mix pellets containing different active substances, which can be an advantage for the patients who then only need to take one formulation at a time (containing more than one active

INTRODUCTION

1195

substance) instead of several formulations. Disadvantages with multiple-unit systems may also exist; for example, the time to develop the multiple-unit systems may well be longer than for monolithic systems. There are greater challenges in the scaleup procedure for the multiple-unit systems since several expensive and specialized types of equipment may be needed [18]. For film-coated formulations, additional changes in the drug release rate may be obtained upon storage as a consequence of aging of the film. However, this change can be suppressed by introducing a curing step in the production [10]. 6.8.1.3

Influence of Drug Properties

Drug properties that are important to consider during development of IR tablets are metabolism, stability, permeability, and solubility [18, 19]. In the development of ER formulations, these aspects are also important, but in addition to IR formulations, they must be considered in relation to the different environments that the ER formulations meet during their passage through the gastrointestinal tract, and some of these aspects will be discussed briefly below. It can be mentioned that, based on these initial properties for drug candidates, Thrombre has constructed a feasibility assessment flow chart for ER formation development [18]. The pharmacokinetic half-life for a drug may give an indication of whether a conventional or ER formulation is to be chosen. For drugs with short biological t50 (less than 1–2 h), devices that continuously release the active substance are required [2, 18]. For drugs with lifetimes longer than about 10 h, ER formulations may not add any benefits compared to IR formulations. However, for drugs with half-lives between these limits, ER formulations may be a good alternative to IR formulations. The stability of a drug in the solid state or in aqueous solution is a critical parameter when selecting an appropriate manufacturing process. A drug in an oral ER formulation reaches aqueous environments with, for example, variations in pH (1– 8), ionic strength, and bile salt concentration, which requires high chemical stability of the drug [19]. Furthermore, the substance should be stable not only against chemical degradation such as hydrolysis but also against enzymatic degradation (metabolism) during the passage from the lumen to the systemic blood circulation. The drug is released from oral ER formulations along the whole gastrointestinal tract. This implies that, in contrast to IR formulations, drug permeability must be good along the whole gastrointestinal tract for drugs in ER formulations [19]. Furthermore, the solubility and the dissolution rate of the drug are extremely important to consider, since these factors will directly influence the release rate for the drugs from ER formulations. The dissolution rate can be described as the flux J of dissolved material from a drug particle and, according to the Noyes–Whitney equation, it is [20, 21]: J=

D (C s − C b ) h

(1a)

where D is the drug diffusion coefficient, h is the thickness of the stagnant diffusion layer around the particles, and Cs and Cb are the concentrations of the drug at the particle surface and in the surrounding bulk media, respectively. For substances in

1196


their most stable solid-state form, the concentration Cs close to the particle surface is equal to the saturated concentration. However, when a substance is in a different polymorphic state or in an amorphous state, Cs can be larger than the saturation concentration. Many drugs are weak acids or bases with one or several pKa values. According to the Henderson–Hasselbalch equation, the solubility of an acidic drug will depend on the pKa and pH as pH = pKa + log

Si S0

(2)

where Si and S0 are the concentrations of the drug’s un-ionized and ionized forms, respectively. Since the pH varies along the gastrointestinal tract, the solubility and the dissolution rate [Equation (1a)] of the drug depend on the position of the drug in the gastrointestinal tract. Furthermore, for some drugs, such as indomethacin, the dissolution of the acid drug may lead to a changed microenvironmental pH within the stagnant layer and thus also influence the dissolution rate [22, 23].

6.8.1.4

Principles for Extended Drug Release

The main principles related to ER systems are as follows: (i) Insoluble matrix formulations (ii) Membrane-coated solid dosage forms including osmotic pump systems (iii) Soluble hydrophilic matrix formulations Below we will discuss each of these formulation principles in terms of basic release mechanisms and the advantages or drawbacks associated with the different formulations and manufacturing processes. However, drug release from all kinds of ER formulations starts with hydration of the formulation and water diffusion into the system. The presence of water in the formulation facilitates the start of the dissolution process of the drug, whereby the dissolved drug can be released from the formulations. The driving forces for transport of water and drug are the differences in chemical potentials between the formulation and its surrounding. Due to the similarities in the driving forces for the dissolution of a drug and the release from an ER formulation, one can modify Equation (1a) to dMt DA ( Cs − C b ) = JA = dt h

(1b)

where Mt is the amount of active drug that is released at the time t, D the drug diffusion coefficient, and J the flux of the drug from the formulation. The other parameters in Equation (1a) have been adjusted to fit the drug release from the formulation and therefore A becomes the surface area of the releasing system in Equation (1b) (e.g., the area of the membrane-coated tablet), h the diffusion pathlength, and Cs and Cb correspond to the concentration of the dissolved active drug at the surface

INSOLUBLE MATRIX TABLETS

1197

of the drug particles/formulation and in the bulk solution surrounding the ER device, respectively. Depending on the exact type of ER system, some modifications in Equation (1b) may be needed to fully describe the drug release. One important factor in the equation is the difference in concentration (Cs − Cb), and Cb is often assumed to be zero due to release under so-called sink conditions. For active substances in their most stable solid state, the remaining concentration, Cs, is equal to the saturation concentration in that medium. However, as mentioned above, Cs can be oversaturated or depend on pH. This means that the drug release from formulations depends on the solid-state properties and pKa of the drug as well as the pH to which the formulation is exposed [24]. Since the pH varies along the gastrointestinal tract, the drug release will be dependent on the position of the formulation in this tract. Several attempts to avoid pH-independent drug release has been made, for example, by including buffers [25–27].

6.8.2 6.8.2.1

INSOLUBLE MATRIX TABLETS Principles of Formulation and Release Mechanisms

The history of insoluble matrix tablets goes back to the beginning of the 1960s, when Hässle and Abbott developed the Duretter and the Gradumet, respectively [2]. Since then, many ER tablets based on this principle have been developed. Looking at the homepage of the U.S. Food and Drug Administration (FDA [28]) and searching for “insoluble matrix tablets” produces more than 140 hits, which indicates that this research area is still active. The term insoluble matrix tablet refers to tablets in which the drug is embedded in an inert carrier that does not dissolve in the gastrointestinal fluids. The carrier material in insoluble matrix tablets can be based on insoluble lipids or polymers, both matrix builders whose function it is to keep the matrix together during the passage through the gastrointestinal tract and thus prolong the diffusion path of the drug before it is released from the formulation. The drug can be dispersed or dissolved or both in a matrix carrier (see Figure 3) and, depending on the formulation, different mechanisms can be regarded to take place: • • • •

Dissolved drug in the matrix diffuses through the matrix. Dissolved drug in the matrix diffuses through pores in the matrix. Dispersed drug dissolves and diffuses through the matrix. Dispersed drug dissolves and diffuses through pores in the matrix.

FIGURE 3 Schematic pictures of insoluble matrix systems. Left: Drug (light gray) molecularly dissolved in carrier material (black); middle: drug particles dispersed in carrier material; right: drug particles dispersed in carrier material at higher drug loading, leading to continuous network of drug.

1198


As early as 1963, Higuchi [29] derived an expression for drug release from insoluble matrix systems. In this historical paper, Higuchi derived two equations for two different geometries, the simple planar sheet matrix system with infinite area and spherical pellets. Furthermore, two special cases were treated, one where the matrix is a homogeneous matrix without pores and another where the matrix contains pores. In the system without pores, the drug is assumed to diffuse through the homogeneous matrix. For matrix systems with pores the efficiency in transport through liquid-filled pores is greater than through the solid matrix carrier. Therefore, the main contribution to the drug release is transport in the pores. This is gained by penetration of the surrounding medium into the pores, where it dissolves the drug. The dissolved drug can diffuse through the pores and be released at the surface of the matrix. The simplest theoretical treatment of the drug dissolved in the matrix carriers assumes the following [30]: • • •

• •

• • •

•

There is no breakage of the matrix. There is no dissolution of the matrix. There is no resistance to drug transport in the boundary layer surrounding the device. There is no accumulation (e.g., adsorption) of the drug in the device. The saturated concentration Cs,m of the drug in the matrix is constant during the process. The drug-loading concentration C0 is larger than Cs,m. The drug concentration around the matrix is zero, Cb = 0 (sink conditions). The diffusion constant Dm in the matrix is independent of the drug concentration. The partition coefficient K between the matrix material and the surrounding release medium is independent of the drug concentration.

A handy derivation of the equation describing the release from planar homogeneous matrix systems can be found in a book by Wu [30]. It is derived for a sheet with the area A and assumes that the concentration of dissolved drug inside the matrix is constant and equal to the saturation concentration Cs,m. Under these assumptions, the amount of drug, Mt, that is released at time t can be predicted as Mt = A [ Dm KCs,m t ( 2C0 − Cs,m )]

0.5

(3)

When the matrix contains a drug-filled network (Figure 3, right image), water can diffuse and dissolve the drug, and this creates a pore structure. The equation describing the drug release from matrices with networks is modified to include information about the created pore structure [Equation (4)]. This can be described by the porosity ε (the volume of the pores in proportion to the total volume of the device) and the tortuosity τ of the pores (a measure of how much the diffusion path is lengthened due to lateral excursions). Also the diffusion coefficient Dm and Cs,m in Equation (3) are replaced with D and Cs in Equation (4), corresponding to the diffusion coefficient and the solubility of the drug in the solution inside the pores, respectively:

INSOLUBLE MATRIX TABLETS

Mt = A ⎡ ⎣⎢

DεCs t ( 2C0 − εCs )⎤ ⎦⎥ τ

1199

0.5

(4)

The most common types of insoluble matrix tablets are those containing pores. From the equations above one can see that the drug-release depends on the solubility of the drug, the drug-loading concentration, and the diffusion coefficient, which is related to the molecular size of the drug. The area of the insoluble matrix tablet also affects the drug release and can be changed by altering the dimensions or the geometry of the tablet. The drug release from insoluble matrix tablets also depends on the porosity and pore structure of the tablet, and the drug release rate increases with increasing porosity. A comparison of Equations (3) and (4) shows that, in both equations, the amounts of released drug are directly dependent on the area of the device, the square root of the time t, the drug-loading concentration C0, the respective saturated drug concentrations, and the drug diffusion coefficients. In addition, the release rate (the time derivate of the amount of released material) depends on the square root of time and can be stated as dMt 1 ∝ dt t

(5)

As pointed out above, for ideal ER formulations, the rate-limiting step for drug absorption is the release rate from the ER formulation. Thus, since the release rate from an insoluble matrix system depends on time, the concentration of drug in the blood plasma will also be time dependent and not constant (Figure 2), which may be a therapeutic drawback. Another factor influencing the concentration of drug in the blood plasma is the gastrointestinal transit times. When the transit times of the formulations vary, the reproducibility between different administration occasions in one patient will be low, and furthermore, great variation in the patient group may be obtained. However, these conclusions are valid for all ER formulations based on matrix systems and not limited to insoluble matrix systems only. The equations above are valid when no depletion of drug occurs inside the device. The equations for release rate will be much more complex when depletion of the drug can occur [30]. However, it has been shown that, when the amount of released material is less than approximately 60%, the release rate will depend on time as t−0.5 [29, 30]. 6.8.2.2

Manufacturing of Insoluble Matrix Tablets

Insoluble matrix tablets need a carrier, which can be a lipid- or polymer-based excipient [7, 31–36]. Some suggestions of carrier materials can be found in Table 1. The table also presents the number of hits found upon searching the FDA’s homepage [37] for the number of times an excipient is registered as a component in oral extended, sustained, or controlled formulations. This list gives an indication of how often these excipients are commercially used in oral ER formulations but does not automatically tell us the exact formulation or exact mechanistic effect of the excipient. The choice of carrier material is important, and one should be aware of possible

1200


TABLE 1 FDA Registered Oral ER Formulations Containing Commonly Used Excipients in Insoluble Matrix Formulations Excipient Lipid based Carnauba wax Stearyl alcohol Glyceryl behenate Castor oil Cottonseed oil, hydrogenated Cetyl alcohol Paraffin Stearic acid Castor oil, hydrogenated Vegetable oil, hydrogenated Mineral oil Microcrystalline wax Insoluble polymer Ethylcellulose Ammonia methacrylate copolymer Polyvinyl acetate Polyethylene Inorganic Calcium phosphate (dibasic)

Number of Hits on FDA Homepage

Content Interval (mg)

9 4 3 2 2 2 2 2 1 1 1 0

46–300 25–244 15–51 23 58–402 44–59 50–150 26–180 295–410 228

9 5 1 0

15–309 37–138 46

6

33–335

Source: http://www.accessdata.fda.gov/scripts/cder/iig/index.cfm.

exposure of lipid-based formulations to erosion, which can be the result of enzymatic degradation of the lipids [38]. This will of course also influence the drug release rate. In the compositions of insoluble matrix systems, excipients other than the carrier material are needed to obtain products with processability and that meet requirements from the pharmacopedias. Examples of categories of excipients included in insoluble matrix tablets are binders, lubricants, glidants, colorant, taste maskers, and channeling agents. As mentioned above, the drug release rate can be regulated by the porosity in the insoluble matrix system. The properties and amounts of drugs and excipients that can create pores will have a large impact on the release rate. Examples of channeling agents are sugars, salts such as sodium chloride, and polyols [2]. The pore structure also depends on other factors such as the particle sizes of the excipients and the drug, the size and porosity of the granules, and the compaction pressure. The choice of process steps depends on the properties of the drug and the chosen excipients. For insoluble matrix tablets one often mixes the active substance with the excipients. Either this mixture can be directly compressed to matrix tablets or the powder mixture can be exposed to a granulation technology to enlarge the particle sizes. One such technique is dry granulation, that is, compaction of the mixture in, for example, a roller compactor, followed by milling to desirable granule sizes. Another granulation technique is melt granulation, where the melted granulation liquid agglomerates the particles to granules. The most common granulation

MEMBRANE-COATED ORAL EXTENDED RELEASE

1201

method for insoluble matrix tablets is probably wet granulation [2, 7] with aqueousbased or organic granulation liquids. The wet granulated masses are dried in fluid bed driers or ovens. In order to increase the drying speed, microwaves can be used. The powder mixtures or granules are compressed in ordinary tableting machines [7]. However, it is an advantage if the compaction pressures can be carefully monitored, since this pressure may influence the porosity and thus the drug release. The final tablet can be coated to, for example, mask the taste. Alternative production methods to the traditional compaction of powder to insoluble matrix tablets are available, some of which are based on melting technologies, but of course these methods rely on the ability of the carrier materials or additives to melt. The drug is commonly dissolved or dispersed in the melt. This melt can be filled into hard gelatin capsules [31] or it can be spray chilled by pressing it through a nozzle into a vessel containing solid carbon dioxide [39]. Hotmelt extrusion of polymer-based systems to form multiple-unit systems has been investigated. The carrier material in these cases can be, for example, Eudragit [40–42].

6.8.3


6.8.3.1

Principles of Formulation and Release Mechanisms

One way to protect the drug from being directly released is to coat the system with an insoluble film. The drug is suspended or dissolved in a reservoir system which can consist of monolithic or multiple-unit systems. The MR films surrounding the reservoir will be insoluble and thus give the system extended drug release properties or they can become soluble by external trigging. The latter case is defined as a delayed-release formulation, which means that the formulation does not release directly after administration. This can be achieved, for example, by enteric coatings, where the film-forming materials are insoluble in aqueous solutions at low pH but soluble at high pH values. This results in delayed release from an enteric-coated formulation, since the pH is low in the stomach. When the units are transported into the intestine, the pH increases, the film dissolves, and the drug can be released immediately. This type of formulation will not be further discussed here, but the work of Hogan is recommended for further information on these systems [43, 44]. In ER reservoir systems, a membrane surrounds a reservoir of the drug, also called the core of the system. The membrane controls the drug release and the driving force is the difference in chemical potential over the membrane, which can be correlated with a concentration gradient over the membrane. The transport of the drug through the ER membranes can be divided into three different mechanisms [45–47]: • • •

Diffusion through the membrane Diffusion through pores and cracks in the membrane Osmotic transport through pores, cracks, or drilled holes

The overall drug release process for common membrane-coated systems has been shown to pass through three different time periods (Figure 4) [48]. During the initial

1202


Fraction released (%)

100.0

80.0

60.0

40.0

20.0

0.0 0

5

10

15

20

25

Time (h) FIGURE 4 Release from membrane-coated reservoir system, where the three different stages are depicted (initial lag period, “steady-state” period, and final depletion period). A schematic picture of a formulation with the drug reservoir (dark grey) surrounded by the membrane (middle grey) is presented at the top. The dissolution medium penetrates the membrane and dissolves the drug (light gray), and pores/cracks are formed through which the drug can be released.

period, the amount of released drug will be low. The water penetrates the membrane and at the same time starts to dissolve the material in existing pores, consisting of water-soluble sugars, salt, or polymers. The water that has penetrated the membrane and reached the core starts to dissolve the active substance in the reservoir [11]. The concentration of the active drug increases continuously until its saturation concentration has been reached and pseudoequilibrium between the solid material and saturated solution inside the membrane has developed. The osmotic pressure in the reservoir will depend on the concentrations of all dissolved species inside the membrane, and an increase of the osmotic pressure may lead to membrane rupture. After an initial lag period, the number of cracks and pores becomes constant, and the osmotic pressure and the concentration of dissolved species in the reservoir and in the membrane reach their steady-state levels. When these parameters, which are the driving forces for the drug release, are constant, the drug release rate will also be constant. Therefore, a second period with time-independent drug release rate will occur. When the solid drug material inside the membrane is completely dissolved and the concentration of the dissolved drug decreases, a final period with declining


1203

release rate will be entered. Ragnarsson et al. [49] have shown that the solid material disappears earlier as the drug solubility increases and that the third stage with time-dependent and decreasing release rate appears earlier compared to drugs with lower solubility. The contribution of diffusion to the release process can be modeled by using Fick’s first law. For diffusion of a substance through the membrane, it will turn out as (assuming sink conditions) dMt D K A D K ACs = JA = m m ( Cs − Cb ) = m m dt h h

(6)

where Km is the partition coefficient for the drug between the membrane and solution, Dm is the diffusion coefficient in the membrane, A is the area of the membrane, h is the thickness of the membrane, and Cs and Cb are the concentrations on the inside of the membrane surface and in the bulk, respectively. The equation for diffusion through pores or cracks resembles Equation (6): dMt DεKA DεKACs ( Cs − C b ) = = JA = dt hτ hτ

(7)

where ε and τ are introduced to describe the porosity and tortuosity in the membrane, respectively. The parameters Dm and Km in Equation (6) are replaced in Equation (7) by D and K, which are the diffusion coefficient in the solution inside the pores and the partition coefficient between the solution and materials surrounding the liquid-filled pores, respectively. Equations (6) and (7) depend on the concentration gradient over the membrane, and both are independent of the time. The osmotic contribution to the drug transport is described by the so-called Kedem–Katchalsky equations (based on nonequilibrium thermodynamics) [50, 51]. A simplified version is ACs Lp σΔΠ dMt = JA = h dt

(8)

where ΔΠ is the osmotic pressure difference over the membrane and Lp and σ are the hydraulic permeability and the reflection, respectively. A comparison of Equations (6)–(8) shows important similarities; they depend on the solubility of the drug, the area of the device, and the thickness of the membrane. This means that an increased solubility, larger area of the membrane, and thinner membranes will facilitate the drug release rate. This can be exemplified by a study by Ragnarsson and Johansson [11], who showed that, for different salt forms of metoprolol, an increased solubility also increased the drug release rate, which was predicted from the equations. Furthermore, Equations (6)–(8) show constant and time-independent release rates. This constant amount of released drug will be a biopharmaceutical benefit since it theoretically makes it possible to achieve a constant concentration of the drug in the blood plasma.

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One special type of ER formulation based on coated reservoir systems is the socalled osmotic-controlled oral drug delivery system or osmotic pump. Pure osmotic systems have semipermeable membranes; that is, water can permeate the membrane but not other substances. These semipermeable membranes can be made of, for example, cellulose acetate [44], and for such formulations the dominating release mechanism is the osmotic pressure [Equation (8)]. The oldest formulation based on osmotic release was OROS from Alza Corporation [47]. In order to achieve drug release through the semipermeable membrane, a laser hole was drilled, but today many osmotic formulations instead use pores filled with water-soluble materials. The aspects of the formulation and different types of commercially available formulations are summarized in the review by Verma et al. [52]. Some advantages with osmotic pumps compared to other ER formulations such as hydrophilic matrix systems are (i) the time-independent drug release (often mentioned as zero-order release), (ii) the superior in vivo–in vitro correlation which facilitates further formulation development, and (iii) less variation between fasted and fed states. Potential drawbacks may be high initial development costs and lack of in-house competence. Drawbacks from an economic point of view may be the necessity to pay royalties and the need for special equipment associated with laser drilling technology. 6.8.3.2

Manufacturing of Oral Membrane-Coated Systems

The first step in the production of membrane-coated systems is to prepare the drug reservoir, the core, of the system. The process steps for producing the core depend on the size of the core. In monolithic membrane-coated systems, the core can be a filled capsule or a tablet which is produced in the traditional ways. This may include mixing of active substance and excipients, possibly granulation and drying, filling into capsules, or compaction into tablets. The production of cores for multiple-unit systems (often termed pellets) is more sophisticated and may be performed in different ways. One, and probably the most common way, is to produce cores for multiple-unit systems by extrusion and spheronizing [53–55]. An alternative methodology is to coat an inert core, e.g. glass or nonpareil beads, with the active substance and the excipients [11]. When the drug-containing core is manufactured, the process continues with the coating of the release-controlling membrane. The composition of the core depends on the properties of the drug and the excipients, the chosen production chain, and whether the systems should be a monolithic or multiple-unit system. The compositions and production steps are reviewed by Tang et al. [56]. In general, the core will include the active substance together with the filler materials and, if necessary, solubilizers and lubricants/glidants. Classical filler materials are lactose and microcrystalline cellulose, but also other materials such as dextrose, mannitol, sorbitol, and sucrose can be used. However, it should be remembered that the dissolution of the filler material might influence the osmotic pressure. The effect of filler solubility has been investigated by Sousa et al. [54], who found a relation between the solubility of the filler materials and drug release rate. For filler materials with large water solubility, there is a great risk that the membrane will rupture due to the development of an excessive osmotic pressure, which will influence the drug release rate.


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The choice of film-forming materials and film-coating techniques is critical for the drug release rate [54]. The ER membrane should remain intact during the release, which implies that it should not dissolve or erode. As film-forming material, water-insoluble substituted cellulose derivatives such as ethylcellulose have been suggested [53, 57] as well as synthetic polymers such as methylacrylates (e.g., Eudragit NE 30D, RS30D or RL30D, where NE stands for nonionic and RS/RL correspond to cationic polymers) [55]. A commercial technology platform is available under the name Eudramode, which is a platform for development of multipleunit systems with extended drug release based on Eudragit [58, 59]. Other film-coating materials such as shellac and zein have been used, but a drawback with these naturally occurring materials is the variation in quality. To obtain a film with satisfying release properties, channeling agents such as the hydrophilic polymers hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), and polyethylene glycol (PEG) or other water-soluble materials such as sodium chloride or lactose may be used [2, 57]. To improve the mechanical properties of the film and thus avoid ruptures and cracks in the film, plasticizers may be added to the formulation. Examples of plasticizers are PEGs, diethyl phthalate, triacetin, mineral oils, glycerol, and chlorobutanol [60]. The film coating may be performed in different types of equipment. For coating of larger units, such as tablets or capsules, a rotating drum is often used, for example an Accela Coater, but other similar equipment may also be used [10, 53, 57, 61]. This type of equipment contains a perforated pan that rotates and thus mixes the bed of units. At the same time, a coating liquid is applied to the moving units by means of a spray gun, where the mixing of the units ensures uniform coverage of the coating. The film coating is dried by blowing a stream of hot air onto the surface of the tablets. For all types of coating processes, there are many parameters, such as the temperature and relative humidity of the inlet air, drum rotation speed, spraying rate, and droplet size of the coating liquid, that have to be adjusted in order to produce good coated films. For coating smaller units, such as pellets, the fluid bed coating technique is used [56]. This is an attractive technique in which the starting material is placed in the coater and heated air is blown through a base plate. This leads to vigorous mixing of the pellet units. By changing the pressure of the incoming air stream, the material becomes suspended in the air. This happens when the bed starts to fluidize and the bed will then have fluidized properties similar to the properties of ordinary liquids. There are different designs of fluid bed: top spray coating, bottom spray coating (or Wurster coating), or tangential coating. They differ with regard to placement of the spray guns. In top spray coating, the liquid is sprayed from the top of the equipment and the droplets hit the particles moving in opposite direction. In the bottom spray coating, the sprayed liquid drops and particles flow in the same direction, which avoids the problem of blocking the spray guns that may occur in top spray equipment. In the more rarely tangential spray coating equipment the base plate rotates and the spray guns are spraying in a tangential direction to the spiral moving particles [10, 62]. A critical parameter for obtaining films with desirable properties is the creation of coating droplets, a process often referred to as atomization, that is, when a bulk liquid is dispersed in air to form a spray or a mist [61]. Atomization is done by letting pressurized air and bulk solution pass simultaneously through a nozzle (spray

1206


gun). The air divides the droplets into smaller units, but in spite of the name atomization, the droplets are dispersed not at atomic scale but rather in the nano- to micrometer-scale range [61]. The mechanism for film formation depends on whether the polymer is dissolved or dispersed as small latex particles in the solution. For both technologies the film formation process starts with wetting and spreading of the coating droplets on the surface of the reservoir system [10]. For the case with polymer dissolved in solution, the film formation mechanism continues with evaporation of the solvent. This leads to an increase of the polymer concentration, and at a certain limit the polymers precipitate and a coated film is formed. In the case of ER films, the most commonly dissolved polymer is ethyl cellulose and the most commonly used solvent is ethanol but other organic solvents can also be used. The use of organic solvent is problematic from the SHE (safety, health, and environmental) point of view and therefore these aspects must be considered before starting to use organic-based coating process technologies. An alternative process methodology is to disperse particles of the film-forming polymer in an aqueous solution [63]. Film formation of dispersed particles undergoes the following steps: evaporation of water, close packing of the particles, deformation of the particles, and annealing of the particles by migration of individual polymer chains between the particles to form a coherent, smooth film coating. The first steps of the process occur in the coating equipment but the last, the annealing step, may continue days after the coated product has left the coating equipment.

6.8.4 6.8.4.1

HYDROPHILIC MATRIX TABLETS Principles of Formulation and Release Mechanisms

Several recent informative review articles on hydrophilic matrix systems have been published [e.g., 24, 64]. Hydrophilic matrix tablets are composed of an active substance, a hydrophilic polymer, release modifiers, lubricants, and glidants. The technology goes back to the mid-1960s when Lapidus and Lordi [65, 66] and Huber et al. [67] determined the drug release from hydrophilic matrix systems. The release mechanism for this type of formulation starts with dissolution of hydrophilic matrix polymers and the formation of a highly viscous polymer layer around the tablet (Figure 5) [64]. This layer is often referred to as a gel layer even though it normally contains only physical entanglements and not chemical cross-linkers, which is traditionally required for gels. However, the gel layer surrounds the inner (more or less dry) part of the tablet and this part is called the core. In traditional hydrophilic tablets, the active substance particles are embedded in the matrix carrier. The dissolution process of the active substance can start when the carrier material has dissolved in water and formed the aqueous gel layer, since without exposure to water the active substance cannot dissolve. Therefore, the “dry” core will shield the active drug from dissolution, which is one reason for the extended drug release from this type of formulation. The release process for hydrophilic matrix tablets can be schematically described as in Figure 5. The left side of the figure shows a hydrophilic tablet undergoing dissolution, swelling, and release. An interface between the solution and gel layer, here

1207

Fraction released (%)

HYDROPHILIC MATRIX TABLETS

100 75 50 25 0 0

6

12

18

24

Time (h)

Swelling front (a)

Diffusion front

Erosion front (b)

(c)

FIGURE 5 (a) Hydrophilic matrix system shown with core (dark gray) and drug particles (small dark gray particles). The swelling, diffusion, and erosion fronts are depicted. (b) Dependencies of volume fraction polymer (solid line) and dissolved drug (broken line) as function of position in matrix system together with swelling, diffusion, and erosion fronts. Top shows how the solid drug particles diminish in size. (c) Examples of drug release as function of time for erosion-controlled (n = 1, solid line) and diffusion-controlled systems (broken line, n = 0.5).

called the erosion front, can be identified, and the polymer chains and drug molecules are released at this front. In the gel layer, the polymer concentration will decrease (Figure 5) from a highly concentrated solution at the swelling front, the interface between the gel layer and more or less dry core, to a diluted polymer solution at the erosion front [68]. Some authors have suggested that the polymer concentration at the erosion front is related to the overlap concentration [68–70]. At the same time, the water content in the gel layer gradually increases from the center of the tablet toward the erosion front, and thus the dissolution of the active substance particles can start already in the gel layer. However, the volume fraction of dissolved drug depends on the amount of available water, which in turn is a function of the position in the gel layer [64, 71]. Assuming that the drug saturation concentration is equal in water solutions and polymer gels, the volume fraction dissolved drug will correlate with the volume fraction water available, and the volume fraction drug will gradually increase with increasing distance from the core. Far from the core, the variations in the concentration of polymer and water are less pronounced and, as long as solid particles coexcite with the saturated drug solution, the volume fraction of the drug will theoretically be almost constant. A third front has also been introduced, the diffusion front, which corresponds to the position in the gel layer where all of the active substance has dissolved. Between the swelling and dissolution front, dissolved and undissolved drug particles will coexist, but between the dissolution and erosion fronts, only dissolved drug molecules occur. In this region, the diffusion of drug out from the matrix will give rise to a decrease in the volume fraction of the drug. Achieving a mechanistic understanding of drug release from hydrophilic matrix tablets is not a trivial task since the release depends on the properties of both the polymer and the drug. The swelling process is directly related to the properties of the polymers, and this is an important factor for drug release, since the polymer swelling process can, for example, transport individual drug particles through the

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gel layer, which has been shown by Adler et al. [72]. Macroscopically, polymer swelling can be observed as an increase in the size of the tablet. On a molecular level, the swelling depends on the dilution and transport of water into the gel layer. This transport is driven by changes in the chemical potential, and the main contribution is the increase of conformational entropy when the polymer chains are diluted [73]. The kinetics of the swelling process may vary. A faster polymer release rate compared to the swelling rate results in a movement of the erosion front toward the center of the tablet and the size of the tablet will diminish. Conversely, the tablet size increases when the polymer release is slower than the swelling process. When the swelling rate is on the same order as the polymer release rate, the position of the erosion front (i.e., tablet size) will remain constant. The swelling front between the gel layer and amorphous (or semicrystalline) core material has traditionally been described as corresponding to a transition of the solid states of the polymers. The polymers in the core, for example in HPMC tablets, are in a glassy state, and the polymer material is transformed to a rubbery state due to the fact that water acts as a plasticizer and decreases the glass transition temperature [74]. This rubbery state can be regarded as a polymer solution, and therefore the glassy-to-rubbery state transition can be regarded as a dissolution process of the polymer, where the dissolution rate will determine the position of the swelling front. A commonly described special case for hydrophilic tablets is the so-called front synchronization, which is when the movements of the swelling front and the erosion front occur equally fast. This special case corresponds to a constant gel layer thickness. Depending on the drug solubility and the dissolution, swelling, and release processes of the polymer, either of two different drug mechanisms can be observed: erosion- or diffusion-controlled drug release (Figure 5). One way to characterize these two mechanisms is to compare the drug and the polymer release. The erosion mechanism is characterized by equal release of the polymer and the drug, whereas when the release of the drug is faster than that of the polymer, this is called diffusion-controlled drug release [64]. The diffusion-controlled mechanism occurs when the diffusion front, the border between undissolved and dissolved drug, is displaced in the gel layer and the drug can efficiently diffuse out from the gel layer. The erosion mechanism dominates when the diffusion and erosion fronts overlap. This means that drug particles may be released from the surface of the gel layer. When this occurs, the drug particles will dissolve faster in the free solution than in the gel layer due to the fact that stirring is more efficient in the free solution, which results in a decreased thickness of the unstirred boundary layer around the particles and thus an increased dissolution rate [Equation (1a)]. Whether the release mechanism will be diffusion or erosion controlled depends on (i) the polymer release rate, which governs the position of the rate erosion front, and (ii) the drug dissolution rate, which governs the position of the diffusion front. The position of the diffusion front and the dissolution rate depend on the solubility of the drug. Lower solubility of the drug gives slower dissolution rates and hence the diffusion front can overlap more easily with the erosion front, which yields erosion-controlled drug release. On the other hand, large solubility of the drug will give diffusion-controlled drug release [75–78]. Traditionally, the drug release rate from hydrophilic matrix systems has been modeled as [79, 80]

HYDROPHILIC MATRIX TABLETS

t

Q = ∫0

dM dt = kt n dt

1209

(9)

where Q is the accumulated amount of released drug and k and n are constants. The values of n have been suggested to describe the drug release mechanisms. Release from a planar surface with n = 1 has been shown to correspond to erosioncontrolled drug release and n = 0.5 to pure diffusion-controlled drug release. This is strictly only valid when the polymer release has n = 1, but this is often the case. In practice, for hydrophilic matrix systems, one often finds n to be between 0.5 and 1, indicating that both the diffusion of the drug and the polymer erosion influence drug release. For other geometric shapes, such as tablet shapes, the limits for n shift to 0.45 and 0.89 for diffusion- and erosion-controlled release, respectively [81–83]. Another popular way to describe the drug release is to characterize the influence of the relative contributions of erosion and diffusion to drug release as [84] Q = at m + bt 2 m

(10)

where a, b, and m are constants. The first factor in Equation (10) should represent the Fickian diffusional contribution and the second term to the erosion contribution to the drug transport. One mechanistic drawback with this approach is that it treats the diffusion and erosion processes as independent of each other, which they are probably not in any practical case. The drug release from hydrophilic matrix tablets has been found to vary with the polymer parameters, the composition of the formulation, and the process parameters. Examples of important polymer-related parameters with significant influence on drug release are the viscosity and the hydrophilicity of the polymer, where polymers of larger viscosity grades give lower release rates and longer durations of the release [85] and an increased hydrophilicity gives larger swelling and faster drug release, which was found by comparing different degrees of substitution of HPMC [86]. The drug release rate also depends on the composition of the formulation. When components with high water solubility, such as lactose, are included in the matrix, the drug release increases, which can be seen as a dilution of the gel-forming material [69, 87]. Similarly, an increased amount of a soluble active substance also increases the release rate of drugs, probably also due to the corresponding decrease of the relative amount of hydrophilic polymer [88, 89]. The size and shape of the tablet also influence drug release, and the release rate increases as the area-tovolume ratio increases [90–93]. The influence of particle size on the drug release rate has also been investigated [89, 94, 95]. The size of polymer particles seems to have low influence on drug release when there is enough gelling polymer available to quickly form a coherent gel layer. In contrast, at low amounts of hydrophilic matrix polymer, gel formation may be too slow, which makes polymer particle size important. In this case, the matrix may disintegrate before it develops a coherent gel layer [95–97]. The effect of drug particle size on the drug release rate depends on the solubility of the substance. Varying the particle size of drugs with high solubility seems to have little influence [89], whereas the release rate may depend on the particle sizes of drugs with moderate solubility [24, 89].

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The effects of process parameters on drug release have been discussed in the literature. Different granulation technologies, such as dry granulation [98–100] or wet granulation [101–103] (which includes fluid bed granulation [104, 105]), have been used. Also direct compression has been used for production of hydrophilic matrix tablets [95]. The effects on the choice of production steps may be critical when the relative fraction of the polymers is low in the formulations. The effect of the compaction pressure on drug release has also been studied. Several authors [89, 88, 106, 107] have found that, while the compaction pressure has a significant effect on the tensile strength of the tablets, it has a minimal influence on drug release. This can be due to the fact that, when a coherent gel layer is formed, only the parameters governing the performance of this gel layer are important, and since parameters such as porosity do not affect the gel layer, they are of low importance for the drug release rate. 6.8.4.2

Manufacturing of Hydrophilic Matrix Tablets

The traditional way of producing hydrophilic matrix tablets resembles the production of the core for membrane-coated tablets and insoluble matrix tablets. It includes a mixing step, possibly a granulation step, a compaction step, and sometimes a coating step. However, one large difference between the production of insoluble and soluble matrix systems is notable; the latter matrix type has strong interactions with water, which complicates the production steps when water is present [108]. Therefore, for hydrophilic matrix systems with large fractions of hydrophilic polymers, traditional wet granulation with water as granulation liquid may cause problems with formation of hard lumps [108]. To avoid this problem, a new technique using foam granulation has been suggested [109, 110]. During granulation, the foams will flow on the top of already foam-wetted particles, which may lead to superior distribution of the granulation liquid. An alternative wet granulation method is to use organic solvents such as ethanol as granulation liquid [111]. However, even if the production of granules with good compaction properties can be maintained in this way, it may, as already mentioned, be an advantage to consider the SHE aspects before choosing organic solvents as granulation liquid. Another alternative to wet granulation is to use dry granulation technologies such as roller compaction [98, 99, 100]. Although this technique has several advantages compared to traditional wet granulation with water, it may result in lower tensile strengths of the tablets [112]. An alternative to granulation technologies is direct compression. This can be done by purchasing special direct-compression qualities of the hydrophilic polymers. A drawback that always arises in relation to direct compression is the difficulty to achieve content uniformity of the tablets, a problem that increases with decreasing doses of the active drug. Therefore, special care should be taken with regard to formulations with low doses of active substance when direct compaction is used [2]. Water-soluble hydrophilic matrix systems may also be extruded, both to monolithic systems and to multiple-unit systems [12, 14–16]. Polyethylene oxide (PEO) and chloropheniramine maleate have, for example, been extruded to a monolithic unit [113]. This manufacturing method proved more feasible for mixtures between low- and high-molecular-weight PEO, since systems containing only high-molecular-weight PEO proved too viscous and difficult to extrude. It was also

COMPARISON AND SUMMARY OF DIFFERENT TECHNOLOGIES

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TABLE 2 FDA Registered Oral ER Formulations Containing Commonly Used Excipients in Hydrophilic Matrix Tablets Hydrophilic Polymera

Fraction of Excipient (%)

Number of Hits on FDA Homepage

Other Names of Excipients Hypromellose, HPMC, MHPC HPC

Hydroxypropyl methyl cellulose [24]

10–80

12

Hydroxypropylcellulose [120, 121] Polyethylene oxide [68, 128] Sodium alginate [134] Xanthan gum [125, 137–140] Hydroxyethyl cellulose [117–119] Carbomer [129]

15–35

4

5–85 5–50 10–99 40–97.5

4 3 3 3

PEO

20–75

1

5–75

1

Carbopol, polyacrylic acid MC

Methyl cellulose [121]

Source: http://www.accessdata.fda.gov/scripts/cder/iig/index.cfm. a Examples of references using them are given.

shown that degradation of PEO to lower molecular weight might occur due to the high temperature used in the extruder. Extrusion to small pellets based on hydrophilic polymers hydroxyethyl cellulose (HEC) and HPMC has been performed, and isopropyl alcohol instead of water was used to avoid lump formation [12]. Also Carbopol [14–16], pectin [114], and xanthan [115, 116] have been extruded and used as matrix carrier materials for extended release. The choice of hydrophilic polymer is one determining factor for the drug release rate, as discussed above. The most common type used for hydrophilic matrix systems is HPMC (hypromellose, hydroxypropyl methyl cellulose) [24, 83] but some alternatives are HEC [117–119], HPC [120, 121], methyl cellulose [121], xanthan [122–127], PEO [68, 128], Carbopol [129–131], pectin [131–133], and alginate [134–136]; see Table 2. HPMCs are available in several approved degrees of substitution [60]. To achieve different release durations, different viscosity grades of the polymers may be used (the higher the viscosity grade, the slower the release rate). If the desired viscosity is not available, one can mix two polymer grades with different viscosity grades [68].

6.8.5

COMPARISON AND SUMMARY OF DIFFERENT TECHNOLOGIES

The main principles for oral extended drug release reviewed here are the membrane-coated reservoir systems and hydrophilic or hydrophobic matrix systems. They all have advantages and drawbacks and Table 3 summarizes some aspects of the different formulation principles. There are a number of commercial variants of the above-mentioned principles. Examples of commercial matrix-based formulations can be found in a review by Varma et al. [137]. One of the oldest commercial hydrophilic matrix systems is

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TABLE 3

Comparison between Three Drug Release Principles

Aspects Drug solubility Release mechanism Release profile Main release dependence (except drug properties) Composition alternatives

Insoluble Matrix Systems Dose dependent [18] Diffusion controlled Q ∝ t0.5 Channeling components and processes parameters Many approved and functional excipients available Pore-channeling excipients may determine drug release Small changes in formulation may change release rate

Manufacturing aspects

Uses traditional production technologies and is inexpensive Release depends on process parameters

In vivo–in vitro correlation

Release depends on fasted and fed state

Membrane-Coated Systems

Hydrophilic Matrix Systems

Dose dependent [18] Diffusion and osmotic Q ∝ t1 Properties of membrane

Whole range

Many approved and functional excipients available Release can be changed by film and core compositions Many polymers are based on natural material which may give large batch-to-batch variation Coating process can be used to control drug release Film coating process dominates release, which may be sensitive in relation to many different process parameters Good

Many grades of available polymers exist Easy to regulate drug release by means of composition and polymer properties Many polymers are based on natural material, which may result in large batch-to-batch variation

Diffusion to erosion controlled Q ∝ tn, 0.4 < n < 1 Properties of polymer carrier

Release is mainly robust against process parameters Granulation with water is difficult

Release depends on fasted and fed state

TIMERx, which is based on xanthan gum mixed with, for example, locus bean gum [138]. A mixture of these polymers has a special property; the different polymers interact strongly with each other and this interaction produces gels with large viscosity. Variants of osmotically controlled ER formulations, some of which are commercially available, are reviewed by Verma et al. [52].

OTHER ORAL ER FORMULATIONS

6.8.6

1213

OTHER ORAL ER FORMULATIONS

Here some new oral ER technologies based on principles other than those mentioned above will be presented. It is beyond the scope of this chapter to cover all systems and details thereof, and the interested reader is recommended to make use of the literature included in the references. One promising technology is to let the active drug interact with the excipients, for example, by covalent binding between the drug and excipient. The azoaromatic cross-linkers between the drug and excipient can exemplify this. These special crosslinkers break due to bacterial degradation in the colon, but not until they are exposed to this bacterial flora [139–141]. This means that the drug is hindered from release before it reaches the colon. This is an example of site-specific delivery to the colon, an area that is reviewed by a special issue of the journal Advanced Drug Delivery Reviews 2005 (volume 57, number 2). Another example of modifications of ER systems is when electrostatic interaction between charged drugs and excipients (with opposite charge) is used. This concept can be valuable for drugs with pH-dependent solubility. If, for example, the drug is a weak base, it will have a large solubility at low pH, and a major part of the drug may be rapidly released already in the stomach, which is not desirable. An introduction of polyions with opposite charge can result in electrostatic interaction between the drugs and the polyions, which can change the solubility or decrease the diffusion rate of the drug through the ER formulation. This concept is, for example, used when propranolol hydrochloride interacts with sodium carboxymethylcellulose (NaCMC) in HPMC matrices [142]. In addition, buffering of the ER systems by organic or inorganic buffers has been used to obtain pH-independent drug release [27, 143, 144]. A recent publication by Riis et al. showed that insoluble inorganic buffers such as magnesium hydroxide and magnesium oxide provided stable drug release over longer periods of time than when more soluble buffer systems were used [26]. Sophisticated systems based on responsive gels are promising alternatives in terms of oral extended release. The principle behind many of these systems is that the formulation should react and undergo some kind of transition due to a trigger signal [145]. In general, the triggers can be various factors such as temperature, light, pressure, electrosensitivity, or interactions with specific molecules such as glucose or antigens. For oral drug delivery systems, the triggers can also be a biological change in the gastrointestinal environment, such as a change in the bacteria flora, as mentioned earlier [139]. It can also be a physiochemical change, such as a pH change in the gastrointestinal tract. Hydrogels composed of copolymers of poly(acrylic acid) and covalently attached Pluronic surfactants is one such example that reacts on pH changes. The cross-linked microgels can be loaded with drug and tailored to collapse in low pH but swell and release the drug at high pH [146]. A novel method for producing ER formulations is a technology called threedimensional printing (TheriForm technology) [147], which is similar to the one used in ordinary printers. The ink is here replaced with an active substance and carrier material. The layer-by-layer “printing” provides controlled placement of the active drug and thus of the release from the device [148]. Even if there have been advances in oral drug delivery technologies during the last 50 years, many highly sophisticated drug delivery systems have failed and have

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not reached the market [149]. This can be due to several reasons and in many cases the formulation is probably not the cause. However, on occasion one may be tempted to agree with Rocca and Park in their review of prospects and challenges in the oral drug delivery: “breakthrough technologies are required to generate novel dosage forms raising drug delivery to higher level” (p. 52) [149].

ACKNOWLEDGMENT Sven Engström, Chalmers University of Technology, is acknowledged for his useful suggestions and comments.

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122. Talukdar, M. M., Michoel, A., Rombaut, P., and Kinget, R. (1996), Comparative study on xanthan gum and hydroxypropyl methyl cellulose as matrixes for controlled-release drug delivery I. Compaction and in vitro drug release behavior, Int. J. Pharm., 129, 233–241. 123. Talukdar, M. M., and Kinget, R. (1997), Comparative study on xanthan gum and hydroxypropyl methyl cellulose as matrixes for controlled-release drug delivery. II. Drug diffusion in hydrated matrixes, Int. J. Pharm., 151, 99–107. 124. Andreopoulos, A. G., and Tarantili, P. A. (2002), Study of biopolymers as carriers for controlled release, J. Macromol. Sci. Phys., B41, 559–578. 125. Andreopoulos, A. G., and Tarantili, P. A. (2001), Xanthan gum as a carrier for controlled release of drugs, J. Biomater. Appl., 16, 34–46. 126. Talukdar, M. M., Rombaut, P., and Kinget, R. (1998), The release mechanism of an oral controlled-release delivery system for indomethacin, Pharm. Dev. Technol., 3, 1–6. 127. Talukdar, M. M., and Kinget, R. (1995), Swelling and drug release behavior of xanthan gum matrix tablets, Int. J. Pharm., 120, 63–72. 128. Maggi, L., Segale, L., Torre, M. L., Ochoa Machiste, E., and Conte, U. (2002), Dissolution behaviour of hydrophilic matrix tablets containing two different polyethylene oxides (PEOs) for the controlled release of a water-soluble drug. Dimensionality study, Biomaterials, 23, 1113–1119. 129. Choulis, N., and Papadopoulos, H. (1975), Timed-release tablets containing quinine sulfate, J. Pharm. Sci., 64, 1033–1035. 130. Huang, L-L., and Schwartz, J. B. (1995), Studies on drug release from a carbomer tablet matrix, Drug Dev. Ind. Pharm., 21, 1487–1501. 131. El-Sayed, G. M., El-Said, Y., Meshali, M. M., and Schwartz, J. B. (1996), Kinetics of theophylline release from different tablet matrixes, STP Pharma Sci., 6, 390–397. 132. Patel, G. N., Patel, G. C., Patel, R. B., Patel, S. S., Patel, J. K., Bharadia, P. D., and Patel, M. M. (2006), Oral colon-specific drug delivery: An overview, Drug Deliv. Technol., 6, 62–71. 133. Liu, L. S., Fishman, M. L., Kost, J., and Hicks, K. B. (2003), Pectin-based systems for colon-specific drug delivery via oral route, Biomaterials, 24, 3333–3343. 134. Liew, C. V., Chan, L. W., Ching, A. L., and Heng, P. W. S. (2006), Evaluation of sodium alginate as drug release modifier in matrix tablets, Int. J. Pharm., 309, 25–37. 135. Shilpa, A., Agrawal, S. S., and Ray, A. R. (2003), Controlled delivery of drugs from alginate matrix, J. Macromol. Sci. Polym. Rev., C43, 187–221. 136. Ostberg, T., and Graffner, C. (1994), Calcium alginate matrixes for oral multiple unit administration: III. Influence of calcium concentration, amount of drug added and alginate characteristics on drug release, Int. J. Pharm., 111, 271–282. 137. Varma, M. V. S., Kaushal, A. M., Garg, A., and Garg, S. (2004), Factors affecting mechanism and kinetics of drug release from matrix-based oral controlled drug delivery systems, Am. J. Drug Deliv., 2, 43–57. 138. McCall, T. W., Baichwal, A. R., and Staniforth, J. N. (2003), TIMERx oral controlledrelease drug delivery system, Drugs Pharm. Sci., 126, 11–19. 139. Friend, D. R. (2005), New oral delivery systems for treatment of inflammatory bowel disease, Adv. Drug Deliv. Rev., 57, 247–265. 140. Ghandehari, H., Kopeckova, P., and Kopecek, J. (1997), In vitro degradation of pHsensitive hydrogels containing aromatic azo bonds, Biomaterials, 18, 861–872. 141. Akala, E. O., Kopeckova, P., and Kopecek, J. (1998), Novel pH-sensitive hydrogels with adjustable swelling kinetics, Biomaterials, 19, 1037–1047.

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142. Takka, S., Rajbhandari, S., and Sakr, A. (2001), Effect of anionic polymers on the release of propranolol hydrochloride from matrix tablets, Eur. J. Pharm. Biopharm., 52, 75–82. 143. Nie, S., Pan, W., Li, X., and Wu, X. (2004), The effect of citric acid added to hydroxypropyl methylcellulose (HPMC) matrix tablets on the release profile of vinpocetine, Drug Dev. Ind. Pharm., 30, 627–635. 144. Varma, M. V. S., Kaushal, A. M., and Garg, S. (2005), Influence of micro-environmental pH on the gel layer behavior and release of a basic drug from various hydrophilic matrices, J. Controlled Release, 103, 499–510. 145. Qiu, Y., and Park, K. (2001), Environment-sensitive hydrogels for drug delivery, Adv. Drug Deliv. Rev., 53, 321–339. 146. Bromberg, L. (2005), Intelligent hydrogels for the oral delivery of chemotherapeutics, Exp. Opin. Drug Deliv., 2, 1003–1013. 147. Rowe, C. W., Wang, C-C., and Monkhouse, D. C. (2003), TheriForm technology, Drugs Pharm. Sci., 126, 77–87. 148. Lee, K-J., Kang, A., Delfino, J. J., West, T. G., Chetty, D., Monkhouse, D. C., and Yoo, J. (2003), Evaluation of critical formulation factors in the development of a rapidly dispersing captopril oral dosage form, Drug Dev. Ind. Pharm., 29, 967–979. 149. Rocca, J. G., and Park, K. (2004), Oral drug delivery: Prospects & challenges, Drug Deliv. Technol., 4, 52–54, 57.

SECTION 7 ROLE OF NANOTECHNOLOGY

7.1 CYCLODEXTRIN-BASED NANOMATERIALS IN PHARMACEUTICAL FIELD Erem Bilensoy and A. Atilla Hincal Hacettepe University Faculty of Pharmacy, Ankara, Turkey


7.1.3

7.1.1

Introduction 7.1.1.1 Cyclodextrins in Pharmaceutical Field Application of Cyclodextrins in Nanoparticles 7.1.2.1 Incorporation of Drug–Cyclodextrin Complexes in Nanoparticulate Delivery Systems 7.1.2.2 Preparation of Nanoparticles from Cyclodextrins 7.1.2.3 Efficacy and Safety of Amphiphilic Cyclodextrin Nanoparticles Conclusion References

INTRODUCTION

Cyclodextrins (CDs) have a wide range of application in the pharmaceutical field due to their unique structure, which allows them to include hydrophobic molecules in their apolar cavity and to mask the physicochemical properties of the included molecule. This results in the enhancement of drug bioavailability by improving aqueous solubility and the physical and chemical stability of the drug, masking undesired side effects such as irritation, taste, or odor, and overcoming compatibility problems or interactions between drugs and excipients. Parallel to the increasing interest and successful licensing and commercialization of nanoparticulate pharmaceutical products, CDs have also been incorporated into nanoparticulate drug delivery systems for several purposes. This can be Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad Copyright © 2008 John Wiley & Sons, Inc.

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CYCLODEXTRIN-BASED NANOMATERIALS IN PHARMACEUTICAL FIELD

achieved by two approaches: (1) complexation of active ingredient with an appropriate CD and entrapment into polymeric nanoparticles to solve problems arising from the drug’s physicochemical properties or (2) modification of CDs to render an amphiphilic character to these molecules, which allows CDs to self-align into nanoparticles in the form of nanospheres, nanocapsules, solid lipid nanoparticles, nanosize liposomes, and nanosize vesicles with or without the presence of surfaceactive agents. In light of current research, this chapter will deal with the following issues concerning the use of CDs and derivatives as nanomaterials for drug delivery: use of CDs (natural and synthetic) derivatives in the pharmaceutical field and application of CDs in nanoparticulate drug delivery systems. A major part of the chapter will be focused on new CD derivatives, amphiphilic CDs, and the characterization, efficacy, and safety of nanoparticles prepared from amphiphilic CDs. 7.1.1.1

Cyclodextrins in Pharmaceutical Field

Natural Cyclodextrins Cyclodextrins are cyclic oligosaccharides obtained by the enzymatic degradation of starch. Major natural CDs are crystalline, homogeneous, nonhygroscopic substances which have a toruslike macroring shape built up from glucopyranose units, as seen in Figure 1 [1–3]. Cyclodextrins are named depending on the number of glucopyranose units. Major industrially produced CDs are named as follows; α-CD, possessing six units, β-CD, possessing seven units; and γ-CD, possessing eight units. Natural CDs have been demonstrated to have a special structure; that is, glucose residues in the CD ring possess the thermodynamically favored 4C1 chair conformation because all substituent groups are in equatorial position. Cyclodextrins behave like rigid compounds with two degrees of freedom: rotation at the glucosidic links C(4)–O(4) and C(1)–O(4) and rotations at the O(6) primary hydroxyl groups at the C(5)–C(6) band. As a consequence of this chair conformation, all secondary hydroxyl groups at C(2) and C(3) are located at the broader side of the CD torus in the equatorial position. Hydroxyl groups on C(2) point toward the cavity and hydroxyl groups on C(3) point outward. The primary hydroxyl groups at the C6 position are located at the narrower side of the torus. These hydroxyl groups ensure good water solubility for the natural CDs. The cavity of the torus is lined with a ring of C−H groups (C3), a ring of glucosidic oxygen atoms, and another ring of C−H groups (C5). Thus, the cavity of CDs exhibits an apolar character. This is accompanied by a high electron density and Lewis base property. The physicochemical characteristics and inclusion behavior of CDs are a direct consequence of these special binding conditions [4, 5]. Certain physicochemical characteristics of natural CDs are given in Table 1. Cyclodextrin Derivatives Natural CDs were reported to form total or partial inclusion complexes with several drugs to improve the aqueous solubility and stability under physiological or ambient conditions, reduce or mask completely the side effects associated with the included drug, and increase compatibility of the drug with other drugs in the formulation or excipients while improving patient compliance by masking the taste or odor of the active ingredient [6–8].

INTRODUCTION

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Primary face OH (6 )

Apolar cavity Modification sites

OH (3)

OH (2) Secondary face CH2OH O

OH

HOCH2

O

HO

O

HO

CH2OH HO

OH

O

O

O

O HO

OH OH

HOCH2

3

HO

2

HO

O CH2OH

O

OH

OH

O CH2OH

OH

O HO

6

O O

CH2OH O

FIGURE 1 Schematic representation of natural CD structure and modification sites. TABLE 1

Some Physicochemical Characteristics of Natural Cyclodextrins

Characteristics Number of glucose units Molecular weight, g/mol Internal diameter, Å External diameter, Å Depth, Å Solubility in water (25°C), g/L Crystal water, w % Approximative cavity volume in 1 mol CD, Å3 Melting point, °C Half-life in 1 M HCl at 60°C, h Crystal forms (from water) Partial molar volumes in solution, mL/mol

α-CD 6 972 4.7–5.2 14.2–15.0 7.9–8.0 145 10.2 174 250–260 6.2 Hexagonal plate 611.4

β-CD 7 1135 6.0–6.4 15.0–15.8 7.9–8.0 18.5 13.2–14.5 262 255–265 5.4 Monoclinic parallelogram 703.8

γ-CD 8 1297 7.5–8.3 17.1–17.9 7.9–8.0 232 8.13–17.7 472 240–245 3.0 Quadratic prism 801.2

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In contrast to the advantageous nature of CDs for molecular inclusion, their surface makes it more difficult for the highly hydrophilic CD molecule to interact with lipophilic biological membranes. For this reason, natural CDs have been chemically modified to alter their water solubility, interaction with biological membranes, and drug release properties. Two of the natural CDs are known to be parenterally unsafe due to nephrotoxic effects [9]. The etiology of the nephrotoxicity of α- and β-CD is unknown but is believed to be related to either CD uptake by kidney tubule cells resulting in disruption of intracellular function or the extraction of lipid membrane components by the CDs. The latter is suggested to be of validity since there seems to be a linear correlation between the ability of some CDs to disrupt cellular membranes and kidney nephrotoxicity [2, 6]. The ability of CDs to cause red blood cell hemolysis and membrane irritation seems also to correlate with their ability to extract lipid membrane components: cholesterol and phospholipids [10, 11]. Modification of natural CDs has been the aim of many research groups to improve safety while maintaining the ability to form inclusion complexes with various substrates. Some groups have also focused on improving the interaction between the pharmaceuticals and the CDs while others have attempted to prepare materials that can be chemically defined more precisely. Methylated CDs are obtained by methylation of CDs on either all C2 secondary and C6 primary hydroxyl groups [dimethyl cyclodextrins (DIMEB)] or all the hydroxyl groups C2, C3, and C6 [trimethyl cyclodextrins (TRIMEB)]. Major disadvantage of methylated CDs is that their solubility decreases with increasing temperature and they are reported to be hemolytic [12]. This is a result of partial methylation of the hydroxyls of β-CD which leads to stronger drug binding but also to stronger hemolysis [12]. Hydroxypropylated CDs are statistically substituted derivatives because hydroxypropylation does not result in selective substitution as with methylation. While the reaction proceeds, the reactivity of the hydroxyl group changes, and this results in a mixture of products with various degrees of substitution. Their dissolution is endothermic so there is no decrease in solubility with increasing temperature [6, 13]. It is necessary to note that degree of substitution in hydroxypropylated CDs is inversely correlated with their inclusion capability [14, 15]. Hydroxyalkylated CDs are commercially available as tablets, ocular collyrs [16], and excipients under the trademarks Encapsin and Molecusol. Sulfobutylether-β-cyclodextrins (SBE-β-CDs) are water soluble and parenterally safe. An advantage over hydroxypropylated-β-CDs (HP-β-CDs) is that higher sulfobutyl group substitution often results in higher drug complexation ability [17]. Inability of the SBE-β-CDs, especially the commercially available product (SBE)7Mβ-CD (Captisol), to form strong complexes with cholesterol and other membrane lipids, arising from their polyanionic nature causing Coulombic repulsions, results in a little or no membrane disruption [6, 13] and reduced hemolysis [18]. Captisol is used in parenteral and ocular systems as well as osmotic tablets and also as a freeze-drying excipient [19]. Branched CDs (mono- or di-glucosyl, maltosyl and glucopyranosyl α- and βCDs) are more resistant to α-amylase than natural CDs. Natural β-CD and monoglucosyl-β-CD are stable in rat blood because they have no linear glycosidic bond. Branched CDs exert a lower hemolytic activity on human erythrocytes and are

APPLICATIONS OF CYCLODEXTRINS IN NANOPARTICLES

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weaker than natural CDs. Their inclusion capability is more or less drug dependent. Steroids were reported to show a slightly higher affinity to branched CDs than to natural CDs [19]. The water solubility of acylated CDs decreases proportionally to their degree of substitution, whereas their solubility in less polar solvents such as ethanol increases. Acylated CDs of hydrophobic nature are useful for controlling the release rates of water-soluble drugs [20]. Ionizable β-CDs possess interesting solubility properties, too. Solubility in water for these derivatives depends on the pH of the solution. Carboxymethylethyl-βcyclodextrin (CME-β-CD) is prepared as an enteric-type carrier system. The presence of a carboxymethyl group causes a pH-dependent solubility range in water, meaning that it is only slightly soluble in the low-pH region, but freely soluble in neutral and alkaline regions due to the ionization of the carboxyl group (pKa about 3–4) [21]. Inclusion-forming capability of CME-β-CD is dependent on drug properties, including size, shape, and charge of the molecule. Sulfated CDs are of anionic nature and are interesting from chemical and biological points of view because of their angiogenic and antiviral properties [22, 23]. Sulfated derivatives are also reported to have a heparin-like activity, resulting in increase in blood-clotting times, which limits their injectable dose [7]. Low-molecular-weight CD polymers (MW 3000–6000 Da) are soluble in water whereas high-molecular-weight CD polymers (MW > 10,000 Da) can only swell in water and form insoluble gels [24, 25]. Insoluble cross-linked bead polymers seem to be applicable as wound-healing agents for the treatment of wounds like burns or ulcers. Iodine has been complexed with such a CD polymer as an antiseptic wound healing agent [24].

7.1.2 APPLICATIONS OF CYCLODEXTRINS IN NANOPARTICLES 7.1.2.1 Incorporation of Drug–Cyclodextrin Complexes in Nanoparticulate Delivery Systems Nanoparticles are of pharmaceutical interest due to their active and passive targeting properties and their ability to deliver poorly solube drugs and drugs with stability problems. Nanoparticles are considered more stable than liposomal delivery systems. However, a major drawback is associated with the drug-loading capacity of polymeric nanoparticles. Classical emulsion polymerization procedures result in considerably low drug-loading capacities. This results in the administration of excessive quantities of polymeric material which may impair the safety of the drug delivery system [26, 27]. For this reason, different techniques are used to improve the drug-loading properties of polymeric nanoparticles. Cyclodextrins are used for this reason to improve water solubility and sometimes the hydrolytic or photolytic stability of drugs for better loading properties [8]. Drug–CD complexes act to solubilize or stabilize active ingredients within the nanoparticles, resulting in increased drug concentration in the polymerization medium and increased hydrophobic sites in the nanosphere structure when large amounts of CDs are associated to the nanoparticles [27, 28].

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The antiviral agent saguinavir was complexed to HP-β-CD to increase saquinavir loading into polyalkylcyanoacrylate nanoparticles by providing a soluble drug reservoir in the polymerization medium that is the basis of nanoparticle formation [29]. A dynamic equilibrium was observed between the complex, dissociated species of the complex, and the forming polymeric nanoparticle. During nanoparticle formation, free drug was believed to be progressively incorporated into a polymer network, driven by the drug partition coefficient between the polymer and polymerization medium. Simultaneous direct entrapment of some CD–drug complex was also suspected [28–30]. Incorporation of the steroidal drugs hydrocortisone and progesterone in complex with β-CD and HP-β-CD reduced the particle size for solid lipid nanoparticles (SLNs) below 100 nm. Steroids were demonstrated to be dispersed in the amorphous state. Compexation to CDs resulted in higher drug-loading properties for the more hydrophobic drug hydrocortisone and lower in vitro release for both drugs when they are complexed to CDs rather than their free form [31]. The in vivo behavior of nanoparticles obtained from drug–CD complexes was also evaluated. HP-β-CD addition in the polymerization medium of polyethylcyanoacrylate (PECA) nanospheres improved the subcutaneous absorption of metoclopramide in rats. PECA nanospheres with HP-β-CD provided the highest drug concentration and enhanced drug absorption compared with those with dextran or with drug solution. However, in addition to drug absorption from subcutaneous sites, HP-β-CD also enhanced the drug elimination by enhancing the drug absorption to reticuloendothelial tissues [32]. Progesterone complexed to HP-β-CD or DM-β-CD was loaded into bovine serum albumin (BSA) nanospheres. Dissolution rates of progesterone were significantly enhanced by complexation to CDs with respect to free drug. Nanospheres of 100 nm loaded with drug–CD complexes provided a pH-dependent release profile and good stability in an aqueous neutral environment [33]. In another approach, CD properties of complexation were combined with those of chitosan. Complexation with CD was believed to permit solubilization as well as protection for labile drugs while entrapment in the chitosan network was expected to facilitate absorption. Chitosan nanoparticles, including complexes of HP-β-CD with the hydrophobic model drugs triclosan and furosemide, were prepared by ionic cross-linking of chitosan with sodium tripolyphosphate (TPP) in the presence of CDs. Nanoparticles were then prepared by ionotropic gelation using the obtained drug–HP-β-CD inclusion complexes and chitosan. Cyclodextrin and TPP concentration largely affected particle size but the zeta potential remained unchanged with different parameters. On the other hand, drug entrapment increased up to 4 and 10 times by triclosan and furosemide, respectively. The release profile of nanoparticles indicated an initial burst release followed by a delayed release profile lasting up to 4 h [34]. Recently a CD–insulin complex was encapsulated in polymethacrylic acid–chitosan–polyether[polyethylene glycol (PEG)–propylene glycol] copolymer PMCP nanoparticles from the free-radical polymerization of methacrylic acid in the presence of chitosan and polyether in a medium free of solvents or surfactants. Particles had a size distribution of 500–800 nm. The HP-ß-CD inclusion complex with insulin was encapsulated into the nanoparticles, resulting in a pH-dependent release profile as seen in Figure 2. The biological activity of insulin was demonstrated with enzyme-


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100 90

Percentage of release

80 70 60 50 HPCDI pH7.4 I pH7.4 HPCDI pH1.2 I pH1.2

40 30 20 10 0 0

50

100

150

200

250

300

350

Time (min)

FIGURE 2 pH-dependent release profile for insulin complexed to HP-ß-CD and encapsulated in nanoparticles. (Reprinted from S. Sajeesh and C. P. Sharma, International Journal of Pharmaceutics, 325, 147–154, 2006, Copyright 2006, with permission from Elsevier.)

linked immunosorbent assay (ELISA). Cyclodextrin complexed to insulin encapsulated into mucoadhesive nanoparticles was believed to be a promising candidate for oral insulin delivery [35]. 7.1.2.2

Preparation of Nanoparticles from Cyclodextrins

Cyclodextrins are used as excipients in the preparation of nanoparticles by three approaches: 1. Preparing nanoparticles from polymers under the presence of CDs in the medium 2. Preparing nanoparticles from polymers incorporating or conjugated to CDs 3. Preparing nanoparticles directly from amphiphilic CDs Nanoparticles consisting copolymers of aminoethylcarbamoyl-β-cyclodextrin (AEC-β-CD) and ethylene glycol diglycidyl ether (EGDGE) were prepared by an interfacial polyaddition reaction in a mini–emulsion system. By combining these two technologies, namely, cross-linking and modification of hydroxyl groups, a novel functional nanoparticle based on β-CD was introduced as a novel material of nanobiotechnology [36]. Nanoparticles prepared fromn CDs are promising targeted delivery systems. Transferrin, an iron-binding glycoprotein ligand for tumor targeting, was conjugated to β-CD polymers and adamantane–PEG5000 through carbohydrate groups selfassembled into sub-100-nm particles in a recent study [37]. A CD-containing polyca-

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tion was used for nucleic acid condensation into nanoparticles [38] and the second component, adamantane-terminated modifier for stabilizing nanoparticles to minimize interactions with plasma and to target cell surface receptors, was incorporated in this system. The particles were demonstrated to mediate transferrin-mediated delivery of nucleic acids to cultured cells [37]. Transferrin-containing CD polymer-based nanoparticles were studied as nucleic acid delivery system that can be modified for targeted delivery of small interfering ribonucleic acid (siRNA) to cancer cells. Molecular studies showed that the siRNA CD nanoparticles reduced levels of Ewing’s transcript by 80% and inhibited growth of cultured Ewing’s tumor cell line. It was also reported that this delivery system indicated a lack of toxicity [39]. A new tadpole-shaped polymer was synthesized via a coupling reaction of PLA onto mono[6-(2-aminoethyl)amino-6-deoxy]-β-cyclodextrin (CDenPLA). A hydrophilic head consisting of the CD group was believed to bind proteins and the PLA tail gave the amphiphilic property [40]. BSA was incorporated into nanoparticles of CDenPLA using both nanoprecipitation and double-emulsion techniques, as can be seen in Figure 3 [40]. A similar process was used to couple PLGA onto aminoβ-CD and ethylenediamino-bridged bis(β-CD) to afford amphiphilic conjugates forming nanoparticles with the nanoprecipitation technique. This approach was believed to be promising for protein delivery since BSA structure was unchanged

0.5 mm

100 nm DE2

DE1

0.5 mm

100 nm NP1

NP2

FIGURE 3 TEM photographs of BSA-loaded ßCDen47PLA nanoparticles prepared with different techniques and their magnified images. (Reprinted from H. Gao, Y. W. Yang, Y. G. Fan, and J. B., Ma, Journal of Controlled Release, 112, 301–311, 2006. Copyright 2006, with permission from Elsevier.)


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after encapsulation into nanoparticles and during its release. Nanoparticles were reported to be stable after freeze drying [40, 41]. Nanoassemblies were formed by mixing solutions of β-CD polymer and dextran hydrophopbically modified with alkyl chains (C12) and loaded with the model hydrophobic drug benzophenone. Nanoassemblies were characterized as 110–190 nm with relatively low drug-loading values ranging between 0.3 to 1.01% w/w. Authors suggested that hydrophobic model drug and hydrophobically modified dextran compete for the apolar CD cavity; however, benzophenone does not impede the hydrophobic dextran to interact with β-CD polymer to form supramolecular assemblies at the nanoscale [42]. Another group has worked on the oligo(ethylenediamino)-β-cyclodextrin modified gold nanoparticles (OEA-CD-NP) as a vector for DNA binding. Possessing many hydrophobic cavities at the outer space, OEACD-NP was believed to have a capability of carrying biological and/or medicinal substrates into cells. Presence of the CD moieties was suggested to be the key parameter in the high affinity to DNA for the gold nanoparticles. In addition, CD moieties were demonstrated to reduce the cytotoxicity of gold nanoparticles arising from the gold clusters that impair plasma membrane functions and lead to cell death [43]. Nucleic acid delivery was also studies by Park et al. using CD-based nanoparticles prepared from β-CD-modified poly(ethylenimine) (CD-PEI). The inclusion-forming capability of β-CD was used in order to immobilize the nanoparticles on solid surfaces (adamantine-modified self-assembled monolayers). CD-PEI nanoparticles were proposed as delivery systems onto solid surfaces to attain specific and high affinity loading. The interaction is schematized in Figure 4 [44].

OH

O

O O OH HO O OH OH HO

OH O HO O

CD-PEI/DNA complex

OH O OH OH = HO O O HO OH HO O HO HO OH HO O OH O O O HO

Specific binding with high affinity

Scheme 1A

CD-PEI nanoparticle

AD-modified surface

+ 50-μm gold layer Adamantane

β-cyclodextrin

Inclusion complex

Glass slide

FIGURE 4 Schematic representation of β-CD–adamantane inclusion complex formation and immobilization of CD nanoparticles on adamantine-modified surface. (Reprinted with permission from ref. 44. Copyright 2006 by the American Chemical Society.)

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Amphiphilic Cyclodextrins Nanoparticles have been obtained spontaneously from modified CDs of amphiphilic structure since the last decade. This approach differs from the previously discussed approaches in that amphiphilic CDs do not require the presence of another polymer or macromolecule or even surfactants. Amphiphilic CDs have been synthesized to solve problems of natural CDs that limit their pharmaceutical applications. The main reasons for the synthesis of amphiphilic CDs are as follows: 1. Enhancement of interaction of CDs with biological membranes through a relative external hydrophobicity 2. Modification or enhancement of interaction of CDs with hydrophobic drugs arising from the high number of long aliphatic chains and by increasing the number of hydrophobic sites for possible interactions with hydrophobic molecules 3. Allowing self-assembly of CDs resulting in the spontaneous formation of nanosize carriers in the form of nanospheres and nanocapsules The unique advantage of amphiphilic CDs is that they possess self-assembling properties that are sufficient to form nanoparticles spontaneously without the presence of a surfactant as well as the capability of including hydrophobic molecules in their cavity and within the long aliphatic chains [45–47]. Amphiphilic CDs can be classified according to their surface charge. Nonionic Amphiphilic Cyclodextrins Nonionic amphiphilic CDs are obtained by grafting aliphatic chains of different length on the primary and/or secondary face of the CD glucopyranose unit. Different derivatives depicted in Figure 5 are named after their structure: 1. Lollipop CDs [48] are obtained by grafting only one aliphatic chain to 6-amino-β-CD. 2. Cup-and-ball CDs were synthesized by the introduction of a voluminous group such as the tert-butyl group which is linked to the end of the aliphatic chain in order to prevent self-inclusion of the pendant group [49, 50].

Lollipop CD

Cup-and-ball CD

Medusa-like CD

Skirt-shaped CD Bouquet-shaped CD

FIGURE 5

Schematic representation of some nonionic amphiphilic CDs.


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3. Medusa-like CDs are obtained by grafting aliphatic chains with length between C10 and C16 to all the primary hydroxyls of the CD molecule [51–53]. 4. Skirt-shaped CDs consist of β- and γ-CDs per-modified with aliphatic esters (C2–C14) on the secondary face [54–57]. 5. Bouquet-shaped CDs result from the grafting of 14 polymethylene chains to 3-monomethylated β-cyclodextrin, meaning seven chains on each side of the CD ring molecule [58]. Per(2,6-di-O-alkyl) CDs where the alkyl chain may be propyl, butyl, pentyl, 3-methylbutyl, or dodecyl also take part in the bouquet family [4]. 6. Cholesteryl CDs were recently introduced as more complicated derivatives [59]. They have been designed assuming that CD is the hydrophilic head group and cholesterol is the hydrophobic part. Interfacial properties of nonionic amphiphilic CDs have been demonstrated by different groups [60–62]. It was found that length, structure, and bond type of the aliphatic chain play important roles upon the surface-active characteristics of amphiphilic CDs. Alignment of the amphiphilic CD molecule at the air–water interface was demonstrated to be aliphatic chains perpendicular to and a CD ring parallel to the film [62]. Inclusion-forming capability of nonionic amphiphilic CDs also has been reported with various model molecules of a different nature. It was suggested that leaving the wider side of the cavity, that is, the secondary face, unsubstituted may facilitate entrance of the drug in the cavity of the amphiphilic CD [63–65]. Cationic Amphiphilic Cyclodextrins Recently cationic amphiphilic CDs were obtained and characterized carrying an amino group as an ionic group. Heptakis(2ω-amino-O-oligo(ethyleneoxide)-6-hexylthio)-β-CD, a “stealth” cationic amphiphilic CD because of the oligoethylene glycol group it carries, was synthesized [66]. The structural properties of cationic amphiphilic CDs were believed to be due to the balance between hydrophobic tails such as thioalkyl chains and hydrophilic components such as ethylene glycol oligomers. The presence of ethylene glycol chains in particular was believed to increase the colloidal stability of the supramolecular aggregates formed by cationic amphiphilic CDs. Amphiphilic alkylamino-α- and β-CDs were also reported regarding their synthesis and characterization [67]. A series of polyamino-β-CDs have been synthesized by Cryan et al. [68] and complete substitution by amine groups at the 6-position. Neutral CDs have been shown to interact with nucleic acids and nucleotides and to enhance their transfection efficiency in vivo. Cationic CDs have shown even greater ability to bind nucleotides and enhance delivery by viral vectors. The major advantage of polycationic CDs and their nanoparticles is their enhanced ability to interact with nucleic acids combined with their self-organizational properties [68]. Anionic Amphiphilic Cyclodextrins Anionic amphiphilic CDs possess a sulfate group in their structure to render an anionic nature to the molecule. An efficient regiospecific synthetic route to obtain acyl-sulfated β-CDs was introduced in which the upper rim is functionalized with sulfates and the lower rim with fatty acid esters [69]. These derivatives were able to form aggregates in aqueous medium. Sulfated amphiphilic α-, β-, and γ-CDs were demonstrated to form 1 : 1 inclusion complexes with the antiviral drug acyclovir. Noncovalent interactions between

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acyclovir and nonsulfated amphiphilic CDs (nonionic amphiphilic CDs) appeared to take place both in the cavity of the CD and inside the hydrophobic zone generated by alkanoyl chains. However, in the case of sulfated anionic amphiphilic CDs, the interactions appear to take place only in the hydrophobic region of the alkanoyl chains [70]. Fluorine containing anionic β-CDs were first introduced by Granger et al. [71] functionalized at the 6-position by trifluoromethylthio groups. They exhibit an amphiphilic behavior at the air–water interface and are good candidates for a new class of amphiphilic carriers. Péroche et al. [72] described the synthesis of new amphiphilic perfluorohexyl- and perfluorooctyl-thio-β-CDs and their alkyl analogue, nonanethio-β-CD. The ability of these products to form nanoparticles was also investigated by photon correlation spectroscopy and imaging techniques such as scanning electron microscopy (SEM) and cryo–transmission electron microscopy (TEM). Fluorophilic CD derivatives have been obtained as a result of combinations of CDs and a linear perfluorocarbon [73]. 2,3-Di-O-decafluorooctanoyl-γ-CD was obtained with a protection–deprotection synthetic method and characterized further by thin-layer chromatography (TLC), Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), elemental analysis, and time-of-flight mass spectrometry (OF-MS). 7.1.2.3

Safety and Efficacy of Amphiphilic Cyclodextrin Nanoparticles

Amphiphilic CDs yield nanoparticles spontaneously in the form of nanospheres or nanocapsules depending on the preparation technique. Nanoparticles have been manufactured using three different techniques. However, the nanoprecipitation technique is generally preferred since it is a simple technique resulting in unimodal distribution. The general preparation techniques for amphiphilic CD nanoparticles are as follows: 1. Nanoprecipitation [74–76] 2. Emulsion/solvent evaporation [77] 3. Detergent removal [78] Nanocapsules are also prepared according to the same techniques. Amphiphilic CD and the oil Miglyol or benzyl benzoate are dissolved in suitable organic solvent (acetone, ethanol). The solution is poured into aqueous phase under constant stirring and the nanocapsules form spontaneously. Organic solvent is then evaporated. Resulting nanocapsules vary in size between 100 and 900 nm according to the preparation process and technological parameters [79]. Particle sizes of nanocapsules are mostly affected by the size of the oil droplet formed during the preparation along with the molar concentration and nature of amphiphilic CD. Nanospheres, on the other hand, are not significantly affected by amphiphilic CD concentration and can be formed with very high concentrations of amphiphilic CDs. The modification site of the CD (primary or secondary face) is influential for nanosphere size since modifications on the secondary face result in a larger surface area. The presence and concentration of a surfactant such as Pluronic F68 do not affect the particle size of nanospheres and nanocapsules [80]. Nano-


(a)

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(b)

FIGURE 6 Cryo-TEM images of 6-O-CAPRO-β-CD nanospheres (a) and nanocapsules (b). [(a) Reprinted from E. Memisoglu, A. Bochot, M. Şen, D. Duchene, and A. A. Hincal, International Journal of Pharmaceutics, 252, 143–153, 2003. Copyright 2003 with permission from Elsevier.)

spheres and nanocapsules of amphiphilic CDs were imaged with different microscopic techniques such as cryo-TEM, atomic force microscopy (AFM), and scanning transmission microscopy (STM). SEM imaging, on the other hand, results in shrinkage or disruption of the nanoparticles due to electron bombardment. Figures 6a and 6b present TEM photomicrographs after freeze fracture for 6-O-CAPRO-β-CD nanospheres and nanocapsules, respectively [62, 66]. Drug loading into amphiphilic nanospheres and nanocapsules is governed by the loading technique used. Amphiphilic CD nanoparticles can be loaded with the following techniques: 1. Conventional Loading Drug solution is added to the organic phase during preparation. 2. Preloading Nanoparticles are prepared directly from preformed drug– amphiphilic CD complexes. 3. High Loading Nanoparticles are prepared directly from preformed drug– amphiphilic CD complexes and further loaded by the addition of drug solution in the organic phase. A high-loading technique results in two- to threefold increase in drug entrapment. Other factors influencing drug loading to amphiphilic CD nanospheres are related to drug physicochemical properties such as drug–CD association constant k1:1, representing the affinity of the drug to the CD cavity, oil/water partition coefficient, and aqueous solubility. The affinity of the drug to the CD cavity is correlated with drug-loading capacity. Lipophilic drugs interact both with the CD cavity and the long aliphatic chains situated on either the primary or the secondary face. Drug release properties of amphiphilic CD nanospheres are affected by various parameters, including drug lipophilicity, drug–CD association constant, and loading technique with release profiles varying from 2 to 96 h depending on the above parameters. Nanocapsules, on the other hand, exert somewhat different drug release profiles that are mostly dependent on lipophilicity and aqueous solubility of the drug. Lipophilicity of the drug is inversely correlated with the rate of release, as seen in Figure 7 [81]. Nevertheless, preparing nanoparticles directly from preformed

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100

nanocapsules)

Cumulative drug (% released from

120

80 60 40 20 0 0

2

4

6

8

10

12

14

16

18

20

22

24

Time (h) HCR CL

HCR HL

TST CL

TST HL

PRO CL

PRO HL

FIGURE 7 In vitro release profiles of steroids with different physicochemical properties from β-CDC6 nanocapsules (HCR HL, hydrocortisone high loaded, HCR CL, hydrocortisone conventionally loaded; TST HL, testosterone high loaded; TST CL, testosterone conventionally loaded; PRO HL, progesterone high loaded; PRO CL, progesterone conventionally loaded).

inclusion complexes helped reduce the initial burst effect observed in general for nanospheres due to their very large surface area. Cancer Therapy Nanoparticles were first prepared with the concept of targeting colloidal carriers of nanosize to tumor tissues via the leaky vasculature in tumor regions. Since then nanoparticulate drug carriers have been associated with cancer therapy through passive and active targeting to cancer cells. Thus, amphiphilic CD nanoparticles were mainly focused on cancer therapy and its different aspects. Tamoxifen, an antiestrogen drug used for the first-line and adjuvant therapy for metastatic breast cancer as long-term chemotherapy, has been incorporated into amphiphilic CD nanoparticles prepared using the amphiphilic CD, β-CDC6 seen in Figure 8 in order to reduce the severe side effects associated with the nonselective cytotoxicity of this drug. Tamoxifen citrate–loaded nanospheres and nanocapsules with approximately 65% entrapment efficiency liberated the drug with a controlledrelease profile up to 6 h when the high-loading technique is used [82]. Anticancer efficacy of tamoxifen citrate–loaded nanospheres and nanocapsules was demonstrated to be equivalent to tamoxifen citrate solution in ethanol against MCF-7 human breast cancer cells. Transcription efficiency of the tamoxifen citrate nanocapsules and nanospheres was evaluated against MELN cells in the presence of 17β-estradiol (E2) for the inhibition of E2-mediated luciferase gene expression. It was found that transcription efficiency of tamoxifen citrate–loaded nanospheres and nanocapsules were concentration dependent [83]. Paclitaxel, an anticancer drug with bioavailability problems arising from its very low aqueous solubility, its tendency to recrystallize when diluted in aqueous media,


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OH 6

OH O O

O 2

3

O

C O

C

O O

C O 6

OH OH

O 2

3

OH O 7

7

(a)

(b)

FIGURE 8 Amphiphilic β-CD derivatives modified with 6C aliphatic esters on (a) secondary face, β-CDC6 and (b) primary face, 6-O-CAPRO-β-CD.

and solubilizers used in its commercially available injectable formulations, has been loaded into nanoparticles prepared from amphiphilic β-CD modified on the primary face with 6C aliphatic esters, 6-O-CAPRO-β-CD, seen in Figure 8. Paclitaxel-loaded 6-O-CAPRO-β-CD nanospheres and nanocapsules were characterized with a diameter of 150 nm for nanospheres and 500 nm for nanocapsules with high entrapment efficiencies. Blank nanoparticles were proven to be physically stable in aqueous dispersion for 12 months. The in vitro release of paclitaxel from nanoparticles was completed in 24 h [84]. Amphiphilic β-CD nanoparticles were compared to the commercial vehicle Cremophor EL in terms of hemolysis and cytotoxicity. 6-O-CAPROβ-CD nanospheres in particular were found to be significantly less hemolytic than paclitaxel solution in the Cremophor vehicle on human erythrocytes. Cytotoxic effects of blank nanoparticles were assessed against L929 mouse fibroblast cells and a vast difference in cytotoxicity of up to 100-fold reduction was observed for amphiphilic CD nanoparticles. Drug-loaded nanoparticles were also evaluated for their safety and efficacy. Paclitaxel-encapsulated 6-O-CAPRO-β-CD nanospheres and nanocapsules were evaluated for their physical stability in a one-month period in aqueous dispersion form with repeated particle size and zeta potential measurements and AFM imaging to evaluate recrystallization in aqueous medium. Paclitaxel-loaded amphiphilic CD nanoparticles were found to be physically stable for a period of one month whereas recrystallization occurs within minutes when diluted for intravenous (IV) infusion [85]. Finally, paclitaxel-loaded amphiphilic nanoparticles were demonstrated to show similar anticancer efficacy against MCF-7 cells when compared to paclitaxel solution in a cremophor vehicle [85]. Our group is currently working on the formulation of another potent anticancer drug, camptothecin, that is clinically inactive due to its very low water solubility and poor stability under physiological pH, which causes the drug to be converted from its active lactone form to its inactive carboxylate form. Two different amphiphilic

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β-CD nanospheres, β-CDC6 and 6-O-CAPRO-β-CD, have succeeded in maintaining camptothecin in its active lactone form with considerable loading values and release profiles prolonged up to 96 h [86, 87]. Cationic amphiphilic CDs, heptakis[2-ω-amino-O-oligo(ethylene oxide] hexylthio-β-CD nanoparticles, have encapsulated anionic porphyrins (TPPS) by entangling these molecules within the aliphatic chains aligning both faces of the cationic amphiphilic CD. These nanoparticles were demonstrated to preserve the photodynamic properties of the entrapped photoactive agent. The photodynamic efficacy of the carrier/sensitizer nanoparticles was proven by in vitro studies on tumor HeLa cells showing significant cell death upon illumination with visible light [88]. Oxygen Delivery Amphiphilic and fluorophilic β-CD derivatives perfluoro-β-CDs were used to prepare nanocapsules with a single-step nanoprecipitation technique. Highly fluorinated materials have multiple properties, such as repellance to water and oil, unique dielectric, rheological, and optical properties, as well as exceptional chemical and biological inertness. The fluorinated chains, due to their strong hydrophobic and flurorophilic character, impart unique properties to the vesicles, including enhanced particle size stability, prolonged intravascular persistence, and increased drug encapsulation capability. Thus, 2,3-di-O-decafluorooctanoyl-β-CD nanoparticles were believed to be a suitable carrier for oxygen solubilization and delivery. Oxygen delivery of perfluorinated amphiphilic CD nanocapsules was compared to water and showed a prolonged delivery of oxygen. Fluorophilic nanocapsules were believed to overcome fluorocarbon emulsions as oxygen carriers due to their higher number of particles in the colloidal solution which will permit a greater rate of dissolved oxygen [73]. Oral Delivery Amphiphilic β-CD nanocapsules loaded with indomethacin have been evaluated in vivo. The nanocapsules have been applied to the rat model. It was reported that the gastrointestinal mucosa of the rat was significantly protected from the ulcerogenic effects of the active ingredient indomethacin in free form. Drug encapsulation yield in the nanocapsules were >98% and the drug content per CD unit was 7.5% w/w [89]. Cytotoxicity The cytotoxicity of nanocapsules was investigated against L929 mouse fibroblast cells and human polymorphonuclear PMNC cells with MTT assay [90]. Cell viability values of different nanocapsule and nanosphere formulations on L929 and PMNC cells indicated that nonsurfactant β-CDC6 nanocapsules were less cytotoxic than nanocapsules containing surfactants. The cytotoxicity of the nanoparticles mostly arises from surfactant presence and was concentration dependent [90]. Nanospheres of β-CDC6 prepared without surfactant and with Pluronic F68 of varying concentrations between 0.1 and 1% were found to be slightly less cytotoxic than nanocapsules to both L929 and human PMNC cells. It was concluded that cytotoxicity increased with increasing concentration of surfactant and the most suitable percentage for surfactant if required was found to be 0.1% [80]. Sterilizability Three different sterilization techniques—autoclaving, filtration, and gamma sterilization—were evaluated for amphiphilic CD nanoparticles of

CONCLUSION

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100

Cumulative TMX citrate (% released)

90 80 70 60 50 40 30 20 10 0 0

1

Nonsterile NC

2

3 Time (h)

Sterile NC

4

Nonsterile NS

5

6

Sterile NS

FIGURE 9 In vitro release profile of tamoxifen from ß-CDC6 nanospheres and nanocapsules before and after gamma sterilization. (Reprinted from E. Memisoglu-Bilensoy and A. A. Hincal, International Journal of Pharmaceutics, 311, 203–208, 2006. Copyright 2006 with permission from Elsevier.)

β-CDC6 loaded with the model drug tamoxifen [90]. It was found that filtration was not suitable for injectable amphiphilic CD nanoparticles since nanoparticle sizes were too close to filter pore sizes of 0.22 μm. Autoclaving did not affect the nanoparticle yield but caused a significant increase in particle size and aggregates. Gamma irradiation realized with a dose of 25 kGy was demonstrated to be a suitable sterilization technique since no significant change was observed for mean diameter, zeta potential, drug entrapment efficiency, and in vitro release profiles for nimodipineloaded β-CDC6 nanospheres and nanocapsules. The in vitro release profile of sterile and nonsterile nanospheres and nanocapsules of ß-CDC6 loaded with nimodipine is seen in Figure 9 [90].

7.1.3

CONCLUSION

Cyclodextrins have been involved in nanoparticulate drug delivery systems by increasing the solubility of the drug via complex formation, forming nanoparticles in the presence of another polymer/macromolecule, forming nanoparticles by conjugation to polymers, or modification of natural CDs to render this molecule an amphiphilic character. This chapter mainly focused on the potential of amphiphilic CDs as promising carriers for anticancer drugs with bioavailability problems, oxygen delivery for the treatment of ischemia, or the safe oral administration of drugs with gastrointestinal side effects.

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Many new studies are available to modify amphiphilic CDs further by giving them “stealth” properties or targeting moieties such as transferring to enable the active targeting of CD-based nanoparticles to tumor tissues. Amphiphilic CD nanocarriers now emerge as promising delivery systems for poorly soluble anticancer drugs, DNA and oligonucleotide delivery, and photodynamic and targeted tumor therapy. These systems are proven to be nonhemolytic and noncytotoxic and are capable of prolonging the release of drugs with different properties.

ACKNOWLEDGMENTS The authors wish to thank the TUBITAK Turkish Council of Scientific and Technical Research, projects SBAG-CNRS-3 and SBAG-CD-66, and the Hacettepe University Research Fund, project 0202301005, for financial support of the amphiphilic cyclodextrin research carried out by our group at Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology.

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65. Tanaka, M., Ishizuka, U., Matsumoto, M., Nakamura, T., Yabe, A., Nakanishi, H., Kawabata, Y., Takahashi, H., Tamura, S., Tagaki, W., Nakahara, H., and Fukuda, K. (1987), Host-guest complexes of an amphiphilic β-cyclodextrin and azobenzene derivatives in Langmuir-Blodgett films, Chem. Lett., 1307–1310. 66. Mazzaglia, A., Angelini, N., Darcy, R., Donohue, R., Lombardo, D., Micali, N., Sciortino, M. T., Villari, V., and Scolaro, L. M. (2003), Novel heterotropic colloids of anionic porphyrins entangled in cationic amphiphilic cyclodextrins: Spectroscopic investigation and intracellular delivery, Chem. Eur. J., 9, 5762–5769. 67. Matsumoto, M., Matsuzawa, Y., Noguchi, S., Sakai, H., and Abe, M. (2004), Structure of Langmuir-Blodgett films of amphiphilic cyclodextrin and water-soluble benzophenone, Mol. Cryst. Liq. Cryst., 425, 197–204. 68. Cryan, S. A., Holohan, A., Donohue, R., Darcy, R., and O’Driscoll, C. M. (2004), Cell transfection with polycationic cyclodextrin vectors, Eur. J. Pharm. Sci., 21, 625–633. 69. Dubes, A., Bouchu, D., Lamartine, R., and Parrot-Lopez, H. (2001), An efficient regiospecific synthetic route to multiply substituted acyl-sulphated ß-cyclodextrins, Tetrahedron Lett., 42, 9147–9151. 70. Dubes, A., Degobert, G., Fessi, H., and Parrot-Lopez, H. (2003), Synthesis and characterisation of sulfated amphiphilic alpha-, beta- and gamma-cyclodextrins: Application to the complexation of acyclovir, Carbohydr. Res., 338, 2185–2193. 71. Granger, C. E., Feliz, C. P., Parrot-Lopez, H., and Langlois, B. R. (2000), Fluorine containing ß-cyclodextrin: A new class of amphiphilic carriers, Tetrahedron Lett., 41, 9257– 9260. 72. Péroche, S., Degobert, G., Putaux, J. L., Blanchin, M. G., Fessi, H., and Parrot-Lopez, H. (2005), Synthesis and characterization of novel nanospheres made from amphiphilic perfluoroalkylthio-ß-cyclodextrins, Eur. J. Pharm. Biopharm., 60, 123–131. 73. Skiba, M., Skiba-Lahiani, M., and Arnaud, P. (2002), Design of nanocapsules based on novel fluorophilic cyclodextrin derivatives and their potential role in oxygen delivery, J. Inclus. Phenom. Macroc. Chem., 44, 151–154. 74. Fessi, H. C., Devissaguet, J. P., Puisieux, F., and Thies, C. (1997), Process for the preparation of dispersible colloidal systems of a substance in the form of nanoparticles, U.S. Patent 5,118,528. 75. Wouessidjewe, D., Skiba, M., Leroy-Lechat, F., Lemos-Senna, E., Puisieux, F., and Duchene, D. (1996), A new concept in drug delivery based on “skirt-shaped cyclodextrins aggregates” present state and future prospects, STP Pharma. Sci., 6, 21–26. 76. Lemos-Senna, E., Wouessidjewe, D., Lesieur, S., Puisieux, F., Couarrazze, G., and Duchene, D. (1998), Evaluation of the hydrophobic drug loading characteristics in nanoprecipitated amphiphilic cyclodextrins nanospheres, Pharm. Dev. Technol., 3, 1–10. 77. Lemos-Senna, E., Wouessidjewe, D., Lesieur, S., and Duchene, D. (1998), Preparation of amphiphilic cyclodextrin nanospheres using the emulsion solvent evaporation method, influence of the surfactant on preparation and hydrophobic drug loading, Int. J. Pharm., 170, 119–128. 78. Lemos-Senna, E. (1998), Contribution a l’etude pharmacotechnique et physicochimique de nanospheres de cyclodextrins amphiphiles comme transporteurs de principes actifs, Ph.D. thesis, University of Paris, Paris. 79. Skiba, M., Wouessidjewe, D., Fessi, H., Devissaguet, J. P., Duchene, D., and Puisieux, F. (1992), Preparation et utilizations des nouveau systemes colloidaux dispersibles a base de cyclodextrines sous forme de nanocapsules, French Patent 92-07285. 80. Memisoglu-Bilensoy, E., Doğan, A. L., and Hincal, A. A. (2006), Cytotoxic evaluation of injectable amphiphilic cyclodextrin nanoparticles on fibroblasts and polymorphonuclear cells: Surfactant effect, J. Pharm. Pharmacol., 58(5), 585–589.

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81. Memişoğlu-Bilensoy, E., Şen, M., and Hincal, A. A. (2006), Effect of drug physicochemical properties on in vitro characteristics of amphiphilic cyclodextrin nanospheres and nanocapsules, J. Microencapsul., 23(1), 59–68. 82. Memişoğlu-Bilensoy, E., Vural, İ., Renoir, J. M., Bochot, A., Duchene, D., and Hincal, A. A. (2005), Tamoxifen citrate loaded amphiphilic β-cyclodextrin nanoparticles: In vitro characterization and cytotoxicity, J. Controlled Release, 104, 489–496. 83. Vural, İ., Memişoğlu-Bilensoy, E., Renoir, J. M., Bochot, A., Duchene, D., and Hincal, A. A. (2005), Transcription efficiency of tamoxifen citrate loaded β-cyclodextrin nanoparticles, J. Drug Deliv. Sci. Technol., 15(5), 339–342. 84. Bilensoy, E., Gürkaynak, O., Ertan, M., Şen, M., and Hincal, A. A., Development of nonsurfactant cyclodextrin nanoparticles loaded with anticancer drug paclitaxel, J. Pharm. Sci., DOI: 10.1002/jps.2111, Published online. 85. Bilensoy, E., Gürkaynak, O., Doğan, A. L., Duman, M., and Hıncal, A. A., Safety and efficacy of amphiphilic cyclodextrin nanoparticles for paclitaxel delivery, Int. J. Pharm., DOI: 10.1016/j.ijpharm.2007.06.05. 86. Çırpanlı, Y., Bilensoy, E., Çalış, S., and Hincal, A. A. (2006), Camptothecin inclusion complexes with natural and modified β-cyclodextrins, in Proceedings of the 33rd Annual Meeting and Exhibition on Controlled Release Society, Vienna, July, 22–26 pp. 988–989. 87. Çırpanlı. Y., Bilensoy, E., Çalış, S., and Hincal, A. A. (2007), Development of camptothecin loaded nanoparticles from amphiphilic β-cyclodextrin derivatives, paper presented at the Pharmaceutical Sciences World Congress PSWC, Amsterdam, April, 22–25. 88. Sortino, S., Mazzaglia, A., Scolaro, L. M., Merlo, F. M., Volveri, V., and Sciortino, M. T. (2006), Nanoparticles of cationic amphiphilic cyclodextrins entangling anionic porphyrins as carrier-sensitizer system in photodynamic cancer therapy, Biomaterials, 27, 4256– 4265. 89. Skiba, M., Morvan, C., Duchene, D., and Puisieux, F., (1995), Evaluation of the gastrointestinal behaviour in the rat of amphiphilic β-cyclodextrin nanocapsules loaded with indomethacin, Int. J. Pharm., 126, 275–279. 90. Memisoglu-Bilensoy, E., and Hincal, A. A. (2006), Sterile injectable cyclodextrin nanoparticles: Effects of gamma irradiation and autoclaving, Int. J. Pharm., 311, 203–208.

7.2 NANOTECHNOLOGY IN PHARMACEUTICAL MANUFACTURING Yiguang Jin Beijing Institute of Radiation Medicine, Beijing, China


7.2.3

7.2.4 7.2.5

7.2.1

Introduction Nanomaterials 7.2.2.1 Types of Nanomaterials 7.2.2.2 Manufacturing and Processing of Nanomaterials Nanotechnology for Drug Delivery 7.2.3.1 Nanocarriers 7.2.3.2 Nanosuspensions 7.2.3.3 Self-Assembled Drug Nanostructures Nanomedicine Perspective References

INTRODUCTION

Nanotechnology is the ability to produce and process nanosized materials or manipulate objects within the nanoscale. The nanoscale commonly indicates the range from 1 to 100 nm [1]. However, some scientists regard the nanoscale range from 1 to 200 nm [2], even to 1000 nm [3]. Making a comparison with a human hair, it is about 80,000 nm wide. Nanotechnology is a broad, highly interdisciplinary, and still evolving field which involves the production and application of physical, chemical, and biological systems. Nanotechnology is likely to have a profound impact on our economy and society in the early twenty-first century, perhaps comparable to that of information technology or advances in cellular and molecular biology. Science and engineering research in nanotechnology promises breakthroughs in areas such Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad Copyright © 2008 John Wiley & Sons, Inc.

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as materials, manufacturing, electronics, medicine, health care, energy, environment, biotechnology, information technology, and national security. It is widely felt that nanotechnology will lead to the next industrial revolution [4]. The idea of nanotechnology was first presented by physicist Richard Feynman. His lecture entitled “Room at the Bottom” in 1959 unveiled the possibilities available in the molecular world. Because bulk matter is built of so many atoms, there is a remarkable amount of space within which to build. Feynman’s vision spawned the discipline of nanotechnology, and his dream is now coming true [5]. Along with continually increasing multidisciplinary applications of nanotechnology, many new terms with nanotechnology characteristics appear, for example, nanomechanics [6], nanooptics [7], nanoelectronics [8], nanochemistry [9], nanomedicine [10], nanobiotechnology [5, 11], nanolithography [12], nanoengineering [13], nanofabrication [14], and nanomanufacturing [15]. A very broad sense term, nanoscience is often used. More and more new words with nano as a prefix will be created to fit for the nowadays nanoworld. In fact, applications of nanotechnology in medicine and biotechnology have made great progresses in the recent two decades. All developed countries including the United States, Japan, and Europe invest a great deal of money in nanotechnology. The National Science Foundation (NSF) of the United States is a leading agency in the national nanotechnology initiative, funding nanotechnology investments at $373 million in 2007, an increase of 8.6% from 2006 and of nearly 150% since 2001 [16]. Developing countries such as China and India also invest a lot in this increasing field so as not to stay far behind developed countries. Cancer therapy and research are hottest applied fields of bionanotechnology. In 2004, the U.S. National Cancer Institute (NCI) launched a $144 million cancer nanotechnology initiative, and the investment increased largely in the following two years [17]. At the same time, investment from public resources or companies is much higher than that from governments. The application of nanotechnology in pharmacy has a long history, before the prevalence of the nanoconcept. It was well known 50 years ago that very small drug particles have a high solubility in solvents, resulting from the too large surface area when particle size decreased to a very small level, that is, the nanoscale, although this scale had not been mentioned yet. In 1965, Banham created liposomes (lipid vesicles) consisting of phospholipids which had a small size, typically ranging from 10 nm to several micrometers. It was soon found that liposomes were excellent drug carriers, and more importantly they had site-specific distribution capability in vivo depending on their size. It is well known that nanosized liposomes are inclined to deposit in the mononuclear phagocyte system (MPS), including liver, spleen, lung, and marrow. Therefore, nanotechnology was introduced in drug delivery very long ago. Now various nanomaterials are used to deliver drugs, and some nanosystems delivering active agents are available on the market. Undoubtedly, nanotechnology plays a key role in future pharmaceutical development and pharmacotherapy. 7.2.2

NANOMATERIALS

7.2.2.1 Types of Nanomaterials Nanomaterial is a general term. Although nanomaterials are defined as solid or liquid materials at the nanoscale, the nanoscale range remains unclear. Many scien-

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tists regard materials that are one dimensional and 1 nm to less than 100 nm as nanomaterials. However, some scientists treat larger materials (e.g., less than 200 nm) as nanomaterials [2]. In spite of the different views, nanomaterials show unique characteristics that are different from those of bulk materials. Rapid development of nanotechnology in varied disciplines helps to create various kinds of nanomaterials. In terms of shape differences, nanomaterials can be classified as nanospheres, nanovesicles, nanoshells, nanotubes, nanohorns, nanofibers, nanowires, nanoribbons, nanorods, nanosticks, nanohelices, and so on, and they can appear in any shape imagined. In terms of state differences, nanomaterials can also be classified as nanoparticles with solid cores, nanoemulsions with liquid cores, and nanobubbles with air cores. Images of some nanomaterials of various shapes are shown in Figure 1. Nanoparticle is the most usually used term, having a broad meaning. From a narrow sense, nanoparticles are always used to indicate all ball-like nanomaterials, and therein the term nanosphere is also used. Nanocapsules are core–shell nanoparticles, wherein trapped drugs are gathered in a core coated with a hard shell, though generally nanoparticles have uniformly dispersed drugs within the whole particle. Nanovesicle is not a familiar term, for example, liposomes have an inner phase and an outer phase (dispersing medium) that exist together in nanovesicles [18]. In light of drug nature, especially solubility, drugs are entrapped in an inner phase or bilayers (shells). In addition, nanosuspension often appears in the pharmaceutical field, meaning drug nanocrystal dispersion in liquid media [19]. Needle-shaped nanocrystals are more common than globe-shaped ones. Nanogels are newly developed based on hydrogels, being similar to nanoparticles after lyophilization [20]. Recently, a special kind of nanomaterial consisting of drugs was created for drug delivery, called self-assembled drug nanostructure (SADN), which is formed by the selfassembly of amphiphilic prodrugs in aqueous media [21, 22]. Some special nanomaterials are of great interest due to their unique properties. Dendrimers are versatile, well-defined, nanosized monodispersing macromolecules which are hyperbranched synthesized polymers constructed by repetitive monomer units. They are perfect nanoarchitectures with size from 1 nm to more than 10 nm depending on the synthesis generation. Drugs can be entrapped into the branches

Nanospheres

Nanorods

Nanovesicles

Nanotubes

Nanofibers

Nanohelices

FIGURE 1

Some typical nanomaterials.

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of dendrimers or conjugated with them on the high reactive surfaces [23]. The first fullerene discovered was the buckyball, also known as buckminsterfullerene. It was discovered by Smalley, Curl, and Kroto in 1985 [24], who shared a Nobel Prize in 1996 for the discovery. Buckyball is roughly spherical cages of 60 carbon atoms (C60) arranged in interlocking hexagons and pentagons, like the patches on a soccer ball. Fullerenes have attracted considerable research interest, partly because of their unique structures and further because, once suitably dissolved, they display a diverse range of biological activity [25]. Quantum dots (QDs) are semiconductor nanocrystals commonly consisting of CdSe or ZnS. Besides their utilization as electronic materials, QDs have recently been applied to biomedical areas after modification. The new generations of QDs have far-reaching potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting, and diagnostics [26]. Although many types of nanomaterials are created continually, the most important and basic issues are nanoscale effects and the subsequent particular functions. Nanomaterials with varied shapes and components provide different platforms to achieve more functions. In the area of pharmaceutical manufacturing, people focus on the drug delivery function of nanomaterials. Furthermore, the rapid development of modern medicine has led to the belief that traditional drug dosage forms such as tablets, capsules, and injections may not treat some vital diseases well, perhaps not at all. Some advanced techniques developed in other disciplines should be considered to apply to medicine. Nanomaterials can load and deliver drugs in vivo as well as display special properties such as high dispersion, adhesive property, and site-specific distribution in vivo. Modified nanomaterials further possess new functions, for example, they may be thermally sensitive, pH sensitive, magnetically sensitive, and ultrasound sensitive. Nanotechnology has a great effect on pharmaceutical manufacturing. The unique functions of nanomaterials promise considerable benefit to pharmacotherapy over traditional drug preparations. When drug-loaded nanomaterials go through the gastrointestinal tract, high dispersion and adhesion can lead to tight contact of nanomaterials with mucous membranes, enhancing drug absorption. Nanomaterials have been applied in all routes of administration, including oral, injection (intravenous, subcutaneous, intramuscular, intra-articular cavity, and other possible injection sites), intranasal, pulmonary inhalation, conjunctiva, topical, and transdermal, possibly showing various required effects. Some of the characteristics and pharmaceutical applications of nanomaterials are given in Table 1. More applications will continue to be developed. 7.2.2.2

Manufacturing and Processing of Nanomaterials

When material dimensions reach the nanoscale, quantum mechanical and thermodynamic properties that are insignificant in bulk materials dominate, causing these nanomaterials to display new and interesting properties. The manufacturing and processing of nanomaterials may become difficult due to the unique properties. The very small size of nanomaterials produces a very large surface-to-volume ratio, that is, a great number of molecules/atoms locate on surfaces. High surface energy leads to nanomaterials easily agglomerating to diminish energy unless enough hindrance

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TABLE 1 Characteristics and Applications of Some Nanomaterials in Pharmacy Types of Nanomaterials

Characteristics

Nanoparticles

Solid nanosized particles consisting of polymers, lipids, or inorganic materials spherically shaped most of the time, entrapped compounds dispersing in the whole particle

Nanocapsules

Core–shell nanoparticles with entrapped compounds gathering in the core

Liposomes

Lipid vesicles with entrapped compounds in inner phase or bilayers depending on physicochemical property Nonionic surfactant vesicles with similar property as liposomes

Niosomes

Nanoemulsions

Nanoscale emulsions

Polymeric micelles

Micelles consisting of amphiphilic polymers

Nanogels

Nanosized hydrogels consisting of crosslinked hydrophilic polymers Well-defined, nanosized, monodispersing macromolecules with hyperbranched structures

Dendrimers

Applications in Pharmacy

References

Loading all kinds of active agents, including drugs, vaccines, diagnostic agents, and imaging agents for good bioavailability, targeted delivery, and controlled release Loading all kinds of active agents for same aims as nanoparticles, possibly protecting entrapped agents Loading all kinds of active agents for good bioavailability, targeted delivery, and controlled release

27, 28

Loading all kinds of active agents for same aims as for liposomes Loading drugs, as a method to prepare nanoparticles Loading hydrophobic drugs in the core for solubilization, targeted delivery, and controlled release Loading various compounds for controlled release or targeting Loading all kinds of active agents for good bioavailability, targeted delivery, and controlled release

32

29

30, 31

33, 34

35, 36

20

23

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TABLE 1 Continued Types of Nanomaterials Carbon nanotubes (CNTs)

Fullerenes

Quantum dots

Nanosuspensions

Self-assembled drug nanostructures

Characteristics Nanosized tubes as if rolling up a single layer of graphite sheet (single-walled CNTs; SWNTs) or by rolling up many layers to form concentric cylinders (multiwalled CNTs; MWNTs) with diameters of ∼1 nm and large length– diameter ratio Very tiny balls consisting of 60 carbon atoms with diameter of ∼0.7 nm

Tiny nanocrystals commonly consisting of semiconductor materials in the range of 2–10 nm, glowing upon ultraviolet (UV) light Drug nanocrystals dispersing in aqueous media commonly stabilized by surfactants Nanostructures consisting of amphiphilic prodrugs

Applications in Pharmacy Linking a wide variety of active molecules with functionalized CNTs

References 37

Water-soluble carboxylic acid C60 derivatives acting as antimicrobials, being linked to a variety of active molecules Mainly as probes to track antibodies, viruses, proteins, or deoxyribonucleic acid (DNA) in vivo

25, 38, 39

Suitable for insoluble drugs to obtain good bioavailability and targeting

19

Suitable for hydrophilic drugs to obtain good bioavailability, targeting, and controlled release

21, 22

26, 40

prevents them from agglomeration. As a result, manufacturing and processing of nanomaterials become hard issues. Anyway, many successful methods have been found to manufacture stable nanomaterials. “Top down” and “bottom up” are two basic ways to manufacture nanomaterials. From its apparent meaning, the top-down method starts with a bulk material and then breaks it into smaller pieces using mechanical, chemical, or other forms of energy. Microchip manufacturing is the most common example of the top-down approach to produce nanomaterials. While this is an efficient approach for some industries, the process is generally labor and cost intensive. In contrast, the bottomup method produces nanomaterials from atomic or molecular species via chemical reactions or physicochemical interactions such as self-assembly, allowing the precur-

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sor molecules/particles to grow in size. Self-assembly leads to gaining the lowest energy state of molecules and makes molecules reorient naturally to obtain ordered aggregates. Carbon nanotubes, liposomes, and the SADNs are examples of nanomaterials that are manufactured using the bottom-up approach. A deep understanding of chemical and physical properties of precursor molecules/particles is needed to design and manufacture nanomaterials using the bottom-up approach. Both topdown and bottom-up approaches can be performed in gas, liquid, supercritical fluid, solid state, or vacuum. Anyway, when bulk materials corrupt, energy is required, and certainly the obtained nanoscale materials stay at a higher energy state than their parents. Whereas in the bottom-up approach molecules self-assemble into ordered aggregates with controlled behavior. Considering the higher energy of selfassembling monomolecules dispersing in media, their aggregation should be an energy-diminishing procedure and proceed spontaneously (Figure 2). One of the largest hurdles of nanomanufacturing is how to scale up production. In the laboratory, manufacturing nanomaterials is difficult enough as highly advanced tools and carefully clean environments are required. Therefore, scale-up manufacturing in factories becomes a great challenge, hard to achieve. The most successful mass nanomanufacturing to date has occurred with computer microprocessors where companies have been able to etch circuit boards at 65 nm or smaller. Most manufacturers are interested in the ability to control (a) particle size, (b) particle shape, (c) size distribution, (d) particle composition, and (e) degree of particle agglomeration. Neither the top-down nor bottom-up approach is superior at the moment. Each has its advantages and disadvantages. However, the bottom-up approach may have the potential to be more cost-effective in the future. Clinical applications require that biomedical nanomaterials have good biocompatibility or biodegradability. Therefore, biodegradable polymers (synthetic or natural), small molecules such as lipids, and some bioabsorptive inorganic salts such as calcium phosphate are preferred. Other materials such as poly(ethylene glycol) (PEG) is eventually excreted from body so they can also be selected. Materials that are nonbiodegradable or not easily removed from the body, such as carbon nanotubes and quantum dots, should be carefully considered as drug carriers, although they have already been used to deliver drugs or genes. More importantly, before any nanomaterial can be used in a clinic, the acute and long-term toxicity and side effects must be estimated in detail. So a novel discipline, nanotoxicology, is of great interest [41]. In addition, problems of large-scale production of nanomaterials, for example, the uniformity and stability of products, cannot be ignored. Some nanomaterials, including liposomes, polymeric or lipid nanoparticles, nanosuspensions, and SADNs, are described in detail in the following sections. The common manufacturing methods of pharmaceutical nanomaterials are listed in Table 2, though some are only used in the laboratory.

Top down

Bulk materials

Bottom up

Nanomaterials

Atoms or molecules

FIGURE 2 Two basic methods to manufacture nanomaterials.

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TABLE 2 Manufacturing Methods of Some Nanomaterials in Pharmacy Types of Nanomaterials Nanoparticles Polymeric nanoparticles

Solid lipid nanoparticles (SLNs)

Inorganic nanoparticles

Nanocapsules

Liposomes

Niosomes

Nanoemulsions

Materials Involveda

Manufacturing Methods

References

Various natural polymers, e.g., albumin, gelatin, alginate, collagen, chitosan; biodegradable synthetic polymers, e.g., poly(lactic acid) (PLA), poly(lactide-coglycotide) (PLGA), poly(ε-caprolactone) (PCL), poly(methyl methacrylate), and poly(alkyl cyanoacrylate); derivatives of cyclodextrin and starch; some modified polymers (e.g., PEGylated polymers) also used Mainly glycerides and fatty acids, surfactants also used

Monomer polymerization, precipitation, solvent evaporation, salting out

42–44

Calcium salts (e.g., calcium carbonate and calcium phosphate), gold Various polymers, e.g., poly(isobutylcyanoacrylate) (PIBCA), PLA, PLGA, PCL Phospholipids and cholesterol, phospholipid derivatives, e.g., PEG– polyethylene (PE), also added Noionic surfactants, e.g., sorbitan monostearate (Span 60) Oil and surfactants

High-pressure homogenization, microemulsion technique, solvent evaporation Precipitation

Interfacial polymerization of monomers or interfacial nanodeposition of polymers Many methods used, mainly film hydration, reversephase evaporation, injection, freeze drying As for liposomes

High-pressure homogenization, ultrasonic emulsification, phase inversion

27

45–47

29, 48, 49

50

32

34

NANOMATERIALS

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TABLE 2 Continued Types of Nanomaterials Polymeric micelles

Nanogels

Dendrimers Carbon nanotubes

Materials Involveda

Manufacturing Methods

References

Poloxamer-like block copolymers; PEG and lipophilic polymer copolymers; PEGylated lipids Cross-linked hydrophilic copolymers, e.g., Pluronic– poly(ethylenimine) (PEI) and polyethylene oxide (PEO)–PEI Dendritic macromolecules with repetitive moieties Carbon, but only the water-soluble derivatives of CNTs used in pharmacy

Dialysis, emulsification, or film method

35, 36

Covalent conjugation of polymers

20, 51, 52

Divergent or convergent synthesis CNTs formed by chemical vapor deposition (CVD) in presence of Fe catalyst, watersoluble CNT derivatives obtained by acid processing followed by conjugation with drugs C60 obtained by arc discharge method using graphite electrodes or in a benzene flame, water-soluble C60 derivatives obtained by acid processing followed by conjugation with drugs QDs obtained via pyrolysis of organometallic precursors, watersoluble QD derivatives obtained by chemical reaction Precipitation, wet milling, homogenization Amphiphilic prodrugs obtained by synthesis, subsequently SADNs obtained by injection method

23, 53

Fullerenes (C60)

Carbon, but only the water-soluble derivatives of C60 used in pharmacy

Quantum dots

Water-soluble derivatives of semiconductor materials (e.g., ZnS, PbS, CdSe, InP) used in pharmacy

Nanosuspensions

Pure drugs and stabilizers (including surfactants or polymers) Polar drugs with proper conformation and lipids with long chains (e.g., glycerides, fatty acids, cholesterol)

Self-assembled drug nanostructures

a

Organic solvents may be involved and subsequently removed.

54, 55

38, 56

57–59

19, 60, 61

21, 22

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7.2.3 7.2.3.1


NANOTECHNOLOGY FOR DRUG DELIVERY Nanocarriers

High-throughput screening technologies in drug discovery present an efficient way to find new potential active agents. But in recent years it has become evident that the development of new drugs alone is not sufficient to ensure progress in pharmacotherapy. Poor water solubility of potential active molecules, insufficient bioavailability, fluctuating plasma levels, and high food dependency are the major and common problems. Major efforts have been spent on the development of customized drug carriers to overcome the disappointing in vivo fate of those potential drugs. For drug carriers the followings are considered: nontoxicity (acute and chronic), sufficient drug-loading capacity, possibility of drug targeting, controlledrelease characteristic, chemical and physical storage stability (for both drugs and carriers), and feasibility of scaling up production with reasonable overall costs. Nanocarriers have attracted great interest because they are desirable systems to fulfill the requirements mentioned above. Over the past decade nanocarriers as nanoparticulate pharmaceutical carriers have been shown to enhance the in vivo efficiency of many drugs both in pharmaceutical research and the clinical setting, including liposomes, micelles, nanocapsules, polymeric nanoparticles and lipid nanoparticles. They perform various therapeutically or diagnostically important functions. More importantly, many useful modifications have been made, including the increased stability and half-life of nanocarriers in the circulation, required biodistribution, passive or active targeting into the required pathological zone, responsiveness to local physiological stimuli such as pathology-associated changes in local pH and/or temperature, and ability to serve as imaging/contrast agents for various imaging modalities (gamma scintigraphy, magnetic resonance imaging, computed tomography, ultrasonography). In addition, multifunctional pharmaceutical nanocarriers have already made a promising progress [62]. Some of those pharmaceutical carriers have already found their way into clinics, while others are still under preclinical investigation. This section presents two of the most promising nanocarriers, that is, liposomes and nanoparticles, especially their manufacturing, characteristics, and applications. Liposomes Liposomes (lipid vesicles) have a relative long history, first discovered by Banham in 1965 [63]. In the following decades, liposomes rapidly became a useful drug carrier. During the 1990s, many liposome-based drugs reached the market in the United States and Europe. The history of liposomes is the procedure of nanotechnology application to biomedicine. Phospholipids have particular structural conformation, leading to their self-assembly into bilayers with lipid chains inside and polar head groups outside during hydration. Importantly, phospholipids are the primary components of cell membranes so that liposomes have good biocompatibility without toxicity. The formation of liposomes is almost spontaneous, wherein a bottom-up procedure is involved [64]. When relatively free phospholipid molecules meet water, their polar head groups have affinity with water while lipid chains repulse water, which subsequently leads to their aggregation due to hydrophobic interaction, and then bilayers consisting of phospholipids are formed spontaneously. Closed vesicles are further formed by bilayer bending (Figure 3). Before phospho-

NANOTECHNOLOGY FOR DRUG DELIVERY

H3C

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CH3 N+ CH3

O O– P O O O

O

O O

H3C CH3 Dipalmitoyl phosphatidylcholine (DPPC)

3D optimized structure of DPPC

Polar head group

Lipid chains

Self-assembly

Bilayer Vesicle (liposome)

FIGURE 3 Structures of phospholipids and formation of liposomes.

lipids become “free,” bulk phospholipids must be dispersed throughout, forming a thin film, dissolution or emulsification, wherein additional energy is sometimes needed. Liposomes may have a size ranging from 10 nm to more than 10 μm mainly depending on composition and manufacturing approaches. A number of reports about the preparation of liposomes can be found in the literature and a detailed description of liposomes is in Chapter 7.1 of this handbook. In this section the

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preparation, characteristics, and applications of nanosized liposomes are presented as well as some modified liposomes and recent progress. Although various methods of manufacturing liposomes are reported, three types are usually involved: hydration of lipid film, interface aggregation of lipid molecules by emulsion-like process, and lipid solutions dispersing into nonsolvents by an injection-like process or controlled mixture. Practical methods are thin-film hydration [65], reverse-phase evaporation [66], ethanol injection [67], polyol dilution [68], double emulsions [69], proliposome method [70], and high-pressure homogenization [71]. Liposomes may have various morphologies related to manufacturing methods, mainly multilamellar vesicles (MLVs), large unilamellar vesicles (LUVs), and small unilamellar vesicles (SUVs). Liposomes can be further processed by sonication, detergent depletion, membrane filtration [72], and lyophilization [73] to make them finer and more uniform or stable. For example, MLVs are sonicated to SUVs. The composition of liposomes is a key factor in their manufacturing. Phospholipids are major components of liposomes. In terms of resources, phospholipids are classified as natural, semisynthetic, and wholly synthetic phospholipids. Natural phospholipids also have different resources (e.g., soybean, egg yolk). In terms of polar head groups, phospholipids are classified as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA), where PC and PE are the most used. Different polar head groups result in varied surface charged liposomes that then influence the stability and in vivo distribution. Because PS, PI, PG, and PA have negative charges, the liposomes containing them are negatively charged. Sometimes, other lipids such as N,N′-dioleoyl-N,N′-dimethylammonium chloride (DODAC) and stearylamine are mixed with phospholipids to prepare positively charged liposomes. Cholesterol is commonly used with phospholipids because cholesterol can make liposomal membranes stronger [50]. The mole percentage of cholesterol in the liposomal composition is commonly not more than 50%. Lecithin (an often used term in the lipid field) as a phospholipid from natural resources (e.g., soybean lecithin and egg lecithin) is often used to manufacture liposomes, which is actually a mixture composed of various kinds of phospholipids though PC dominates. The long-chain fatty acids constituting phospholipids also have many types, such as lauric (C12), myristic (C14), palmitic (C16), and stearic (C18). In general, unsaturated fatty acids occur in natural phospholipids. Dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), and dipalmitoyl phosphatidylethanolamine (DPPE) are the most common synthetic phospholipids. The length of the lipid chain influences the gel– liquid crystalline phase transition temperature (Tc) of phospholipids, wherein longer chained lipids lead to higher Tc. For example, DPPC has a Tc of 41°C while DSPC has a Tc of 54°C [50]. Drug entrapment is an important parameter in manufacturing liposomes which is influenced by many factors: the types, molecular weights, and physicochemical properties of drugs; the types, sizes, and compositions of liposomes; and the manufacturing methods. In addition, entrapped drugs may leak during storage. Drugs may be entrapped in one of two parts of liposomes, the inner phase or bilayers, depending on the physicochemical property of the drugs. Water-soluble drugs prefer the aqueous inner phase while lipophilic drugs prefer the hydrophobic environment of bilayers. Macromolecules such as peptides and proteins can adsorb onto bilayers,


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wherein electrostatic interaction can influence entrapment. Those drugs insoluble in both water and oil are hard to entrap. Rather than common manufacturing approaches, more promising methods are adopted to improve drug entrapment. Ionic gradient methods can increase the entrapment efficacy of some ionic drugs, including the pH gradient method [74], the ammonium sulfate gradient method [75, 76], the acetate gradient method [77], and the manganese ion gradient method [78]. Lyophilization of liposomes is a good preservation method which can prevent entrapped drugs from leaking, liposome precipitation and agglomeration due to gravity and thermal movement, and possible hydrolysis of phospholipids (resulting in production of toxic lyso-phospholipids). Generally, lipophilic drugs have a high entrapment efficacy, though drug loading is limited, because they insert into bilayers tightly. Therefore, an efficient method increasing entrapment efficacy is to prepare the lipophilic derivatives of hydrophilic drugs [79]. The manufacturing of nanosized liposomes can be performed using the methods mentioned above. However, the small size of nanoliposomes is difficult to achieved by methods such as film hydration. Molecular self-assembly occurs in the injection method, and then the size and morphology of obtained liposomes can be well controlled. In fact, liposomes that result from the injection method are uniform and small enough, to the nanoscale, and usually SUVs are obtained. Because of the very low toxicity of ethanol, the ethanol injection method is usually used and is described as follows to show the process of manufacturing liposomes [50]. A scale-up manufacturing process of the ethanol injection method has been established [80–82]. The obtained liposome size is mostly less than 300 nm: (a) Handling and storage of lipids is important. Store organic solutions of phospholipids in a sealed glass container layered with argon or nitrogen below −20°C, preferably at −78°C. When transferring a portion of the material, allow it to reach room temperature before opening the bottle. Saturated phospholipids, that is, lipids composed of completely saturated fatty acids, such as DPPC, are stable as powders. However, storage of these lipids as described above is highly recommended. Unsaturated phospholipids are extremely hygroscopic as powders, which will quickly absorb moisture and become gummy upon opening the storage container. Always dissolve such lipids in a suitable solvent (preferably chloroform) and store it in a glass container at −78°C. (b) Prepare materials such as phospholipids, cholesterol, other additives, ethanol, injector, beaker, agitation machine, and evaporation device before manufacturing. Calculate the amount of these agents according to the request of the last products. A fine-gauge needle to a 1-mL glass syringe is preferred. Dissolve lipid components (including lipophilic drugs) in ethanol. Dissolve water-soluble drugs in water or aqueous media as dispersing media. (c) Rapidly inject the ethanol solutions into agitated aqueous media with the tip under the surface. A homogeneous and almost transparent liquid will be obtained. Repeat this process and notice that the percentage of ethanol in the last product is not more than 7.5%. Collect all liquids and remove ethanol by evaporation, dialysis, or gel filtration. The last liposomal suspensions can be further concentrated through evaporating water. Sterilize them by autoclave. They may be lyophilized when needed.

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Separation of nonentrapped drugs from liposomes (or purification of liposomes) is an important process after manufacturing. The size difference between liposomes and unincorporated materials is the basis of separation. Gel chromatography, dialysis, and centrifugation are usual approaches. The drug entrapment percentage of liposomes can be obtained after separation. The whole drugs in liposomal suspensions or the entrapped drugs can be determined by dissolving liposomes with organic solvents or solubilizing liposomes with detergents to release drugs. The morphology of liposomes can be investigated by negatively stained transmission electron microscopy, cryo-electron microscopy, or freeze-fracture electron microscopy. The size distribution of liposomes is usually analysized by photon correlation spectroscopy (laser light scattering) [50]. Beyond conventional liposomes, functional liposomes are designed to achieve various therapeutic effects. Conventional liposomes manufactured by natural phospholipids or commonly used synthetic phospholipids such as PC and PE are negatively charged. However, cationic liposomes can form complexes with peptides or nucleic acids through electrostatic interaction and prefer to adsorb onto the surfaces of cell membranes, subsequently improving interaction with cells and penetrating into cytosol or phagocytosis. Therefore, cationic liposomes have become a standard transfection agent in cell manipulation [83]. Furthermore, they become primary nonviral gene delivery carriers [84]. Liposomes show site-specific distribution in the MPSs after intravenous (IV) administration due to opsonization by the complement system [85]. The diseases in MPSs can benefit from the drug targeting. But this is a bad result for diseases in other tissues. Long-circulating liposomes are then developed for targeting to nonMPS tissues. The long-circulating effect results from hydrophilic polymers coated on liposomes. For example, the half-life of the long-circulating liposomes can be extended to 20 h in rat. They are also called sterically stable liposomes or Stealth liposomes. The lipid conjugate of PEG, PEG–DSPE, is commonly used and inserts into bilayers and hinders plasma protein adsorption. The enhanced permeability and retention (EPR) effect of solid tumors makes long-circulating liposomes a very useful tool for anticancer therapy [86]. However, in recent years it was reported that in most cases PEGylated liposomes were cleared very rapidly from circulation with repeated injection. But doxorubicin PEGylated liposome is an exception. The production of anti-PEG immunoglobulin (Ig) M following injection is the major reason, and the spleen also plays a key role [87]. However, a more recent case has appeared. A modified phospholipid–methoxy(polyethylene glycol) conjugate was recently synthesized through the methylation of phosphate oxygen moiety which could prevent PEGylated liposomes from being activated by a complement system in vivo followed by achieving a true long-circulating effect [88]. Other functional liposomes are mainly stimuli-responsive liposomes. The pH-sensitive liposomes contain pH-sensitive lipids such as 1,2-dioleoyl-sn-3phosphatidylethanolamine (DOPE) showing an inverted hexagonal configuration in a low-pH environment and release entrapped drugs in the low-pH environment of tumor tissues due to liposomal membrane destabilization [89]. Temperature-sensitive liposomes are prepared from special lipids such as DPPC whose phase transition temperature (Tc = 41°C) is proper to perform clinical anticancer therapy. When up to Tc, the fluidity of liposomal membranes increases sharply, followed by


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entrapped drugs releasing [90]. Some thermosensitive polymers can also be used to manufacture temperature-sensitive liposomes [91]. Magnetoliposomes load ultrafine magnetite, preferring to accumulate in the local tissue within the magnetic field [92]. Immunoliposomes load attached monoclonal antibodies to treat some severe diseases such as cancer [93]. Liposomes have been successfully applied to many drugs, diagnostic agents, imaging agents, transfection agents, vaccines, and so on. Liposomes have been tried in almost all routes of administration: oral, injection (intravenous, subcutaneous, intramuscular, intra-articular cavity, and other possible injection sites), intranasal, pulmonary inhalation, conjunctiva, topical, and transdermal. The most significant application field of liposomes is still anticancer therapy. After a long-time research for 30 years, some liposomal products have reached the market (Table 3). The major problems in manufacturing liposomes are scale-up production, efficient sterilization, and stable storage. TABLE 3

Liposomal Drugs Approved for Clinical Application Composition of Liposomes and Other Major Excipients

Drug

Product Name

Daunorubicin

DaunoXome

DSPC, cholesterol

Doxorubicin

Mycet

Egg PC, cholesterol

Doxorubicin

Doxil/Caelyx

MPEG–DSPE, HSPC, cholesterol, ammonium sulfate, sucrose, histidine

Amphotericin B

AmBisome (lyophilized product)

Cytarabine

DepoCyt

Morphine

DepoDur

HSPC, cholesterol, DSPG, αtocopherol, sucrose, disodium succinate DOPC, DPPG, cholesterol, triolein DOPC, DPPG, cholesterol, tricaprylin, triolein

Indication

Company

Kaposi’s sarcoma Combinational therapy of recurrent breast cancer Refractory Kaposi’s sarcoma; ovarian cancer; recurrent breast cancer Fungal infections

Gilead Sciences Zeneus

Alza/SP Europe

Gilead Sciences

Lymphomatous meningitis

SkyePharma

Pain following major surgery

SkyePharma

MPEG = methyl PEG; HSPC = hydrogenated soy phosphatidylcholine; DSPG = disteroylphosphatidylglycerol; DOPC = dioleoylphosphatidylcholine; DPPG = dipalmitoylphosphatidylglycerol.

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Nanoparticles Nanoparticles attract much attention of pharmaceutical scientists because of their controllable manufacturing, uniform preparations, and low cost. The major difference between nanoparticles and liposomes is that the former has a solid core while the latter only has inner aqueous phase and thin bilayers. In addition, in the case of liposomes, the entrapped water-soluble drugs exist only in solutions of the inner phase, while lipophilic drugs are only limited in the small space of bilayers. Therefore, the drug-loading efficiency (drug–lipid ratio) of liposomes is always limited. In the case of nanoparticles, drugs exist in the solid state, and high drug loading is possibly achieved. Unlike liposomes, nanoparticles may be composed of various materials, and biodegradable materials are preferably used. Furthermore, modified materials based on traditional natural and synthetic materials are also frequently used to manufacture nanoparticles to achieve more functions, which then highly enlarges the lists of used materials. In addition, more manufacturing methods of nanoparticles are optional than liposomes. Therefore, nanoparticles are relatively ideal nanocarriers for most drugs. Nanoparticles can be classified as three types, polymeric nanoparticles, lipid nanoparticles, and inorganic nanoparticles, depending on the major components. Polymeric nanoparticles are exploited earlier, while lipid nanoparticles are of great interest in recent years due to very good biocompatibility. The development of polymeric nanoparticles is highly related to polymer science. Besides a great deal of natural polymers, more and more biodegradable polymers are synthesized, which allows pharmaceutical scientists to have enough optional subjects. Solid lipid nanoparticles (SLNs) composed of solid lipids have a profound advantage of no biotoxicity [94]. Inorganic nanoparticles are currently exploited only a little [45–47], and the major problems may be their poor biodegradability and relatively low drugloading efficiency. Polymeric Nanoparticles Polymeric materials for manufacturing nanoparticles include synthetic poly(lactic acids) (PLA), poly(lactide-co-glycolide) (PLGA), poly(ε-caprolactone) (PCL), poly(methyl methacrylates), and poly(alkyl cyanoacrylates); natural polymers (albumin, gelatin, alginate, collagen), and modified natural polymers (chitosan, starch). Polyesters, alone and in combination with other polymers, are most commonly used for the formulation of nanoparticles. PLGA and PLA are highly biocompatible and biodegradable. They have been used since the 1980s for numerous in vivo applications (biodegradable implants, controlled drug release). The U.S. Food and Drug Administration (FDA) has approved PLGA for human therapy [95]. More recently, formulations based on natural polymers have been developed and are on the market. For example, a wonderful natural polymer, chitosan, has permeability enhancer abilities, allowing the preparation of organic solvent free mucoadhesive particles [42]. Nanoparticles of synthetic polymers are usually manufactured by dispersion of preformed polymers. Although many methods can be used, they may be classified as monomer polymerization, nanoprecipitation, emulsion diffusion/solvent evaporation, and salting out. An appropriate method is selected mainly depending on polymer and drug natures. Polymerization of polymer monomers has been developed usually using poly(alkyl cyanoacrylate) [96, 97]. Organic solvents are usually used in polymerization. A detailed description of this method is not provided here.


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The nanoprecipitation method is commonly adopted to entrap lipophilic drugs, and low polydispersity is probably achieved [42]. In general, the organic solution containing drugs and polymers is added a nonsolvent to lead to polymers precipitating together with drugs. The size of formed nanoparticles can be adjusted by the polymer and nonsolvent amounts in the organic phase. Nanoparticles can be separated from solvents and unincorporated drugs with centrifugation followed by spray drying or freeze drying when needed. The stability and drug recovery yield of nanoparticles depend on the ratio of drugs to polymers [98]. Recently, this technique has also been used to entrap hydrophilic compounds into PLGA and PLA nanoparticles [99, 100], especially peptides and proteins [101]. Another common method to manufacture polymeric nanoparticles is the emulsion diffusion or solvent evaporation technique, which is used to entrap hydrophobic or hydrophilic drugs. Generally, the polymer and hydrophobic drugs are dissolved in a partially water miscible organic phase (e.g., benzyl alcohol, propylene carbonate, and ethyl acetate). The organic solution is emulsified in aqueous media containing a suitable surfactant [i.e., anionic sodium dodecyl sulfate (SDS), nonionic poly(vinyl alcohol) (PVA) or cationic didodecyl dimethyl ammonium bromide (DMAB)] under stirring. The diffusion of the organic solvent and the counterdiffusion of water into the emulsion droplets induce polymeric nanoparticle formation. The organic solvent is evaporated. Also hydrophilic drugs could be entrapped into a water-in-oil (W/O) emulsion containing polymers and then undergo the above process. Then a water-in-oil-in-water (W/O/W) emulsion is obtained. After evaporation of total organic solvent, the drug-loaded nanoparticles can be separated. Polymer nature, polymer concentration, solvent nature, surfactant molecular mass, viscosity, phase ratio, stirring rate, temperature, and flow of water all affect nanoparticle size [102]. The salting-out method is also used. Polymers are dissolved in water-miscible organic solvents such as acetone or tetrahydrofuran (THF). The organic phase is emulsified in an aqueous phase that contains the emulsifier and salts of high concentration. Typically, the salt solution used contains 60% (w/w) magnesium chloride hexahydrate or magnesium acetate tetrahydrate with a polymer-to-salt ratio of 1 : 3. In contrast to the emulsion diffusion method, no diffusion of solvents occurs due to the presence of high concentrated salts. The fast addition of pure water to the O/W emulsion under mild stirring reduces the ionic strength and leads to the migration of the organic solvent to the aqueous phase, inducing nanoparticle formation. The final step is purification by cross-flow filtration or centrifugation to remove the salting-out agent. Common salting-out agents are electrolytes (sodium chloride, magnesium acetate, or magnesium chloride) or nonelectrolytes, such as sucrose. Polymer concentration and molecular weight, stirring rate and time, and the nature and concentration of surfactant and solvent are all important parameters. This method would allow avoiding the use of organic chlorinated solvents and large amounts of surfactants [102]. Furthermore, formulation of nanoparticles with natural polymers is performed by ionic gelation (chitosan), coacervation (chitosan, gelatin), and desolvation (gelatin) [102, 103]. These mild methods have the advantage of producing organic solvent-free formulations. Additional advantages can be obtained by changing nanoparticle surface properties, for example, good stability, mucoadhesion, and long circulation time. For example, the in vivo long-circulating effect is achieved either by coating

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nanoparticle surfaces with hydrophilic polymers/surfactants or by incorporating biodegradable copolymers containing a hydrophilic moiety. Like long-circulating liposomes, PEG-containing polymers are frequently used to manufacture long-circulating nanoparticles. PEGylated copolymers (PLA–PEG, PLGA–PEG and PCL–PEG) are used [42, 104], and the long-circulating effect also results from the adsorption or covalent conjugation of some hydrophilic polymers with the hydrophobic surface of nanoparticles [105, 106]. Moreover, the active targeting of nanoparticles can be achieved by incorporating the conjugate of the polymer and target-directed molecule, such as (Arg-Gly-Asp) RGD, (trans-activator transcription) TAT peptides [107], and monoclonal antibody [108]. Recently, self-assembled nanoparticles have aroused great interest, consisting of amphiphilic macromolecules, such as hydrophobically modified glycol chitosan, which can also entrap drugs or peptides [109]. Before nanocarriers go into clinical applications, some issues must be considered, including drug-loading capacity, possibility of drug targeting, in vivo fate of the carrier (interaction with the biological surrounding, degradation rate, accumulation in organs), acute and chronic toxicity, scaling up of production, physical and chemical storage stability, and overall costs. A certain advantage of polymer systems is the wealth of possible chemical modifications. Possible problems of polymeric nanoparticles derive from residues of organic solvents used in the production process, polymer cytotoxicity, and the scaling up of production. Polymer hydrolysis during storage has to be taken into account and lyophilization is often required to prevent polymer degradation [94]. Solid Lipid Nanoparticles The outstanding advantage of lipid nanoparticles is perfect biocompatibility because their raw materials are the components of our body, preferring to be used or degraded by the body. Solid lipids are usually used as the major component of lipid nanoparticles—hence the name solid lipid nanoparticles. However, the used solid lipids generally become liquid at a high temperature to adapt to the preparation of SLNs. Compared with polymeric nanoparticles, the materials used for SLNs are simpler. The frequently used lipids are glycerides of various fatty acids, which also exist in the emulsions for parenteral nutrition. Large-scale production of SLNs can be achieved in a cost-effective and relatively simple way using high-pressure homogenization and microemulsion. Another useful method is solvent emulsification/evaporation. The SLN introduced in 1991 represents an alternative carrier system to traditional colloidal carriers, such as liposomes and polymeric nanoparticles. SLNs combine advantages of the traditional systems but avoid some of their major disadvantages [27]. The proposed advantages of SLNs include [94]: • • • • • • •

Possibility of controlled drug release and drug targeting Increased drug stability High drug payload Incorporation of lipophilic and hydrophilic drugs feasible No biotoxicity of the carrier Avoidance of organic solvents No problems with respect to large-scale production and sterilization


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Solid lipids, emulsifiers, and water are generally the ingredients involved for manufacturing SLNs. The term lipids is used in a broader sense and includes triglycerides (e.g., stearin), partial glycerides (e.g., Imwitor), fatty acids (e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate). All categories of emulsifiers may be used to stabilize the lipid dispersion, and the combination of emulsifiers prevents particle agglomeration more efficiently. The choice of the emulsifier depends on the administration route and is more limited for parenteral administration. High-pressure homogenization (HPH) has emerged as a reliable and powerful technique for the preparation of SLNs. Homogenizers of different sizes are commercially available from several manufacturers at reasonable prices. In contrast to other techniques, scaling up of HPH is out of the question in most cases. Highpressure homogenizers push a liquid with high pressure (100–2000 bars) through a narrow gap (in the range of a few micrometers). The fluid accelerates over a very short distance to very high velocity (over 1000 km/h). Very high shear stress and cavitation forces disrupt the particles down to the submicrometer range. Typical lipid contents are 5–10%, though higher lipid concentrations (up to 40%) may be used. Two general approaches of HPH, hot and cold homogenization, can be used for manufacturing SLNs (Figure 4) [27]. Microemulsions (transparently appearing with droplet size less than 100 nm) are thermodynamically stable systems, and the choice of optimal formulation containing oil, surfactant, cosurfactant, and oil–water ratio is key [110]. Generally, the solid lipid of low melting point (e.g., stearic acid) melts at a high temperature (e.g., 65– 70°C), and then hot microemulsions are prepared using it. SLNs can be obtained after the hot microemulsions are rapidly cooled by injecting them into cold water (e.g., 2°C) under stirring. Emulsifiers in a formulation typically include Tween 20/60/80, lecithin, and sodium taurodeoxycholate, and coemulsifiers include alcohols and sodium monooctylphosphate. The typical volume ratios of hot microemulsions to cold water are from 1 : 25 to 1 : 50. The very low solid concentration of SLN suspensions is the disadvantage of the microemulsion method. Rapid temperature decrease in hot microemulsions is key to obtaining homogeneous and small-sized nanoparticles. A high temperature gradient can also ensure rapid lipid crystallization and prevent aggregation [94]. The solvent emulsification/evaporation method involves lipid precipitation in O/W emulsions. Solid lipids are dissolved in a water-immiscible organic solvent (e.g., cyclohexane) followed by emulsification in an aqueous medium. Upon evaporation of the solvent, the nanoparticle dispersion is formed due to lipid precipitation. Residue of organic solvents is the major problem of this method [94]. However, the microemulsion and solvent emulsification/evaporation methods can be performed conveniently in the laboratory without specific apparatuses. During research of SLNs, some problems have continually appeared, for example, very low drug loads, drug expulsion during storage, and high water content of SLN dispersions. The α and β′ crystallines of higher energy state mainly appear in conventional SLNs manufactured by hot-homogenization technique. However, these crystallines prefer to transform to the more ordered β modification of low energy state during storage. The high ordered degree improves the crystal imperfections, diminishing further to lead to drug expulsion. To solve this problem, a new kind of lipid nanoparticle was developed, called a nanostructured lipid carrier (NLC). NLCs

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NANOTECHNOLOGY IN PHARMACEUTICAL MANUFACTURING Melt lipid and dissolve/disperse drugs in lipid

Hot homogenization technique

Cold homogenization technique

Disperse drug-loaded lipid in hot surfactant aqueous solution

Solidification of drug-loaded lipid in liquid nitrogen or dry ice

Mix thoroghly using stirrer to form coarse preemulsions

Grind in powder mill (50–100 μm)

High-pressure homogenization at temperature above melting point of lipid to form hot o/w nanoemulsions

Disperse powder in surfactant aqueous solution rind in powder mill (50–100 μm)

Solidification of nanoemulsions by cooling down to room temperature

High-pressure homogenization at room temperature or below

Solid lipid nanoparticles (SLNs)

FIGURE 4 High-pressure homogenization for manufacturing SLNs.

are composed of spatially very different lipid molecules, that is, solid lipids and liquid lipids (oils). The matrix remains solid at body temperature though its melting point is lower than one of the original solid lipids. No crystallization happens in NLCs so that the drug loads can be increased and the expulsion during storage is avoided [111]. New functions can be obtained by modifications of SLNs. Incorporation of Tween 80 and Poloxamer 188 can stabilize SLNs to achieve long-circulating or crossing blood–brain barrier effects [112]. Recently, novel nanoparticles called polymer– lipid hybrid nanoparticles (PLNs) were developed [113]. They can entrap cationic anticancer agents (e.g., doxorubicin) effectively by incorporation of an anionic lipophilic polymer into lipids to treat multidrug-resistant (MDR) cancers. In general, SLNs are used to entrap hydrophobic drugs due to their lipid nature, but a few reports show that hydrophilic drugs can also be entrapped. A hydrophilic peptide, gonadorelin, and monostearin were dissolved in acetone and ethanol at 50°C followed by pouring the resultant organic solution into an aqueous medium containing 1% PVA under agitation to obtain peptide-loaded SLNs that were sub-


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sequently separated by centrifugation. Up to 69% of gonadorelin was incorporated. The in vitro release of gonadorelin from SLNs was slow [114]. The W/O/W multipleemulsion technique was also used to manufacture peptide-loaded SLNs. Insulin is a model peptide located in the inner water phase of the W/O/W emulsion, tripalmitin is the core of SLNs, and the surfaces are modified with PEG 2000–stearate. The insulin-loaded SLNs show good stability upon the low pH of the gastric medium and the pancreatic enzymes in intestinal medium [115]. Perspective of Nanoparticles As drug nanocarriers, nanoparticles have unique advantages: for example, high dispersing, adhesive property, targeting in vivo. Like liposomes, anticancer therapy is a major function of nanoparticles [116]. Easy modification of nanoparticles also makes them platforms to perform more functions, for example, delivering drugs across the blood–brain barrier (BBB) [117], lymphatic targeting [118], and gene delivery [119]. Abraxane is a successful paradigm of nanoparticle application. It is an albumin nanoparticle loading paclitaxel developed by American Pharmaceutical Partners (APP) and American BioScience. The outstanding advantage of Abraxane is no significant side effects, not like the traditional paclitaxel preparation with Cremophor EL (polyethoxylated castor oil) and ethanol. More nanoparticle products will reach the market in the future. Other Nanocarriers Nanoemulsions Lipid nanoemulsions were introduced in the 1950s as parenteral nutrition. Vegetable oils (e.g., soy oil) or middle-chain triglycerides are used, typically occupying 10–20% of the emulsion. Other ingredients include phospholipids as stabilizers and glycerol as osmolar regulation agent. In recent years this system has been further developed to load lipophilic drugs and several formulations are commercialized. Examples are etomidate (Etomidat-Lipuro), diazepam (Diazepam-Lipuro and Stesolid), propofol (Disoprivan), and dexamethasone palmitate (Lipotalon). In comparison to previous, solubilization-based formulations of these drugs, reduction of the local and systemic side effects (e.g., pain during injection) has been achieved. The possibility of controlled drug release from nanoemulsions is restricted due to the small size and the liquid state of the carrier. Most drugs show a rapid release from them. Advantages of nanoemulsions include toxicological safety and a high content of the lipid phase as well as the possibility of large-scale production by high-pressure homogenization [94]. Microemulsions Microemulsions are nanoemulsions which are optically isotropic, transparent or translucent, low-viscous, and thermodynamically stable liquid solutions, mainly containing tiny liquid droplets less than 100 nm. The manufacturing of microemulsions as a self-formed system is relatively simple. They are bicontinuous systems essentially composed of water and oil with surfactant and cosurfactant separating. A very low interfacial tension to 0 mN/m is found in microemulsions despite the large oil–water interfacial areas. A prominent example is the Sandimmun Optoral/Neoral preconcentrate for microemulsions. Now microemulsions are usually limited to dermal and peroral applications due to their high surfactant content. Because they only exist in narrow regions of phase diagrams, they are very restricted in tolerance to quantitative formulation changes [120].

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Polymeric Micelles Polymeric micelles composed of amphiphilic copolymers, that is, polymers consisting of a hydrophobic block and a hydrophilic block, are gaining increasing attention. They show high stability both in vitro and in vivo and good biocompatibility, and more importantly they can solubilize a broad variety of poorly soluble drugs in their inner core. Many of these drug-loaded micelles are currently at different stages of preclinical and clinical trials. Due to their hydrophilic shell and small size, they prefer to exhibit prolonged circulation times in vivo and can accumulate in tumor tissues. Polymeric micelles are formed by block copolymers consisting of hydrophilic and hydrophobic monomer units with the length of a hydrophilic block exceeding to some extent that of a hydrophobic one. If the length of a hydrophilic block is too high, copolymers exist in water as unimers (individual molecules), while molecules with very long hydrophobic block prefer to form structures with nonmicellar morphology, such as rods and lamellae. Diblock copolymers with an A–B structure and tri- or multiblock copolymers such as poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) (PEO–PPO–PEO) (A–B–A) may self-organize into micelles. The amphiphilic copolymers commonly have the critical micelle concentration (CMC) values as low as 10−6 M, which is about two orders of magnitude lower than that of such surfactants as Tween 80. As potential drug carriers, the hydrophobic core of polymeric micelles generally consists of a biodegradable polymer such as poly(β-benzyl-l-aspartate) (PBLA), PLA, or PCL which serves as a reservoir for an insoluble drug, protecting it from contact with the aqueous environment. The core may also consist of a water-soluble polymer [e.g., poly(aspartic acid; P(Asp)], which is rendered hydrophobic by the chemical conjugation of a hydrophobic drug or is formed through the association of two oppositely charged polyions (polyion complex micelles). Another special group of polymeric micelles is formed by lipid-core micelles, that is, micelles formed by conjugates of soluble copolymers with lipids (e.g., PEG–PE) [35, 36]. Three methods are used to manufacture polymeric micelles: dialysis, emulsification, and film methods. In the dialysis method, the drug and copolymer are dissolved together in a water-miscible solvent (e.g., ethanol) followed by dialysis against water. During the process (possibly several days), the insoluble drugs are incorporated into the formed micellar core. In the emulsification method, an O/W emulsion is first prepared using an aqueous solution of the copolymer and the drug solution in a water-insoluble volatile solvent (e.g., chloroform). The drug-loaded micelle is formed as solvent evaporation. In the film method, the copolymer solution and the drug solution are dissolved separately in miscible volatile organic solvents and are mixed followed by evaporating solvents to form a polymer/drug film. The film is hydrated in water or buffers, and then the micelle is formed by intensive shaking. If the amount of a drug exceeds the solubilization capacity of micelles, the excess drug precipitates in a crystalline form and is removed by filtration. The loading efficiency for different compounds varies from 1.5 to 50% by weight. The major driving force behind self-association of amphiphilic polymers is the decrease of free energy of the system due to removal of hydrophobic fragments from the aqueous surroundings with the formation of micelle core stabilized by hydrophilic blocks exposed to water [35, 36]. Various drugs, for example, diazepam and indomethacin, doxorubicin, anthracycline antibiotics, and polynucleotides, were effectively solubilized in polymeric micelles. Also polymeric micelles can carry various reporter (contrast) groups and


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become the imaging agents. Besides targeted drug delivery due to the EPR effect of tumor, specific polymeric micelles having stimuli-responsive amphiphilic block copolymers, targeting ligand molecules, or monoclonal antibody molecules are also manufactured [35, 36]. Nanogels Nanogels are colloidal stable particles made from hydrogels with nanosized hydrophilic polymeric networks. Hydrogels are the simple gels swelling strongly in aqueous media, typically composed of hydrophilic polymer components cross-linked into a network by either covalent (chemical cross-linking) or noncovalent (physical cross-linking) interactions. It is the cross-linking that provides for dimensional stability, while the high solvent content gives rise to the fluidlike transport properties. Cross-links are important to maintain the network structure of the hydrogels and prevent dissolution of the hydrophilic chains [121]. Two methods, emulsification–evaporation and the micelle/nanoparticle approach, are used to manufacture nanogels. In the former method, bis-activated PEG in dichloromethane is added dropwise to the aqueous solution of polyethylenimine (PEI) and then sonicated. The resulting white emulsion is evaporated in vacuum, producing a clear, slightly opalescent solution. This solution is stirred for less than one day at room temperature and much debris is separated by centrifugation. The nanogel suspension is obtained after dialysis against water [51]. This procedure is convenient except for using organic solvents, a vacuum evaporation step, and the obtained particles with a wide size distribution. Another method involves surface preactivated micelles or nanoparticles followed by reaction with other polymers on the surface in aqueous media. None of the organic solvents involved are of benefit. For example, a Pluronic block copolymer both ends of which are activated by 1,1′carbonyldiimidazole is dissolved in water at a concentration above its CMC. A diluted aqueous solution of PEI is then added dropwise to the micellar solution, stirring overnight. During this procedure a covalently linked cationic polymer PEI layer is formed on the Pluronic micelles, producing nanogels with narrow size distribution. After dialysis the resulting nanogel suspension can be further lyophilized. Using this procedure, the nanogels based on Pluronic P85/PEG and F127/PEG are obtained with final yields of 55 and 70% by weight and average hydrodynamic diameters of 100 and 180 nm, respectively [20, 52]. Many drugs can be entrapped into nanogels, for example, valproic acid, nucleoside analogues, antisense oligonucleotides, adenosine triphosphate (ATP), and small interfering ribonucleic acid (siRNA). Because macromolecular drugs such as peptides and proteins need to locate in a hydrophilic environment to maintain their activity, the particular hydrophilic property of nanogels would be of benefit. Special functions such as cellular targeting, crossing the BBB, and controlled release may also be achieved by the nanogel technique. In addition, the nanogel materials should be biodegradable. In cationic nanogels, PEI and PEG are cross-linked via urethane bonds, usually considered as stable links. However, due to the presence of highly protonated PEI, hydrolysis of these bonds was significantly accelerated, and the polymer network of nanogels could rapidly degrade in aqueous solution at the physiological pH during a period of about two weeks [20]. Dendrimers Dendrimers attracted much attention after they were first investigated by Tomalia 20 years ago [122, 123], and they have become the star molecules

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in recent years. Dendrimers possess perfect nanoarchitectures from 1 nm to more than 10 nm, consisting of repetitive chemical moieties with tree architecture. According to repetitive folds, dendrimers with the same basic cores are divided into a series of generations. The higher generation of dendrimers represents more repetitive units. Dendrimers are hyperbranched macromolecules that can be subdivided into three architectural parts: (a) the multivalent surface, with a high number of potential reactive sites; (b) the “outer shell” just beneath the surface, having a welldefined microenvironment protected from the outside by the dendrimer surface; and (c) the core, which in higher generation dendrimers is protected from the surroundings, creating a microenvironment surrounded by dendritic branches. Therefore, the interior of dendrimers is well suited for entrapment of guest molecules. The multivalent surfaces on a higher generation dendrimer can contain a very high number of functional groups. This makes the dendritic surfaces and outer shell well suited to host–guest interactions. Dendrimers can be tailored specifically for the desired purposes, for example, as dendritic sensors, drug vehicles, or even drugs [23]. Dendrimers are synthesized through a stepwise repetitive reaction sequence, wherein a convergent or divergent approach is used. On the one hand, the most divergent dendrimer syntheses require excess monomer loading and lengthy chromatographic separations, particularly at higher generations. On the other hand, convergent synthesis strategies are generally limited to the construction of only lower generation dendrimers due to the nanoscale steric issues that are encountered when attaching the dendrons to the molecular-level core [124]. Currently much of the work on dendrimers has been based on the commercially available Starburst poly(amidoamine) (PAMAM) dendrimers that are extensively studied as drug carriers. PAMAM may be synthesized from an ammonia or ethylenediamine core (EDA) by the divergent approach, involving Michael addition followed by amidation with methyl acrylate and resulting in the production of a dendrimer family (G = 0–7), and half-generation dendrimers are carboxyl terminated and full-generation dendrimers are amine terminated (e.g., G = 5.0, 5.3 nm in size) [122, 123]. Dendrimers have been evaluated as drug nanocarriers, gene transfection agents imaging agents, and nanodrugs [124]. Also many surface-modified dendrimers have been synthesized to obtain more functions such as active targeting and gene delivery. Dendrimers may be used as drugs for antibacterial and antiviral treatment and as antitumor agents. VivaGel, a topical water-based gel based on sulfonated naphthyl-modified poly(lysine) dendrimers, has been evaluated against human immunodeficiency virus (HIV) and other sexually transmitted diseases (STDs). The cationic dendrimers prefer to destabilize cell membranes and cause cell lysis and the cytotoxicity is generation dependent with higher generation dendrimers being the most toxic. The degree of substitution as well as the type of amine functionality is important, with primary amines being more toxic than secondary or tertiary amines. Another common dendrimer, poly(propylenimine) (PPI), shows similar behavior. However, anionic dendrimers show significantly lower cytotoxicity than cationic ones. PEG or fatty acid surface-modified dendrimers can reduce the cytotoxicity of cationic dendrimers [124]. Carbon Nanotubes, Fullerenes, and Quantum Dots Carbon nanotubes and fullerenes are carbon-based nanomaterials, and quantum dots are semiconductor nano-


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crystals. All of them show hydrophobic property. The possibility of cytotoxicity of these materials with inorganic nature should not be ignored although low toxicity is shown [25, 40, 125, 126]. However, these seemingly good results may be partly attributed to their poor solubility in polar solvents, which subsequently makes investigation of their biological properties difficult. They hardly load any drugs unless the surface is modified hydrophilically. Because these nanomaterials are mainly produced in laboratories with the special devices, their modifications and subsequent pharmacological investigations are limited. However, a number of functional derivatives have been synthesized, and it is found that the modified products have potent and selective pharmacological effects on organs, cells, enzymes, and nucleic acids [25, 37, 57]. 7.2.3.2

Nanosuspensions

Nanosuspensions of drugs are submicrometer colloidal dispersions of pure particles of drug which are stabilized by surfactants. A surprisingly large proportion of new drug candidates emerging from drug discovery programs are water insoluble, and therefore poorly bioavailable, leading to development efforts being abandoned. More than 40% of active substances during formulation development by the pharmaceutical industry are poorly water soluble. A substantial factor that prevents the development of such substances is the limited dissolution rate. Nanosuspensions are promising in addressing these so-called brickdust candidates. During the process of overcoming issues involving solubility, the additional pharmacokinetic benefits of drugs formulated in nanosuspensions come to be appreciated [19, 61]. Nanosuspensions can be used for those water-insoluble and oil-soluble compounds (high log P), although other lipidic carriers such as liposomes and emulsions can be used to formulate these compounds as well. However, nanosuspensions can be used to address other problems, such as compounds that are insoluble in both water and oil. Nanosuspensions can maintain the drug in a preferred crystalline state of size sufficiently small for pharmaceutical acceptability. For reasons of convenience to the patients, aqueous nanosuspensions can also be transformed to tablets or capsules after spray drying or freeze drying. Moreover, utilization of the dense, solid state confers an additional advantage of higher mass per volume loading. This is crucial when high dosing is required, for example, low-volume intramuscular and ophthalmic applications. Conventional approaches often attempt to solubilize insoluble drugs with the use of excessive amounts of cosolvents, but this often brings problems of toxicity. Besides, very large doses of drugs must be administered to animals when acute toxicity is investigated in preclinical research. As a result, the interference of toxic side effects caused by cosolvents cannot be ignored if using them [19]. Nanosuspensions are not nanocarriers so that what is emphasized during manufacturing is not materials but the manufacturing techniques. Only drugs and stabilizers (usually surfactants) participate in manufacturing nanosuspensions so that the process may be simple depending on drug instincts but sometimes it is not easy. The bottom-up and top-down approaches may be used in manufacturing nanosuspensions depending on drug nature and in-house devices. Antisolvent precipitation is a bottom-up method wherein two phases are involved: the initial creation of crystal nuclei of drugs and the subsequent growth. Formation

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of a stable suspension with the smallest particle size requires a high nucleation rate but low growth rate. Both process rates are dependent on temperature and supersaturation. The optimum temperature for nucleation might lie below that for growth. A high-supersaturation condition is achieved by adding small amounts of a watermiscible organic solution of the drug to the nonsolvent (water) under rapid mixing, which leads to spontaneous nucleation. At high-supersaturation levels, the crystal habit or external appearance is changed to a needlelike or dendritic morphology. These crystals are easily broken, forming new smaller nuclei. Rapidly grown crystals tend to be more imperfect and often incorporate impurities and dislocations. This effect is more pronounced for flexible molecules that have many degrees of freedom [19]. The presence of stabilizing surfactants is generally necessary to assist in forming submicrometer particles, and hydrophilic groups in the surfactants lead to rapid wetting of the high-surface-area particles in aqueous media, for example, in the case of oral administration. It is well known that the unprotective surfaces of nanoparticles show a high energy that leads to particle agglomeration. Therefore, the nanoparticles must be protected by, for example, steric hindrance and electrostatic pulsion. In the case of itraconazole (ITZ) nanosuspension manufacturing, a mixture solution of ITZ and Poloxamer (P407) in THF at room temperature was mechanically injected into a P407 aqueous solution at 3°C. Magnetic stirring was utilized to enhance heat and mass transfer. Nanosuspensions containing sub-300-nm particles were obtained with drug loads as high as 86% [60]. Top-down methods are also commonly used to manufacture nanosuspensions, including wet milling and homogenization. In pearl/ball milling, the active agent, in the presence of surface stabilizer(s), is comminuted by milling media. Particle size is determined by stress intensity and the number of contact points. The drug macrosuspensions are poured into a milling container containing milling pearls from, for example, glass, zircon oxide or special polymers such as hard polystyrene derivatives. The drugs are ground to nanocrystals between the pearls. The nanosuspensionderived products, Rapamune (sirolimus tablets) and Emend (aprepitant capsules), were approved by the FDA and launched in 2000 and 2003, respectively. They are manufactured by Elan’s NanoCrytal technology using a proprietary wet-milling technique. A general problem of pearl mills is potential erosion of materials from the milling pearls leading to product contamination. A polymer as substitution may minimize erosion. Scaling up with pearl mills is possible; however, there is a certain limitation in the size of the mill due to its weight. Up to about two-thirds of the mill volume are the pearls lead to heavy weight of the machinery, thus limiting the maximum batch size [127]. Homogenization can be divided into two types. One is the forcing of a suspension under pressure through a narrow-aperture valve (microfluidization). The other is high-pressure homogenization of particles in water or other media (piston gap). Microfluidization is a jet stream principle. The suspension is accelerated and passes a specially designed homogenization chamber with a high velocity. In the Z-type chamber, the suspension changes the direction of its flow a few times, leading to particle collision and shear forces, while in the Y-type chamber, the suspension stream is divided into two streams colliding frontally. Sometimes it is necessary to pass through the microfluidizer many times to minimize particle size [127]. In piston-gap homogenization, suspension contained in a cylinder passes a very thin gap with an extremely high velocity. Bubbles of water vapor are produced for


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compensation followed by collapsing in the valve. Cavitation-induced shock waves occur and crack the particles. Homogenization can also be utilized to further reduce the size of particles made by precipitation. Commonly dendritic crystals made by precipitation are more susceptible to rupture by the subsequent mechanical shock of homogenization. In addition, the mechanical energy supplied by the homogenizer can change the initially formed, unstable amorphous particles to a stable state through subsequent crystallization. The size of drug nanocrystals depends mainly on (a) power density of the homogenizer, (b) number of homogenization cycles, and (c) temperature. Another important determining factor for the final size of drug nanocrystals is the hardness of drugs. A relatively soft drug, paclitaxel, can reach 250 nm in size, which is less than harder drugs. The size should be homogeneous as achieved with a homogenizer to avoid physical destabilization. Stabilizers have an effect on long-term physical stability but not on maximum dispersity or the nanocrystal shape. Contamination from the production equipment is typically below 1 ppm, which is within a suitable range. Besides water, water-free media and water mixtures are used preferably due to advantages of easy evaporation or homogenization at higher temperature (with subsequent more cavitation). Oils, propylene glycol, and PEG with varieous molecular weights can be used. For PEG being solid at room temperature (e.g., PEG 1000, 6000), the obtained drug nanocrystals disperse in PEG particles at room temperature and can conveniently be put into hard capsules [19, 127]. The lyophilized drug nanosuspensions can be transferred to a final dry oral dosage form such as tablets or reconstituted prior to administration. Drug nanosuspensions can be directly used as parenteral products. A shelf life of up to three years was shown for selected nanosuspensions. Sterilization can be achieved by aseptic processing of previously sterilized components, membrane filtration for particles sufficiently small or for drugs that can withstand it, steam sterilization, or γ-irradiation. 7.2.3.3

Self-Assembled Drug Nanostructures

It is well known that liposomes are composed of amphiphilic phospholipids. The formation of liposomes is actually a procedure of molecular self-assembly. Furthermore, great amounts of amphiphilic compounds, natural or synthesized rather than phospholipid-like surfactants, can also self-assemble into ordered aggregates in aqueous media or organic solvents [128–130]. The formed aggregates are mostly nanoarchitectures with various shapes such as vesicle, rod, ribbon, fiber, tube, or helix [131–134]. They can remain relatively stable in certain environments. Many of them may become drug nanocarriers, such as liposomes, or even perform as active agents [109, 135, 136]. The research of self-assembled nanocarriers seems to go into the field of supramolecular chemistry. But these results also give us some useful information for developing new approaches of drug delivery. A novel idea may relate to why we do not try to construct a nanostructure from drugs themselves. Twenty years ago the cardiovascular drug pindolol was conjugated with stearyl glycerol via succinyl as linker followed by forming maleate salt to obtain pindolol diglyceride. Vaizoglu and Speiser used the word “pharmacosomes” to describe the colloidal dispersions prepared from drug–lipid conjugates with or without additional surfactants [137]. Pindolol pharmacosomes (vesicle-like) were prepared from

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pindolol diglyceride, which showed good stability and useful pharmacokinetic parameters. Unfortunately, no more detailed research is being done about pharmacosomes, possibly because no appropriate theory supports the new dosage form and no proper drugs and lipids are selected. The idea of manufacturing nanostructures from drugs may be resourced from liposomes, pharmacosomes, and other molecular self-assemblies in supramolecular chemistry. More importantly, this novel idea resulted from long-term efforts to work on drug delivery and to solve the disadvantages of current drug carriers. Almost all current delivery systems (usually called carriers) passively load drugs so as to always lead to low entrapment efficiency and possible drug leakage in preparation, preservation, and transportation in vivo [30, 73], and these carriers might have been destroyed in vivo before reaching target sites. In addition, lipophilic biomembranes, including cell membranes, usually prevent hydrophilic drugs from entering into target sites. If carriers cannot override cell membranes except for endocytosis/ phagocytosis by cells/macrophages, the loaded hydrophilic drugs are probably released only on target surfaces, not entering. In summary, a majority of drugs could not eventually reach and get into target sites due to poor properties of carriers and drugs. A novel technology involving prodrug, molecular self-assembly, and nanotechnology was developed to address the problems of drugs and classical carriers. The nanostructures are formed by molecular self-assembly of amphiphilic prodrugs in aqueous media generally without additional excipients. The self-assembled drug nanostructures not only possess the amphiphilic property of monomolecular drugs, benefiting to cross biomembranes, but also deliver themselves in vivo without “carriers” and then prefer to release active parent agents with a sustained rate. They may overcome some deficiencies of traditional nanocarriers such as liposomes, for example, low efficiency of drug entrapment and loading, rapid drug leakage in vitro/in vivo, and bad stability [22]. Self-assembled drug nanostructure is not a proprietary term in pharmacy currently. Herein this term is defined as the ordered nanosized self-aggregates of amphiphilic drugs in aqueous media. It is abbreviated as SADN. Another term, self-assembled drug delivery system (SADDS), introduced by Jin [22] obviously includes SADN. Unfortunately, most current drugs do not occupy an amphiphilic and self-assembling nature [138], so they must be modified in chemical structures before manufacturing SADNs. Then the prodrug technique is selected. In contrast to nanosuspension technology, SADN technology is mainly applied to hydrophilic or polar drugs. These drugs are rationally modified to their amphiphilic prodrugs by lipid derivation. Molecular self-assembly in aqueous media is the key to manufacturing SADNs. According to the principles of supramolecular chemistry, the amphiphilic molecules forming self-assemblies should have proper structural conformation. The morphology of assemblies also depends on the structure of amphiphiles and the surrounding environment. Some parameters, including the optimal head area ao, the volume v of fluid hydrophobic chain, and the maximum efficient chain length lc, are used to describe the conformation of amphiphiles. The critical packing parameter (CPP), equal to v/aolc, can be applied to direct selfassembly behavior. The amphiphiles prefer to form vesicles when the CPP is –1. Generally, single-chain lipids with small head group areas (e.g., SDS in a low-salt solution) are cone shaped, prone to form spherical micelles, while double-chain


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lipids with large head group area and fluid chains (e.g., phosphatidylcholine) are truncated-cone shaped, prone to form flexible bilayers, vesicles [139]. Therefore, the lipids used for drug covalent conjugation are rationally selected from long-chain alkyl lipids, for example, fatty acids, lipid alcohols, lipid amines, long-chain glycolipids, and cholesterol. Furthermore, too large or small polar drugs are not appropriate for preparation of self-assembling prodrugs. Antiviral nucleoside analogues such as acyclovir, didanosine, and zidovudine were used to prepare their long-chain glyceride or cholesteryl derivatives in Jin’s laboratory [140–142]. All the derivatives showed amphiphilic property and some of them self-assembled into ordered aggregates in water. Amphiphilic prodrugs were subsequently used to manufacture self-assemblies using the bottom-up approach, such as liposomes, and the self-assembly may be driven by a hydrophobic interaction, hydrogen bonding, and so on [21, 143]. The monomolecular amphiphilic prodrug is prone to incorporate into the assemblies and not to depart so that almost no drugs leak from SADNs. The whole self-assemblies are nearly composed of amphiphilic drugs, leading to high drug loading. When SADNs reach targets in vivo, the continual dissociation of aggregates and the sustained degradation of prodrugs provide controlled drug release. Acyclovir self-assembled nanoparticles as SADNs were manufactured which showed strong targeting effect in vivo (mainly in the MPSs) and sustained release at target sites [22]. Based on this paradigm, a general process to manufacture SADNs is as follows [21, 22]: (a) To obtain an amphiphilic prodrug with proper molecular structure, stearyl glyceride was selected to conjugate with acyclovir. Succinyl acyclovir (SACV) was synthesized and subsequently conjugated with stearyl glyceride by acylation reaction. The amphiphilic prodrug stearyl-glyceride-succinyl acyclovir (SGSA) was obtained. (b) The injection method was used to manufacture SADNs. SGSA was dissolved in the water-miscible solvent THF. The solution containing 5 mg/mL SGSA was slowly and continually injected into vortexed water under surface via a 100-μL microsyringe. A homogeneous and slightly opalescent suspension was obtained, which was acyclovir self-assembled nanoparticles (SANs). (c) The organic solvent was removed from the suspension through evaporation by heating, and the suspension can further be concentrated by removing water under heating until the appropriate prodrug concentration is obtained. The concentrated suspension was transferred into ampoules and sealed. It may be sterilized by autoclave. (d) Acyclovir SANs were characterized. They were cuboidlike shaped based on transmission electron microscopy and were nanoscale with an average size of 83 nm based on dynamic light scattering. The zeta potential of −31 mV indicated the nanoparticles had negative surface charge. Hydrophobic interaction of alkyl chains improves SGSA molecules to form bilayers, and then cuboidlike nanoparticles were achieved by layer-by-layer aggregation based on inter-bilayer hydrogen bonding. The gel–liquid crystalline phase transition was about 50°C, and the mechanism of configuration changes on phase transition was analyzed [144].

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(e) The in vitro and in vivo behavior of acyclovir SANs was investigated. The SANs kept the physical state stable upon centrifugation or exposure of some common additives Autoclave and bath heat for sterilization hardly influenced the state of SANs. SGSA in SANs showed good chemical stability in weak acidic or neutral buffers, although they were very sensitive to alkaline solutions and carboxylester enzymes. The SANs were rapidly removed from blood circulation after bolus IV administration to rabbits and mainly distributed in liver, spleen, and lung followed by slow elimination in these tissues. Because nucleoside analogues are important and plentiful agents in antiviral and anticancer therapy, other polar drugs can simulate the above process to manufacture SADNs. In addition, macrophages as the reservoirs of HIV or other viruses prefer to carry viruses throughout the whole body even to the central nervous system. How to deliver drugs to macrophages has become a key issue in antiviral therapy [145]. SADNs prefer to show macrophage-specific distribution. Therefore, the antiviral SADNs show the advantages of high drug loading, controlled release, and targeting macrophage, which may provide a useful and promising way to treat increasing viral diseases. In the future SADNs will be modified to get more functions, such as long circulating effect, pH sensitivity, and use in antiviral, anticancer, and gene therapy.

7.2.4

NANOMEDICINE

Nanomedicine is a concept with broad implications. According to the definition of the European Science Foundation (ESF), the field of nanomedicine is the science and technology of diagnosing, treating, and preventing disease and traumatic injury, of relieving pain, and of preserving and improving human health using molecular tools and molecular knowledge of the human body. It is perceived as embracing five main subdisciplines that in many ways are overlapping and underpinned by the following common technical issues: (a) analytical tools, (b) nanoimaging, (c) nanomaterials and nanodevices, (d) novel therapeutics and drug delivery systems, and (e) clinical, regulatory, and toxicological issues. The ESF’s scientific forward look on nanomedicine warns that nanomedicine benefits will be lost without major investment and calls for a coordinated European strategy to deliver new nanotechnology-based medical tools for diagnostics and therapeutics [146]. From a view of narrow sense, nanomedicine can be defined as the use of nanoscale or nanostructured materials in medicine that have unique medical effects according to their structure. In addition, nanostructures up to 1000 nm in size are adopted because from a technical point of view the control of materials in this size range not only results in new medical effects but also requires novel, scientifically demanding chemistry and manufacturing techniques [147]. The increasing research in nanomedicine has led to many publications, accounting for about 4% of publications on nanotechnology research (about 34,300 documents in 2004) worldwide. Also commercialization efforts in nanomedicine are increasing. About 207 companies (including 158 small- and medium-size enter-

NANOMEDICINE

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prises) visibly pursue nanomedicine activities and devote either all or a significant share of their business to the development of nanomedicines. A characterizing feature of nanotechnology is its enabling function to add new functionality to existing products, making them more competitive. For example, Ambisome (Gilead), a liposomal formulation of the fungicide Fungizone (Bristol-Myers Squibb) that shows reduced kidney toxicity, had total sales of $212 million in 2004. The total sales of the 38 identified nanomedicine products from all sectors of nanomedicine are estimated to be $6.8 billion in 2004. The market is predicted to further grow to ∼$12 billion by the year 2012. Currently, nanomedicine is dominated by drug delivery systems, accounting for more than 75% of the total sales. Twenty-three nanoscale drug delivery systems are available on the market, but within this group, three polymer therapeutics alone account for sales of $3.2 billion: (i) Neulasta (pegfilagrastim; recombinant methionyl human granulocyte colony stimulating factor and PEG), (ii) Pegasys (PEGylated interferon α 2a), and (iii) PEG-Intron (PEGylated interferon α 2a), all protein therapeutics to which nanoscale polymer strings of PEG have been attached to reduce immunogenicity and to prolong plasma half-life. The most widely used nanotechnology product in the field of in vitro diagnostics is colloidal gold in lateral flow assays, rapid tests for pregnancy, ovulation, HIV, and other indications. Magnetic nanoparticles are also used for cell-sorting applications in clinical diagnostics. In the field of biomaterials, the commercial status of nanotechnology-based dental restoratives is most advanced. Furthermore, nanohydroxyapatite-based products for the repair of bone defects have been successfully commercialized. Nanotechnology-based contrast agents are a market with estimated sales of about $12 million. All of the marketed contrast agents consist of superparamagnetic iron oxide nanoparticles for magnetic resonance imaging. Nanostructured electrodes are used to improve the electrode tissue contact, and nanomaterials are used to increase the biocompatibility of implant housings. Pacemakers with nanostructured (fractal) electrodes are the only active implants currently on the market that contain a nanotechnology-enabled component [147]. In spite of the great success, the safety of nanomedicine is maintained as a worrying issue. A new discipline appears to exploit the toxicological problem in nanotechnology applications, called nanotoxicology. Nanotoxicology can be defined as safety evaluation of engineered nanostructures and nanodevices. Nanomaterials could be deposited in all regions of the respiratory tract after inhalation. The small size facilitates uptake into cells and transcytosis across epithelial and endothelial cells into the blood and lymph circulation to reach potentially sensitive target sites such as bone marrow, lymph nodes, spleen, and heart. Access to the central nervous system and ganglia via translocation along axons and dendrites of neurons has also been observed. Nanomaterials could also penetrate the skin via uptake into lymphatic channels [41]. Although possible damages of those biodegradable nanomaterials for drug delivery need consideration, too much fear is needless. Usually they would be ultimately degraded nearly without any trace. However, hard or nonbiodegradable materials, including carbon nanotubes, fullerenes, quantum dots, polystyrene, and metal nanoparticles, should be thoroughly investigated about their toxic effects on our body before clinical application.

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7.2.5


PERSPECTIVE

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126. Smart, S. K., Cassady, A. I., Lu, G. Q., and Martin, D. J. (2006), The biocompatibility of carbon nanotubes, Carbon, 44, 1034–1047. 127. Keck, C. M., and Muller, R. H. (2006), Drug nanocrystals of poorly soluble drugs produced by high pressure homogenisation, Eur. J. Pharm. Biopharm., 62, 3–16. 128. Guerin, C. B. E., and Szleifer, I. (1999), Self-assembly of model nonionic amphiphilic molecules, Langmuir, 15, 7901–7911. 129. Choi, S. K., Vu, T. K., Jung, J. M., Kim, S. J., Jung, H. R., Chang, T., and Kim, B. H. (2005), Nucleoside-based phospholipids and their liposomes formed in water, ChemBioChem, 6, 432–439. 130. Snip, E., Shinkai, S., and Reinhoudt, D. N. (2001), Organogels of a nucleobase-bearing gelator and the remarkable effects of nucleoside derivatives and a porphyrin derivative on the gel stability, Tetrahedr. Lett., 42, 2153–2156. 131. Engberts, J. B. F. N., and Hoekstra, D. (1995), Vesicle-forming synthetic amphiphiles, Biochim. Biophys. Acta, 1241, 323–340. 132. Giulieri, F., and Krafft, M. P. (2003), Tubular microstructures made from nonchiral single-chain fluorinated amphiphiles: Impact of the structure of the hydrophobic chain on the rolling-up of bilayer membrane, J. Coll. Interf. Sci., 258, 335–344. 133. Giorgi, T., Lena, S., Mariani, P., Cremonini, M. A., Masiero, S., Pieraccini, S., Rabe, J. P., Samori, P., Spada, G. P., and Gottarelli, G. (2003), Supramolecular helices via self-assembly of 8-oxoguanosines, J. Am. Chem. Soc., 125, 14741–14749. 134. Yanagawa, H., Ogawa, Y., Furuta, H., and Tsuno, K. (1989), Spontaneous formation of superhelical strands, J. Am. Chem. Soc., 111, 4567–4570. 135. Zemel, A., Fattal, D. R., and Ben-Shaul, A. (2003), Energetics and self-assembly of amphipathic peptide pores in lipid membranes, Biophys. J., 84, 2242–2255. 136. Chabaud, P., Camplo, M., Payet, D., Serin, G., Moreau, L., Barthelemy, P., and Grinstaff, M. W. (2006), Cationic nucleoside lipids for gene delivery, Bioconjugate Chem., 17, 466–472. 137. Vaizoglu, M. O., and Speiser, P. P. (1986), Pharmacosomes—A novel drug delivery system, Acta Pharm. Suec., 23, 163–172. 138. Schreier, S., Malheiros S. V. P., and de Paula, E. (2000), Surface active drugs: Selfassociation and interaction with membranes and surfactants. Physicochemical and biological aspects, Biochim. Biophys. Acta, 1508, 210–234. 139. Israelachvili, J. N. (1992), Intermolecular and Surface Forces with Applications to Colloidal and Biological Systems, Academic, London. 140. Jin, Y., Li, M., Tong, L., Wang, L., and Peng, T. (2003), Lipid derivatives of nucleoside analogues and their salts, China Patent CN1259331. 141. Jin, Y., and Ai, P. (2004), Cholesteryl derivatives of nucleoside analogues, China Patent CN1566130. 142. Jin, Y., Du, L., Xing, L., and Xin, R. (2006), Cholesteryl phosphoryl derivatives of nucleoside analogues, China Patent Application 2006101122957. 143. Jin, Y., Qiao, Y., and Hou, X. (2006), The effects of chain number and state of lipid derivatives of nucleosides on hydrogen bonding and self-assembly through the investigation of Langmuir-Blodgett films, Appl. Surf. Sci., 252, 7926–7929. 144. Jin, Y. (2007), Effect of temperature on the state of the self-assembled nanoparticles prepared from an amphiphilic lipid derivative of acyclovir, Coll. Surf. B Biointerf., 54, 124–125.

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145. Aquaro, S., Calio, R., Balzarini, J., Bellocchi, M. C., Garaci, E., and Perno, C. F. (2002), Macrophages and HIV infection: Therapeutical approaches toward this strategic virus reservoir, Antivir. Res., 55, 209–225. 146. European Science Foundation (2005), Nanomedicine, An ESF-European Medical Research Council (EMRC) forward look report, European Science Foundation, Strasbourg. 147. Wagner, V., Dullaart, A., Bock, A.-K., and Zweck, A. (2006), The emerging nanomedicine landscape, Nat. Biotechnol., 24, 1211–1217.

7.3 PHARMACEUTICAL NANOSYSTEMS: MANUFACTURE, CHARACTERIZATION, AND SAFETY D. F. Chowdhury University of Oxford, Oxford, United Kingdom

Contents 7.3.1 7.3.2 7.3.3 7.3.4

7.3.5

7.3.6

7.3.7

Definition 7.3.1.1 Top-Down and Bottom-Up Approaches to Nanotechnology Taxonomy of Nanomedicine Technologies Nano–Pharmaceutical Systems Description of Nanosystems 7.3.4.1 Polymeric Systems 7.3.4.2 Quantum Dots and Quantum Confinement 7.3.4.3 Metal Nanoparticles and Surface Plasmon Resonance 7.3.4.4 Self-Assembled Systems 7.3.4.5 Nanostructures Based on Carbon Manufacturing Technologies 7.3.5.1 Nanoscale Assembly Methods 7.3.5.2 Nano-structuring processes for polymeric materials Characterization Techniques 7.3.6.1 Nanoparticle Characterization Methods and Tools 7.3.6.2 Scanning Probe Technologies Toxicology Considerations 7.3.7.1 Lung Toxicity 7.3.7.2 Systemic Uptake 7.3.7.3 Skin Permeation of Nanoparticles References Suggested Reading


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7.3.1

PHARMACEUTICAL NANOSYSTEMS

DEFINITION

Nanotechnology is an enabling technology and one which is generally manifest at the primary level in the form of nanomaterials. The definition of nanotechnology therefore focuses on materials and how manipulation at the nanoscale leads to novel properties and therefore potentially new uses. The pharmaceutical industry has yet to adopt strict guidelines for what falls under the remit of nanotechnology, with numerous definitions in existence. For the purpose of this chapter, the current U.S. Food and Drug Administration (FDA) definition for nanotechnology as applied to pharmaceuticals is deemed most appropriate. The FDA describes nanotechnology as technology that includes the following [1]: 1. Research and technology development or products regulated by the FDA that are at the atomic, molecular or macromolecular levels and where at least one dimension that affects the functional behavior of the product is in the length scale range of approximately 1–100 nm 2. Creating and using structures, devices, and systems that have novel properties and functions because of their small and/or intermediate size 3. Ability to control or manipulate at the atomic scale Nanotechnology is therefore essentially about understanding and manipulating materials at the atomic, molecular, and macromolecular level in a way that imparts properties to the material that would otherwise not exist either as individual atoms or as bulk processed macroscopic systems. Properties that can be exploited to provide novel and unique properties to materials include surface and quantum effects, for example, van der Waals forces; electrostatic interaction; ionic, covalent and hydrogen bonding; and quantum confinement. Additionally nonconventional means of molecular assembly and atomic manipulation can lead to novel material properties. Control and exploitation of these effects can lead to new and useful changes to the thermal, magnetic, electrical, optical and mechanical, and biological and physicochemical properties of materials.

7.3.1.1 Top-Down and Bottom-Up Approaches to Nanotechnology There are generally two approaches to nanotechnology, the top-down and bottomup approaches. As the names suggest, the top-down approach utilizes ultraprecision machining and nanolithographic techniques among others to achieve very high definition structures with nanolevel accuracy, usually either by removing material from the surface of a larger structure until the desired structure with desired features is achieved or through deposition of material with almost atomic-scale precision and control. The bottom-up approach involves the assembly of atoms, molecules, or nanoscale components to assemble a larger structure within the nanoscale range. There are numerous methods by which this can be achieved, including conventional bulk chemical processing methods and exploitation of chemical and biological selfassembly techniques. The pharmaceutical industry is primarily involved in the application of nanomaterials rather than the discovery and development of new materials, though as this chapter will indicate, there are often areas of overlap between what is a new material and a construct of a novel material.

NANO-PHARMACEUTICAL SYSTEMS

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7.3.2 TAXONOMY OF NANOMEDICINE TECHNOLOGIES A useful starting point would be to gauge the breadth of technologies falling under the classification of nanomedicine. Table 1 provides a means of classifying materials and processes derived from nanotechnology as relating to pharmaceuticals and medicine in general.

7.3.3

NANO–PHARMACEUTICAL SYSTEMS

Having gauged the huge scope of nanotechnology in medicine, the scope of this chapter is limited to pharmaceuticals. The term pharmaceutical is intended in the context of systems pertaining to drugs or dosage formulations. Nano–pharmaceutical systems generally imply products that may in their own right or in combination with another moiety bring about therapeutic benefit. They may also include engineered nanostructured systems that may act as a carrier for drugs or a delivery vehicle or a delivery system for drugs and therapeutic agents. The definition is extended to include imaging systems which may be used alone or in conjunction with therapeutic agents given the numerous nanosystems that have found application in diagnostics and imaging. The various nanoscale architectures that can be achieved using nanotechnology include spheres (solid or hollow), tubes, porous particles, solid particles, and branched structures, and with the rapid evolution of lithographic techniques, threedimensional objects of almost any desired shape can be achieved from both metals and polymers. Given the vast spectrum of materials of construction, size, shape, and form covered by nanosystems, a simple means of classification is needed for effective differentiation between systems. Nanosystems can be classified in a number of ways, for example, according to their elemental composition, according to size, structure, and function, or perhaps according to a structure–function relationship. It can be said that nanosystems fall into the broader category of nanostructures, which can be generalized into the following categories: Particulate nanostructures Capsular nanostructures Crystalline nanostructures Polymeric nanostructures It can be readily appreciated that there will be an element of overlap between the broad categories above and in particular with evolving complex hybrid systems. The classification is intended as a point of reference and for ease of understanding the vast possibilities that exist with nanosystems without the need for constant reclassification as far as possible. These structures may be further differentiated according to their primary composition: Organic Inorganic Organic/inorganic hybrid Carbon based

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TABLE 1 Partial Nanomedicine Technologies Taxonomy Raw nanomaterials Nanoparticle coatings Nanocrystalline materials Nanostructured materials Cyclic peptides Dendrimers Detoxification agents Fullerenes Functional drug carriers Magnetic resonance (MR) scanning (nanoparticles) Nanobarcodes Nanoemulsions Nanofibers Nanoparticles Nanoshells Carbon nanotubes Quantum dots Artificial binding sites Artificial antibodies Artificial enzymes Artificial receptors Molecularly imprinted polymers Cell simulations and cell diagnostics Cell chips Cell stimulators DNA manipulation, sequencing, diagnostics Genetic testing Deoxyribonucleic acid (DNA) microarrays Ultrafast DNA sequencing DNA manipulation and control Tools and diagnostics Bacterial detection systems Biochips Biomolecular imaging Biosensors and biodetection Diagnostic and defense applications Endoscopic robots and microscopes Fullerene-based sensors Imaging (e.g., cellular) Lab on chip Monitoring Nanosensors Point-of-care diagnostics Protein microarrays Scanning probe microscopy

Control of surfaces Artificial surfaces, adhesive Artificial surfaces, nonadhesive Artificial surfaces, regulated Biocompatible surfaces Biofilm suppression Engineered surfaces Pattern surfaces (contact guidance) Thin-film coatings Nanopores Immunoisolation Molecular sieves and channels Nanofiltration membranes Nanopores Separations Biological research Nanobiology Nanoscience in life sciences Drug delivery Drug discovery Biopharmaceuticals Drug delivery Drug encapsulation Smart drugs Synthetic biology and early nanodevices Dynamic nanoplatform “nanosome” Tecto-dendrimers Artificial cells and liposomes Polymeric micelles and polymersomes Nanorobotics DNA-based devices and nanorobots Diamond-based nanorobots Cell repair devices Intracellular devices Intracellular assay Intracellular biocomputers Intracellular sensors/reporters Implant inside cells BioMEMS Implantable materials and devices Implanted bio—microelectromechanical systems (MEMSs), chips, and electrodes MEMS/nanomaterial-based prosthetics Sensory aids (e.g.,) Microarrays Microcantilever-based sensors Microfluidics Microneedles Medical MEMS MEMS surgical devices

DESCRIPTION OF NANOSYSTEMS

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TABLE 1 Continued Molecular medicine Genetic therapy Pharmacogenomics Artificial enzymes and enzyme control Enzyme manipulation and control Nanotherapeutics Antibacterial and antiviral nanoparticles Fullerene-based pharmaceuticals Photodynamic therapy Radiopharmaceuticals

Biotechnology and biorobotics Biological viral therapy Virus-based hybrids Stem cells and cloning Tissue engineering Artificial organs Nanobiotechnology Biorobotics and biobots


7.3.4


There is clear evidence from Table 2 that some of the nanosystems indicated are based on conventional colloidal chemistry, and their characteristics are well established and understood. The descriptions below deal mainly with systems deemed nonconventional and cover some of the key novel properties derived from or utilized as part of their construction. 7.3.4.1

Polymeric Systems

Polymer-based systems offer numerous advantages, such as biocompatibility, biodegradability, and ability to incorporate functional groups for attachment of drugs. Drugs can be incorporated into the polymer matrix or in the cavity created by the polymeric architecture, from which the drug molecule can be released with an element of temporal control, and controlled pharmacokinetic profile with almost zero-order release achievable. Dendrimers are large complex globular polymeric molecules [46] with welldefined chemical structure, size, and shape [47]. They consist of characteristic threedimensional branched structures. The key components of dendrimers are their core, branches, and end groups, and precise control over these features is possible during the bottom-up synthesis process, thus allowing control over size composition, and final chemical reactivity. A more advanced form of dendrimer is the hyperbranched dendrimer, where precision control over the architectural construct is lost during the synthesis process [48]. Dendrimers are produced from monomers through an iterative sequence of reaction steps [49] using either convergent [50, 51] or divergent [52, 53] step growth polymerization [54–56] and have potential applications in gene and cancer therapy and drug delivery through complexation or encapsulation [57–59]. 7.3.4.2

Quantum Dots and Quantum Confinement

Quantum dots are inorganic semiconductor nanocrystals that possess physical dimensions smaller than the exciton Bohr radius, giving rise to the unique phenom-

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TABLE 2 Classification of Nanostructures According to Composition and Perceived Applications Composition Organic

Inorganic

Organic/ inorganic hybrid

Carbon based

Type of Nanostructure

Applications

Polymer micelles [3, 4] Polymeric spheres [5] Polymer nanoparticles [6, 7] Polymer vesicles/containers [8–10] Lipid nanovesicles [11] Lipid emulsions [12] Ring peptides [13] Lipid nanospheres [14, 15] Lipid nanoparticles [16, 17] Lipid nanotubes [18] Peptide nanoparticles [19, 20] Nanobodies [21] Dendrimers [22–24] Palladium/platinum nanoparticles [25] Silicon nanoneedles [26] Porous silicon [27, 28] Gold nanoparticles [29] Iron oxide nanoparticles [30] Gold nanoshells [31] Quantum dots [32] Metallic nanoshells [33] Nanocrystals [34, 35] Nanocomposites [36] Nanosphere–metallic particle composite [37] Carbon nanotube clusters [38]

Drug delivery Drug delivery Drug delivery Drug delivery Drug delivery Drug delivery Drug delivery Drug delivery Drug delivery Drug delivery Drug delivery Therapeutic, diagnostic Drug delivery, gene delivery Drug delivery Drug delivery Drug delivery Drug delivery Imaging Imaging agent, thermal ablation Imaging, cell targeting Imaging, thermal ablation Drug delivery Drug delivery Drug delivery, imaging

Core–shell structures [39, 40] Fullerenes [41–43] Carbon nanotubes [44, 45]

Drug delivery, imaging, thermal ablation Imaging, thermal ablation Drug delivery, prodrug Drug delivery, imaging, thermal ablation

enon known as quantum confinement. Quantum confinement is the spatial confinement of charge carriers (i.e., electrons and holes) within materials. It leads to unique optical and electrical properties that are not common for bulk solids. Quantum dots have novel and unique optical, magnetic [60], and electronic properties, exceptional imaging properties due to high color intensity, with up to 20 fluorophores, high resistance to photobleaching, and narrow spectral line widths. Their size and composition allow for tunable emission that can be excited using a single wavelength [61–64]. These properties have led to uses as fluorescent imaging probes, detection of cell signaling pathways, and cell targeting. Low depth of light penetration and relatively high background fluorescence are the key limitations of quantum dots in in vivo clinical applications. Quantum dots are generally of the size rage 2– 8 nm in diameter [65] and have large molar extinction coefficients [66], thus making them very bright in vivo probes.


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Applications of quantum dots include optical detection of genes and proteins in animal models and cell assays and tumor and lymph node visualization through imaging [67, 68]. 7.3.4.3

Metal Nanoparticles and Surface Plasmon Resonance

Surface plasmons, also known as surface plasmon polaritons or packets of electrons, are surface electromagnetic waves that propagate parallel along a metal–dielectric interface [69, 70]. Surface plasmons exist where the complex dielectric constants of the two media are of opposite sign. The excitation of surface plasmons by light of a wavelength matching the resonant frequency of the electrons is termed surface plasmon resonance (SPR) for planar surfaces and localized surface plasmon resonance where nanometer-sized metallic structures are concerned [71, 72]. Surface plasmon effects result in useful photothermal effects [73] and have been used to enhance the surface sensitivity of various spectroscopic measurements [74], including fluorescence, Raman scattering, and second-harmonic generation. Metallic nanostructures exhibiting SPR are composed of a dielectric core and metallic shell, for example, gold sulfide dielectric core and gold shell. By varying the core–shell thickness ratio, the surface plasmon resonance is shifted from the visible to the infrared range [75], spanning a range that is mostly transparent to human tissue, that is, has a high physiological transmissivity. Additionally, control over the particle diameter allows control over light scattering and light absorption at particle diameters below approximately 75 nm. Potential applications of the photothermal effects of engineered nanoparticles include the following: Controlled drug delivery [76] Analysis of controlled drug release from a matrix [77] DNA sensor [78, 79] Deep tissue tumor cell thermal ablation [80] Real-time assessment of drug action [81, 82] Immunosensor applications [83, 84] 7.3.4.4

Self-Assembled Systems

Molecular self-assembly is a synthetic technique that has been widely used to produce nano- and microstructures in a quick and efficient manner. It has become all the more crucial to the formation of nanostructures due to the control attainable over the end product and the relative ease with which nanostructures of defined structure and function can be produced using bulk manufacturing methods. The basic principle of self-assembly is based on the simultaneous coexistence of two parallel forces [85, 86], long-range repulsive forces and short-range attractive interactions. The types of structures attainable using molecular self-assembly are referred to as micellar structures [87] and can take on various sizes and shapes: Direct spherical micelles [88] Inverse spherical micelles [89, 90]

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Lamellar sheets [91] Vesicles (hollow or concentric) [92] Body-centerd-cubic [92] Hexagonally packed cylinders/tubes [92] Gyroids [93] Hollow spheres [94] These systems have found widespread use as drug delivery vehicles, and the more advanced nanosystems are termed smart nano-objects due to their ability to sense local variations in physiological conditions, such as pH and temperature, and respond to the stimulus accordingly. 7.3.4.5

Nanostructures Based on Carbon

Nanotubes The structure of carbon nanotubes as observed by scanning tunneling microscopy is that of rolled grapheme sheets where endpoints of a translation vector are folded one onto another [95]. Single-walled carbon nanotubes (SWCNTs) were first reported by Iijima and Ichihashi [96] in 1993. Enormous interest in CNTs has centered around their unique properties, including high electrical conductivity, thermal conductivity, high strength and aspect ratio, ultralight weight, and excellent chemical and thermal stability. The most common method for the production of carbon nanotubes is hydrocarbon-based chemical vapor deposition (CVD) [97] and adaptations of the CVD process [98, 99], where the nanotubes are formed by the dissolution of elemental carbon into metal nanoclusters followed by precipitation into nanotubes [100]. The CVD method is used to produce multiwalled carbon nanotubes (MWCNTs) [101] and double-walled carbon nanotubes (DWCNTs) [102] as well as SWCNTs [103]. The biomedical applications of CNTs have been made possible through surface functionalization of CNTs, which has led to drug and vaccine delivery applications [104, 105]. Fullerenes Fullerenes were first discovered in 1985 [106] and are large molecules composed exclusively of carbon atoms and manifest physically in the form of hollow spherical cagelike structures. The cages are in the region of 7–15 Å in diameter with the most common form being C60, though other forms exist too, such as C70, C76, and C84, depending on the number of carbon atoms making up the cage. Fullerenes can be produced using combustion [107] and arc discharge methods [108]. Fullerenes offer numerous points of attachment and allow precise bonding of active chemical groups in three-dimensional (3D) conformations and positional control with respect to matching conjugated fullerene compounds with a given target. Water-soluble fullerenes have shown low biological toxicity both in vitro [109] and in vivo [110]. Some of the potential applications of fullerenes in pharmaceuticals include their use in neurodegenerative and other disease conditions where oxidative stress is part of the pathogenesis due to their powerful antioxidant properties [111] and in nuclear medicine for binding of toxic metals ions, increasing therapeutic potency of radiation therapy and reducing adverse events as fullerenes do not undergo biochemical degradation within the body. Fullerene applications in photodynamic tumor therapy have also been shown [112].

MANUFACTURING TECHNOLOGIES

7.3.5

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There are a diverse range of technologies being applied to the manufacture of nanosystems for pharmaceutical applications. Some of these are derived from conventional pharmaceutical technologies, such as colloidal processing, and many have been adopted from the semiconductor industry, whereby precision spatial control is achieved over the production of nanosystems and particles using fabrication techniques. To add to this, new technologies are constantly evolving through the adaptation and amalgamation of existing technologies in different fields or through pure innovation leading to completely new processes. It is outside the remit of this chapter to cover in any depth all those manufacturing technologies that may be applied to pharmaceutical manufacturing. The summary in Table 3 provides a detailed synopsis of the different types of manufacturing processes and types of technologies for each process. This is followed by a brief introduction to some of the technologies, with the omission of silicon- and carbon-based fabrication processes, which are beyond the scope of this chapter, to provide the reader with a starting point for further detailed study and investigation into those processes and technologies that may be most suited to their particular product or concept.

TABLE 3 Summary of Manufacturing Processes and Technologies for Producing Nanosystems Manufacturing Process Nanoscale assembly

Nanostructuring processes for polymeric materials

Nanostructuring processes for silicon

Nanostructuring processes for carbon

Technology Self-assembling micellar structures [113, 114] Bio-self-assembly and aggregation [115, 116] Nanomanipulation [117, 118] Soft lithography [119, 120] Molecular imprinting [121, 122] Layer-by-layer electrostatic deposition [123, 124] Chemical vapor deposition [125] Mold replication [126, 127] Colloidal lithography [128, 129] X-ray lithography [130] Interfacial polymerization [131, 132] Nanoprecipitation [133, 134] Emulsion solvent evaporation [135] Nanoimprinting [136] Electrospinning [137, 138] Photolithographic fabrication X-ray lithography [130] Electron beam lithography [139] Chemical etching [140] Physical and chemical vapor deposition [141] Electric arc discharge [142, 143] Laser ablation [144, 145] Chemical vapor deposition [146] Combustion [147]

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7.3.5.1

Nanoscale Assembly Methods1

Self-Assembly through Micelle Formation Self-assembly at the nanoscale is deemed important to be able to produce commercially viable products and processes, since it offers a mode of bulk production with control over features such as size, shape, and morphology at the nanoscale. The basic principle of self-assembly is based on the simultaneous coexistence of two parallel forces: Long-range repulsive interactions between incompatible domains Short-range attractive interactions If we take the example of an amphiphilic diblock copolymer, the polymer is composed of two blocks, a hydrophobic block and a hydrophilic block. When introduced to a solvent beyond a minimum concentration, the critical micelle concentration (CMC), the monomers begin to orientate such that the block that is soluble in the solvent orients itself toward the periphery, in contact with the continuous media, and the insoluble portion turns toward the core in an attempt to minimize contact with the continuous phase, thus leading to the formation of a micelle. The long-range repulsive forces arise from the relative solubilities of the blocks in the solvent, and the short-range attractive forces arise from the covalent link between the two blocks. The basic theory of micelle formation using block copolymers is outlined below since nanosystem and nano-object self-assembly is likely to be facilitated by such polymer systems, and similar principles will apply or aid toward developing selfassembling systems. Key factors that affect micelle formation are as follows: Equilibrium constant Solvent type Solvent quality Critical micelle temperature (CMT) Critical micelle concentration Overall molar mass of the micelle, MW Micelle aggregation number, Z Copolymer architecture Relative block lengths Relative geometries of copolymer blocks Polymer composition Core–corona interfacial tension These key factors will influence the following micelle characteristics: Hydrodynamic radius of micelles formed, RH Radius of gyration, RG 1

The description in this section has been summarized and adapted from J. Rodriguez-Hernandez et al., Toward “smart” nano-objects by self-assembly of block copolymers in solution, Progress in Polymer Science, 30 (2005), 691–724 [148].


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Ratio of hydrodynamic radius, RH, to radius of gyration, RH Micelle core radius, RC Micelle corona thickness, C Micellar structures can be produced either by addition of the polymer solution or addition of the powdered material to the desired solvent and stirring at the optimum temperature and monomer concentration. The CMC can be determined by ultraviolet (UV) absorption or light scattering techniques such as static light scattering (SLS), dynamic light scattering (DLS), or small-angle X-ray scattering (SAXS). At the concentration at which monomers form micelles, there will be a radical drop in monomer concentration in the bulk. The stability of micellar systems depends upon the ability to ensure the aggregated monomers do not deaggregate and that individual micelles do not coalesce to form larger aggregates. This is inevitable over a period of time, but steps can be taken to prolong the stability of the systems through various techniques, and those listed below are some of the methods used for spherical micellar systems: Steric stabilization using emulsifiers and surfactants Shell or core cross-linking Viscosity-enhanced stabilization Amine cross-linked stabilization Thermodynamic stabilization The types of systems that can be produced using micelle formation include spheres, shells, capsules, vesicles, clusters, and particles of various shapes and sizes, such as spheres, rods, planar structures, and layered structures. Further processing can be undertaken to add rate-controlling polymer membranes to the outer shell and to incorporate different molecules to the surface (e.g., for receptor recognition). Biological Self-Assembly Using DNA as Construction Tool This is a technique that has been adopted to produce 2D or 3D nanosystems by utilizing the basepairing affinity of DNA [149, 150]. Biological self-assembly using DNA can be described as a process that allows the systematic assembly of molecules with high levels of precision and accuracy without external constraints or influences. This allows the construction of nanoscale objects to the desired structure, conformation, and composition very rapidly and without the need for complex processing techniques and conditions. DNA is a copolymer composed of a phosphate and sugar backbone and four types of bases that branch off from the backbone, A (adenine), G (guanine), C (cytosine), and T (thymidine). During DNA replication two strands of DNA come together to form a helical structure through complementary base pairing which is highly specific, whereby thymidine pairs with adenine and guanine with cytosine. When strands of DNA come together where the ends of the strands are noncomplementary, a portion of the strand extends beyond the complementary base-paring region leading to an overhang, otherwise known as a “sticky end”. The natural mechanism of DNA base pairing can be used to assemble synthetic sequences of DNA molecules by synthesizing DNA molecules such that they form

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stable branches [150, 151] with arms that form sticky ends that in turn can assemble to form supramolecular structures [152, 152a]. This approach may be used to produce complex 3D assemblies through sequential or layer-by-layer self-assembly and may incorporate other materials, such as particles and proteins [153–156]. The advantages of this method are as follows: Specificity and geometry of intermolecular interactions that can be predicted Precision control over the final structure at the nanoscale Simple manufacturing process without external restrictions Complex structures that can be built with defined topologies Potential for creation of nanodevices The types of structures that may be constructed are Branched planar/2D quadrilateral structures [157], cubes [158], octahedrons [159], and complex 2D and 3D periodic structures [160]. DNA Synthesis for Nanoconstruction Single strands of DNA, otherwise known as oligomers, are most commonly produced using a solid-support synthesis process [161, 162]. This is a cyclic process where each nucleotide is sequentially coupled to form a nucleotide chain (working from the 3′ end to the 5′ end). The 3′ end is initially covalently linked to a solid support and the nucleotide monomers are added sequentially. This is a well-established process and its key parameters and critical process steps are well documented in the literature [163, 164]. The DNA strands can be tailored according to the desired nanoconstruction scheme and target structure [165]. Nano Manipulation As the name suggests, this is quite literally a technique for physically manipulating matter at the nanoscale. Scanning probe microscopy (SPM) techniques have been most widely used to achieve this using the scanning probe tip as an implement for assembling atoms, molecules, or nanoparticles according to the desired spatial conformation [166–169]. Soft Lithography Lithography is essentially a process for printing features on a planar surface. Nanolithography tools, commonly referred to as soft lithography, allow precisely defined nanoscale features to be produced on a substrate, which can be removed from the substrate as free-standing 3D nano-objects. A number of techniques fall within the field of soft lithography, primarily for construction of micrometer-sized objects: Replica molding Micromolding in capillaries (MIMIC) Microtransfer molding Solvent-assisted microcontact molding (SAMIM) Microcontact printing Near-field phase shift lithography is a soft lithographic technique used to produce geometric shapes with size features at the nanoscale (approximately 40–80 nm). This involves the production of a polymer mask containing the desired pattern to be


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replicated on the substrate, with nanoscale features usually patterned by X-ray or electron beam exposure. The mask is then placed on the surface of the substrate and exposed to near-field light, the intensity of which leads to replication of the pattern on the mask on to the substrate. Complex geometries, shapes, and features can be produced on the substrate which can subsequently be removed to give freestanding particles or objects [170, 171]. Molecular Imprinting Molecular Imprinting is a process used to imprint or copy recognition sites from desired molecules on to polymer structures [172, 173]. The recognition sites can be produced on organic or inorganic polymers and inorganic materials such as silica and biomaterials such as proteins. A template molecule is dissolved in solvent with polymerizable monomers which undergo bond formation with the template molecule forming either noncovalent bonds through electrostatic interactions, hydrogen bonds or hydrophobic interactions, or reversible covalent bonds. The monomers are then polymerized to form a cast or semirigid polymeric structure which maintains the steric conformation of the molecule template and its recognition site upon removal of the template molecule. As a result, the molecular template affinity for molecules and analyte is mimicked by the “imprinted” polymer [174]. This has applications in chromatography and drug discovery and potential applications in targeted drug delivery. Layer-by-Layer Electrostatic Deposition Electrostatic deposition utilizes the electrostatic bonding affinities of materials imparted by their surface charge to build highly ordered multilayered films or structures on a substrate. The process involves the successive deposition of oppositely charged polyions, exploiting the Coulombic long-range electrostatic interactions between the oppositely charged molecules, allowing formation of multilayers over a large distance. This technique can be used to build multilayer composite films on particles incorporating molecular fragments such as polymer–polymer, polymer–organic, polymer bimolecular, and polymer– mineral composition [175–177]. Chemical Vapor Deposition CVD is a crystal growth process whereby a solid material is deposited from the gas phase onto a controlled substrate using a suitable mixture of volatile precursor materials which react to produce the desired deposit on the substrate surface (Table 4). Types of films and structures that can be produced include the following: Polycrystalline Amorphous Epitaxial silicon Carbon fiber Filaments Carbon nanotubes Silicon dioxide Tungsten Silicon nitride Titanium nitride

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TABLE 4

Chemical Vapour Deposition Methods and Their Key Features

CVD Method Atmospheric pressure CVD [178, 179] Atomic layer CVD (atomic layer epitaxy) [180, 181] Aerosol-assisted CVD [182, 183] Direct liquid injection CVD [184, 185] Hot-wire CVD [186]

Low-pressure CVD [187] Metal organic CVD [188] Microwave plasma-assisted CVD [189] Plasma-enhanced CVD [190] Rapid thermal CVD [191]

Remote plasma-enhanced CVD [192]

Ultrahigh vacuum CVD [193]

7.3.5.2

Key Features Operates at atmospheric pressure High-precision film thickness and uniformity requirements For use with involatile precursors High film growth rates possible High growth rate, low temperature, and use of inexpensive materials such as plastics as substrate Improved film uniformity Uniform and conformal deposition No external heating required Reduced substrate temperatures can be used Conformal coverage over high-aspect-ratio features is possible, i.e., improved control of interfacial properties Excellent conformal coverage of complex structures Can produce multilayer and graded layers with tailored functional group attachment Reduced surface contamination

Nanostructuring Processes for Polymeric Materials

Numerous microfabrication techniques have been used to produce a wide range of implantable and oral drug delivery systems using materials ranging from silicon, glass, silicone elastomer, and plastics. Fabrication techniques have rapidly evolved to produce nanoscale objects and therapeutic systems using polymeric materials as the substrate due to their biodegradable nature. There are a number of different synthetic polymer systems that have been developed for this type of application, and the most common ones are listed below: Poly(d-lactic acid) (PDLA) Poly(ε-caprolactone) (PCL) Poly(vinyl alcohol) (PVA) Polyalkylcyanoacrylates (PACA) Poly(l-lactide) (PLLA) Poly(lactide-co-glycolide) (PLGA) Polymethylcyanoacrylate (PMCA) Techniques for the production of micrometer-sized features using polymers are well established and apply primarily to device construction. The techniques listed


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below focus primarily on attaining submicrometer, nanoscale features, and geometries using polymers such as those listed above. Nanomold Replication A physical mold is produced that has nanoscales on the order of tens or a few hundred nanometers. To achieve such fine features with precision and repeatability, electrodeposition is used to produce the molds, otherwise referred to as a nanostamp [194]. The stamp is then use as a master stamp to duplicate the image or object by casting or embossing the polymeric material. Colloidal Lithography Colloidal lithography is a process whereby an electrostatically self-assembled array of monodispersed colloidal nanospheres is used as a mask to construct nanoscale objects and features through deposition or etching processes. The monodisperse colloidal spheres, for example, surface-charged latex, selforganize or assemble into periodic arrays on the substrate, glass, for example, and do not aggregate due to the surface charge repulsion. This method can be used to produce 2D [195, 196] and 3D [197] nanostructures, arrays of rings, dots, honeycomb structures, pillars, and chemical patterns [198] with a high level of control over structure and conformation. Interfacial Polymerization Interfacial polymerization is a process whereby very thin films or membranes, on the order of nanometer thickness, are produced by reacting two monomers at the interface between two immiscible solutions [199]. Nanoparticles [200] and aqueous core capsules with very thin membranes have been produced using this method for drug delivery applications. Nanoprecipitation Nanoprecipitation is a self-assembly directed nanoparticle formation method. There are three key steps involved in this process: rapid micromixing of the solutes, the creation of a high level of supersaturation to instigate rapid nucleation and growth of precipitate, and the kinetic control and termination of growth using copolymer stabilizers. One of the drawbacks using this method is the poor incorporation of water-soluble drugs [201]. However, the main advantage associated with the production of nanospheres for drug delivery using this technique is the high degree of control attainable over particle size [202]. Emulsion Solvent Evaporation The basic concept of the emulsion solvent evaporation technique producing nanoparticles is very straightforward. The particles are formed as an emulsion of a polymer–surfactant mixture and dispersed in an organic solvent. The solvent is then evaporated to leave behind the individual emulsion droplets which form stable free nanoparticles [203]. This method is far easier and more preferable over methods such as spray drying and homogenization and operates under ambient conditions and mild emulsification conditions. The size and composition of the final particles are affected by variables such as phase ratio of the emulsion system, organic solvent composition, emulsion concentration, apparatus used, and properties of the polymer [204]. Nanoimprinting This is a lithographic technique similar to soft contact lithography discussed earlier, with the main difference being that nanoimprinting uses a

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hard mold to produce nanoscale features down to sub-10 nm resolution [205] by directly imprinting onto the polymer surface at high temperatures. More recently, molds produced from carbon nanotubes have been used to achieve molecular-scale resolution. Molds are generally made using electron beam lithography; however, high-definition molds are produced using molecular beam epitaxy. Some of the technical issues associated with this technique include sticking, adhesion, and material transport during imprinting [206]. Electrospinning Electrospinning is a process that uses electrostatic force to produce nanofibers from a charged polymer. An electrode is placed into a spinning polymer solution/polymer melt and the other electrode is attached to a collector plate. A high-intensity electric field is created by applying a high voltage such that the polymer solution is discharged as a jet, and during travel of this charged polymer jet toward the grounded collector plate, solvent evaporation leaves a charged polymer fiber which deposits on the collector plate [207]. These fibers have high specific surface areas and are highly flexible, and applications include the preparation of controlled drug release membranes [208].

7.3.6 7.3.6.1

CHARACTERIZATION TECHNIQUES Nanoparticle Characterization Methods and Tools

A summary of some key properties that may be assessed as part of a characterization schedule for nanoparticles and nanostructures and a comprehensive but not exhaustive list of tools and techniques that may be used are presented in Table 5 [209–223]. The degree of characterization and method used will be determined by the intended application of the nanomaterials. Characterization of micellar and supramolecular structures and their counterparts often require different or additional tools and techniques [224–232] and a summary is provided in Table 6 of various characterization parameters for micellar and supramolecular structures and components and analytical tools that may be applied. 7.3.6.2

Scanning Probe Technologies

Scanning probe microscopy has almost become synonymous with nanomaterial characterization [233]. This is a family of techniques that have evolved from the use of a sharp proximal probe to scan a surface in order to ascertain its properties down to atomic-scale resolution based on tip–surface interaction. There are two main SPM techniques, scanning tunneling microscopy (STM) [234, 235] and AFM [236]. Nearfield scanning optical microscopy (NSOM) [237–239] also falls within the SPM family of techniques; however, this uses a subwavelength near-field light source as the scanning probe, achieving resolutions down to 50 nm, and is not discussed further here. A host of techniques have evolved from STM and AFM, primarily involving adaptations to instrumentation depending on the material and parameter under

CHARACTERIZATION TECHNIQUES

TABLE 5

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Characterization Parameters and Tools for Nanoparticles and Nanostructures

Characterization Parameter Composition

Particle diameter Size distribution Surface area Porosity (pore size, volume, and distribution)

Core–shell thickness Surface structure and morphology

Surface charge density

Shape Concentration distribution Crystallinity, Bulk Crystallinity, Local Magnetic properties Electrical properties Optical properties

Analytical Tool Liquid chromatography, e.g., high-performance liquid chromatography (HPLC), size exclusion chromatography (SLC)–HPLC Field flow fractionation (FFF) UV–visible spectrophotometry Refractive Index Inductively coupled plasma–optical emission spectrometry (ICP–OES) Fourier transform infrared spectrometry Mass spectrometry X-ray fluorescence Extended X-ray absorption fine structure (EXAFS) spectroscopy X-ray absorption near edge (XANES) spectroscopy Static and dynamic laser light scattering Scanning probe technologies Static and dynamic laser light scattering Photon correlation spectroscopy BET method (Brunauer, Emmett, and Teller method) Physical gas sorption Chemical gas sorption Helium picnometry Mercury intrusion porometry (MIP) Small-angle scattering of polarized neutrons (SANSPOL) Small-angle neutron scattering (SANA) Proton nuclear magnetic resonance (1H NMR) spectroscopy Scanning electron microscoscopy (SEM), atomic force microscopy (AFM), energy dispersive X-ray (EDXA), transmission electron microscopy (TEM), scanning probe microscopy (SPM), auger electron spectroscopy (AES), X-ray diffraction (XRD), X-ray photoelectron microscopy (APS), X-ray photoelectron spectroscopy (XPS), Vertical scanning phase shifting interferometry Zeta potential using Electrostatic light scattering (ELS) Zeta potential using multifrequencyelectro acoustics Zeta potential using phase analysis light scattering (PALS) Electron microscopy Scanning probe technologies Energy dispersive X-ray spectrometry (EDS) combined with SEM or scanning X-ray diffraction TEM/–selected area diffraction (SAD) Differential scanning calorimetry (DSC) Scanning probe technologies Scanning probe technologies UV–visible Spectroscopy

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TABLE 6 Characterization Parameters and Analytical Tools for Micellar and Supramolecular Structures Characterization Parameter

Analytical Tool

Critical micelle concentration

Aggregation number Radius of gyration, RG Hydrodynamic radius, RH Core/corona size, micelle structure, overall micelle size Overall shape, cross section Size, shape, and internal structure

Flurimetric methods Static light scattering Dynamic Light Scattering Fluorescence correlation spectroscopy Small-angle X-ray scattering Photon correlation spectroscopy Small-angle X-ray scattering

Small-angle neutron scattering Transmission electron microscopy Scanning probe technologies Average molecular weight Membrane and vapor pressure osmometry Monitor equilibrium state, stability monitoring Light-scattering methods Structure elucidation, polymer architecture, Nuclear magnetic resonance polymer interactions

investigation. It should be noted that technological advances continue unabated and new techniques are constantly being developed within the scanning probe family to cater for the characterization of new and novel materials and nanoscopic constructs. Table 7 gives a current synopsis of these techniques. Scanning Tunneling Microscopy The scanning tunneling microscope was first described by Nobel Prize winners Binnig and Rohrer in 1982 [249] and consists of an atomically sharpened tip usually composed of tungsten, gold, or platinum–irridium. The tip is scanned within atomic distance (about 6–10 Å) of the sample under study under very high vacuum, and a bias voltage is applied between the sample and the scanning probe tip, resulting in a quantum mechanical tunneling current across the gap. The magnitude of the tunnelling current is related exponentially to the distance of separation and the local density of states (i.e., electron density in a localized region of a material) [250, 251]. The relationship between tunneling current and separation is given as: I = Cρtρse d 0.5 where

I = tunnelling current C = constant (linear function of voltage) ρt = tip electron density ρs = sample electron density ed0.5 = separation (governed by exponential term)

The tip is scanned across the sample surface using a piezoelectric transducer in one of two modes, topographic mode or current mode. In the topographic mode a con-

CHARACTERIZATION TECHNIQUES

TABLE 7

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Summary of Scanning Probe Technologies

SPM Technique Atomic force microscopy Noncontact AFM [240]

Contact AFM [241, 242]

Piezoresponse Lateral force

Scanning thermal Intermittent AFM (tapping mode) [243] Phase imaging AFM

Lift mode AFM techniques [244–246] Magnetic Force Electrical Force Surface Potential Scanning Capacitance Force modulation Scanning tunnelling microscopy [247]

Spin-polarized STM/STS [248]

Property Measured Visualisation and measurement of surface features Insulating substrates, atomic resolution Molecular systems, atomic resolution Biocluster and biomolecular imaging Imaging and spectroscopic data in liquid environments Nanoscale charge measurement Nanoscale magnetic properties Topographic imaging of solid substrates Mechanical properties Local adhesive properties Characterization and domain engineering of ferroelectric materials Fine structural detail Transitions between components on surface, e.g., polymer composites Defects in sample based on thermal differences Biological systems: DNA/RNA analysis, protein–nucleic acid complexes, molecular crystals, biopolymers, ligand– receptor binding Two phase polymer blends Surface contaminants Biological samples Topography Magnetic properties/regions Electrical properties Surface potential Material capacitance Elasticity Surface imaging Three-dimensional profiling with vertical resolutions to 0.1 Å Measurement of electronic and magnetic properties Surface electronic state Mapping surface magnetism at atomic scale

stant distance is maintained between the tip and sample surface using a feedback loop operated with the scanner. In the current mode, variations in current with changes in surface topography are monitored by switching off the feedback loop, thus providing a 3D image of the surface under study. The key features and limitation of STM are as follows: Features of STM [252, 253] Can undertake topographical imaging of surfaces with atomic-scale lateral resolution, down to 1 Å There-dimensional profiling possible with vertical resolutions down to 0.1 Å Wide range of materials can be analyzed

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Surface electronic properties may be measured Large field of view, from 1 Å to 100 μm Vibrational isolation allows highly sensitive measurements to be undertaken Ultrahigh vacuum (in the range 10−11 torr) minimizes sample contamination and reduces oxide layer growth, thus allowing for high sensitivity measurements Limitations of STM Can be difficult to differentiate between a composite of materials on the surface Tip-induced desorption of surface molecules may occur Ultrahigh vacuum requirements Vibrational isolation requirements lead to increased installation costs Low scanning speed Atomic Force Microscopy Atomic force microscopy is a direct descendant of STM and was first described in 1986 [254]. The basic principle behind AFM is straightforward. An atomically sharp tip extending down from the end of a cantilever is scanned over the sample surface using a piezoelectric scanner. Built-in feedback mechanisms enable the tip to be maintained above the sample surface either at constant force (which allows height information to be obtained) or at constant height (to enable force information to be obtained). The detection system is usually optical whereby the upper surface of the cantilever is reflective, upon which a laser is focused which then reflects off into a dual-element photodiode, according to the motion of the cantilever as the tip is scanned across the sample surface. The tip is usually constructed from silicon or silicon nitride, and more recently carbon nanotubes have been used as very effective and highly sensitive tips. In noncontact-mode AFM the cantilever is oscillated slightly above its resonant frequency and the tip does not make contact with the sample surface but instead oscillates just above the adsorbed fluid layer on the surface, maintaining a constant oscillation. The resonant frequency of the cantilever decreases due to van der Waals forces extending from the adsorbed fluid layer. This changes the amplitude of oscillation, the variations of which are detected using sensitive alternating current (AC) phase-sensitive devices, providing topographical information. In contact mode, AFM the tip remains in contact with the sample surface, and the feedback loop maps the vertical vibrational changes. In tapping mode, the cantilever is oscillated above the sample surface such that it intermittently contacts the sample surface. The key features and limitations of AFM are a follows: Features of AFM [255–257] High scan speeds Atomic-scale resolution possible Rough sample surfaces can be analyzed High lateral resolutions possible Soft samples (e.g., biological tissue) can be measured

TOXICOLOGY CONSIDERATIONS

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Limitations of AFM [255–257] Potential for image distortion due to lateral shear forces (in contact mode) May be reduced spatial resolution due to sample scraping (in contact mode) Tapping mode has lower scan speed compared to contact mode, though there is less susceptibility for sample damage and image distortion

7.3.7 TOXICOLOGY CONSIDERATIONS Nanomaterials may in their own right possess novel and useful properties or as a composite of the same or different materials to form larger useful structures. Safety consideration is therefore of paramount importance since completely inert materials have the ability to exhibit toxic effects by virtue of a reduction in their size and associated increase in surface area–mass ratio, let alone materials manipulated specifically to impart novel properties. Two obvious routes of human contact with nanoparticulates are the skin and via inhalation. Given the size of the particles, there may be a propensity for absorption into the systemic circulation. In some cases the nanosystems are engineered to achieve enhanced systemic absorption. The established methodology for toxicological assessment of new materials should be adhered to, and the discussion below is intended only to touch upon some of the immediate safety concerns that should be understood and addressed when dealing with nanomaterials. 7.3.7.1

Lung Toxicity

The safety of ultrafine particles remains to be clearly elucidated and requires the collaborative input of toxicologists (animal, cellular, molecular), epidemiologists, clinicians (pulmonary, cardiovascular, neurological), and atmospheric scientists. There are several published studies to indicate that ultrafine particles pose a higher toxicity risk compared with their larger counterparts [258–261]. Figure 1 outlines the potential effects of ultrafine particles on respiratory mucosa, the cardiovascular system, and central and peripheral nervous systems, upon inhalation. Hohfeld et al. [262] hypothesized the toxic effects of ultrafine particles to be attributable to the following: • • •

•

High efficiency of deposition in the alveolar region due to particle size Large surface area Decreased phagocytosis leading to interaction of the particles with the epithelium, resulting in the development of conditions such as chronic diffuse interstitial fibronodular lung disease Dislocation from the alveolar space, leading to potential systemic effects

The hypothesis affirms the need to characterize the material’s physical and chemical properties, including morphological analysis. The latter has significant ramifications on the aerodynamics of the particulate matter and hence its ultimate disposition.

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PHARMACEUTICAL NANOSYSTEMS Particle translocation Inhalation

Mediators

Respiratory tract deposition

s ron

u Ne CNS (effects ?)

m

iu

Autonomic nervous system

tit

s er

t

In

Blood vessel dysfunction

Lung inflammation

Systemic inflammation

Circ

ula

tion Extrapulmonary organs Liver Heart

White blood cell activation

Heart effects Modifying factors: Age, gender, underlying disease, copollutants

FIGURE 1 Potential mechanisms of effects of inhaled ultrafine particles. (Reproduced with permission from G. Oberdörster, Inhaled nano-sized particles: Potential effects and mechanisms, paper presented at the Symposium, Health Implications of Nanomaterials, October 2004.)

Carbon nanotubes are a class of materials finding increasingly widespread applications in drug discovery and development and may be classed as a form of ultrafine material. It has been shown that single-wall carbon nanotubes do not produce any significant respirable aerosol levels due to agglomeration resulting from the very high surface area–volume ratio and associated electrostatic interaction between the nanotubes [263]. A good deal of research has focused on developing methods for the dispersion of nanotubes for further downstream processing for conversion to useful applications. The liquid dispersions do not pose the same level of hazards posed by the dry powder material, and most of the work in the pharmaceutical industry with carbon nanotubes is focused on liquid dispersions whereby nanotubes are being functionalized and conjugated with drugs and possibly other carriers for therapeutic intent [264]. However, it has at the same time been shown that dry powder carbon nanotubes can persist in the lungs and have the potential to induce inflammatory and fibrotic reactions, evident in the form of collagen-rich granulomas in the bronchi and interstitium [265]. This emphasizes the need for caution and further work to establish the exact cause of these effects given the propensity of nanotubes to agglomerate. 7.3.7.2

Systemic Uptake

Nanoparticles, by virtue of size, have a tendency to evade phagocytosis. Uptake into the systemic circulation is thus thought to be through diffusion and via the endo-

REFERENCES

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thelial cells, the epithelium, interstitium, and blood vessels. Translocation into the blood is thought to be through enhanced epithelial or endothelial permeability imparted by inflammatory mediators. Systemic hypercoagulation may be triggered by the inflammatory mediators in response to the diffusion of the nanoparticles through endothelium and vasculature [260]. 7.3.7.3

Skin Permeation of Nanoparticles

The stratum corneum provides a formidable barrier to the entry of chemical and particulate matter into human tissue and systemic circulation. It provides a first-line defence to the ingress of foreign agents. However, there are indications that particles up to 1 μm are able to penetrate the skin’s barrier and deposit in the epidermis where the antigen-presenting cells reside [266]. It follows therefore that submicrometer particles in the nanometer range have the potential to cross the stratum corneum and illicit an inflammatory response. Once again, however, the tendency of fine particles to agglomerate will to some extent inhibit penetration into the skin, in particular where the agglomerates are macroscopic. A correlation must however be drawn to establish any potential link between nanoparticle affinity for skin penetration and particle physical and morphological characteristics or indeed whether the novel and unique properties of the engineered particle in any way impart enhanced skin permeation properties and, if so, their nature and mechanisms.

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252. Fontaine, P. A., et al. (1998), Characterization of scanning tunneling microscopy and atomic force microscopy-based techniques for nanolithography on hydrogen-passivated silicon, J. Appl. Phys., 84(1), 1776–1781. 253. Wiesendanger, R. (1994), Contributions of scanning probe microscopy and spectroscopy to the investigation and fabrication of nanometer-scale structures, J. Vac. Sci. Technol. Microelectron. Nanometer Struct., 12(2), 515–529. 254. Binnig, G., et al. (1986), Atomic force microscopy, Phys. Rev. Lett., 56, 930–933. 255. de Souza Pereira, R. (2001), Atomic force microscopy as a novel pharmacological tool, Biochem. Pharmacol., 62(8), 975–983. 256. Rajagopalan, R. (2000), Atomic force and optical force microscopy: Applications to interfacial microhydrodynamics, Coll. Surf. Physicochem. Eng. Aspects, 174(1–2), 253– 267. 257. Santos, N. C., and Castanho, M. A. R. B. (2004), An overview of the biophysical applications of atomic force microscopy, Biophys. Chem., 107(2), 133–149. 258. Ferin, J., et al. (1992), Pulmonary retention of ultrafine and fine particles in rats, Am. J. Respir. Cell Mol. Biol., 6, 535–542. 259. Li, X. Y., et al. (1999), Short term inflammatory responses following intratracheal instillation of fine and ultrafine carbon black in rats, Inhal. Toxicol., 11, 709– 731. 260. Oberdorster, G., et al. (1995), Association of particulate air pollution and acute mortality: Involvement of ultrafine particles? Inhal. Toxicol., 7, 111–124. 261. Li, X. Y., et al. (1997), In vivo and in vitro pro-inflammatory effects of particulate air pollution (PM10), Environ. Health Perspect., 105, 1279–1283. 262. Brüske-Hohlfeld, I., et al. (2004), Epidemiology of nanoparticles, in Proc. of First International Symposium on Occupational Health Implications of Nanomaterials. 263. Warheit, D. B., et al. (2004), Pulmonary bioassay toxicity study in rats with single wall carbon nanotubes, in Proc. of First International Symposium on Occupational Health Implications of Nanomaterials. 264. Klumpp, C., et al. (2006), Functionalized carbon nanotubes as emerging nanovectors for the delivery of therapeutics, Biochim. Biophys. Acta, 1758, 404–412. 265. Muller, J. (2004), Respiratory toxicity of carbon nanotubes, in Proc. of First International Symposium on Occupational Health Implications of Nanomaterials. 266. Tinkle, S. (2004), Dermal penetration of nanoparticles, in Proc. of First International Symposium on Occupational Health Implications of Nanomaterials.

SUGGESTED READING General Dai, L. (2004), Intelligent Macromolecules for Smart Devices: From Materials Synthesis to Device Applications, Springer. Di Ventra, M., Evoy, S., and Heflin, J. R. (2004), Introduction to Nanoscale Science and Technology, Springer. Lyshevski, S. E. (2005), Nano- and Mirco-Electromechnical Systems: Fundamentals of Nano and Microengineering, CRC Press, Boca Raton, FL. Roco, M. C., and Bainbridge, W. S. (2003), Converging Technologies for Improving Human Performance: Nanotechnology, Biotechnology, Springer.

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Nano–Pharmaceutical Materials and Structures Hirsch, A., and Brettreich, M. (2004), Fullerenes: Chemistry and Reactions, Wiley, Hoboken, NJ. Cao, G. (2004), Nanostructures & Nanomaterials: Synthesis, Properties & Applications, Imperial College Press. Haley, M. M., and Tykwinski, R. R. (2006), Carbon-Rich Compounds: From Molecules to Materials, Wiley, Hoboken, NJ. Harris, P. J. (2001), Carbon Nanotubes and Related Structures, Cambridge University Press. Kumar, C. S. S. R. (2005), Biofunctionalization of Nanomaterials, Wiley, Hoboken, NJ. Kumar, C. S. S. R. (2006a), Nanomaterials for Cancer Therapy, Wiley, Hoboken, NJ. Kumar, C. S. S. R. (2006b), Biological and Pharmaceutical Nanomaterials, Wiley, Hoboken, NJ. Lazzari, M., Liu, G., and Lecommandoux, S. (2006), Block Copolymers in Nanoscience, Wiley, Hoboken, NJ. Schmid, G. (2004), Nanoparticles: From Theory to Application, Wiley, Hoboken, NJ. Tadros, T. F. (2005), Applied Surfactants: Principles and Applications, Wiley, Hoboken, NJ. Vogtle, F. (2000), Dendrimers III: Design, Dimension, Function, Springer. Zhang, J., Zhang, J., Wang, Z., Liu, J., and Chen, S. (2002), Self-Assembled Nanostructures, Springer.

Manufacturing Technologies Butt, H.-J., Graf, K., and Kappl, M. (2006), Physics and Chemistry of Interfaces, Wiley, Hoboken, NJ. Champion, Y., and Fecht, H. (2004), Nano-Architectured and Nanostructured Materials: Fabrication, Control and Properties, Wiley, Hoboken, NJ. Elimelech, M., Jia, X., Gregory, J., and Williams, R. (1998), Particle Deposition & Aggregation: Measurement, Modelling and Simulation, Elsevier, New York. Hoch, H. C., Jelinski, L. W., and Craighead, H. G. (1996), Nanofabrication and Biosystems, Cambridge University Press. Köhler, M., and Fritzsche, W. (2004), Nanotechnology: An Introduction to Nanostructuring Techniques, Wiley, Hoboken, NJ. Komiyama, M., Takeuchi, T., Mukawa, T., and Asanuma, H. (2003), Molecular Imprinting: From Fundamentals to Applications, Wiley, Hoboken, NJ. Kroschwitz, J. I. (1989), Polymers: Biomaterials and Medical Applications, Wiley, New York. Kumar, C. S. S. R., Hormes, J., and Leuschner, C. (2005), Nanofabrication Towards Biomedical Applications: Techniques, Tools, Applications, and Impact, Wiley, Hoboken, NJ. Osada, Y., and Nakagawa, T. (1992), Membrane Science and Technology, Marcel Dekker, New York. Pierson, H. (1999), Handbook of Chemical Vapor Deposition: Principles, Technologies and Applications, William Andrew. Ramakrishna, S., Fujihara, K., Teo, W., Lim, T., and Ma, Z. (2005), An Introduction to Electrospinning and Nanofibers, World Scientific. Rotello, V. M. (2004), Nanoparticles: Building Blocks for Nanotechnology, Springer. Sotomayor Torres, C. M. (2004), Alternate Lithography: Unleashing the Potentials of Nanotechnology, Springer. Wise, D. L. (2000), Handbook of Pharmaceutical Controlled Release Technology, Marcel Dekker, New York.

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Characterization Methods Binks, B. P. (1999), Modern Characterization Methods of Surfactant Systems, Marcel Dekker, New York. Brittain, H. G. (1995), Physical Characterization of Pharmaceutical Solids, Marcel Dekker, New York. Brown, W., Ed. (1996), Light Scattering: Principles and Development, Oxford University Press. Buyana, T. (1997), Molecular Physics, World Scientific. Chung, F. H., and Smith, D. K., Eds. (1999), Industrial Applications of X-Ray Diffraction, Marcel Dekker, New York. Jena, P. B., and Hoerber, J. K. H., Eds. (2006), Force Microscopy: Applications in Biology and Medicine, Wiley, Hoboken, NJ. Jenkins, R., and Jenkins, J. (1995), Quantitative X-Ray Spectrometry, Marcel Dekker, New York. Meyer, E., Hug, H. J., and Bennewitz, R. (2003), Scanning Probe Microscopy, Springer. Pethrick, R. A., and Viney, C., Eds. (2003), Techniques for Polymer Organisation and Morphology Characterisation, Wiley, Hoboken, NJ. Rosoff, M. (2002), Nano-Surface Chemistry, Marcel Dekker, New York. Sharma, A., and Schulman, S. G. (1999), Introduction to Fluorescence Spectroscopy, Wiley, New York. Sibilia, J. P. (1988), A Guide to Materials Characterization and Chemical Analysis, Wiley, New York. Watts, J. F., and Wolstenholme, J. (2004), An Introduction to Surface Analysis by XPS and AES, Wiley, Hoboken, NJ. Wyckoff, R. W. G. (1971), Crystal Structures, Wiley, New York.

Toxicology Fan, A. M. (1996), Toxicology and Risk Assessment: Principles, Methods & Applications, Marcel Dekker, New York. Gad, S. C. (2004), Safety Pharmacology in Pharmaceutical Development and Approval, CRC Press, Boca Raton, FL. Gardner, D. E. (2005), Toxicology of the Lung, CRC Press, Boca Raton, FL. Kumar, C. S. S. R. (2006), Nanomaterials: Toxicity, Health and Environmental Issues, Wiley, Hoboken, NJ. Meeks, R. G. (1991), Dermal and Ocular Toxicology: Fundamentals and Methods, CRC Press, Boca Raton, FL.

7.4 OIL-IN-WATER NANOSIZED EMULSIONS: MEDICAL APPLICATIONS Shunmugaperumal Tamilvanan* University of Antwerp, Antwerp, Belgium


7.4.3 7.4.4 7.4.5

7.4.6

7.4.1

Introduction Generations of Oil-in-Water Nanosized Emulsions 7.4.2.1 First-Generation Emulsion 7.4.2.2 Second-Generation Emulsion 7.4.2.3 Third-Generation Emulsion 7.4.2.4 Unique Property of Third-Generation Emulsion Preparation Methods for Drug-Free/Loaded Oil-in-Water Nanosized Emulsions Excipient Inclusion: Oil-in-Water Nanosized Emulsions Medical Applications of Oil-in-Water Nanosized Emulsions 7.4.5.1 Parenteral Routes 7.4.5.2 Ocular Routes 7.4.5.3 Nasal Route 7.4.5.4 Topical Route Future Perspective References

INTRODUCTION

It is estimated that 40% or more of bioactive substances being identified through combinatorial screening programs are poorly soluble in water [1, 2]. Consequently, the drug molecules belonging to these categories cannot be easily incorporated into aqueous-cored/based dosage forms at adequate concentrations, and thus the clinical

*Current address: Department of Pharmaceutics, Arulmigu Kalasalingam College of Pharmacy, Anand Nagar, Krishnankoil, India Pharmaceutical Manufacturing Handbook: Production and Processes, edited by Shayne Cox Gad Copyright © 2008 John Wiley & Sons, Inc.

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OIL-IN-WATER NANOSIZED EMULSIONS

efficacy of highly lipophilic drugs is being impeded. Furthermore, it is well established that the pharmaceutical industry will face more difficulties in formulating and developing novel dosage forms of new chemical entities since 50–60% of these molecules are lipophilic in nature and often exhibit hydrophobic character. Among the different innovative formulation approaches that have been suggested for enhancing lipophilic drug absorption and then clinical efficacy, lipid-based colloidal drug delivery systems such as nanosized emulsions recognized particularly for overcoming the formulation and bioavailability-related problems of such drug molecules. Other nomenclatures are also being utilized often in the medical literature to refer to nanosized emulsions, including miniemulsions [3], ultrafine emulsions [4], and submicrometer emulsions [5, 6]. The term nanosized emulsion [7] is preferred because in addition to giving an idea of the nanoscale size range of the dispersed droplets, it is concise and avoids misinterpretation with the term microemulsion (which refers to thermodynamically stable systems). Hence, nanosized emulsions are heterogenous dispersions of two immiscible liquids [oil-in-water (o/w) or waterin-oil (w/o)], and they are subjected to various instability processes such as aggregation, flocculation, coalescence, and therefore eventual phase separation according to the second law of thermodynamics. However, the physical stability of nanosized emulsions can substantially be improved with the help of suitable emulsifiers that are capable of forming a mono- or multilayer coating around the dispersed liquid droplets in such a way as to reduce interfacial tension or increase droplet–droplet repulsion. Depending on the concentrations of these three components (oil–water– emulsifier) and the efficiency of the emulsification equipment/techniques used to reduce droplet size, the final nanosized emulsion may be in the form of o/w, w/o, macroemulsion, micrometer emulsion, submicrometer emulsion, and double or multiple emulsions (o/w/o and w/o/w). Preparation know-how, potential application, and other information pertinent to w/o emulsions [8], macroemulsions [9–11], microemulsions [12, 13], and multiple emulsions [14] are thoroughly covered elsewhere. In addition, some studies have compared the performance of different emulsified systems (macroemulsions, microemulsion, multiple emulsions, and gel emulsions) prepared with similar oils and surfactants for applications such as controlled drug release [15] or drug protection [16]. Similarly the state of the art of so-called oxygen carriers or perfluorocarbon emulsions, dispersions containing submicrometer/nanosized fluoroorganic particles in water, is also thoroughly covered in the literature [17–19] and readers can refer to these complete and interesting articles. Possible usefulness as carriers stems from the nanosized emulsion’s ability to solubilize substantial amounts of hydrophilic/hydrophobic drug either at the innermost (oil or water) phase or at the o/w or w/o interfaces. While hydrophilic drugs are contained in the aqueous phase of a w/o-type emulsion or at the w/o interface of the system, hydrophobic drugs could be incorporated within the inner oil phase of an o/w-type emulsion or at the o/w interface of the system. It appears that the choice of the type of emulsion to be used therefore depends, to a large extent, upon the physicochemical properties of the drug. Between w/o and o/w types, the o/w type of nanosized emulsions would be preferred in order to successfully exploit the advantages of an emulsion carrier system. Additionally, within the o/w type, simple modifications on surface/interface structures of emulsions can be made. For instance, incorporating an emulsifier molecule alone or in a specific combination that is capable of producing either positive or negative charges over the emulsified droplets surface will lead to the formation of surface-modified emulsions. Based on these

GENERATIONS OF OIL-IN-WATER NANOSIZED EMULSIONS

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surface modifications, the o/w-type nanosized emulsions can be divided into cationic and anionic emulsions. The o/w nanosized emulsions have many appealing properties as drug carriers. They are biocompatible, biodegradable, physically stable, and relatively easy to produce on a large scale using proven technology [20]. Due to their subcellular and submicrometer size, emulsions are expected to penetrate deep into tissues through fine capillaries and even cross the fenestration present in the epithelial lining in liver. This allows efficient delivery of therapeutic agents to target sites in the body. Not only considered as delivery carriers for lipophilic and hydrophobic drugs, nanosized emulsions can also be viewed nowadays as adjuvants to enhance the potency of deoxyribonucleic acid (DNA) vaccine. For instance, Ott et al. [21] prepared a cationic o/w emulsion based on MF59 (commercially termed Fluad), a potent squalene in water and a cationic lipid, 1,2-dioleoyl-sn-glycero-3-trimethylammonium propane (DOTAP). It is shown that an interaction of cationic emulsion droplets with DNA and the formed DNA-adsorbed emulsion had a higher antibody response in mice in vivo while maintaining the cellular responses equivalent to that seen with naked DNA at the same doses. Another example of o/w emulsion-based adjuvants resulting from U.S. patent literature is the Ribi adjuvant system (RAS) [22–24]. Depending on the animal species used, RAS can be classified into two types: one for use in mice, termed monophosphoryl-lipid A + trehalose dicorynomycolate emulsion (MPL + TDM emulsion), and another for use in rabbits, goats, and larger animals, called monophosphoryl-lipid A + trehalose dicorynomycolate + cell wall skeleton emulsion (MPL + TDM + CWS emulsion). Strikingly, the MPL + TDM and MPL + TDM + CWS emulsions are prepared based on 2% oil (squalene)– Tween 80–water. These adjuvants are derived from bacterial and mycobacterial cell wall components that have been prepared to reduce the undesirable side effects of toxicity and allergenicity but still provide potent stimulus to the immune system. Another example is the syntex adjuvant formulation (SAF) that contains a preformed o/w emulsion stabilized by Tween 80 and Pluronic L121 [25]. Keeping in mind the potential of o/w nanosized emulsions, the purpose of this chapter is to classify the emulsions and provide a short overview on the preparation of the new- (second- and third-) generation emulsions followed by a description of the unique property of the third-generation emulsion and examples of selected excipients used for emulsion preparation. Given a specific interest especially on the parenteral route and then on the ocular topical route, o/w nanosized emulsions for both routes share a common platform on strict criteria concerning the maximum globule size and requirement of sterility in the final emulsions. Nasal and topical routes are also covered based on published research works with nanosized emulsions. It is emphasized that the chapter focuses only on preformed nanosized emulsions (having size distribution ranging between 50 and 1000 nm with a mean droplet size of about 250 nm), which should not be confused with self-microemulsifying drug delivery systems or preformed microemulsions that are transparent, thermodynamically stable dosage forms.

7.4.2


In this chapter, the o/w nanosized emulsions are classified into three generations (see Figure 1) based on their development to ultimately make a better colloidal

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OIL-IN-WATER NANOSIZED EMULSIONS Nanosized emulsion, NE

First-generation ne (phospholipid based)

Second-generation NE, with PEGylation on droplet surface

Third-generation NE, with PEGylation and positive charge on droplet surface

FIGURE 1

Flow chart of three generations of emulsion.

carrier for a target site within the organs/parts of the body and eye, thus allowing site-specific drug delivery and/or enhanced drug absorption. 7.4.2.1

First-Generation Emulsion

To be healthy with a quality life style is every human’s desire. According to documented Indian scriptures dating back to 5000 b.c., nutritional status has always been associated with health [26]. Because nutritional depletion due to either changes in the quality or amount of dietary fat intake or abnormalities in lipid metabolism results in immunosuppression and therefore host defense impairment, favoring increased infection and mortality rates. Traditionally depletion in dietary fats in malnourished or hypercatabolic patients is compensated through intravenous feeding using a solution containing amino acids, glucose, electrolytes, and vitamins as well as nanosized emulsions. Structurally, an o/w-type emulsion is triglyceride (TG) droplets enveloped with a stabilizing superficial layer of phospholipids [27]. Emulsions for parenteral use are complex nutrient sources composed not only of fatty acids but also substances other than TG, such as phosphatidylcholine, glycerol, and α-tocopherol in variable amounts. The emulsions also had a complex inner structure and consisted of particles with different structures, namely, oil droplets covered by an emulsifier monolayer, oil droplets covered by emulsifier oligolayers, double-emulsion droplets, and possibly small unilamellar vesicles. Commercially available nanosized emulsions used as intravenous high-calorie nutrient fluids have particle size normally around 160– 400 nm in diameter and, typically, their surfaces are normally negatively charged. Larger droplets can also be detected in commercially available emulsions [28]. Lutz et al. [29] reported that the mean diameter of particles in the 20% emulsions is larger than in the 10% emulsions. The TG in nanosized emulsions may be presented structurally in long or medium chains respectively, named LCT and MCT. The mean diameter of particles in LCT emulsions is greater than that in MCT emulsions [29]. LCT contains fatty acid chains with 14, 16, 18, 20, and 22 carbon atoms and sometimes with double bonds. The number of double bonds present defines the fatty acids in LCT as saturated, monounsaturad, or polyunsaturated. If the first double bond is on carbon number 3, 6, or 9 from the methyl end of the carbon chain, then the fatty acid is n-3, n-6, or n-9, respectively. Purified soybean or safflower oil contains LCT with a high proportion


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of n-6 polyunsaturated fatty acids whereas olive oil has LCT with n-9 monounsaturated fatty acids. Fish oil includes LCT with 20 or more carbon atoms where the first double bond is located between the third and fourth carbons from the methyl terminal of the fatty acids chain (omega-3 or n-3). On the other hand, MCT is derived from coconut oil and has saturated fatty acids with chains containing carbon atoms at the 6, 8, 10, or 12 positions. Both LCT and MCT either alone or MCT in combination with LCT are known for their long-term commercial acceptability in parenteral emulsions and are found in several U.S. Food and Drug Administration (FDA)–approved products. Also in Europe emulsion containing LCT/MCT enriched with fish oil became available for research. With MCT/LCT combinations in a specific ratio, nanosized emulsions appear to provide a more readily metabolizable source of energy [30]. However, LCT emulsion has been used in clinical practice for over 40 years. But for drug solubilization purpose, MCT is reported to be 100 times more soluble in water than LCT and thus to have an escalated solubilizing capability. 7.4.2.2

Second-Generation Emulsion

An easy and substantial association of lipophilic bioactive compound with the MCT or other vegetable oil–based emulsions, however, allows the emulsions to be used as vehicles/carriers for the formulation and delivery of drugs with a broad range of applications. These applications extend from enhanced solubilization or stabilization of the entrapped drug to sustained release and site-specific delivery. Hence the emulsions used for these applications are termed second-generation emulsions. Fittingly the o/w-type nanosized emulsions containing either positive or negative charge can be administered by almost all available routes, that is, topical, parenteral, oral, nasal, and even aerosolization into the lungs [31]. Despite differences in routes of administration, examples of commercially available emulsion-based formulations utilized for medical and nonmedical applications purposes are given in Table 1. The lipid-induced enhancement in oral bioavailability of many drugs having poor water solubility is a well-known documented fact when the drugs are incorporated into emulsions [32, 33]. However, direct intravascularly or locally administered conventional first- and second-generation emulsions could be taken up rapidly by the circulating monocytes for clearance by reticuloendothelial cells (through organs such as the liver, spleen, and bone marrow) [34]. Furthermore, the extent of clearance is enhanced by the adsorption of opsonic plasma proteins onto emulsion surfaces. However, the oily hydrophobic particles of the emulsions can also be taken up by macrophages without the necessity of opsonization provided the oil phase is liberated from the emulsion through the destabilization process occurring inside the blood compartment immediately after emulsion administration intravascularly or locally. Although the core of o/w emulsions is indeed hydrophobic, the emulsion envelope is not. The exposure of the hydrophobic part to the aqueous medium will therefore destabilize the emulsion. Moreover, Sasaki et al. [35] have assumed that when the castor oil–based emulsions interact with the tears in the eye, the electrolytes in the tears elicit a physical emulsion instability resulting in some release of the oil. The electrolytes present in blood or cellular fluids can also cause a similar type of emulsion destabilization process, resulting in separation of the oil and water

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Abbott Pharmacia-Upjohn B. Braun — B. Braun B. Braun Fresenius Clintec/Baxter Clintec/Baxter Green Gross Pharmacia-Upjohn Baxter SA

Producer

Fluosol DA Imagent Oxygent

Product

Green Gross, Osaka Alliance Pharmaceutical Alliance Pharmaceutical

Producer

Blood supplement or O2 carrier Contrast agent to image heart Blood supplement or O2 carrier

Application

Braun Melsungen AstraZeneca Braun Melsungen Merckle Dumex Novartis Novartis Abbott Abbott Allergan Allergan Chiron

Producer

Intravenous Intravenous Intravenous Intra-arthr. Intravenous Oral Oral Oral Oral Ocular topical Ocular topical Parenteral

Application

Daivonex cream and ointment Voltaren emulgel EMLA cream

Product

Laboratoire Leo Ciba-Geigy Astra, Swedan

Producer

Selected Topical Formulations Based on o/w or w/o Emulsions

Diazepam Propofol Etomidate Dexamethasone palmitate Diazepam Cyclosporin A Cyclosporin A Cyclosporin A Ritonavir Cyclosporin A Drug free Adjuvant

Drug

Registered Emulsions (o/w Type) Containing Drugs

Diazepam-Lipuro Diprivan Etomidat-Lipuro Lipotalon (Limethason) Stesolid Sandimmune Neoral Gengraf Norvir Restasis Refresh Endura Fluad (MF59)

Perfluorocarbon Emulsions (Fluorocarbon-in-Water Emulsions)

Abbolipid/Liposyn Intralipid Lipofundin N or Endolipide Lipofundin MCT/LCT Medialipide/Vasolipid Medianut Lipovenos Ivelip/Salvilipid Clinoleic Intralipos Kabimix Trivè 1000

Product

Product

Selected Marketed Medical and Nonmedical Emulsions

Parenteral Fat Emulsions (o/w Type) for Nutrition

TABLE 1


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Promonocyte (bone marrow)

Monocyte (blood) Macrophages (tissues) highly phagocytic Connective tissue Liver Lung Spleen Lymph node Bone marrow Serous cavity Bone tissue Nervous system

FIGURE 2

(histiocyte) (Kupffer cell) (alveolar macrophage) (free and fixed macrophages, sinusoidal lining cell) (free and fixed macrophage) (macrophages, sinusoidal lining cell) (peritoneal macrophage) (osteoclast) (microglia)

Mononuclear phagocyte system.

phases from the parenterally administered emulsions. It is thus reasonable to say that the resultant oily hydrophobic particles of the emulsions would also be taken up by macrophages independent of an opsonization process. An opsonization process is the adsorption of protein entities capable of interacting with specific plasma membrane receptors on monocytes and various subsets of tissue macrophages (see Figure 2), thus promoting particle recognition by these cells. Classical examples of opsonic molecules include various subclasses of immunoglobulins [36, 37], complement proteins such as C1q and generated C3 fragments (C3b, iC3b) [38], apolipoproteins [36, 37], von Willebrand factor, thrombospondin, fibronectin [39], and mannose-binding protein. When given by other parentral routes, for example, intraperitoneally, subcutaneously, or intramuscularly, the majority of emulsion droplets enter the lymphatic system and eventually the blood circulation where particles behave as if given intravenously. Liver, spleen, and bone marrow uptake is significantly lower. Indeed, relative to the emulsion droplet size, lymph nodes take up a much greater (over 100-fold) proportion than any other reticuloendothelial system (RES) tissue. There is increasing interest in developing injectable emulsions that are not cleared quickly from the circulation when they are designed to reach non-RES tissues in the vascular system or extravascular sites of action or to act as circulating drug reservoirs. Earlier approaches for making long-circulating emulsions concentrate mainly on changes in the oil phase of the emulsion such as MCT versus LCT [40], use of structured lipids (SLS) having medium-chain (C8–C10) fatty acids (SLM) and short-chain (C4) fatty acids (SLS) [41], addition of sphingomyelin [42–45] and cholesterol [46] to the emulsion, and use of hydrogenated castor oil (HCO) with at least 20 oxyethylene units (HCO20) [47–52]. Using the further established formulation approaches by which the emulsion droplet surfaces could be altered might, however, be more realistic and even more useful for a wide array of drug-targeting purposes. Steric barrier or enhanced hydrophilicity effect exerted by a polyoxyethylene (POE) chain having surfactants when added as coemulsifier into the phospholipidstabilized first-generation emulsion allows, to some extent, the passive/inverse drug targeting to the lung, kidneys, and areas of inflammation [53, 54]. Addition of POEbased surfactants into the otherwise amphipathic phospholipid-stabilized emulsion

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is particularly effective against plasma protein adsorption onto emulsion surfaces because of the hydrophilicity and unique solution properties of POE-based surfactants, including minimal interfacial free energy with water, high aqueous solubility, high mobility, and large exclusion volume [54]. In addition, colloidal particles presenting hydrophilic surfaces with a low contact angle will be almost ignored by phagocytic cells [55], although emulsion particles are not supposed to be recognized as foreign by the body to some extent. Examples of POE chains containing surfactants employed so far in emulsions are Tween 80, Span 80, Brij, and Poloxamer 188 (commercially named Pluronic F-68 or Lutrol F-68). The effectiveness of these polymeric surfactant molecules to intercalate at the oil–water interface with strong bonding to the phospholipid molecules could also be checked/judged through an in vitro monolayer experiment [56]. In general, the modification of particulate carriers using amphipathic polyethylene glycol (PEG)–containing molecules results in a prolongation of their blood circulation time [57, 58]. A phosphatidylethanolamine derivative with polyethylene glycol (PEG–PE) is widely used to increase the plasma retention of particulate carriers such as liposomes [59–61], polystyrene microspheres [62], and nanospheres [63]. Therefore, similar to POE, the PEG–PE is also incorporated as a coemulsifier into emulsions (termed PEGylated emulsion) to augment its circulation half-life [64]. Liu and Liu [53] studied the biodistribution of emulsion particles coated with phosphatidylethanolamine derivatives with three different molecular weight PEGs (MW 1000, 2000, and 5000). Among them, PEG-2000 was able to prolong the circulation time of emulsion probably due to the increased hydrophilicity of the droplet surface and/or the formation of a steric barrier. However, Tirosh et al. [65] assumed, while characterizing the PEG-2000-grafted liposome by differential scanning calorimetry, densitometry, and ultrasound velocity and absorption measurements, that the steric stabilization is much more than increasing hydrophilicity. In addition, PEG-containing compounds also decrease the lipolysis of emulsion particles [47] and prevent the uptake by the mononuclear phagocytes [66]. A dipalmitoyl phosphatidylcholine (DPPC)–stabilized emulsion was prepared by Lundberg et al. [67] and the effect of addition of PEG–PE, polysorbate 80, or Pluronic F-68 on the metabolism of DPPC-stabilized emulsion was studied. Two different radioactive markers were used to investigate the fate of emulsion particles following injection into the tail vein of female BALB/c inbred mice. While 14C- triolein (TO) is susceptible to the action of lipoprotein lipase (LPL), 3H-cholesteryl oleate ether (CO ether) is not. Hence the removal of 14C-TO represents the triglyceride metabolism, whereas the other one is a core marker to represent whole particle removal by RES organ uptake. The emulsions with DPPC as sole emulsifier were rapidly cleared from the blood with only 10–11% of CO or TO left in circulation after 1 h. However, addition of PEG–PE gave a prolonged clearance rate, especially during first 3 h. A further addition of cosurfactant polysorbate 80 or Pluronic F-68 resulted in a marked extension of the circulation lifetime during the first 6 h. The notable effects of polysorbate 80 and Pluronic F-68 can apparently be attributed mainly to the decrease in droplet size, although an additional influence due to the increased hydrophilicity may not be ruled out. The in vivo disposition of emulsions administered as nutrients (surfaceunmodified first-generation emulsion) as well as administered as drug carriers (surface-modified second-generation emulsion) would depend on the particle prop-


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erties, such as the size [68–71], zeta potential (see Sections 7.4.2.3 and 7.4.5), and compositions of phospholipids and oil phase (see the above paragraphs), which may vary among different products and the batches of each product. The size of particulate carriers such as liposomes is known to influence both the phagocytic uptake by the mononuclear phagocyte system (MPS) [68–70] and the binding of apolipoprotein (apo) to emulsions [71]. Furthermore, the particle size is a major determinant of the transfer to extravascular spaces from the blood compartment. The capillaries of the vascular system can be classified into three categories: continuous, fenestrated, and discontinuous (sinusoidal) [72]. Particulate carriers including nanosized emulsions are considered to pass through capillaries and reach extravascular cells only in organs having discontinuous capillaries such as liver, spleen, and bone marrow. In such tissues, the extravasation of particles should be regulated by their size since the largest pores in the capillary endothelium is reported to be about 100 nm [73]. In addition, tumor capillaries have unique characteristics in their structures and functions in comparison with normal tissues such as muscle [74, 75], which results in the enhanced distribution of particulate carriers to tumor tissues [76–78]. The distribution of emulsions within a tumor tissue was also regulated by the size of particulate carriers [79]. Obviously, because of the submicrometer size range (175–400 nm in diameter) of the emulsions, the more they circulate, the greater their chance of reaching respective targets. More specifically, growing solid tumors as well as regions of infection and inflammation have capillaries with increased permeability as a result of the disease process (e.g., tumor angiogenesis [74]). Pore diameters in these capillaries can range from 100 to 800 nm. Thus, drug-containing emulsion particles are small enough to extravasate from the blood into the tumor interstitial space through these pores [80]. Normal tissues, by and large, contain capillaries with tight junctions that are impermeable to emulsions and other particles of this diameter. This differential accumulation of emulsion-laden drug in tumor tissues relative to normal cells is the basis for the increased tumor specificity for the emulsion-laden drug relative to free (nonemulsion) drug. In addition, tumors lack lymphatic drainage and therefore there is low clearance of the extravasated emulsion from tumors. Thus, long-circulating lipid carriers, such as POE/PEG-coated nanosized emulsions, tend to accumulate in tumors as a result of increased microvascular permeability and defective lymphatic drainage, a process also referred to as the enhanced permeability and retention (EPR) effect [81]. Table 2 lists various formulation factors affecting the metabolism as lipoproteins, the recognition by the MPS, and the elimination from the blood circulation of both second- and third-generation nanosized emulsions after parenteral administration. On the other hand, essential requirements of this “active” targeting approach include identification of recognition features (receptors) on the surface of the target and the corresponding molecules (ligands) that can recognize the surface. Indeed, emulsions with appropriate ligands anchored on their surface must be able to access the target, bind to its receptors, and, if needed, enter it. Furthermore, in order to bring the colloidal carrier closer to otherwise inaccessible pathological target tissues, homing devices/ligands such as antibodies and cell recognition proteins are usually linked somehow onto the particle surfaces. Various methods have been employed to couple ligands to the surface of the nanosized lipidic and polymeric carriers with reactive groups. These can be divided into covalent and noncovalent couplings. Noncovalent binding by simple physical association of targeting ligands to the

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TABLE 2 Formulation Factors Affecting Metabolism as Lipoproteins, Recognition by Mononuclear Phagocyte System (MPS), and Elimination from Blood Circulation of Second- and Third-Generation Nanosized Emulsions after Parenteral Administration Metabolism as Lipoproteins Factor Particle size Emulsifier

Coemulsifier

Cationic lipid Oil phase

Recognition by MPS

Elimination from Blood Circulation

Poor

Extensive

Poor

Extensive

Slow

Rapid

Large DPPC DSPC SM Poloxamers HCO-60 PEG–PE Polysorbates Solutol SA/OA LCT —

Small EYPC — — — — — — — — MCT —

Small DPPC SM — Poloxamers HCO-60 PEG–PE Polysorbates Solutol SA/OA — —

Large DSPC — — — — — — — — — —

Small DPPC SM — Poloxamers HCO-60 PEG–PE Polysorbates Solutol SA/OA LCT SLS

Large EYPC DSPC — — — — — — — MCT SLM

Note: DPPC, dipalmitoylphosphatidylcholine; DSPC, distearoylphosphatidylcholine; SM, sphingomyelin; EYPC, egg yolk phosphatidylcholine; HCO-60, polyoxyethylene-(60)-hydrogenated castor oil; PEG–PE, phosphotidylethanolamine derivative with polyethylene glycol; SA, stearylamine; OA, oleylamine; LCT, long-chain triglyceride; MCT, medium-chain triglyceride; SLS, structured lipid with short-chain fatty acids, C8–C10; SLM, structured lipid with medium-chain fatty acids, C4.

nanocarrier surface has the advantage of eliminating the use of rigorous, destructive reaction agents. Common covalent coupling methods involve formation of a disulfide bond, cross-linking between two primary amines, reaction between a carboxylic acid group and primary amine, reaction between maleimide and thiol, reaction between hydrazide and aldehyde, and reaction between a primary amine and free aldehyde [82]. For antibody-conjugated second-generation anionic emulsions, the reaction of the carboxyl derivative of the coemulsifier molecule with free amine groups of the antibody and disulfide bond formation between coemulsifier derivative and reduced antibody were the two reported conjugation techniques so far [83–85]. However, by the formation of a thio-ether bond between the free maleimide reactive group already localized at the o/w interface of the emulsion oil droplets and a reduced monoclonal antibody, the antibody-tethered cationic emulsion was developed for active targeting to tumor cells [86]. It should be added that the cationic emulsion investigated for tumor-targeting purpose belongs to the thirdgeneration emulsion category (Section 7.4.2.3). Apart from non-RES-related disease treatment through target-specific ligand conjugation, the second-generation emulsions may also be useful for RES-related disease treatment. Certain lipoprotein or polysaccharide moiety inclusion into the emulsions would help to achieve this concept. In general, uptake of small colloidal drug carriers by the phagocytotic mononuclear cells of RES in the liver can be exploited to improve the treatment of parasitic, fungal, viral and bacterial diseases such as, for example, leishmaniasis, acquired immunodeficiency syndrome (AIDS), and hepatitis B. The approach to use emulsions as a drug carrier against microbial


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diseases is superior to free antimicrobial agents in terms of both distribution to the relevant intracellular sites and treating disseminated disease states effectively. As already discussed, conventional emulsion particles are capable of localizing in liver and spleen, where many pathogenic microorganisms reside. Rensen et al. [87] demonstrated the active/selective liver targeting of an antiviral prodrug (nucleoside analogue, iododeoxyuridine) incorporated in an emulsion complexed with ligands such as recombinant apolipoprotein E (apoE) using the Wistar rat as animal model because its apoE–receptor system is comparable to that of humans [88]. Whereas the parent drug did not show any affinity for emulsion due to its hydrophilic property, derivatization with hydrophobic anchors allowed incorporation of at least 130 prodrug molecules per emulsion particle without imparting any effect on the emulsion structure and apoE association to emulsion droplets. The authors did not describe where the 130 prodrug molecules reside in the emulsion and what is the emulsion/medium partition coefficient of the prodrug. The prodrug molecules might have reasonably higher solubility in the oil or o/w interface of the emulsion possibly due to a high partition coefficient value. Plausibly, this high partition value for prodrug molecules will determine the kinetic parameter koff (desorption rate of an emulsion component from the assembly), as suggested by Barenholz and Cohen [89] for liposomal technology. Furthermore, without being bound by theory, the apoE component helps to disguise the emulsion particles so that the body does not immediately recognize it as foreign but may allow the body to perceive it as native chylomicrons or very low density lipoproteins (VLDL). The small size and the approximately spherical shape allow the emulsion particles to exhibit similar physicochemical properties to native chylomicrons or VLDL (hydrolyzed by LPL) whereas the incorporated prodrug remained associated with the emulsion remnant particles following injection into the blood circulation of the rat [87]. Because the carrier particles are not recognized as foreign, the systemic circulation of the drug increases, thus increasing the likelihood of drug delivery to the target tissues (up to 700 nM drug concentration in liver parenchymal cells). Additionally, the clearance rate of the drug decreases, thereby reducing the likelihood of toxic effects of the drug on clearance tissues since accumulation of the drug in another part of the clearance tissues is reduced. Thus, specific organs may be targeted by using nanosized emulsion particles as described above due to target cells comprising high levels of specific receptors, for example, but not limited to apoE receptors. To address this issue, the saccharide moieties of glycolipids and glycoproteins on the cell surface are considered to play an important role in various intercellular recognition processes. For instance, Iwamoto et al. [90] investigated the influence of coating the oil droplets in emulsion with cell-specific cholesterol bearing polysaccharide, such as mannan, amylopectin, or pullulan, on the target ability of those formulations. They observed a higher accumulation of mannan-coated emulsion in the lung in guinea pigs. Thus selective drug targeting through emulsion-bearing ligands not only leads to an improved drug effectiveness and a reduction in adverse reactions but also offers the possibility of applying highly potent drugs. Hence, the composition of the emulsion plays an important role concerning intercellular cell recognition processes and, indeed, cell recognizability is also being improved by incorporation of apoproteins or galactoproteins onto the emulsion particles to enhance their specificity [91].

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Overall, although second-generation emulsion is usually used as a means of administering aqueous insoluble drugs by dissolution of the drugs within the oil phase of the emulsion, employing surface modification/PEGylation by the attachment of targeting ligands (apoE, polysaccharide, and antibody) onto the droplet surface of emulsions may be useful for both passive and active drug-targeting purposes. Thus receptor-mediated drug targeting using ligands attached to emulsions seems to hold a promising future to the achievement of cell-specific delivery of multiple classes of therapeutic cargoes, and this approach will certainly make a major contribution in treating many life-threatening diseases with a minimum of systemic side effects. 7.4.2.3 Third-Generation Emulsion In order to increase cellular uptake, a cationization strategy is applied particularly on the surfaces of nonviral, colloidal carrier systems such as liposomes, nano- and microparticulates, and nanocapsules [92]. To make the surface of these lipidic and polymeric carrier systems a cationic property, some cationic lipids/polymers are usually added into these systems during/after preparation. But, adding only the cationic substances in phospholipids-stabilized first-generation emulsions does not help to obtain a physically stabilized emulsion for a prolonged storage period. However, using different cationic lipids as emulsifier and additional helper lipids as coemulsifier, for example, DOTAP, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine-N[poly(ethylene glycol)2000] (PEG2000PE), reports are available to prepare emulsions with positive charges on their droplet surfaces [93, 94]. Alternatively, inclusion of cation-forming substances such as lipids (stearyl or oleyl chain having primary amines) [95, 96], polymers (chitosan) [97, 98], and surfactants (cetyltrimethylammonium bromide) [99] during the preparation of second-generation emulsion allows the formation of a stabilized system with positive charges over on it. Further, the positive charge caused by stearylamine was also confirmed by a selective adsorption of thiocyanate. Its adsorption was correlated with increasing stearylamine concentration [95]. So, nanosized emulsion consisting of complex emulsifiers, that is, phospholipid–polyoxyethylene surfactant-cationized primary amine or a polymer combination, can conveniently be termed third-generation emulsion. The extemporaneous addition of the solid drug or drug previously solubilized in another solvent or oil to the preformed first- and second-generation emulsions is not a favored approach as it might compromise the integrity of the emulsion. Since therapeutic DNA and single-stranded oligonucleotides or small interfering ribonucleic acid (siRNA) are water soluble due to their polyanionic character, the aqueous solution of these compounds need to be added directly to the preformed third-generation emulsion in order to interact electrostatically with the cationic emulsion droplets and thus associate/link superficially at the oil–water interface of the emulsion [100, 101]. During in vivo conditions when administered via parenteral, nasal, and ocular routes, the release of the DNA and oligonucleotides from the associated emulsion droplet surfaces should therefore initially be dependent solely on the affinity between the physiological anions of the biological fluid and the cationic surface of the emulsion droplets. For instance, the mono- and divalent anions con-


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taining biological fluid available in the parenteral route is plasma and in ocular topical route is tear fluid, aqueous humor, and vitreous humor. Moreover, these biofluids contain a multitude of macromolecules and nucleases. There is a possibility that endogenous negatively charged biofluid components could dissociate the DNA and oligonucleotides from cationic emulsion. It is noteworthy to conduct, during the preformulation development stages, an in vitro release study for therapeutic DNA and oligonucleotide-containing emulsion in these biological fluids, and this type of study could be considered an indicator of the strength of the interaction that occurred between DNA or oligonucleotide and the emulsion particles. However, it is interesting to see what could happen when the third-generation emulsion is applied to in vitro cell culture models in the presence of serum. The serum stability of the emulsion–DNA complex was reported [102, 103]. Further interesting investigations using third-generation emulsions in gene delivery purposes are briefly summarized in a review article [104]. 7.4.2.4

Unique Property of Third-Generation Emulsion

To enhance the drug-targeting efficacy of colloidal carriers such as nanospheres and liposomes, a PEGylation/cationization strategy is traditionally made over the surface of these carriers. While surface PEGylated colloidal carriers exhibit a prolonged plasma residence time through an escaping tendency from RES uptake following parenteral administration, surface-cationized colloidal carriers can facilitate the penetration of therapeutic agents into the cell surface possibly via an endocytotic mechanism. These two facts are proved in both liposomes and nanospheres when they possess separately the cationic and PEGylatic surface modifications on them. However, a cationic emulsion colloidal carrier system developed in Simon Benita’s laboratory at Hebrew University of Jerusalem, Israel, differs significantly in such a way that it holds a combination of cationic and PEGylatic surface properties on it. Benita’s group have prepared a novel cationic emulsion vehicle using a combination of emulsifiers consisting of Lipoid E 80, Poloxamer 188, and stearylamine and have found the formulation suitable for parenteral use, ocular application, nasal drug delivery, and topical delivery [105]. It has been reported in an ocular pharmacokinetic study of cyclosporin A incorporated in deoxycholic acid–based anionic and stearylamine-based cationic emulsions in rabbit that, when compared to anionic emulsion, the cationic emulsion showed a significant drug reservoir effect of more than 8 h in corneal and conjunctival tissues of the rabbit eye following topical application [106]. Since cornea and conjunctiva are of anionic nature at physiological pH [107], the cationic emulsion would interact with these tissues electrostatically to implicate the observed cyclosporin A reservoir effect. This hypothesis is supported, in principle, by an ex vivo study which showed that cationic emulsion carrier exhibited better wettability properties on rabbit cornea than either saline or anionic emulsion carrier [108]. Studies [109, 110] have shown that small changes in physical properties of emulsions can influence the elimination rate of these formulations from the blood. Indeed, an organ distribution study of stearylamine-based cationic or deoxycholic acid–based anionic nanosized emulsions and Intralipid, a well-known commercial anionic emulsion, containing 14C-CO was carried out following injection into the tail vein of male BALB/c mice (20–26 g) at a volume of 5 mL/kg [111, 112]. Since CO

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(cholesteryl oleate) is one of the most lipophilic compounds used in biopharmacy and is not prone to degradation in the body (which remains within particles even after lipolysis of emulsion), its in vivo behavior can be regarded as reflecting that of the injected nanosized emulsion in the early phase [42, 113]. Following intravenous administration of the various emulsions having 14C-CO to BALB/c mice, the 14 C-CO was found to accumulate in organs such as lung and liver. Furthermore, it was observed that the concentration of 14C-CO in the lung decreased but was again elevated over time for both the developed cationic and anionic emulsion formulations, with a concomitant decrease in the concentration of the radiolabeled compound in the liver. However, within the various emulsion distribution patterns observed in liver, a lower 14C-CO concentration was observed for stearylaminebased cationic emulsion when compared to Intralipid while for deoxycholic acid– based anionic emulsion the observed concentration of 14C-CO was relatively very low when compared to cationic emulsion and Intralipid. In addition, in comparison to both anionic emulsions, the stearylamine-based cationic emulsion elucidated a much longer retention time of 14C-CO in the plasma, clearly indicating a long circulating half-life for cationic emulsion in the blood. Thus, the cationic nanosized emulsion can be considered a stealth long-circulating emulsion. The above two studies clearly described the unique characteristics of thirdgeneration emulsion in enhancing ocular drug bioavailability; on the other hand, the same emulsion has the property of circulating for a longer time in blood following parenteral administration. Excess positive charge at the oil–water interface in conjunction with the projection of highly hydrophilic POE chain (due to the presence of Poloxamer 188) toward the aqueous phase of the o/w-type nanosized emulsion is the main reason behind the emulsion attaining its unique property, which is absent in first- and second-generation emulsions. However, a better understanding of the structure of the third-generation emulsion in terms of forces involved in its formation and stabilization must ultimately be obtained in an effort to provide a clearly understood physical basis for the uniqueness in its biological efficacy following parenteral and ocular administration. It should be added that the use of stearylamine in intravenous administered emulsion might be problematic. Stearylamine is a single-chain amphiphile having relatively high critical micellar concentration, although the concentration used in the studied emulsion is much higher than the critical micellar concentration. Therefore, due to the dilution in plasma as well as plasma lipoproteins and blood cells, there is a high probability that the emulsion will lose its stearylamine almost instantaneously. To substantiate indirectly this issue, Klang et al. [114] showed the lack of potential induced toxicity of stearylamine-based cationic emulsion in animal models in vivo and Korner et al. [115] investigated the surface properties of mixed phospholipid–stearylamine monolayers and their interaction with a nonionic surfactant (poloxamer) in vitro. Despite the presence of the stearylamine, which may be suspected of being an irritant in pure form, in the emulsifier combination, the hourly instillation of stearylamine-based cationic emulsion vehicle into rabbit eye was well tolerated without any evidence of any toxic or inflammatory response to the ocular surface during the 5 days of the study (40 single-drop instillations between 8 AM and 4 PM each day) [114]. Following 0.2-, 0.4-, and 0.6-mL single-bolus injections of the same emulsion vehicle, representing a huge single administered dose of 30 mL/kg, no animal deaths were noted over a period of 30 days, apparently indicat-

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ing the absence of marked acute toxicity [114]. Furthermore, the same stearylaminebased cationic emulsion vehicle did not cause acute neurotoxicity in rats when a continuous intravenous infusion (3.3 mL) for 2 h at a rate of 27.4 μL/min was administered through the jugular vein [114]. An another study from Benita’s laboratory suggests that long-term subchronic toxicity examination of the rabbit eye (healthy) following thrice-daily single-drop topical instillation of the stearylamine-based emulsion elicited an almost similar nonirritating effect to eye tissues in comparison to the thrice-daily single-drop topical instillation of the normal saline–treated control rabbit eyes (unpublished data). Thus, overall results clearly indicated that the stearylamine was strongly bound at a molecular level to the mixed interfacial film formed by Lipoid E 80 and poloxamer 188 at the oil–water interface system [115]. Such an intercalation between the emulsifiers is responsible for emulsified oil droplet stability and, in fact, prevented the stearylamine from leaking and exerting any intrinsic possible local or systemic adverse effects in model animals.

7.4.3 PREPARATION METHODS FOR DRUG-FREE/LOADED OIL-IN-WATER NANOSIZED EMULSIONS To get a better idea of how to formulate the nanosized emulsion delivery systems suitable for parenteral, ocular, percutaneous, and nasal uses, the reader is referred to more detailed descriptions of methods of nanosized emulsion preparation [6, 116]. A hot-stage high-pressure homogenization technique or combined emulsification technique (de novo production) is frequently employed in order to prepare nanosized emulsions with desired stability even after subjection to autoclave sterilization. Therefore, the steps involved in this technique in making blank anionic and cationic emulsions were arranged in the following order: 1. Weigh the oil- and water-soluble ingredients in separate beakers. 2. Heat both oil and water phases separately to 70°C. 3. Add the oil phase to the water phase and continue the heating up to 80°C with constant stirring to form a coarse emulsion. 4. Mix at high shear to make a fine emulsion. 5. Cool the fine emulsion formed in ice bath. 6. Homogenize the fine emulsion. 7. Cool the homogenized emulsion in ice bath. 8. Filter the emulsion using a 0.5 μm membrane filter. 9. Adjust the emulsion to 7 using 0.1 N hydrochloric acid or 0.1 N sodium hydroxide solution. 10. Pass nitrogen gas into the vials containing the emulsion. 11. Sterilize the emulsion using an autoclave. The traditional droplet size–reducing steps involved during the preparation include constant mild stirring using a magnetic stirrer when initially mixing oil and water phases, rapid Polytron mixing at high speed, and final droplet size homogenization using a two-stage homogenizer valve assembly. The initial heating is vital for the

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TABLE 3 Typical Formula to Make o/w Anionic and Cationic Nanosized Emulsions Oil Phase Natural/semisynthetic oils Phospholipid mixture Stearylamine/oleylaminea Deoxycholic acid/oleic acidb Vitamin E

Water Phase Poloxamer 188 Glycerol Double-distilled water

a

Necessary ingredient for cationic emulsions. Necessary ingredient for anionic emulsions.

b

effective solubilization of the respective oil and water phase components in their corresponding phases. Mixing the two phases with constant mild stirring and subsequently raising the temperature to 85°C are needed to form an initial coarse emulsion and to localize the surfactant molecules for better adsorption at the oil– water interface, respectively. A typical formula to make anionic and cationic nanosized emulsions is given in Table 3. There are three different approaches to incorporate lipophilic drugs into the oil phase or at the o/w interface of the nanosized emulsions, namely, extemporaneous drug addition, de novo emulsion preparation, and an interfacial incorporation approach, which includes the recently developed SolEmul technology [117]. In principle, the lipophilic drug molecules should however be incorporated by a de novo process. Thus, the drug is initially solubilized or dispersed together with an emulsifier in suitable single-oil or oil mixture by means of slight heating. The water phase containing the osmotic agent with or without an additional emulsifier is also heated and mixed with the oil phase by means of high-speed mixers. Further homogenization takes place to obtain the needed small droplet size range of the emulsion. A terminal sterilization by filtration, steam, or autoclave then follows. The emulsion thus formed contains most of the drug molecules within its oil phase. This is a generally accepted and standard method to prepare lipophilic drug–loaded nanosized emulsions for parenteral, ocular, percutaneous, and nasal uses, as illustrated in Figure 3. This process is normally carried out under aseptic conditions and nitrogen atmosphere to prevent both contamination and potential oxidation of sensitive excipients.

7.4.4 EXCIPIENT INCLUSION: OIL-IN-WATER NANOSIZED EMULSIONS In general, nanosized emulsions should be formulated with compatible vehicles and additives. The components of the internal and external phases of emulsion should be chosen to confer enhanced solubility and/or stability to the incorporated biologically active lipophilic drug. In addition, it should also be designed to influence biofate or therapeutic index of the incorporated drug following administration via parenteral, ocular, percutaneous, and nasal routes. In this section, general considerations concerning excipient selection and optimum concentrations are comprehen-

EXCIPIENT INCLUSION: OIL-IN-WATER NANOSIZED EMULSIONS

FIGURE 3

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Preparation of o/w nanosized emulsion (de novo method).

sively presented mainly in their relation to the oil phase, the aqueous phase, and the emulsifiers. Prior to the formulation design of the emulsions, data are needed concerning the drug solubility in the oil vehicle. Additionally, prerequisite information is needed about compatibility of the oil vehicle with other formulation additives and the established ocular/skin tissues–oil vehicle matching before the dosage form can be prepared. Table 4 lists the common emulsion excipients and the oils suitable for dissolving or dispersing lipophilic drugs of ocular/parenteral interests. Since oils are triglycerides, care must be taken to minimize or eliminate oxidation. Therefore, antioxidants such as α-tocopherol (0.001–0.002% w/w) should be included in a typical emulsion formulation for medical applications. The final oil-phase concentration in emulsions meant for ocular use is now widely accepted to be at or below 5% w/w taking into account that the emulsion must be kept in a low-viscosity range of between 2 and 3 centipoises, which also is the optimal viscosity for ocular preparations [118]. However, for all other medical uses, the amount of oil may be varied but generally is within 5–20% w/w. Sometimes, a mixture of oils rather than a single oil is employed since drug solubilization in the oil phase is a prerequisite to exploiting the emulsion advantages. Jumaa and Müller [98, 119] reported the effect of mixing castor oil with MCT on the viscosity of castor oil. The oil combination at the ratio of 1 : 1 (w/w) led to a decrease in the viscosity of castor oil and simultaneously to a decrease in the interfacial tension of the oil phase. This was related to the free fatty acids contained in castor oil, which can act as a coemulsifier resulting in lower interfacial tension and, simultaneously, in a more stable formulation in comparison with the other oil phases. In addition to the digestible oils from the family of triglycerides, including soybean oil, sesame seed oil, cottonseed oil, and safflower oil, which are routinely used for making medical emulsions, alternative biocompatible

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TABLE 4

Excipients Used for Formulation of o/w Nanosized Emulsions

Oils Sesame oil Castor oil Soya oil Paraffin oil Paraffin light Lanolin Vaseline Corn oil Glycerin monostearate Medium-chain monoglycerides Medium-chain triglycerides Squalene Vitamin E

Emulsifiers Cholesterol Phospholipids (Lipoid) Polysorbate 80 and 20 (Tween 80 and 20) Transcutol P Cremophor RH Poloxamer 407 Poloxamer 188 Miranol C2M and MHT Tyloxapol TPGS

Cationic Lipids and Polysaccharide Stearylamine Oleylamine Chitosan

Miscellaneous α-Tocopherol Glycerin Xylitol Sorbitol Thiomersal EDTA Methyl paraben Propyl paraben

Note: TPGS, α-tocopheryl-polyethylene glycol-1000-succinate; EDTA, ethylenediamine tetraacetic acid.

oils such as α-tocopherol and/or other tocols were also investigated for drug delivery purposes via o/w emulsions [120, 121]. But the emulsions formed from tocols are often considered as microemulsion systems with few exceptions being the nanosized emulsions [122, 123]. Unlike spontaneously forming thermodynamically stable microemulsion systems that require a high surfactant concentration (20% and higher), the kinetically stable nanosized emulsions can be prepared by using relatively lower surfactant concentrations. For example, a 20% o/w nanosized emulsion may only require a surfactant concentration of 5–10%. Traditionally, lecithins or phospholipids are the emulsifiers of choice to produce nanosized emulsions. However, emulsifiers of this kind are not suitable to produce submicrometer–sized emulsion droplets or to withstand the heat during steam sterilization. Therefore, additional emulsifiers preferably dissolved in the aqueous phase are usually included in the emulsion composition. A typical example of the aqueous soluble emulsifiers is nonionic surfactants (e.g., Tween 20) after taking into consideration their nonirritant nature when compared to ionic surfactants. The nonionic block copolymer of polyoxyethylene–polyoxypropylene, Pluronics F68 (Poloxamer 188), is included to stabilize the emulsion through strong steric repulsion. However, amphoteric surfactants Miranol MHT (lauroamphodiacetate and sodium tridecethsulfate) and Miranol C2M (cocoamphodiacetate) were also used in earlier ophthalmic emulsions [124]. It should be added that commercially available cyclosporin A–loaded anionic emulsion (Restasis) contains only polysorbate 80 and carbomer 1342 at alkali pH to stabilize the drug-loaded anionic emulsion. To prepare a cationic emulsion, cationic lipids (stearyl and oleylamine) or polysaccharides (chitosan) are added to the formulation. Strikingly, if chitosan is a choice of cation producing polysaccharide emulsifier molecules, there is no need to add amphoteric or nonionic surfactants to the phospholipid or lecithin-stabilized

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emulsion [125]. Conversely, a cationic emulsion based on an association of poloxamer 188 and chitosan without lecithin was prepared and also showed adequate physicochemical properties regarding stability and charge effects [97, 98]. Oil-inwater emulsion compositions based on a tocopherol (or a tocopherol derivative) as the disperse phase have been described in a patent granted to Dumex [126]. The emulsion is intended for use with compounds that are sparingly soluble in water. Interestingly, the emulsifying agent used to make tocol-based emulsions are restricted to vitamin E TPGS. Additives other than antioxidants such as preservatives (e.g., benzalkonium chloride, chlorocresol, parabens) are regularly included in emulsions to prevent microbial spoilage of multidose medical emulsions. α-Tocopherol is a good example of an antioxidant used to obtain a desirable stabilized emulsion under prolonged storage conditions. The presence of components of natural origin such as lecithin or oils with high calorific potential renders the emulsion a good medium to promote microbial growth when it is packed in multidose containers. Pharmaceutical products when distributed into multidose containers, especially for parenteral and ocular administrations, should be properly preserved against microbial contamination and proliferation during storage in normal conditions and proper use. Incorporation of preservatives in single-dose vials is also a common procedure if filtration is used as a sterilization method. Sznitowska et al. [127] studied the physicochemical compatibility between the lecithin-stabilized emulsion and 12 antimicrobial agents over two years of storage at room temperature. Preliminary physicochemical screening results indicate that addition of chlorocresol, phenol, benzyl alcohol, thiomersal, chlorhexidine gluconate, and bronopol should be avoided due to the occurrence of an unfavorable pH change followed by coalescence of lecithin-stabilized droplets of the emulsion. Furthermore, the efficacy of antimicrobial preservation was assessed using the challenge test according to the method described by the European Pharmacopoeia. Despite good physicochemical compatibility, neither parabens nor benzalkonium chloride showed satisfying antibacterial efficacy in the emulsion against the tested microorganisms and consequently did not pass the test. Therefore, higher concentrations of antimicrobial agents or their combination may be required for efficient preservation of the lecithin-stabilized emulsion probably because of unfavorable phase partitioning of the added antimicrobials within the different internal structures of the emulsion. It is interesting to note that benzalkonium chloride, a highly aqueous soluble drug, did not pass the standard challenge test even when incorporated in a cationic emulsion, particularly the third-generation category (unpublished data). This finding clearly indicates that the possible electrostatic attraction between the negatively charged lipid moieties of the mixed emulsifying film formed around the anionic emulsified oil droplets [127] and the quaternary cationic ammonium groups of the preservative is not the plausible cause for the reduced activity of the benzalkonium chloride. Thus, the possible intercalation of this surfactant in either the cationic or anionic interfacial mixed emulsifying film is likely to occur, preventing benzalkonium chloride from eliciting its adequate preservative action. Overall, it is preferable to formulate nanosized emulsions devoid of preservative agents and fill it in sterile single-dose packaging units to prevent potential contamination due to repeated use of multidose packaging. It should be pointed out that the two available ocular emulsion products (Refresh Endura and Restasis, Allergan, Irvine, CA) on the market are preservative free and packed in

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single-use vials only. Currently there is no commercial parenteral emulsion which contains preservatives and research concerning the problem of preservation of nanosized emulsion is very limited [128–131].

7.4.5 MEDICAL APPLICATIONS OF OIL-IN-WATER NANOSIZED EMULSIONS It has been shown in a number of studies that the incorporation of drug in o/w nanosized emulsions significantly increased the absorption of the drug when compared with the equivalent aqueous solution administered orally [132–135]. However, the use of emulsions for oral application is limited since other attractive alternatives, such as self-emulsifying oil delivery systems, which are much less sensitive and easy to manufacture, are available [136, 137]. Thus the potential of nanosized emulsions after administration with parenteral and traditional nonparenteral topical routes such as ocular, percutaneous, and nasal is covered in this section. 7.4.5.1

Parenteral Routes

The o/w nanosized emulsion formulations of lipophilic drugs, such as propofol, etomidate, dexamethasone palmitate, and diazepam, were already developed and marketed (Table 1). Furthermore, various research groups across the world are currently undertaking projects to exploit the potential of o/w emulsions for parenteral delivery of a myriad of investigational drugs as well as other lipophilic drugs by receptor-mediated targeting to cancer cells. The important technical and clinical points to keep in mind before the use of the emulsion systems for this kind of work are given below. It has to be clear that, once diluted and injected (or administered in ocular and other routes), the emulsion stability and fate are determined by three measurable parameters. The first is the partition coefficient of each emulsion component (including added drugs and agents) between the emulsion assembly and the medium. To some extent this partition coefficient is related to oil–water and/or octanol–water partition coefficients. For example, it was well demonstrated that per component of which log P is lower than 8, the stability upon intravenous (IV) injection is questionable [42, 138]. The other two parameters are koff, a kinetic parameter which describes the desorption rate of an emulsion component from the assembly, and kc, the rate of clearance of the emulsion from the site of administration. This approach is useful to decide if and what application a drug delivery system will have a chance to perform well [89]. Stability in plasma is an important requirement for IV emulsions as flocculated droplets may result in lung embolism. It was found that tocol-based emulsions stabilized by sodium deoxycholate/lecithins flocculated strongly when mixed with mouse, rat, and sheep plasma and serum, whereas soya oil–based emulsions with the same emulsifiers did not [123]. It was hypothesized that this effect was caused by the adsorption of plasma proteins onto the tocol droplets (opsonization). Indeed, the steric stabilization of emulsions by incorporation of emulsifiers like poloxamer 188 or PEGylated phospholipids such as PEG5000PE proved to be effective in the stabilization of tocol-based emulsions in plasma. Conversely, in vitro studies were

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conducted on plasma protein adsorption onto the blank second- and thirdgeneration emulsion droplets [37, 139] to mimic the in vivo opsonization phenomenon responsible for the rapid clearance of the emulsion droplets from the blood. According to these authors, the adsorption of many protein species such as apoAs, apoCs, apoE albumin, fibrinogen, and gamma globulin onto the emulsion droplet surfaces is detectable by two-dimensional polyacrylamide gel electrophoresis. 7.4.5.2

Ocular Routes

For the eye, the method of drug delivery is important. However, when nanosized emulsion is used as a vehicle for ocular drug delivery purposes, both topical/local and intraocular routes of administration can be possible (though no data concerning intraocular drug delivery through emulsion are currently available). The o/w nanosized emulsions having both anionic and cationic charges provide a liquid-retentive carrier for ocular active agents, particularly when topically instilled into the eye. It is interesting to add here that thermodynamically stable and optically isotropic colloidal systems such as the w/o microemulsion is also designed nowadays for ocular topical. As delivery the w/o microemulsion system comprises both aqueous and oily components into its multistructure, it has the ability to incorporate considerable amounts of both hydrophilic and lipophilic drugs [140]. In fact, in comparison to ocular inserts or implants and semisolid ocular preparations, the liquid-retentive nature gives impetus to investigating further the emulsion-based ophthalmic drug delivery as it has the benefit of being comfortable to use for both ophthalmologists and patients. In addition, through topical instillation of emulsions possessing ocular active substances, the delivery of drug molecules even to the posterior portion of the eye might be of possible. In this context, the third-generation emulsion is being designed by adsorbing electrostatically the therapeutic oligonucleotides onto its surface for modulating functions of retinal pigment epithelium (RPE) cells effectively in order to treat blindness associated with age-related macular degeneration (AMD), proliferative vitreoretinopathy, retinal and choroidal neovascularization, and retinitis pigmentosa. To achieve this, it becomes necessary to know first the ocular protective mechanisms and other concomitant factors to be faced by emulsion droplets following ocular topical application. This point is further developed below. Considerations of ocular drug delivery are not detailed in this section. Pertinent information concerning factors affecting drug permeation or retention as well as eye anatomy and physiology can be found in several reviews [141–146]. From a medical point of view, o/w nanosized emulsions for ophthalmic use aim at enhancing drug bioavailability either by providing prolonged delivery to the eye or by facilitating transcorneal/transconjunctival penetration. Drugs incorporated in o/w nanosized emulsions are lipophilic in nature, and depending on the extent of lipophilicity, either the corneal or the conjunctival/scleral route of penetration may be favored [147]. For the more lipophilic drugs the corneal route was shown to be the predominant pathway for delivering drugs to the iris, whereas the less lipophilic drugs underwent conjunctival/scleral penetration for delivery into the ciliary body [147]. Thus, transcorneal permeation has traditionally been the mechanism by which topically applied ophthalmic drugs are believed to gain access to the internal ocular structures. Relatively little attention has been given to alternate routes by which drugs may enter the eye. It was reported that drugs may be absorbed by the

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noncorneal route and appeared to enter certain intraocular tissues through the conjunctiva/sclera [148–150]. Indeed when compared to the cornea, drug penetration through the conjunctiva has the advantage of a larger surface area and higher permeability, at least for drugs which are not highly lipophilic. Furthermore, the lasting presence of drug molecules in the lower conjunctival cul-de-sac of the eye could result in a reservoir effect. Nevertheless, the o/w nanosized emulsions more or less physically resemble a simple aqueous-based eye drop dosage form since more than 90% of the external phase is aqueous irrespective of the formulation composition. Hence, following topical administration, nanosized emulsions would probably face almost similar ocular protective events as encountered with conventional eye drops into the eye. The o/w nanosized emulsions are likely to be destabilized by the tear fluid electrolytic and dynamic action. Because of constant eyelid movements, the basal tear flow rate (1.2 μL/min), and the reflex secretion induced by instillation (up to 400 μL/min depending on the irritating power of the topical ocular solutions [35]), topical eye drop dosage forms are known for being rapidly washed out from the eye. Therefore, the water phase of the emulsion is drained off while, probably, the oil phase of the emulsion remains in the cul-de-sac for a long period of time and functions as a drug reservoir [35]. If the volume of instilled emulsion in the eye exceeds the normal lachrymal volume of 7–10 μL, then the portion of the instilled emulsion (one or two drops, corresponding to 50–100 μL) that is not eliminated by spillage from the palpebral fissure of conjunctiva is drained quickly via the nasolacrimal system into the nasopharynx. In other words, the larger the instilled volume, the more rapidly the instilled emulsion is drained from the precorneal area. Hence the contact time of the emulsion with the absorbing surfaces (cornea and conjunctiva) is estimated to be a maximum of a few minutes, well beyond the short residence time of conventional eye drops. In order to verify the extension of the residence time of the emulsion in the conjunctival sac, Beilin et al. [151] added a fluorescent marker to the formulations. One minute after the topical instillation into eye, 39.9 ± 10.2% of the fluorescence was measured for the nanosized emulsions whereas only 6.8 ± 1.8% for regular eye drops. In addition a study was carried out in male albino rabbits to compare the corneal penetration of indomethacin from Indocollyre (a marketed hydro-PEG ocular solution) to that of negatively and positively charged emulsions [108]. By this comparison, it was intended to gain insightful mechanistic comprehension regarding the enhanced ocular penetration effect of the emulsion as a function of dosage form and surface charge. The contact angle of one droplet of the different dosage forms on the cornea was measured and found to be 70° for saline, 38° for the anionic emulsion, and 21.2° for the cationic emulsion. Respectively, the values of the calculated spreading coefficient were −47, −8.6, and −2.4 mN/m. It can clearly be deduced, owing to the marked low spreading coefficient values elicited by the emulsions, that both nanosized emulsions had better wettability properties on the cornea compared to saline. The emulsion may then prolong the residence time of the drop on the epithelial layer of the cornea, thereby enabling better drug penetration through the cornea to the internal tissues of the eye, as confirmed by animal studies [108]. It is therefore believed that drug is not released from the oil droplet and equilibrates with the tears but rather partitions directly from the oil droplets to the cell membranes on the eye surface. Therefore, it is reasonable to consider that nanosized emulsions have a real advantage since they elicit an increased ocular residence time in com-


TABLE 5

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Selected List of o/w Nanosized Emulsions for Ocular Topical Drug Delivery

Emulsion Type Anionic emulsion

Cationic emulsion

Drug Used

Reference

Δ8-THC Pilocarpine base and indomethacin Adaprolol maleate Indomethacin Synthetic HU-211 and pilocarpine base Pilocarpine base Cyclosporin A Piroxicam Indomethacin Miconazole Cyclosporin A

154 163 161, 162 159, 160 124, 155, 156 157, 158, 164 168, 165–177 178 108 112 106, 179

Note: Δ8-THC and synthetic HU-211 are derivatives of Cannabis sativa.

parison to conventional eye drops and will significantly improve the ocular drug bioavailability [152]. This is also confirmed in numerous cited papers that are listed in Table 5. In spite of a relatively rapid removal of conjunctivally absorbed emulsion from the eye by local circulation, direct transscleral access to some intraocular tissues cannot be excluded, especially if an electrostatic attraction does occur between the cationic oil droplets of emulsion and anionic membrane moieties of the sclera, as shown by some authors [108]. There is no doubt that the drug absorption from emulsion through the noncorneal route needs to be investigated further as it may elicit useful information on the potential of nanosized emulsions to promote drug penetration to the posterior segment through a mechanism which bypasses the anterior chamber. In addition to the above-described protective and elimination mechanisms of the eye, nanosized emulsions remaining in the precorneal area may be subject to protein binding and to metabolic degradation in the tear film. In conjunction with blood plasma, although low, tear film, aqueous humor, and vitreous humor also have varying amounts of relatively detectable proteins such as albumin, globulin, and immunoglobulins (e.g., IgA, IgG, IgM, IgE) and the enzyme lysozyme. Additional studies (at least in vitro) are necessary to understand clearly the nanosized emulsion interaction with the ocular fluid components. Although it is unlikely to happen because of the low emulsion volume remaining in the conjunctival sac, the fluid dynamics may be moderately altered by the physical and chemical properties of nanosized emulsions, which include tonicity, pH, refractive index, interfacial charge, viscosity, osmolality, and irritant ingredients. Thus, formulations of ophthalmic drug products must take into account not only the stability and compatibility of a drug in the emulsion but also the influence of the emulsion on precorneal fluid dynamics. All of the concepts exposed in this section may ultimately result in transcorneal/conjunctival absorption of 1–2% or less of the drug applied topically through the emulsions. In summary, the rate of loss of drug/emulsion from the eye can be 500–700 times greater than the rate of absorption into the eye. Thus, conventional topical delivery using emulsions cannot achieve adequate intracellular concentrations of drugs or other substances such as oligonucleotides or genes for the treatment of endophthalmitis or other sight-threatening intraocular diseases (e.g., AMD).

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In order to achieve a high concentration of drug within the eye using an emulsion delivery vehicle, an approach that bypasses physiological and anatomical barriers (e.g., blood–ocular) of the eye is a more viable and attractive option. One such approach is to administer emulsion through direct intraocular injections such as periocular (subconjunctival and sub-Tenon), intracameral, intravitreal, intracapsular, or subretinal. Moreover, it is likely that intraocularly administered emulsion is able to both significantly increase drug half-life and minimize intraocular side effects that appear following intraocular injection of drug alone. In general, drugs encapsulated within emulsion are less toxic than their free counterparts. Additionally, there is a possibility of obtaining slow drug release from an intraocularly injected emulsion. Taking into account the nonphagocytic character of neural retinal cells and the ability of RPE cells to take up large molecules, including oligonucleotides, the third-generation emulsion for intravitreal or subretinal injections is more likely to be a successful approach in future. Moreover, intravitreally administered drug molecules are able to bypass the blood–ocular barrier to achieve constant therapeutic levels in the eye while minimizing systemic side effects. However, the hyalocytes, the main cellular components of the vitreous, have been classified in at least one report [153] as macrophages and thus may play a role in the uptake of intravitreally injected emulsion. It should be added that no studies are focused so far on injecting emulsion intraocularly and significant work should be devoted to generate this novel idea into a fruitful solution in ophthalmic drug delivery applications. Over the last decade, o/w nanosized emulsions containing either anionic or cationic droplets have been recognized as interesting and promising ocular topical delivery vehicles for lipophilic drugs. Complete details are available elsewhere [117]. As an overview of this topic, important results on emulsion-based ocular topical drug delivery are covered below and are listed in Table 5. The in vivo data obtained from studies of early formulations confirm that o/w nanosized anionic emulsions can be effective topical ophthalmic drug delivery systems [154] with a potential for sustained drug release [155]. Naveh and coworkers [156] have also noted that the intraocular pressure (IOP)–reducing effect of a single, topically administered dose of pilocarpine-loaded anionic emulsion lasted for more than 29 h in albino rabbits whereas that of the generic pilocarpine lasted only 5 h. Zurowska-Pryczkowska et al. [157] studied how nanosized emulsion as a vehicle influences the chemical stability of pilocarpine and the effect the drug has on the physical stability of nanosized emulsions. In a subsequent paper [158] from the same group on in vivo evaluation using normotensive rabbits, it was shown that the nanosized emulsion formulated with pilocarpine hydrochloride at pH 5.0 could be indicated as a preparation offering prolonged pharmacological action (miotic effect) together with satisfactory chemical stability. However, the ocular bioavailability arising from such a formulation is not significantly improved when compared to an aqueous solution. Calvo et al. [159, 160] observed an improvement in indomethacin ocular bioavailability when the drug was incorporated in an emulsion dosage form with respect to the commercial aqueous drops following topical application into rabbit eye. In order to verify the extension of the residence time of the emulsion in the conjunctival sac, Beilin et al. [151] added a fluorescent marker to the formulations, as mentioned previously. From that observation, it is reasonable to consider that an emulsion formulation has the real advantage of increasing ocular residence time in


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comparison to eye drops. Anselem et al. [161] and Melamed et al. [162] prepared a nanosized emulsion containing adaprolol maleate, a novel soft β-blocking agent, and observed a delayed IOP depressant effect in human volunteers. A similar pharmacological effect was also observed in human volunteers by Aviv et al. [163] using pilocarpine base-loaded emulsion. Another randomized human trial conducted by Garty et al. [164] compared the activity of the pilocarpine base-laden nanosized emulsion instilled twice daily with a generic dosage form instilled four times a day to 40 hypertensive patients for seven days. No local side effects were observed. The IOP decreased 25% in both formulations during this time period. No significant difference was noticed between the two treatments. These results proved that the anionic emulsion extended the action of the drug and two daily administrations have the same result as four instillations of regular eye drops. A novel nanosized anionic emulsion incorporating the immunomodulatory agent cyclosporin A was developed and the clinical efficacy was investigated for the treatment of moderate to severe dry-eye disease in humans [165–167]. The novel cyclosporin A ophthalmic dosage form represents a real breakthrough in the formulation of a complex, highly lipophilic molecule such as cyclosporin A within an o/w nanosized emulsion. Following thorough validation of this formulation through several clinical studies in various countries [165–175], an anionic o/w emulsion containing cyclosporin A 0.05% (Restasis, Allergan, Irvine, CA) was approved for the first time by the FDA, on December 23, 2002. In addition, this anionic emulsion having cyclosporin A is now available at pharmacies in the United States for the treatment of chronic dry-eye disease (available at www.restasis.com and www.dryeye.com). Furthermore, an over-the-counter (OTC) product that features an emulsion formula, Refresh Endura, is already launched in the U.S. market for eye-lubricating purposes in patients suffering from moderate to severe dry-eye syndrome. The effect of Restasis on contact lens comfort and reducing dry-eye symptoms in patients with contact lens intolerance was evaluated in comparison to rewetting drops (carboxymethylcellulose 0.5%, Refresh Contacts) [176]. Both formulations were applied twice per day before and after lens wear. Symptoms were assessed by lens wear time, use of rewetting drops during lens wear, subjective evaluation of dryness, and completion of the ocular surface disease index questionnaire. The results of this pilot study indicate that cyclosporin 0.05% is beneficial for contact lens wearers with dry eye and reduces contact lens intolerance [176]. Furthermore, Sall et al. [177] have recently evaluated the efficacy of marketed artificial tears (Systane and Restasis) in relieving the signs and symptoms of dry eye when used as supportive therapy to a cyclosporin-based ophthalmic emulsion (i.e., Restasis + Systane vs. Restasis + Refresh). Significant differences were seen in favor of Restasis + Systane versus Restasis + Refresh for less ocular burning, stinging, grittiness, and dryness. Systane was better than Restasis + Refresh for less burning, dryness, and scratchiness. Results indicate that the choice of concomitant therapy used with Restasis has significant effects on outcome measures and both supportive therapies were compatible with Restasis [177]. When compared to either saline or anionic emulsions, the nanosized cationic emulsions were shown to enhance the ocular bioavailability of indomethacin [108], piroxicam [178], and cyclosporin A [106, 179] following one single-drop dose instillation into the rabbit eye (Figure 4). A significant drug reservoir effect was noted in the cornea and conjunctiva even for more than 8 h following the instillation [106].

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OIL-IN-WATER NANOSIZED EMULSIONS 2800

Cornea positive emulsion Cornea negative emulsion

2400

Conjunctiva positive emulsion CsA concentration, ng/g

Conjunctiva negative emulsion 2000

1600

1200

800

400

0 15

30

60

120 Time (min)

180

480

FIGURE 4 Influence of emulsion surface charges cyclosporin A (CsA) concentrations in ocular surface tissues (cornea and conjunctiva) following one single-dose (50-μL) instillation of positively (cationic) and negatively (anionic) charged CsA-loaded nanosized emulsions into albino rabbit eye.

This long residence time may help reduce evaporation of the limited volume of natural tears present in patients with dry eye. This was probably due to the adhesion of the positively charged oil droplets to the negatively charged corneal surface moieties as a result of electrostatic attraction. This hypothesis was supported by data from an ex vivo study which showed that cationic emulsion exhibited better wettability properties on albino rabbit eye cornea than either saline or anionic emulsion [108]. Associated with Poloxamer and phospholipids, a cationic primary amine, stearylamine, has been used to obtain the above-described third-generation cationic emulsions. Additionally, a cationic emulsion based on an association of Poloxamer 188 and chitosan was prepared and also showed interesting physicochemical properties on stability and charge effects [97, 98]. Moreover, the stability and ocular tolerance following topical instillation into the eye of these cationic emulsion vehicles were investigated [98, 114]. The overall studies hence stress the effectiveness of nanosized cationic emulsion, which promotes ocular drug absorption via internalization possibly through an endocytic process [112]. 7.4.5.3

Nasal Route

The nasal route is still receiving great attention due to a number of advantages over parenteral and oral administration [180], particularly when first-pass metabolism makes the drug ineffective. The approach of an o/w emulsion formulation of the drug may increase absorption by solubilizing the drug in the inner phase of the emulsion and prolonging contact time between emulsion droplets and nasal mucosa. One of the first examples for systemic delivery of peptides concerned a lipidsoluble rennin inhibitor [181]. The peptide was solubilized in the oil phase of an o/w emulsion containing membrane adjuvants such as oleic acid and mono- and diglyc-


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erides. Emulsion formulations have been proposed to simultaneously increase the solubility of the peptide and to enhance membrane permeability through interaction between the membrane and the oil components. Enhanced and prolonged in vivo nasal absorption of the rennin inhibitor was observed in emulsion formulation compared to aqueous suspension. From morphological studies, the emulsions did not provoke any significant changes to the nasal mucosa [181]. Such a formulation approach was also used for the administration of a steroidal male sex hormone testosterone [182]. The steroid was solubilized in the oil phase of the o/w emulsion and the ionic composition of the aqueous phase was modified in order to produce electrically positive, negative, and neutral droplets. Droplets with a surface charge led to better bioavailability than neutral droplets, but contrary to the above-described topical applicabilities of cationic emulsions over anionic emulsions, positively charged droplets did not provide the best results [182]. However, the emulsion approach was advantageous since it helped to overcome the solubility problem of the hydrophobic compounds. In another study which does not involve peptide drugs, various emulsion formulations were prepared in order to modulate the partitioning of the drug between the aqueous phase and the oil phase [183]. The disappearance of a drug from the nasal cavity was determined by an in situ perfusion technique. When the drug was solubilized in the aqueous phase, the formulation did not have a significant effect on the drug disappearance rate. However, partitioning of the drug in the oil phase resulted in delaying absorption. It was suggested that oil droplets containing medium-chain triglycerides formed a pseudo–oily layer on the mucous membrane, which slowed down the drug disappearance from the nasal cavity [183]. Another interesting study reported nasal delivery of insulin formulated in both o/w and w/o emulsions [184]. As insulin partitions into the aqueous phase of the emulsion, the peptide is either incorporated within the continuous phase of the o/w emulsion or encapsulated in the aqueous droplets of the w/o emulsion. Following nasal perfusion experiments, plasma insulin concentration profiles showed enhanced insulin absorption when the peptide was formulated as an o/w emulsion compared to an aqueous solution. However, a w/o emulsion did not cause any significant increase in plasma insulin concentration. Delivery of insulin by administration of nasal drops also revealed a large dose-dependent increase in plasma insulin concentration. It also needs to be pointed out that the emulsifier mixture alone did not promote any absorption. It was suggested that insulin molecules probably were adsorbed at the surface of the oil droplets. Adhesion of the oil droplets on the mucosal membrane then induced a local increase in insulin concentration at the membrane surface. However, the number of droplets in contact with the surface had to be small enough. Otherwise, a stagnant oil layer is formed which acts as an additional barrier to the transport, as was observed with the w/o emulsion [183]. Other recent applications of emulsion formulation involve mucosal gene and vaccine delivery [185–187] and the preparation of polymeric microspheres by the w/o emulsification solvent extraction technique [188]. 7.4.5.4 Topical Route Many drugs exhibit low skin penetration, which results in poor efficacy. As opposed to common chemical skin penetration enhancers, organic solvents, which are

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generally associated to some degree with skin irritation, toxicity and sensitization, a solvent-free topical vehicle based on drug entrapment in o/w emulsion droplets of submicrometer size is more efficacious in terms of percutaneous absorption and possibly devoid of adverse effects. In addition, the uniqueness of the large internal hydrophobic oil core of o/w nanosized emulsion droplets allows high solubilization capacity for water-insoluble topically active medicaments and also aids in carrying water, an excellent softener, to the skin. The concept of using anionic nanosized emulsion vehicles for enhanced percutaneous absorption of nonsteroidal anti-inflammatory drugs (NSAIDs) and diazepam was clearly proven [189, 190]. NSAIDs and diazepam in a nanosized emulsion vehicle also demonstrated noticeable systemic activity. The o/w emulsion was tested for primary irritation in humans in a 48-h trial. Low irritancy and excellent human acceptance were observed, subsequently making the further development of a nanosized emulsion vehicle very attractive. Even though emulsion vehicles increase dermal drug delivery of lipophilic drugs in humans, one of the problems for topical drug delivery is the difficulty of applying these vehicles to the skin because of their fluidity. Rheological properties are studied in transdermal formulations and different results are given. Realdaon and Ragazzi [191] have investigated different mechanical emulsifying conditions on o/w emulsion formulations containing methyl nicotinate. The influence of these procedures on rheological properties and in vivo availability of methyl nicotinate was evaluated. Even if various viscosities were obtained, differences between batches did not compromise drug availability. On the contrary, Welin-Berger and co-workers [192, 193] concluded in their study on nanosized emulsions containing model compounds that both release and permeation rates decrease when the apparent yield stress (i.e., the macroviscosity) increases by addition of gelling polymers. Because a topical anesthetic agent will induce a pain-suppressing anesthesia, the eutectic mixture of local anesthetics (EMLA) has proven to be a useful medication for children. It is an emulsion containing a mixture of lidocaine and prilocaine. This cream gives an effective deep sedation and can be applied half an hour to 1 h prior to the procedure. Local side effects with this emulsion are very mild [194, 195]. Systemic activity of nanosized emulsions containing diazepam was compared with regular creams or ointments by Schwarz et al. [190]. Their efficiency was tested on protection against pentamethylenetetrazole, which induces convulsive effects in mice. Diazepam applied topically in emulsion creams was strongly dependent on oil droplet size. Furthermore, nanosized emulsions increased transdermal drug delivery and prolonged protective activity for up to 6 h. Many formulations for topical emulsions are available in the scientific literature, in patents, and on the market. Progresses made in the last years in this field are concentrated on the various aspects of drug release and the influence of droplet size. Third-generation cationic emulsions were suggested as drug carriers for topical use in the skin. It was found that α-tocopherol-loaded cationic emulsion was able to prevent oxidative damage of cultured fibroblast cells [196]. In addition, the same cationic formulation was able to protect rat skin against oxidative stress induced by ultraviolet (UV) irradiation significantly better than either the corresponding anionic emulsion or the cationic blank emulsion, as measured with a noninvasive

FUTURE PERSPECTIVE

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evaluation of the lipid hydroperoxidation process of the rat skin. However, no difference was found between cationic or anionic nanosized emulsions of α-tocopherol as assessed by endogenous peroxyl radical scavenging ability. Taken together, these results suggest that the cationic emulsion allows the α-tocopherol to remain on the surface of the skin because of electrostatic interactions between the negatively charged sites of the superficial layers of the skin and the positively charged oil droplets. Although the extent of α-tocopherol incorporation into the skin is similar for both cationic and anionic emulsions, the prolonged skin surface residence time of the cationic emulsion allows an enhanced protective effect against oxidative stress. In contrast to these results, an in vitro percutaneous absorption study on hairless rat skin found that the antifungal drugs econazole and miconazole nitrate incorporated into a similar cationic emulsion formulation were more effective in terms of skin penetration than the corresponding anionic emulsion [197]. The enhanced rate of diffusion of these antifungal drugs through the skin by the cationic emulsion suggests a new approach for dermal drug penetration enhancement [197].

7.4.6

FUTURE PERSPECTIVE

Based on the performances in previous and present decades, o/w-type nanosized emulsions can conveniently be classified into three generations. First-generation emulsions are considered primarily as nutrient carriers to be administered via intravenous routes to bed-ridden patients. Second-generation emulsions start initially as drug carrier systems by solubilizing considerable amounts of lipophilic drugs at the oil phase or at the oil–water interface of the emulsion. This particular merit of emulsions is specifically exploited even commercially for both ocular and parenteral active drugs. Modifications made either in the oil phase or at the o/w interfacial film forming emulsifier molecules allow the emulsions to be able to escape from lipolysis by lipoprotein lipase, apo adsorption, and liver uptake. Such a surface-modified emulsion would prolong the circulation time in plasma and thereby an alteration in in vivo disposition of incorporated drugs following parenteral administration. Attachment of homing devices such as antibody and apoE to the surfaces of emulsions makes the selective/active delivering of emulsionincorporated drugs to target sites such as a tumorized organ or hepatic system. Active targeting increases the affinity of the carrier system for the target site, while passive targeting minimizes the nonspecific interaction with nontargeted sites by the RES. Having together a positive charge and a steric stabilizing effect led to the development of third-generation emulsions that contain a unique property: plasma half-life prolongation and electrostatic adhesion to ocular surface tissues after topical instillation into eye. Furthermore, the third-generation emulsion shows a potential of carrying a wide range of lipophilic, amphiphilic, and polyanionic compounds, including DNA and oligonucleotides for transdermal and nasal routes. Accumulating knowledge thus suggests that constant progress in better understanding the principles and processes governing the various issues related to o/w nanosized emulsions has surely brought major improvements in the efficacy of parenteral or nonparenteral drug delivery systems.

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INDEX

Abortifacients, 850 Acyclovir, 1037–1042 Adjuvants, 635–637 ADMET, 8–9 AERx, 709–710 AIDS, 4 Alginate nanoparticles, 540–541 Alginic acid, 295 Anticancer drug delivery, 485–506 Antimicrobials, 845–846 API, 5 Artificial neural networks, 1016 Aseptic compounding, 107–108 Auxiliary excipients, 894–895 Avonex, 47 Aztirelin, 620 Bentonite, 295 Benzodiazepines, 623–626 Betaseron, 47 Bioadhesion, 305–306 Bioburden considerations, 26 Bioconversion, 566, 572–574 Biodegradable microspheres, 419–426 Biodegradable polymeric nanoparticles, 536–543 Biodrug, 565–566 Biogenerics, 35

Biological half-life, 356 Biopharmaceuticals Classification System (BCS), 237–238, 961 Boron Neutron Capture Therapy (BNCT), 489 Breast cancer, 492–497 Breath actuation, 698–699 CaCo-2, 960 Caclyx, 497 Calcitonin, 613–617 Cancer therapy, 1238–1240 Capillary Aerosol Generator (CAG), 710–711 Carbomer, 295–296 Carbxymethyl cellulose, 655 Carnauba wax, 274 Carr index, 908 Carrageenan, 296, 833 Challenges in ocular drug delivery, 730–737 Characteristics of radiopharmaceuticals, 60–61 Chemical penetration enhancers, 803 Chemically induced release, 384–385 Chitosan nanoparticles, 541 Chitosan, 608, 636, 655, 657, 658, 661–662, 665–666, 833


1367

1368

INDEX

Cholera toxin, 637 Ciliotoxicity, 668 Classification of hygroscopicity, 912 Climatization, 1085–1086 CMV, 481 Coated tablets, 244–245 Colloidal silicon dioxide, 296 Colorants, 243 Compactibility, 1138–1141 Compendial gels, 307 Compendial ointments, 289–291 Compressibility, 917–918 Container closure systems, 17–18 Controlled-release delivery systems, 11 Cryogenic spray drying, 401–402 Cytotoxicity, 1240 Defi nitions of density terms, 1178 Dendrimers, 1272 Depyrogenation, 117–120 Diluents, 240–241 Diphtheria Toxoid (DT), 420–421 DirectHaler, 602–604 Disintegration, 920–922 DPI, 689 Drug product stability, 21–25 Drug-excipient compatibility, 969–970 Dry Powder Inhalers (DPIs), 684, 700–706 Effective half life, 61 Effervescent tablets, 251–252 Electroresponsive release, 381–383 Ethylcellulose, 297 Excipients, 1344, 19–21, 239–244, 243–244, 410–412, 412–419, 695, 822–823, 883– 896, 884–885, 897 Exubera, 704, 705–706 Fair Packaging and Labeling Act, 190–195 FDA-approved transdermal patches, 794 First-uterine-pass effect, 821 Fluidized-bed coating, 1102–1103 FluMist, 592 Formulation approaches to improve ocular bioavailability, 737–753 Formulation assessment, 7–8 Formulation development, 15–16, 238 Fortical, 52–53, 55 Friability, 928 Fullerenes, 1272, 1296–1297

Gas and vapor sterilization, 119 Gastrointestinal tract and absorption, 356–357 Gelatin capsules, 245–251 Gelatin, 539 Gelling agents, 293–301 GELS, 288–310 Giladin, 540 Glucose-responsive insulin release device, 384–385 Glycerol behenate, 298–299 Group B streptococcus vaccine, 420 Guar gum, 297 Hammer Mill, 1169 Hausner ratio, 908 Herpes simplex virus, 481 HFA reformulation, 690–692 High-throughput Screening (HTS), 934 Human Growth Hormone (HGH), 34 Hyaluronic acid, 499, 655,657, 833 Hydralazine, 627 Hydrogels, 291–292 Hydrophilic matrix tablets, 1210–1211 Hydroxyethyl Cellulose (HEC), 297–298 Hydroxypropylmethyl Cellulose (HPMC), 298 Immunity after intranasal immunization, 634–635 Immunogenicity, 50, 53–54 Inhalation drug products, 179 Injectable microspheres, 407–408 Insulin, 424–426 Ionophoresis, 804 Japanese Encephalitis Virus (JEV), 423–424 Kurve Technology, 601 Labor inducers, 850 Lanolin, 271 Lipinski Rule of Five, 934 Liposomal drugs approved for clinical application, 1263 Liposome-based products currently under clinical testing, 484 Liposomes, 365–367, 636, 747–748 Liquid dosage forms, 338 Low-molecular-weight heparins, 617–620 Lozenges, 252–253

INDEX

Lung cancer, 497–502 Lung toxicity, 1309–1310 Lyophilization, 127–128 Magnetically induced release, 383–384 Marked medical and nonmedical emulsions, 1332 MDI, 689 Mechanisms of protein and peptide degradation, 22–23 Metal as packaging material, 170–171 Metered-dose Inhalers (MDIs), 684, 690–700 Microbicides, 843–845 Microbiological quality, 334–335 Microcrystalline cellulose (MCC), 653, 655 Microemulsions, 1267, 748–750 Microencapsulation, 358 Microneedles, 803–804 Milestones in early biologics regulation, 38 MLVs (multilamellar vesicles), 444 Mononuclear phagocyte system, 1333 Mucoadhesion, 840 Mucoadhesive microspheres, 657 Mucoadhesive polymers, 744 Mucosal toxicity screening method using the slug arion lusitanicus, 667 Mucosal-associated lymphoid tissue (MALT), 635 Musciliary clearance, 596 Nanocapsules, 363 Nanocarriers, 1258–1273 Nanoemulsions, 1269 Nanogels, 1271 Nanomaterials in pharmacy, 1253–1254 Nanomaterials, 1250–1252 Nanomedicine technologies taxonomy, 1292–1293 Nanomedicine, 1278–1279 Nanoparticles, 1231–1236, 1264–1269, 536, 746–747 Nasal delivery, 481–482 Nasal delivery of nonpeptide molecules, 622–630 Nasal delivery of vaccines, 633–637 Nasal dry powder formulations, 652–655 Nasal route, 1352–1353 Nasal vaccination delivery systems, 636–637 Nasal vasculature, 594–595 Nebulizers, 706–707

1369

Niosomes, 367, 748 Nitroglycerin, 627–628 Noncovalent binding of ligands, 465–466 Nose-associated Lymphoid Tissue (NALT), 635 Nose-to-Brain Delivery, 632 Ocular delivery, 477–481 Ocular drug delivery, 738–741, 784–785 Ocular routes, 1347–1352 Official creams, 282 Official gels, 304 Oil-in-water nanosized emulsions, 1329–1341 Ointments and creams, 269–270 Omnitrope, 51, 53–56 OptiNose, 601–602 Oral drug delivery, 781–782 Oral ER formulations, 1193–1195 Orally disintegrating tablets, 259–262 Organogels, 292 Ovarian cancer, 502–506 Pan coating, 1102 Parenteral drug delivery, 783–784 Parenteral routes, 1346–1347 Partition coefficient, 352, 956–957 PEGylated liposomes, 469–472 Percolation theory, 1013–1016, 1030–1042 Permeability enhancement methods, 964 Preservatives, 20–21 PET radiopharmaceuticals, 83 Petrolatum, 272 Photostability, 23 pH-sensitive polymeric nanoparticles, 547 Physiochemical properties of liposomes, 449–456 Plastic additives, 164 Plastic as packaging material, 166–170 Poloxamer, 299 Poly (lactic acid), 543–544 Polyethylene oxide, 299–300 Polymorphism, 936–942 Polysaccharides, 539–540 Polyvinyl Alcohol (PVA), 300 Povidone, 300 Preformulation approaches for tablet production, 883 Principles for extended drug release, 1196–1197 Principles of radiation protection, 63–64 Production of radionuclides, 75–76

1370

INDEX

Production of radiopharmaceuticals, 78–88 Propylene Glycol Alginate (PGA), 300

Synthetic cervical mucus, 816 Systemic uptake of nanoparticles, 1310–1311

Quantum dots, 1293–1295 Radiation sterilization, 119 Radioactive decay, 61–63 Radiochemical purity, 90–91 Radionuclides, 65 Reaction calorimetry, 141–142 Respmat, 708–709 Route of administration, 8–10 Salmon calcitonin, 52 Salt selection, 952–956 Scanning tunneling microscopy, 1306–1308 Selected drugs administered in vagina, 853 Selection guideline of pharmaceutical excipients, 895–896 Selection of microemulsion ingredients, 773 Sodium alginate, 300–301, 538, 655 Soft Mist Aerosols, 707–708 Solubility characteristics, 950–965 Sonophoresis, 804 Spermicides, 849–850 Stability, 336–337 Stability-indicating methodologies, 14–15 Stability of liposomes, 455–456 Sterile products, 169–170 Sterilization, 117–120 Sterilization by fi ltration, 119–120 Sterilization of radiopharmaceuticals, 73–74 Surface hydrophilicity, 550 Synthesis of PET radiopharmaceuticals, 86

Tablet coating methods, 1102–1103 Tablet tooling terminology, 1147 Tableting machines, 1058–1067 Tableting process, 1055–1056 Tetanus toxoid, 421–423 Thermoresponsive drug release dosage forms, 379–381 Thermosensitive polymeric nanoparticles, 546–547 TNO gastrointestinal tract model, 569–571 Topical route, 1353–1355 Toxicological effects of dry powder formulation, 666–667 Tragacanth, 301 Transdermal drug delivery, 368, 782–783 Ultrasonic atomization, 403 Ultrasound-assisted tableting, 1043–1045 United States Pharmacopoeia Center for the Advancement of Patient Safety, 195 U.S. Pharmacopoeia, 177 USP, 281–282, 304, 903 Vaccines, 420–424, 851–852 Vaginal and uterine controlled-release dosage forms, 371 Vaginal fi lms, 831 Vaginal fluid stimulant, 816 Vaginal foams, 831 Vaginal rings, 826–830 Vaginal sponges, 832 Vibrio cholerae vaccine, 423

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