Bisphosphonates in Bone Disease: From the Laboratory to the Patient

Bisphosphonates in Bone Disease

To My wife, Maria Pia My children, Marie-Gabrielle, Isabelle DSsirde, and Marie-Laure My father, Alfred Fleisch, who taught me scientific thinking and experimental rigor William F. Neuman, who introduced me to the bone

Bisphosphonates in Bone Disease From the Laboratory to the Patient

Herbert Fleisch Professor Emeritus University of Berne Berne, Switzerland

ACADEMIC PRESS A Harcourt Science and Technology Company

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NOTICE The information contained in this book has been compiled from the available literature. Although every effort has been made to report faithfully, the author and publisher cannot be held responsible for its correctness. The book is not intended to be and should not be construed as medical advice. For any use the package inserts of the various drugs should be consulted. The author and publisher disclaim any liability arising directly or indirectly from the use of the compounds, drugs, techniques or procedures described in this book.

This b o o k is printed on acid-free paper. |

Copyright 9 2000, 1997, 1995, 1993 by Herbert Fleisch All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Inc., 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 A c a d e m i c Press A Harcourt Science and Technology Company 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.academicpress.com A c a d e m i c Press Harcourt Place, 32 Jamestown Road, London NW1 7BY, UK http://www.hbuk.co.uk/ap/ Library of Congress Catalog Card Number: 00-100337 International Standard Book Number: 0-12-260371-0 (casebound) International Standard Book Number: 0-12-260370-2 (paperback) PRINTED IN THE UNITED STATES OF AMERICA 00 01 02 03 04 05 CO 9 8 7 6 5 4

3

2

1

Contents

Note: The arrows (~) that appear in the margins indicate the text to which the reader is referred by the captions, also in the margins. Preface

.....................................................

xi

1

lo

Bone and mineral

1.1. 1.1.1. 1.1.2.

Bone physiology ......................................... Morphology ............................................ C o m p o s i t i o n of b o n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mineral ............................................. Organic matrix ....................................... B o n e cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Osteoblasts ........................................ L i n i n g cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Osteocytes ........................................ Osteoclasts ........................................ O t h e r cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modeling and remodeling ................................ Calcium homeostasis .................................... B o n e as a n o r g a n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A s s e s s m e n t of b o n e t u r n o v e r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R e c o m m e n d e d selected r e a d i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 3 3 4 5 5 7 7 8 12 12 16 19 21 21

Bisphosphonates

27

1.1.3. 1.1.4. 1.1.5. 1.1.6.

o

2.1.

2.2.

metabolism

mpreclinical

.........................

.......................

1

B a c k g r o u n d to t h e p h a r m a c o l o g i c a l d e v e l o p m e n t . . . . . . . . . . . . . .

27

R e c o m m e n d e d selected reading . . . . . . . . . . . . . . . . . . . . . . . . . . . .

29

Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

30 33

R e c o m m e n d e d selected r e a d i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.

Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

34

2.3.1.

P h y s i c o c h e m i c a l effects

34

..................................

Contents

2.3.2.

Biological effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I n h i b i t i o n of b o n e r e s o r p t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . In vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.4. 2.4.1. 2.4.2. 2.4.3. 2.4.4.

2.5. 2.5.1. 2.5.2.

0

Intact animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A n i m a l s w i t h e x p e r i m e n t a l l y increased b o n e resorption ..................................... Relative activity of b i s p h o s p h o n a t e s . . . . . . . . . . . . . . . . . . . . M e c h a n i s m s of a c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D i r e c t effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I n d i r e c t effect t h r o u g h o t h e r cells . . . . . . . . . . . . . . . . . . . I n h i b i t i o n of m i n e r a l i z a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ectopic m i n e r a l i z a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Normal mineralization .............................. M e c h a n i s m s of a c t i o n in the inhibition of calcification . . . . . . O t h e r effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R e c o m m e n d e d selected r e a d i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . .

39 40 41 43 45 48 48 49 50 50 51

Pharmacokinetics ....................................... Intestinal a b s o r p t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distribution ........................................... Renal clearance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . O t h e r m o d e s of a p p l i c a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R e c o m m e n d e d selected r e a d i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . .

56 56 57 59 60 60

Animal toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acute toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N o n a c u t e toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alendronate ........................................ Clodronate ......................................... Etidronate .......................................... Pamidronate ........................................ Other bisphosphonates ................................ R e c o m m e n d e d selected r e a d i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . .

63 63 63 64 64 65 65 65 66

Bisphosphonates--

67

3.1.

Introduction

3.2.

Paget's disease

3.2.1. 3.2.2. 3.2.3. 3.2.4.

3.2.5. 3.2.6.

34 34 34 36

clinical ..........................

...........................................

......................................... Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology .......................................... Pathophysiology ........................................ Clinical m a n i f e s t a t i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signs a n d s y m p t o m s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Laboratory ......................................... Dia g n o s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F o l l o w - u p of p r o g r e s s i o n of the disease . . . . . . . . . . . . . . . . . . . . T r e a t m e n t w i t h drugs o t h e r t h a n b i s p h o s p h o n a t e s . . . . . . . . . . . . . . Treatment with bisphosphonates ........................... Preclinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vi

67 68 68 68 68 69 69 70 71 71 72 72 72

Contents Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

73

Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment regimens ................................

73 76

Alendronate ....................................

78

Clodronate

....................................

79

Etidronate ..................................... Olpadronate ................................... Pamidronate ................................... Risedronate .................................... Tiludronate ....................................

79 80 80 81 82

Other bisphosphonates

83

...........................

Conclusion ....................................... R e c o m m e n d e d selected r e a d i n g . . . . . . . . . . . . . . . . . . . . . . . . . .

83 84

3.3.

O s t e o l y t i c t u m o r - i n d u c e d b o n e disease . . . . . . . . . . . . . . . . . . . . . . .

88

3.3.1.

Definition .............................................

88

3.3.2.

Pathophysiology ........................................ Local bone destruction ................................ Generalized bone destruction ........................... M e c h a n i s m s of h y p e r c a l c e m i a . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical manifestations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signs a n d s y m p t o m s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

88 88 90 90 92 92

3.3.3.

3.3.4. 3.3.5.

3.4. 3.4.1. 3.4.2. 3.4.3. 3.4.4. 3.4.5.

Laboratory ......................................... F o l l o w - u p of p r o g r e s s i o n of t h e disease . . . . . . . . . . . . . . . . . . . . Treatment with drugs other than bisphosphonates .............. Treatment with bisphosphonates ........................... Preclinical s t u d i e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypercalcemia .................................. Urinary parameters .............................. O t h e r effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment regimens ............................... Clodronate ................................... Etidronate .................................... Ibandronate ................................... Pamidronate .................................. Other bisphosphonates .......................... C o m p a r i s o n of t h e v a r i o u s b i s p h o s p h o n a t e s . . . . . . . . . . . .

93 94 94 95 95 98 98 98 99 101 104 104 105

Conclusion ........................................ R e c o m m e n d e d selected r e a d i n g . . . . . . . . . . . . . . . . . . . . . . . . . . .

110 111

Non-tumor-induced hypercalcemia ........................ Definition ............................................

118

Pathophysiology .......................................

118

Clinical manifestations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

118 119 119 119

Treatment with drugs other than bisphosphonates ............. Treatment with bisphosphonates .......................... Preclinical s t u d i e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

106 106 108 109

118

Contents

3~ 3.5.1. 3.5.2. 3.5.3. 3.5.4.

Clinical s t u d i e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

119

Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment regimens ............................... Conclusion ........................................ R e c o m m e n d e d selected r e a d i n g . . . . . . . . . . . . . . . . . . . . . . . . .

120 120 121 121

Osteoporosis ......................................... Definition ............................................ Epidemiology ......................................... Pathophysiology ....................................... Clinical m a n i f e s t a t i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signs a n d s y m p t o m s Laboratory Diagnosis

.................................

........................................ .........................................

123 123 123 124 128 128 128 129

F o l l o w - u p of p r o g r e s s i o n of the disease . . . . . . . . . . . . . . . . . . . Treatment with drugs other than bisphosphonates ............. Treatment with bisphosphonates .......................... Preclinical s t u d i e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical s t u d i e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect on b o n e m i n e r a l d e n s i t y . . . . . . . . . . . . . . . . . . . . . Effect o n b o n e t u r n o v e r . . . . . . . . . . . . . . . . . . . . . . . . . . Effect o n f r a c t u r e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects a f t e r d i s c o n t i n u a t i o n of the d r u g . . . . . . . . . . . . . . V a r i o u s o t h e r effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment regimens ............................... Alendronate ................................... Clodronate ................................... Etidronate .................................... Ibandronate ................................... Pamidronate .................................. Risedronate ................................... Tiludronate ................................... Conclusion ...................................... R e c o m m e n d e d selected r e a d i n g . . . . . . . . . . . . . . . . . . . . . . . . .

129 130 133

3.6.2. 3.6.3.

H e t e r o t o p i c calcification and ossification . . . . . . . . . . . . . . . . . . . . Definition ........................................... Pathophysiology ....................................... Clinical m a n i f e s t a t i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

160 160 160 160

3.6.4.

Treatment with drugs other than bisphosphonates .............

161

3.6.5.

Treatment with bisphosphonates

161 161 161

3.5.5. 3.5.6.

3.6. 3.6.1.

..........................

Preclinical s t u d i e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical s t u d i e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H e t e r o t o p i c calcification . . . . . . . . . . . . . . . . . . . . . . . . . . . Soft tissue c a l c i f i c a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . Urolithiasis ................................... Dental calculus

................................

viii

133 136 136 137 143 144 148 148 149 149 150 150

151 151 151 152 152 152

161 161 162 162

Contents

3.7.

H e t e r o t o p i c ossification . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fibrodysplasia (previously myositis) ossificans p r o g r e s s i v a . . . . . . . . . . . . . . . . . . . . . . . . . . O t h e r h e t e r o t o p i c ossifications . . . . . . . . . . . . . . . . . . . . Conclusion ........................................ R e c o m m e n d e d selected reading . . . . . . . . . . . . . . . . . . . . . . . . . . .

162

Other diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

166 166

Diseases with enhanced resorption Osteogenesis imperfecta

......................

..............................

162 163 164 164

166

O t h e r diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R e c o m m e n d e d selected reading . . . . . . . . . . . . . . . . . . . . . . . . . . .

166 167

3.8.

Adverse events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.8.1.

B i s p h o s p h o n a t e s in g e n e r a l

168 168

3.8.2.

Individual bisphosphonates .............................. Alendronate ....................................... Clodronate ........................................ Etidronate ......................................... Pamidronate .......................................

..............................

169 169 170

171 173

Risedronate ........................................ Tiludronate ........................................ Other bisphosphonates ............................... R e c o m m e n d e d selected reading . . . . . . . . . . . . . . . . . . . . . . . . . . .

174 175 175 175

3.9.

Contraindications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R e c o m m e n d e d selected reading . . . . . . . . . . . . . . . . . . . . . . . . . . .

178 180

3.10.

Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

181

0

Index

Commercially

available bisphosphonates

....................................................

ix

..............

182 207

This Page Intentionally Left Blank

Preface

The bisphosphonates are a new class of drug that has been developed in the past three decades for use in various diseases of bone and calcium metabolism. Seven bisphosphonates are commercially available today. Those available, as well as the indications for which they are registered, vary from country to country. A substantial number are in preclinical or clinical development, so in the near future, specialists and practitioners will have the opportunity to choose the most suitable drug and the best regimen to treat an individual patient. Information for the doctor is available today in original articles, reviews, and documentation distributed by the companies selling the various compounds. No complete, easy-to-read publication is available where the practicing doctor can quickly find the necessary information on all bisphosphonates. This book has been written to cover this deficit. It starts with a chapter giving a small aper~u of the physiology of bone. In the preclinical section, it then covers the chemistry, mechanisms of action, and animal toxicology of these compounds. Before addressing the use of the bisphosphonates, which is the main aim of this book, the clinical section discusses the diseases treated by these compounds with respect to their pathophysiology, clinical picture, and treatment with other drugs. After a chapter on adverse events, the book ends with a table containing the trade names of the commercially available bisphosphonates, the registered indications, and the available forms for each country. In order to keep this book concise, it was necessary to simplify many of the issues and therefore to make choices. It is hoped that the result nevertheless faithfully represents the state of the art. Literature had to be kept very restricted, the reader being referred when possible to ones, for further information, the references of original articles being limited to the earliest ones, for historical background, and to the most current articles on the subject.

xi

Preface I express here my gratitude to the late Professor W. F. Neuman, Radiation Biology Department, Rochester, New York, USA, where the seeds of this work were planted; to the late Professor A. Fleisch; to Professor M. Allg6wer; to Professor M. E. Miiller; and to the Canton of Berne, who gave me the opportunity to pursue and develop this research in the Department of Physiology of the University of Lausanne, the Laboratory for Experimental Surgery, Davos, and the Department of Pathophysiology of the University of Berne. I also thank Dr. M. D. Francis, with whose collaboration the bisphosphonates were born; my collaborators over these many years, who have allowed an idea to become reality; my colleagues S. Adami, J. P. Bilezikian, J.-J. Body, P. D. Delmas, T. A. Einhorn, J. L. Ferretti, J. A. Kanis, T. J. Martin, P. J. Meunier, G. R. Mundy, S. E. Papapoulos, L. G. Raisz, R. Rizzoli, G. A. Rodan, R. Rubens, R. G. G. Russell, R. K. Schenk, E. Seeman, F. R. Singer, and E. S. Siris, who have read and improved various parts of this work. The first edition appeared in 1993. Since then second and third updated English versions, as well as Italian, Japanese, Spanish, and German versions, have been published. In view of the very rapid development in this field, it seemed appropriate to prepare an updated fourth English edition.

Herbert Fleisch Av. Ddsertes 5 CH-1009 Pully, Switzerland

xii

1.

1.1. 1.1.1.

Bone and mineral metabolism

BONE

PHYSIOLOGY

Morphology

Macroscopically, bone can be divided into an outer part called cortical or compact bone, which makes about 80% of the total skeleton, and an inner part named cancellous, trabecular, or spongy bone. This structure, an outer cortical sheath and an inner three-dimensional trabecular network, allows optimal mechanical function under customary loads.

Biomechani, adaptation pp. 19-20

Bone is a superb engineering construction with an outer compact sheath and an inner trabecular scaffold allowing optimal mechanical properties. -+

Microscopically, woven and lamellar bone can be distinguished. Woven bone is the type formed initially in the embryo and during growth, and is characterized by an irregular array of loosely packed collagen fibrils. It is then replaced by lamellar bone, so that it is practically absent from the -+ adult skeleton, except under pathological conditions of rapid bone formation, such as occur in Paget's disease, fluorosis, or fracture healing. In contrast, lamellar bone is the form present in the adult, both in cortical and in cancellous bone. It is made of well-ordered parallel collagen fibers, organized in a lamellar pattern.

Histologically bone formed during growth is of the woven type; in the adult it is lamellar, except in certain diseases with rapid formation.

Paget's disea p. 71

1. Bone and mineral metabolism

Bone is made of basic units called bone structural units (BSUs). In cortical bone these are called osteons or Haversian systems, which represent its basic structural building blocks. These are hollow cylinders of a median length of 2 mm, but which can reach 8 mm, and 200 btm in diameter, made of concentric lamellae, between which the osteocytes are located. In the center is a canal containing the nutrient blood vessels. These anastomose with vessels from other osteons so that the various osteons are in communication with one another. The diameter of the osteon is always about 200 ~tm, regardless of species, the maximal distance of any part from the central vessel being no more than 100 btm, this being the largest transport distance for nutrients. Osteons are separated from one another by so-called cement lines.

Compact bone Interstitial

Vascular canal

I

~ ~'2~J ' "t ,,a'~ ' ~ ,

, ~ ; ~ " ~' ~i~' , X q g .~ -~" ." ,.f., ;- ol

Fig. 1.1-1 Cross section of compact bone showing osteons with osteocytes (left), and--in polarized lightmwith collagen lamellae (right). [From Schenk, R. K., et al. (1993). Reproduced from Royce, P. M., and Steinmann, B. (eds.) (1993 ). Connective Tissue and Its Heritable Disorders. Molecular Genetic, and Mineral Aspects, pp. 85-101, with copyright permission from the author and John Wiley & Sons, Inc.]

4b.

The osteon is the basic u n i t o f the Haversian bone o f the cortex.

Remodeling packets and BMUs p. 13

Osteoporosis p. 124

The trabeculae also consist of structural units, which in this location § are called packets. They are separated, as are the osteons of the cortex, by cement lines. When they are on the surface and not yet terminated, they are called bone multicellular units (BMUs). However, BMUs and packets are also found on the inner surface of the cortex, which therefore looks very much like trabecular bone. These two locations, trabeculae and inner cortex, are those that are affected predominantly by osteoporosis. Trabeculae generally possess no vessels and are therefore nourished from the surface. This explains why they cannot become much thicker than about 2 0 0 - 3 0 0 btm, twice the distance of 100 ~tm over which transport of nutrients is possible.

1.1. Bone physiology Fig. 1.1-2 Trabecular bone showing individual packets separated by cement lines. (Courtesy of Dr. R. K. Schenk.)

Trabecular bone ~

,~

,

~.~

Packets

Cement lines

1.1.2.

Composition of bone

Bone is made up of mineral, a fibrillar organic matrix, cells, and water. Fig. 1.1-3 of bone.

Composition

Composition of bone Mineral -65%

Matrix -35% Cells

Hydroxyapatite

Collagen -90% Other prQteins Lipids Osteoblasts Lining cells Osteocytes Osteoclasts

Water

Mineral Mineral accounts for about two-thirds of the total dry weight of bone. It is made of small crystals of about 2 0 0 - 4 0 0 A x 35-75 fii x 1 0 - 4 0 ~i in the shape of plates, located within and between the collagen fibrils. Chemically it is a calcium deficient apatite, containing, however, many other constituents, among others H P O 4 - , carbonate, citrate, magnesium, sodium, fluoride, and strontium. These are either incorporated into the crystal lattice, or adsorbed onto the crystal surface. For this reason, the more general term calcium phosphate will be used in this book for bone mineral. Some substances, such as tetracyclines, polyphosphates, and bisphosphonates, have a special affinity for calcium phosphate and hence for bone. They are deposited in preference on the mineral at sites of new bone formation, but can also be deposited at other sites such as resorption areas. This bone seeking property has been utilized in the case of tetracyclines

Deposition of bisphosphonates in bone p. 57

1. Bone and mineral metabolism

Paget's disease p. 70

in order to label newly formed bone, thus enabling the assessment of bone formation. Indeed, by administering tetracycline, a fluorescent molecule, twice or more at known time intervals, it is possible to measure in bone biopsies the distance between the two lines of deposition of the fluorochrome, thus enabling the quantification of the bone formed during the time interval. The binding of polyphosphates and bisphosphonates, when linked to 99mTc, is used in nuclear medicine to visualize hot spots of bone formation by scintigraphy. This technique is especially useful for detecting skeletal metastases and the bone lesions in Paget's disease. Lastly, the strong binding of bisphosphonates to bone mineral is fundamental to their pharmacological activity. The binding of these substances is usually reversible at sites where the bone surface is accessible to the extracellular fluid. However, it is irreversible at sites which become buried by new bone formation, until the bone with the bone seeker is destroyed again during modeling or remodeling.

The bone mineral is made essentially of impure calcium apatite. Foreign substances such as tetracyclines, polyphosphates, and bisphosphonates can also be incorporated with high affinity.

M D and bone turnover p. 136

The mineral crystals are deposited within and between the collagen crystals in a manner which gives the bone tissue its compressive strength and stiffness. The process of mineralization proceeds rapidly initially, to proceed subsequently over months and years with decreasing speed, a process called secondary mineralization. This property explains why old bone is more mineralized and has a higher mineral density when measured by DXA than a younger one, and why a decrease in bone turnover is accompanied by an increase in bone density. The mineralization process is under the modulation of both activators and inhibitors. Thus, the collagen fibrils themselves as well as other proteins can act as activators, while pyrophosphate and proteins such as matrix gla-protein can act as inhibitors.

Organic matrix

Measurement of bone turnover p. 21

The matrix amounts to about 35 % of the dry weight of bone. It consists of 90% collagen, which is thus by far the most abundant bone protein. Its complex three-dimensional structure, comparable to that of a rope, gives bone tissue its tensile strength. The remainder of the bone matrix is made up of various noncollagenous proteins, the role of which is not yet well understood. The most abundant are osteonectin, osteocalcin, previously called bone gla-protein (BGP), osteopontin, and bone sialoprotein. Because some of them are synthesized and deposited almost exclusively in bone, their urinary excretion and plasma or serum levels are used clinically to assess bone turnover.

1.1. Bone physiology The organic matrix also contains a large amount of various growth factors, especially transforming growth factor [3 (TGF[3) and insulin-like growth factor II (IGF II). These are thought to play a role after their release during bone resorption in the local modulation of bone formation during the turnover of the BSUs, and in the growth of tumor cells in bone metastases.

Effect on bone metastases p. 89

Bone matrix is made up of 90% collagen and about 10% ol:various noncollagenous proteins. It contains many growth factors which may play a role, when released, in bone turnover and in tumorinduced bone disease. Bone cells Osteoblasts The osteoblasts, which derive from mesenchymal progenitors, are the cells that synthesize the bone matrix. They form an epithelial-like structure at the surface of the bone and are connected by gap junctions containing connexins. These and the cell adhesion molecules of the cadherin superfamily are thought to play an essential role in the control of osteoblast formation and function. The osteoblasts secrete unidirectionally the osseous organic matrix which, in a second step, then calcifies extracellularly. As a consequence of the time lag between the formation of the matrix and its calcification, there is a layer of unmineralized matrix osteoid under the osteoblasts. This osteoid seam diminishes in width when the rate of bone matrix formation decreases, but it widens when mineralization is delayed. This widening is most prominent when there is an arrest in mineralization, such as in osteomalacia.

Bone formation Marrow Osteoblasts ,

~ o~';~t ..

Osteoid.....f..-'r ~ - ~ , ,

..

~.

~,

Calcified bone Fig. 1.1-4 Lamellar bone formation with osteoblasts and osteoid seam. (Courtesy of Dr. R. K. Schenk.)

Osteomalacia pp. 171-172

1. Bone and mineral metabolism

Remodeling and modeling p. 12

Fluoride in osteoporosis p. 132

Corticosteroidinduced osteoporosis p. 126

The modulation of bone formation is still poorly understood. Histologically it seems to occur at the level of the recruitment of new osteoblasts as well as through modification of the activity of the mature osteoblasts. Although many hormones and cytokines influence osteoblasts in vitro, among them the insulin-like growth factors (IGFs), transforming growth factor [3 (TGFf3), acidic and basic fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF), bone morphogenetic proteins (BMPs), and prostaglandins, their individual roles in vivo are not yet clear. Some of them are thought to mediate cell to cell messages that stimulate or inhibit bone formation and resorption in specific sites. They are the consequence to the strains produced in bone by mechanical use. This results not only in bone remodeling, but also in bone modeling, both during growth and in the adult. Very recently it was found that a peptide made by fat cells, called leptin, decreases bone formation through a yet unknown central hypothalamic pathway. This opens a fascinating new aspect in our understanding of the regulation of bone mass. One of the main aims of current research is to develop molecules that will increase bone formation. Up to now the only substances that are active in this direction, when given systemically, are fluoride, parathyroid hormone, and certain cytokines such as prostaglandins and IGF-1. Of these only fluoride has been used therapeutically to date in clinical practice, namely, in osteoporosis. However, the increase in bone mass produced by fluoride did not induce a decrease in fracture incidence. Parathyroid hormone is under investigation and looks very promising. When locally administered in the proximity of bone in animals, various growth factors such as TGFf3, basic FGF, PDGF, and BMPs induce bone formation at the site of injection. These, as well as others, such as the IGFs, could prove useful in the future for such indications as improving fracture healing, filling osseous defects, and possibly inducing ridge augmentation in periodontology. Whether systemic administration will become possible is unknown. Effects on other organs are likely to make this development difficult. However, it might be possible to modulate their local synthesis. Thus, HMG Co-A reductase inhibitors, compounds called statins which reduce serum cholesterol, increase bone formation in vitro and in vivo, apparently by increasing BMP-2 production. Recently it was reported that the use of statins may be associated with higher BMD and lower fracture risk in older women. It is also hoped that the discovery of transcription factors for osteoblast differentiation, such as Cbfal, will open new aspects in our search for stimulators of bone formation. In contrast, corticosteroids inhibit bone formation, possibly because they induce osteoblast apoptosis (programmed cell death), explaining why chronic administration of these compounds leads to osteoporosis both in animals and in humans.

1.1. Bone physiology Fig. 1.1-5 Possible physiological and pharmacological modulators of bone formation.

S~ste~e Fluoride PTH Prostaglandins Cytokines

BMPs TGF[3 IGFs

Corticosteroids

FGFs PDGF Prostaglandins

Bone is formed by the osteoblasts. Their modulation and therefore

the modulation of bone formation are still little understood. Lining cells When the osteoblasts are not in the process of forming bone, they are flat and are called resting osteoblasts or lining cells. Active and resting osteoblasts form a membrane at the surface of the bone tissue, which may be important in constituting some kind of blood-bone barrier able to assure a characteristic osseous milieu intSrieur.

Resting osteoblasts at the surface of the bone are called lining cells and constitute a blood-bone barrier. Osteocytes At a certain moment some of the osteoblasts stop synthesizing matrix and become embedded within bone. They are then called osteocytes. Despite the fact that the osteocytes are the most numerous cells in bone, their function is still poorly understood. They are located in lacunae and are interconnected by long cytoplasmic processes among themselves and with the osteoblasts and the lining cells. Gap junctions at the membrane contact sites make a functional syncytium, allowing bone to respond to stimuli over large areas. These cell processes are located within canaliculi, which contain, together with the lacunae, the so-called bone fluid. As the surface

Morphology of cell connections p. 2

1. Bone and mineral metabolism of these lacunae and canaliculi is very large, in humans about 1000 m 2, the bone fluid is in immediate contact with the mineral, with which it is in equilibrium. The osteocytes are thought to influence the composition of this bone fluid. Since the latter is also related to the extracellular fluid and therefore to blood, the osteocytes may play a role in the regulation of plasma minerals, especially calcium. Osteocytes are also well located for responding to mechanical strain and are thought today to play a key role in transducing mechanical loads into changes in bone formation and bone resorption. Like other cells osteocytes undergo apoptosis.

The role of the osteocytes is still little understood. They are probably involved in the homeostasis of bone fluid and consequently in the homeostasis or:plasma calcium, and in the adaptation of bone in response to mechanical influences. Osteoclasts

Remodeling p. 13

The fourth type of cell in bone is the osteoclast. It originates from a different lineage to that of the other bone cells, namely from the hemopoietic compartment, more precisely from the granulocyte-macrophage colonyforming unit (GM-CFU). Osteoclasts are usually large multinucleated, sometimes mononucleated cells that are situated either on the surface of the cortical or trabecular bone, often in depressions called Howship's lacunae, or within the cortical bone. They are located at the tip of the remodeling units, drilling the vascular canals in which the new osteons will be formed. The role of osteoclasts is to resorb bone. This is performed in a closed, sealed-off microenvironment located between the cell and the bone, delimited by a peripheral actin-rich ring of tight adherence between the cell membrane and bone matrix. This specialized adhering site of the cell, called the clear zone, involves cell membrane receptors, called integrins, which recognize specific peptide sequences in the matrix. Covering this microenvironment is another specialized part of the cell membrane, called the ruffled border, which secretes two types of products, both leading to bone destruction. The first, the H § ions, which dissolve the bone mineral, originate from HECO 3 as a result of the action of carbonic anhydrase and are secreted by means of a proton ATPase. The second category includes various proteolytic enzymes, such as cathepsins, especially cathepsin K, and possibly collagenases, such as matrix metalloproteinase 1, which digest the matrix. Current investigations are directed toward the development of specific inhibitors of these various processes, with the aim to develop drugs that will decrease bone destruction.

1.1. Bone physiology

Osteodast

Clear zone Nucleus Ruffled border Bone Fig. 1.1-6 Electron micrograph of an osteoclast. [Reproduced from Schenk, R. K. (1974). Verh. Dtsch. Ges. Pathol., 58, 72-83, with permission from the author and publisher.] Nucleus

? .~--~7-------.~:~ Ca, P, Peptides

Mitochondrion~~

. .-~

Clear z o n e - . . . - _ . ~ ~ : ~ ~ ~ / ! ~ ,.,~,.~:. g l ? ' / / A ~ 9

'~:"

'" ....

"

..... ,

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Fig. 1.1-7 Diagram of an osteoclast. N, nucleus. [Adapted from Schenk, R. K. (1974). Verh. Dtsch. Ges. Pathol., 58, 72-83, with permission from the author and publisher.] Bone resorption can be modulated by altering three basic processes, namely the recruitment of new osteoclasts, the life span of the latter which is programmed by the time when they will undergo apoptosis, and the activity of mature osteoclasts. All three processes are influenced by a series of cytokines and hormones. Recent results indicate that all three processes seem to be under the control of cells of osteoblastic lineage, which synthesize factors influencing directly the osteoclasts and their precursors.

Bone resorption is modulated by altering recruitment of osteoclasts, their life span, and their activity.

1. Bone and mineral metabolism

Osteoclast recruitment and activation

Fig. 1.1-8 Modulation of bone resorption: role of the osteoblast lineage cells.

Recruitment Precursor

~i

ResorbingOsteoclast ~) Osteoblast lineage cell

Activation Inactive Osteoclast

Resorbing Osteoclast

The three main hormones modulating bone resorption are parathyroid *hormone (PTH), 1,25(OH)2 D (calcitriol), and calcitonin, the first two increasing, the latter decreasing resorption. Furthermore, estrogens in women and testosterone in men inhibit bone resorption. This is why menopause and ovariectomy, as well as orchidectomy, induce an increase in bone resorption, mediated possibly in part by an increase in IL-1, IL6, and tumor necrosis factor a (TNFa). PTH-related protein, a peptide which has a resemblance to PTH, and binds to one of its receptors, also increases bone resorption. Its main role is probably during fetal life where it modulates, among others, chondrocyte differentiation in the growth plate. In the adult it plays a major role Tumor bone in tumor bone disease, favoring bone resorption and hypercalcemia. disease Whether it contributes to plasma calcium homeostasis and to bone homepp. 88, 91 ostasis in the adult is not known. Among the most important cytokines that can increase bone resorption, at least in vitro, and are possibly involved in this process in vivo, are the interleukins 1, 3, 6, and 11 (IL-1, IL-3, IL-6, IL-11), tumor necrosis factor a and 13 (TNFa, TNFf3), macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), and prostaglandins. Interferon T (IFNT), TGFf3, IL-4, and IL-13 as well as IL-1 receptor antagonist on the other hand, decrease bone resorption. Some of these cytokines are produced by the cells of the osteoblastic lineage and are therefore possibly involved in the "osteoblast- osteoclast axis. Recently a novel factor inhibiting osteoclast formation and activity, called osteoprotegerin (OPG), has been discovered. It is a soluble glycoprotein of the TNF superfamily, which is produced by stromal cells, osteoblastic lineage cells, and pre-B cells. Acting as a decoy receptor, it binds Calcium homeostasis pp. 17-18

10

1.1. Bone physiology to an osteoclastic differentiation factor called RANK ligand (RANKL), or ODF, which is located on the membrane of the osteoblastic lineage cells. By doing so, it inhibits the binding of the latter to its transmembrane receptor on the osteoclast (RANK), an osteoclast differentiation and activation stimulator, and thus inhibits osteoclast recruitment, involving the transcription factor NF-•I3. Osteoprotegerin inhibits bone resorption both in vitro and in vivo. Its synthesis, and that of RANKL, are modulated by hormones and cytokines affecting bone resorption, and it is likely that they play a major role in the modulation of osteoclastic bone resorption. All these proteins have various names in the literature, the above being used most commonly today. Fig. 1.1-9 Possiblemodulators of bone resorption.

Bone resorption Increase

Decrease Systemic

PTH PTHrP Calcitriol Thyroxin

Calcitonin Estrogen Testosterone

Local IL-1 IL-6 IL-11 IL-17 FGFs Prostaglandins RANKL

TNF(z TNFI3 TGF(z M-CSF GM-CSF SCF

TGF]3 IFN7 IL-4 IL-10 IL-13 IL-18 IL-lra Osteoprotegerin

Bone resorption is performed by the osteoclast. Its recruitment, activity, and apoptosis are under the modulation of a series of hormones and cytokines and under the control of the cells of osteoblastic lineage. Recently a new inhibiting mechanism involving a glycoprotein from the TNF superfamily called osteoprotegerin has been described.

11

1. Bone and mineral metabolism

Other cells Bone marrow contains many other cells, among them hemopoietic, immune, and stromal cells. They are not only important for the formation of the osteoclasts, but also interrelate with the bone cells through various cytokines and appear to be involved thus in the modulation of both the formation and resorption of bone. Thus it is not fortuitous that bone marrow and bone are in close proximity.

1.1.3. Modeling and remodeling

Biomechanical adaptation p. 19

Calcium homeostasis p. 17

Osteoporosis pp. 124-125

Bone is built during fetal life, youth, and adolescence by two fundamental mechanisms: endochondral ossification, whereby bone is built on a cartilaginous scaffold, and intramembranous ossification where it is formed directly without the latter. The correct elaboration of the definitive form is under the control of numerous genes which are gradually being elucidated. Once formed, the shape and structure of the bones is continuously renovated and modified by the two processes of modeling and remodeling. In the modeling, which takes place principally during growth, new bone is formed at a location different from the one destroyed. This therefore results in a change in the shape of the skeleton. It allows not only the development of a normal architecture during growth, but also the modulation of this architecture in the adult when the mechanical conditions change. Furthermore it is the cause of the increase in size of the bones during life. Modeling is the main process by which the skeleton can increase its volume and its mass. In remodeling, which is the main process in the adult, the two processes are coupled in space and time, so that no change occurs in the shape of the bone. Both modeling and remodeling, however, result in the replacement of old bone by new bone. This allows the maintenance of the mechanical integrity of the skeleton, which is illustrated by the fact that in diseases where bone resorption is impaired, such as in osteopetrosis, bone becomes fragile and fractures occur. The turnover also allows the bone to play its role as an ion bank. The remodeling rate is between 2 and 10% of the skeletal mass per year. It can be either a systemic process which occurs more or less randomly, or a targeted one, occurring at special sites of the skeleton. The former is increased by parathyroid hormone, thyroxin, growth hormone, and 1,25(OH)2 D, and decreased by calcitonin, estrogen, and glucocorticosteroids. It is also stimulated by microfractures and modulated by the mechanical strain environment. The cancellous bone, which represents about 20% of the skeletal mass, makes up 80% of the turnover, while the cortex, which represents 80% of the bone, makes up only 20% of the turnover. This explains why osteoporosis, which is the result of

12

1.1. Bone physiology an abnormal turnover and balance, is seen first and mainly in cancellous bone.

Bone is continuously turned over by modefing and remodeling, the rates of which are under hormonal and mechanical influence. Cancellous bone accounts for 80% of the turnover, although it represents only 20% of the skeleton. The morphological dynamic structure of turnover is the basic multicellular unit (BMU), also called bone remodeling unit (BRU). The morphological entity formed when the process is terminated is called the bone structural unit (BSU), which corresponds to the packet in cancellous bone and to the osteon in cortical bone. Both in the cortex and in the trabeculae, the process of remodeling starts in the same way by bone being eroded by osteoclasts. In a second step, the resorption sites are refilled by the osteoblasts. The tight coupling of bone resorption followed by bone formation emphasizes the necessity for a previous occurrence of bone resorption to trigger bone formation at the same site. The linear resorption rate of osteoclasts is about 50 ~tm per day. The formation rate is slower, about 1 ~tm per day for lamellar, more for woven bone. The time required for the completion of a new BSU is between 3 and 5 months.

Cortical remodeling unit Old bone

Osteoclasts Vasculargap Osteoblasts Osteoid "~ Fig. 1.1-10 Cortical bone remodeling. Osteoclasts located at the tip of the cutter cone erode a canal within the bone. Osteoblasts present on the lateral walls will refill it and form the osteon. [From Schenk, R. K. et al. (1993). Reproduced from Royce, P. M., and Steinmann, B. (eds.) Connective Tissue and Its Heritable Disorders. Molecular Genetic, and Mineral Aspects, pp. 85-101. Copyright 9 1993, by permission of the author and John Wiley & Sons, Inc.]

13

BMUs BSUs p. 2

1. Bone and mineral metabolism Trabecular remodeling Osteoclast

, ~r g

| ,. ",~

~

a,~ ~ "

.,.

Bone

Fig. 1.1-11 Cancellous bone remodeling. The process is similar to that in the cortex but occurs at the surface. Osteoclasts erode a burrow which is then refilled by osteoblasts. [From Schenk, R. K., et al. (1993). Reproduced from Royce, P. M., and Steinmann, B. (eds.) Connective

Tissue and Its Heritable Disorders. Molecular Genetic, and Mineral Aspects, Osteoblasts

.... ,

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......"

~- f ~ .~ ~.

~:

pp. 85-101. Copyright 9 1993, by permission of the author and John Wiley & Sons, Inc.]

Osteoid ~

Cement line "Packet" of

~ ~

ewbone

tDone structural unit)

~.~ .~,.~,

The basic dynamic unit of bone remodeling is the basic multicellular unit, called usually just BMU.

It was generally t h o u g h t in the past that, while the erosion in the cortex was a longitudinal process along the long axis of the bone, this was not the case for the trabeculae. There the osteoclasts w o u l d just erode a pit perpendicularly to the surface, which w o u l d then be refilled by osteoblasts. Recent evidence showed that this is not the case and was due to a misinterpretation of the morphological appearance. Indeed, the osteoclasts also erode bone longitudinally in the trabeculae, excavating a burrow, this time at the surface of the bone. Once refilled, this is called a

14

1.1. Bone physiology hemiosteon by analogy with the intracortical osteon. This new concept has important practical implications. The amount of bone resorbed will depend not only on the number of new BMUs formed and on the depth of the burrow, but also on its length, that is on the duration of life of the osteoclasts.

At the surface of the trabeculae the erosion process is similar to that occurring in the cortex, namely, the formation followed by refilling of a burrow, called then a bemiosteon. Normally the amount of bone formed during bone remodeling equals the amount destroyed, so that the balance is zero. The mechanism underlying the balance and the coupling between the resorption and the formation processes is still unknown and the subject of intensive investigation. It is thought that both local formation of cytokines and their local release during resorption play a major role in healthy as well as in diseased bone. The elucidation of these mechanisms may help to explain the cause of bone loss in osteoporosis, in which more bone is being destroyed than formed, resulting in a negative balance. This imbalance is seen, for example, after the menopause or during immobilization. Similarly this local interplay also probably has a major role in the development of osseous metastases.

Osteoporosi p. 127 Tumor bon~ disease p. 88

In the steady state, the amount of bone formed in the remodeling process equals the amount destroyed. If more bone is destroyed than formed, bone loss occurs and osteoporosis may develop. A special situation arises in periods where turnover rates change, for example, after the administration of an inhibitor of bone resorption. Since there is a time interval between the beginning of the inhibition of the resorption process and the start of the secondary decrease in formation, the ongoing bone formation will produce a transient net gain of bone whenever the turnover rate decreases. This time interval explains why after the administration of an inhibitor of bone resorption, such as a bisphosphonate, markers of bone resorption such as urinary hydroxyproline and pyridinium cross-links decrease sooner than markers of bone formation such as plasma alkaline phosphatase and osteocalcin. The amount of bone gained in this way, sometimes called the remodeling space, may account for up to a total of 2%, more for trabecular bone volume, or when the turnover is rapid, less for the cortex.

15

Turnover m arkers

p. 21

1. Bone and mineral metabolism Fig. 1.1-12 Influence of changes in turnover on calcium balance.

Inhibition of bone turnover Resorption inhibitor Bone formation .

.

.

.

/ ! .

.

.

.

.

I

J

Bone resorption

+

Balance

\

. . . . . .

Time

Inhibition of resorption and BMD p. 15

Furthermore, after inhibition of turnover, osteons have more time to complete their mineralization, so that some calcium will still be taken up by the not yet completely mineralized matrix, accounting for another 1 - 2 % increase in calcium. Thus a total elevation of 3 - 4 % in skeletal calcium, and therefore of bone mineral density, usually called BMD, may occur, possibly more if turnover is higher. However this increase of BMD does not represent faithfully an increase of bone mass, since part of it is due just to an increase in the mineralization of the bone present, and not to an increase of the whole bone tissue. Thus some of the increase in BMD seen with various inhibitors of bone resorption such as estrogens, calcitonin, and bisphosphonates are a true durable augmentation neither of bone mass, nor density, and will be reversed if the inhibition is stopped. This fact has often been neglected in the literature. In contrast, the gain in BMD may remain if bone turnover is maintained at the lower level.

When turnover rate is decreased, a transient increase of bone mineral density, called BMD, occurs, which is, however, at least partially reversed when turnover normalizes again.

1.1.4.

C a l c i u m homeostasis

Calcium concentration in plasma is around 10 mg per 100 ml. About 40% of this amount is bound to proteins and 10% to ultrafilterable ions, so that approximately only one-half is ionized. The level of ionized plasma calcium and not of total calcium is the fraction that is tightly controlled throughout the animal world. This has led some authors to call ionized

16

1.1. Bone physiology calcium one of nature's physiological constants. This constancy is explained by the importance of extracellular calcium for many biological processes. Nevertheless, for clinical purposes, and as long as there is no disturbance in plasma proteins and plasma pH, measurement of total calcium is usually sufficient. In some conditions, however, such as in tumor bone disease, plasma proteins are disturbed to such an extent that a correction is necessary.

Plasma ionized calcium, and not total calcium, is the fraction that is tightly controlled. The level of ionized calcium, and therefore of plasma calcium, is set by the interaction of the three target organs: intestine, bone, and kidney. The level will depend on the fluxes between extracellular fluid and therefore blood, and these three organs. For bone the relative roles played by bone resorption and by the equilibrium at the surface of the apatite crystals is still debated. Probably the latter is more relevant for the short-term steady state regulation. Fig. 1.1-13 Calcium homeostasis in the body.

Calcium homeostasis

Gastrointestinal Tract Blood

~

Bone

Kidney I

The level of ionized calcium is set by the interaction of the three target organs: intestine, bone, and kidney. In bone, the equilibrium at the surface of the crystals is probably also important. The fluxes of calcium are controlled mainly by three hormones, parathyroid hormone (PTH), the vitamin D metabolite 1,25(OH)2 D (calcitriol), and possibly calcitonin, although the last has not yet been proved to act physiologically. All three are directly regulated by plasma calcium levels through a feedback mechanism. Whether the three hormones, or at least some of them, also control the bone surface equilibrium is likely, but not proven.

17

Correction t plasma proteins p. 93

1. Bone and mineral metabolism Blood calcium is regulated mainly by the three hormones parathyroid hormone, 1,25(0H)2 D, and possibly calcitonin. Of the three hormones, the most important is parathyroid hormone, which increases plasma calcium by action on all three target organs. Thus it increases bone resorption, increases the intestinal absorption of calcium, although indirectly through an elevation of 1,25(OH)z D, and increases renal tubular reabsorption of calcium. Since the production of parathyroid hormone is inversely related to plasma calcium and is rapidly modulated through a calcium-sensing receptor located in the parathyroid gland, this hormone provides an excellent rapidly working negative feedback mechanism. The hormone 1,25(OH)2 D increases intestinal calcium absorption and bone resorption and enhances the capacity of PTH to increase renal Ca reabsorption. Since its production is also stimulated by low plasma calcium, it provides a regulatory feedback mechanism too. However, in contrast to parathyroid hormone, which is modulated and acts within minutes, 1,25(OH)2 D requires hours. Vitamin D metabolites are also required for normal mineralization of bone, 1,25(OH)2 D and possibly 24,25(OH)z D forms being the most important. Finally, calcitonin inhibits bone destruction. This property is used therUse in Paget's apeutically in diseases with increased bone resorption, such as Paget's disease disease and osteoporosis. Since its production is rapidly modulated by p. 72 plasma calcium through positive feedback, this hormone could also theUse in oretically provide good feedback regulation. Its relevance in humans for osteoporosis p. 132 calcium homeostasis is, however, not yet established. Bone resorption p. lO

Fig. 1.1-14 Roleof parathyroid hormone, 1,25(OH)2 D, and calcitonin in calcium homeostasis.

Hormones and calcium homeostasis

Gastrointestinal Tract ~, ~" 1,25D+

1,25D+ one

~;'"/" ~

PTH+I~

PTH+ 1,25D+ CT-

Kidney ~

Plasma calcium levels regulate parathyroid hormone, 1,25(0H) 2D as well as calcitonin, providing an excellent homeostatic feedback mechanism for maintaining plasma calcium homeostasis. 18

1.1. Bone physiology Thus the three target organs, intestine, kidney, and bone, are intimately linked with respect to calcium homeostasis. Consequently, a disturbance in one of these organs will affect the others. Because 99% of the body cal-+ cium is located in the skeleton, this organ will act as a reserve of the ion. In the case of calcium shortage, the homeostatic mechanisms will work to the detriment of the bone in order to maintain plasma calcium, which seems to have absolute priority. It is not yet clear what the daily intake of calcium should be for humans. It is thought today that at least I g is necessary in adult life, somewhat more in adolescents, and 1.5 g in the elderly as well as during pregnancy and lactation. If this is true, a large part of the population would be chronically calcium deficient, since its daily intake is usually only about 0.5 g. It is interesting that in the animal kingdom, humans have by far the lowest calcium intake in relation to body weight, and that our ancestors in the Stone Age ate apparently about three times as much calcium as we do. Calcium deficiency may possibly explain in part the bone loss during the second part of life, since calcium absorption decreases in the elderly, who may have vitamin D deficiency or a disturbance in its metabolism and action. A decrease in calcium absorption due to malabsorption, or a loss in the urine such as in renal hypercalciuria, may also induce bone loss. 1.1.5.

B o n e as an o r g a n

In addition to these homeostatic mechanisms at the service of plasma calcium, other mechanisms exist which allow bone to maintain its own in-+ tegrity. First, bone architecture is under the control of a biomechanical cybernetic system, called a mechanostat, that controls bone's modeling of its spatial organization, and its load carrying capacity and ability to transfer loads. To allow this, the strains induced by external mechanical influences appear to play an essential role by altering both modeling and remodeling. This system also has a feed-back loop. Indeed, a loss of bone, or a deterioration of the mechanical property of the bone tissue itself, will increase the strains induced, leading to increased bone formation and/or decreased bone resorption, and therefore a subsequent change in bone mass. These mechanisms allow bone to adapt its structure to function and to fulfill its mechanical role optimally. They explain why the trabeculae of cancellous bone are oriented along the prevailing lines of pressure and traction and why they change if these are altered, for example, after an orthopedic operation that alters the axis, or locally after destruction of individual trabeculae (Fig. 1.1-15). Besides these mechanisms, the bone ensures its own integrity during aging. Apart of the generalized remodeling, which is under endocrine control, the microcracks, which occur constantly during life, stimulate bone remodeling and therefore their own repair. This allows old bone to 19

Calcium in osteoporosis p. 132

1. Bone and mineral metabolism be replaced by young bone and permits the skeleton to keep its mechanical strength.

Bone structure and function

R/

]vl,! i

-'3"

~

o.~

Osteocytes p. 8

Fig. 1.1-15 Adaptation of bone structure to mechanical function along lines of compression and tension. M, muscle pull; R, down direction of load. [Adapted from Pauwels, F. (1960). Z. Anat. Entwickl. Gesch., 121,478-515, with permission from the publisher.]

,,,

,

If the mechanostat does not sense sufficient input, such as in immobi- *-lization, rapid and massive bone loss will occur. The magnitude of the bone loss can be such as to disturb calcium homeostasis and lead to hypercalciuria and even hypercalcemia. Some bone loss also occurs during weightlessness in astronauts. Therefore, one of the rules in osteoporotic patients is to avoid immobilization. The cellular mechanisms underlying these events are yet little understood but are likely to involve the osteocytes and local cytokines. It has been proposed that the mechanostat can be disturbed in some of the metabolic diseases, an interesting hypothesis which deserves further study.

Mechanical forces influence bone turnover and allow the skeleton to maintain an optimal structure to fulfill its mechanical function. Immobilization induces bone loss.

20

1.1. Bone physiology 1.1.6.

Assessment of bone turnover

Bone turnover can be assessed in vivo, although only indirectly. For bone formation the measurements most commonly used are serum alkaline phosphatase, preferentially the bone isoenzyme, and serum osteocalcin. Alkaline phosphatase is produced by the osteoblasts during bone formation. Osteocalcin is specific to bone and is liberated during the formation of matrix by the osteoblasts. Its level in serum is a good index for bone formation, except, for unknown reasons, in Paget's disease. More recently the appearance of certain propeptides liberated during collagen synthesis, such as procollagen I N-terminal extension peptides (PINP), has been used. Bone resorption is evaluated by measuring the urinary excretion of bone collagen breakdown products such as hydroxyproline, pyridinium cross-links, and certain collagen fragments. Collagen contains 10% hydroxyproline. When bone is destroyed, some of this amino acid is excreted in the urine, so that urinary hydroxyproline can be used as an index of bone destruction. However, because urinary hydroxyproline can also originate from nonosseous collagen, and because some of the amino acid is metabolized, this index is not ideal. Furthermore hydroxyproline is also absorbed from dietary collagen, so that the measurements must be performed under a collagen-free diet. The procedure can be simplified by measuring the hydroxyproline/creatinine ratio of the first 2-h morning fasting urine specimen obtained after the urine of the night is voided, and with no intake of collagen the evening before. Better markers of bone resorption, which have now practically replaced hydroxyproline, are the pyridinium cross-links present in collagen. These are formed by the linkage of two collagen molecules to form pyridinoline and deoxypyridinoline. Of the two, the latter is more specific to bone, but both measurements seem to give similar results, so that often both are measured together. No dietary precautions are necessary. Until recently this determination was performed only in specialized laboratories by means of high-pressure liquid chromatography, but the development of immunoassays makes it now more widely available. More recently antibodies have been developed against pyridinium-containing peptides both from the N-terminal and C-terminal telopeptide. These appear in the urine, and their measurement gives excellent results. They appear to be the most useful markers available today. Very recently, assays in plasma have also been developed. Bone formation is assessed by measuring serum alkaline phospha-

tase and osteocalcin; bone resorption previously by measuring urinary hydroxyproline, which has been replaced by pyridinoline cross-links or preferentially peptides containing these cross-links. 21

Markers in Paget's dise~ p. 70

1. Bone and mineral metabolism

Recommended selected reading Books Avioli, L. V., and Krane, S. M. (eds.) (1998). Metabolic Bone Disease and Clinically Related Disorders. (San Diego: Academic Press) Bilezikian, J. P., Raisz, L. G., and Rodan, G. A. (eds.) (1996). Principles of Bone Biology. (San Diego, London: Academic Press) Favus, M. J. (ed.) (1999). Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 4th Ed. (Philadelphia: Lippincott Williams & Wilkins) Marcus, R., Feldman, D., and Kelsey, J. (eds.) (1996). Osteoporosis. (San Diego: Academic Press) Mundy, G. R. (1995). Bone Remodeling and Its Disorders. (London: Martin Dunitz) Mundy, G. R., and Martin, T. J. (eds.) (1993). Physiology and Pharmacology of Bone. Handbook of Experimental Pharmacology, vol. 107. (Berlin, Heidelberg, New York: Springer-Verlag) Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) (1999). Dynamics of Bone and Cartilage Metabolism. (San Diego, London: Academic Press)

Reviews

Morphology Einhorn, T. A. (1996). Biomechanics of bone. In Bilezikian, J. P., Raisz, L. G., and Rodan, G. A. (eds.) Principles of Bone Biology, pp. 25-37. (San Diego, London: Academic Press) Einhorn, T. A. (1996). The bone organ system: Form and function. In Marcus, R., Feldman, D., and Kelsey, J. (eds.) Osteoporosis, pp. 3-22. (San Diego: Academic Press) Eriksen, E. F., Vesterby, A., Kassem, M., Melsen, F., and Mosekilde, L. (1993). Bone remodeling and bone structure. In Mundy, G. R., and Martin, T. J. (eds.) Physiology and Pharmacology of Bone. Handbook of Experimental Pharmacology, vol. 107, pp. 67109. (Berlin, Heidelberg, New York: Springer-Verlag) Marks, S. C., Jr., and Hermey, D. C. (1996). The structure and development of bone. In Bilezikian, J. P., Raisz, L. G., and Rodan, G. A. (eds.) Principles of Bone Biology, pp. 314. (San Diego, London: Academic Press) Monier-Faugere, M.-C., Langub, M. C., and Malluche, H. H. (1998). Bone biopsies: A modern approach. In Avioli, L. V., and Krane, S. M. (eds.) Metabolic Bone Disease and Clinically Related Disorders, pp. 237-273. (San Diego: Academic Press) Ott, S. (1996). Theoretical and methodological approach. In Bilezikian, J. P., Raisz, L. G., and Rodan, G. A. (eds.) Principles of Bone Biology, pp. 231-241. (San Diego, London: Academic Press) Parfitt, A. M. (1992). The physiologic and pathogenetic significance of bone histomorphometric data. In Coe, F. L., and Favus, M. J. (eds.) Disorders of Bone and Mineral Metabolism, pp. 475-489. (New York: Raven) Parfitt, A. M., Mundy, G. R., Roodman, G. D., Hughes, D. E., and Boyce, B. F. (1996). A new model for the regulation of bone resorption, with particular reference to the effects of bisphosphonates. J. Bone Miner. Res., 11,150-159 Schenk, R. K., Felix, R., and Hofstetter, W. (1993). Morphology of connective tissue: Bone. In Royce, P. M., and Steinmann, B. (eds.) Connective Tissue and Its Heritable Disorders. Molecular, Genetic, and Medical Aspects, pp. 85-101. (New York: Wiley-Liss)

Chemistry Boskey, A. L. (1999). Mineralization, structure, and function of bone. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 153-164. (San Diego, London: Academic Press)

22

1.1. Bone physiology Gehron Robey, P., and Boskey, A. L. (1996). The biochemistry of bone. In Marcus, R., Feldman, D., and Kelsey, J. (eds.) Osteoporosis, pp. 95-183. (San Diego: Academic Press) Glimcher, M. J. (1998). The nature of the mineral phase in bone: Biological and clinical implications. In Avioli, L. V., and Krane, S. M. (eds.) Metabolic Bone Disease and Clinically Related Disorders, pp. 23-50. (San Diego: Academic Press) Gundberg, C. M., and Nishimoto, S. K. (1999). Vitamin K-dependent proteins of bone and cartilage. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 43-57. (San Diego, London: Academic Press) Hardingham, T. E. (1999). Proteoglycans and glycosaminoglycans. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 7181. (San Diego, London: Academic Press) Heinegard, D., Saxne, T., and Lorenzo, P. (1999). Noncollagenous proteins: Glycoproteins and related molecules. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 59-69. (San Diego, London: Academic Press) Robins, S. P. (1999). Fibrillogenesis and maturation of collagens. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 591604. (San Diego, London: Academic Press) Von der Mark, K. (1999). Structure and biosynthesis of collagens. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 3-29. (San Diego, London: Academic Press)

Cells Aarden, E. M., Burger, E. H., and Nijweide, P. J. (1994). Function of osteocytes in bone. J. Cell. Biochem., 55,287-299 Clark, I. M., and Murphy, G. (1999). Matrix proteases. In Seibel, M. J., Robins, S. P., and Bilezikian, J. p. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 137-150. (San Diego, London: Academic Press) Helfrich, M. H., and Horton, M. A. (1999). Integrins and adhesion molecules. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 111-126. (San Diego, London: Academic Press) Henthorn, P., Mill~n, J. L., and Leboy, P. (1999). Acid and alkaline phosphatases. In Seibel, M. J., Robins, S. P., and Bilezikian, J. p. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 127-136. (San Diego, London: Academic Press) Lian, J. B., and Stein, G. S. (1996). Osteoblast biology. In Marcus, R., Feldman, D., and Kelsey, J. (eds.) Osteoporosis, pp. 23-59. (San Diego: Academic Press) Lian, J. B., and Stein, G. S. (1999). The cells of bone. In Seibel, M. J., Robins, S. P., and Bilezikian, J. p. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 165-185. (San Diego, London: Academic Press) Martin, T. J., Findlay, D. M., Heath, J. K., and Ng, K. W. (1993). Osteoblast: Differentiation and function. In Mundy, G. R., and Martin, T. J. (eds.) Physiology and Pharmacology of Bone. Handbook of Experimental Pharmacology, vol. 107, pp. 149-183. (Berlin, Heidelberg, New York: Springer-Verlag) Martin, T. J., Romas, E., and Gillespie, M. T. (1998). Interleukins in the control of osteoclast differentiation. Crit. Rev. Eukariot. Gene Expr. 8, 107-123 Nijweide, P. J., Burger, E. H., Klein Nulend, J., and Van der Plas, A. (1996). The osteocyte. In Bilezikian, J. p., Raisz, L. G., and Rodan, G. A. (eds.) Principles of Bone Biology, pp. 115-126. (San Diego, London: Academic Press) Raisz, L. G., and Rodan, G. A. (1998). Embryology and cellular biology of bone. In Avioli, L. V., and Krane, S. M. (eds.) Metabolic Bone Disease and Clinically Related Disorders, pp. 1-22. (San Diego: Academic Press) Roodman, G. D. (1996). Advances in bone biology: The osteoclast. Endocr. Rev., 17, 3083a2. Suda, T., Takahashi, N., Udagawa, N., Jimi, E., Gillespie, M. T., and Martin, T. J. (1999). Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr. Rev., 20, 345-357 Teitelbaum, S. L., Tondravi, M. M., and Ross, F. P. (1996). Osteoclast biology. In Marcus,

23

1. Bone and mineral metabolism R., Feldman, D., and Kelsey, J. (eds.) Osteoporosis, pp. 61-94. (San Diego: Academic Press)

Mechanism of bone formation and resorption and its modulation Canalis, E. (1996). Skeletal growth factors. In Marcus, R., Feldman, D., and Kelsey, J. (eds.) Osteoporosis, pp. 261-279. (San Diego: Academic Press) Croucher, P. I., and Russell, R. G. G. (1999). Growth factors. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 83-95. (San Diego, London: Academic Press) Dempster, D. W. (1999). New concepts in bone remodeling. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 261-273. (San Diego, London: Academic Press) Khosla, S., Spelsberg, T. C., and Riggs, B. L. (1999). Sex steroid effects on bone metabolism. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 233-245. (San Diego, London: Academic Press) Lorenzo, J. A., and Raisz, L. G. (1999). Cytokines and prostaglandins. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 97-109. (San Diego, London: Academic Press) Manolagas, S. C., and Jilka, R. L. (1995). Bone marrow, cytokines, and bone remodeling. N. Engl. J. Med. 332, 305-311 Martin, T. J., Findlay, D. M., and Moseley, J. M. (1996). Peptide hormones acting on bone. In Marcus, R., Feldman, D., and Kelsey, J. (eds.) Osteoporosis, pp. 185-204. (San Diego: Academic Press) Mundy, G. R. (1993). Hormonal factors which regulate bone resorption. In Mundy, G. R., and Martin, T. J. (eds.) Physiology and Pharmacology of Bone. Handbook of Experimental Pharmacology, vol. 107, pp. 185-214. (Berlin, Heidelberg, New York: SpringerVerlag) Mundy, G. R., Boyce, B. F., Yoneda, T., Bonewald, L. F., and Roodman, D. G. (1996). Cytokines and bone remodeling. In Marcus, R., Feldman, D., and Kelsey, J. (eds.) Osteoporosis, pp. 302-313. (San Diego: Academic Press) Oursler, M. J., Kassem, M., Turner, R., Riggs, B. L., and Spelsberg, T. C. (1996). Regulation of bone cell function by gonadal steroids. In Marcus, R., Feldman, D., and Kelsey, J. (eds.) Osteoporosis, pp. 237-260. (San Diego: Academic Press) Raisz, L. G., and Rodan G. A. (1998). Embryology and cellular biology of bone. In Avioli, L. V., and Krane, S. M. (eds.) Metabolic Bone Disease and Clinically Related Disorders, pp. 1-22. (San Diego: Academic Press) Reddi, A. H., and Sampath, T. K. (1996). Bone morphogenetic proteins: Potential role in osteoporosis. In Marcus, R., Feldman, D., and Kelsey, J. (eds.) Osteoporosis, pp. 281-287. (San Diego: Academic Press) Rodan, G. A. (1996). Coupling of bone resorption and formation during bone remodeling. In Marcus, R., Feldman, D., and Kelsey, J. (eds.) Osteoporosis, pp. 290-299. (San Diego: Academic Press) C a l c i u m homeostasis Azria, M., and Avioli, L. V. (1996). Calcitonin. In Bilezikian, J. p., Raisz, L. G., and Rodan, G. A. (eds.) Principles of Bone Biology, pp. 1083-1097. (San Diego, London: Academic Press) Carmeliet, G., Verstuyf, A., Daci, E., and Bouillon, R. (1999). The vitamin D hormone and its nuclear receptor: Genomic mechanisms involved in bone biology. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 217-231. (San Diego, London: Academic Press) Civitelli, R., Ziambaras, K., and Leelawattana, R. (1998). Pathophysiology of calcium, phosphate, and magnesium absorption. In Avioli, L. V., and Krane, S. M. (eds.) Meta-

24

1.1. Bone physiology bolic Bone Disease and Clinically Related Disorders, pp. 165-205. (San Diego: Academic Press) Feldman, D., Mallon, P. J., and Gross, C. (1996). Vitamin D: Metabolism and action. In Marcus, R., Feldman, D., and Kelsey, J. (eds.) Osteoporosis, pp. 205-235. (San Diego: Academic Press) Fitzpatrick, L. A., and Bilezikian, J. P. (1996). Actions of parathyroid hormone. In Bilezikian, J. P., Raisz, L. G., and Rodan, G. A. (eds.) Principles of Bone Biology, pp. 339346. (San Diego, London: Academic Press) Fitzpatrick, L. A., and Bilezikian, J. P. (1999). Parathyroid hormone: Structure, function, and dynamic actions. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 187-202. (San Diego, London: Academic Press) Holick, M. F., and Adams, J. S. (1998). Vitamin D metabolism, and biological function. In Avioli, L. V., and Krane, S. M. (eds.) Metabolic Bone Disease and Clinically Related Disorders, pp. 123-164. (San Diego: Academic Press) Hruska, K., and Gupta, A. (1998). Disorders of phosphate homeostasis. In Avioli, L. V., and Krane, S. M. (eds.) Metabolic Bone Disease and Clinically Related Disorders, pp. 207236. (San Diego: Academic Press) Karaplis, A. C., and Goltzman, D. (1999). PTHrP: Of molecules, mice and men. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 203-216. (San Diego, London: Academic Press) Martin, T. J., Findlay, D. M., Moseley, J. M., and Sexton, P. M. (1998). Calcitonin. In Avioli, L. V., and Krane, S. M. (eds.) Metabolic Bone Disease and Clinically Related Disorders, pp. 95-121. (San Diego: Academic Press) Moseley, J. M., and Martin, T. J. (1996). Parathyroid hormone-related protein: Physiological actions. In Bilezikian, J. P., Raisz, L. G., and Rodan, G. A. (eds.) Principles of Bone Biology, pp. 363-376. (San Diego, London: Academic Press) Parfitt, A. M. (1993). Calcium homeostasis. In Mundy, G. R., and Martin, T. J. (eds.) Physiology and Pharmacology of Bone. Handbook of Experimental Pharmacology, vol. 107, pp. 1-65. (Berlin, Heidelberg, New York: Springer-Verlag) Potts, J. T., Jr., and J~ippner, H. (1998). Parathyroid hormone and parathyroid hormonerelated peptide in calcium homeostasis, bone metabolism, and bone development: The proteins, their genes, and receptors. In Avioli, L. V., and Krane, S. M. (eds.) Metabolic Bone Disease and Clinically Related Disorders, pp. 51-84. (San Diego: Academic Press) Rizzoli, R., and Bonjour, J.-P. (1999). Physiology of calcium and phosphate homeostasis. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 247-260. (San Diego, London: Academic Press)

Measurement of bone t u r n o v e r Brixen, K., and Eriksen, E. F. (1999). Validation of local and systemic markers of bone turnover. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 427-436. (San Diego, London: Academic Press) Christiansen, C., Hassager, C., and Riis, B. J. (1998). Biochemical markers of bone turnover. In Avioli, L. V., and Krane, S. M. (eds.) Metabolic Bone Disease and Clinically Related Disorders, pp. 313-326. (San Diego: Academic Press) Eyre, D. (1996). Biochemical basis of collagen metabolites as bone turnover markers. In Bilezikian, J. P., Raisz, L. G., and Rodan, G. A. (eds.) Principles of Bone Biology, pp. 143153. (San Diego, London: Academic Press) Garnero, P., and Delmas, P. D. (1996). Measurements of biochemical markers: Methods and limitations. In Bilezikian, J. P., Raisz, L. G., and Rodan, G. A. (eds.) Principles of Bone Biology, pp. 1277-1291. (San Diego, London: Academic Press) Kraenzlin, M. E., and Seibel, M. J. (1999). Measurement of biochemical markers of bone resorption. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 411-426. (San Diego, London: Academic Press) Naylor, K. E., and Eastell, R. (1999). Measurement of biochemical markers of bone forma-

25

1. Bone and mineral metabolism tion. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 401-410. (San Diego, London: Academic Press) Risteli, J., and Risteli, L. (1999). Products of bone collagen metabolism. In Seibel, M. J., Robins, S. P., and Bilezikian, J. P. (eds.) Dynamics of Bone and Cartilage Metabolism, pp. 275-287. (San Diego, London: Academic Press) Seibel, M. J., and Pols, H. A. P. (1996). Clinical application of biochemical markers of bone metabolism. In Bilezikian, J. P., Raisz, L. G., and Rodan, G. A. (eds.) Principles of Bone Biology, pp. 1293-1311. (San Diego, London: Academic Press) B o n e as an o r g a n

Rodan, G. A. (1997). Bone mass homeostasis and bisphosphonate action. Bone, 20, 1-4

26

2. Bisphosphonates preclinical

2 . 1 . BACKGROUND TO THE PHARMACOLOGICAL DEVELOPMENT Our knowledge of the biological characteristics of bisphosphonates dates from nearly 30 years ago, the first report having appeared in 1968. The concept was derived from earlier studies in our laboratory on inorganic pyrophosphate. We had found that plasma and urine contained compounds inhibiting calcium phosphate precipitation, and that part of this inhibitory activity was due to inorganic pyrophosphate, a substance that had not been described previously in these fluids. Fig. 2.1-1 Chemical structure of inorganic pyrophosphate.

Pyrophosphate O-I

O-I

o-P-O-P=O I

I

o-

o-

Pyrophosphate is the simplest of the polyphosphates, also called condensed phosphates, compounds which have been used extensively in industry because of their property of inhibiting the precipitation of calcium carbonate. Their main applications were as antiscaling additives in washing powders or water and oil brines to prevent deposition of calcium carbonate scale. 27

2. Bisphosphonatesmpreclinical

Polyphosphates as antiscaling agents

Fig. 2.1-2 Effectof polyphosphates on the deposition of calcium carbonate in a water pipe. [Adapted from Rudy, H. (1960). Altes und Neues

iiber kondensierte Phosphate. (Ludwigshafen am

Rhein: J.A. Benckiser, GmbH). Reproduced with permission from the publisher.]

No addition

With addition

Inorganic pyrophosphate, a compound of the family of the polyphosphates, used industrially [:or their property or: inhibiting calcium carbonate, is present in biological fluids. We then found that pyrophosphate binds very avidly to calcium phos- *-phate and impairs both the formation of calcium phosphate crystals in vitro and their dissolution. Pyrophosphate was then shown to inhibit calcification, also in vivo. Various types of ectopic calcification were efficiently prevented by the parenteral but not the oral administration of the compound. In contrast, no effect was found on bone resorption. This was explained by the possible hydrolysis of pyrophosphate when given orally and at the sites of bone destruction. These results led us to propose that pyrophosphate might be a physiological regulator of calcification and perhaps also of decalcification in vivo, its local concentration being determined by the activity of local phosphatases and other pyrophosphatases.

Inorganic pyrophosphate inhibits both the formation and the dissolution of calcium phosphate in vitro. In vivo it prevents ectopic calcification. It might be a physiological regulator of mineral deposition and dissolution. Because of its failure to act when given orally as a result of its rapid hydrolysis, pyrophosphate found a therapeutic use only in two indications. In view of its strong affinity for calcium phosphate and therefore for bone mineral, it is used, when linked to 99roTc, in skeletal scintigraphy. Furthermore, it is used as antitartar agents in toothpastes.

Pyrophosphate is used in scintigraphy and as an antitartar agent in toothpastes.

28

2.1. Background

This restricted use prompted us to search for analogs which would display similar physicochemical activity, but which would resist enzymatic hydrolysis and would therefore not be broken down metabolically. We found that the bisphosphonates fulfilled these conditions. In the last 30 years our group has worked, in collaboration with various pharmaceutical companies, on the development of the bisphosphonates and the elucidation of their mode of action, which is primarily the inhibition of bone resorption. In the 1980s and 1990s various companies have synthesized new bisphosphonates and developed them for clinical use in diseases c h a r a c t e r i z e d by increased b o n e r e s o r p t i o n , a n d by b o n e loss.

The bisphosphonates are analogs o f pyrophosphate and are used today primarily in diseases with increased bone resorption and with bone loss.

Recommended selected reading Reviews Fleisch, H., and Russell, R. G. G. (1970). Pyrophosphate and polyphosphate. In Ency-

clopaedia (Int.) of Pharmacology and Therapeutics, Section 51. Pharmacology of the Endocrine System and Related Drugs, pp. 61-100. (Oxford, New York: Pergamon) Original articles Fleisch, H., and Bisaz, S. (1962). Isolation from urine of pyrophosphate, a calcification inhibitor. Am. J. Physiol., 203,671-675 Fleisch, H., and Neuman, W. F. (1961). Mechanisms of calcification: Role of collagen, polyphosphates and phosphatase. Am. J. Physiol., 200, 1296-1300 Fleisch, H., Russell, R. G. G., and Straumann, F. (1966). Effect of pyrophosphate on hydroxy-apatite and its implications in calcium homeostasis. Nature (London), 212, 901-903 Fleisch, H., Russell, R. G. G., Bisaz, S., Casey, P. A., and M/ihlbauer, R. C. (1968). The influence of pyrophosphate analogues (diphosphonates) on the precipitation and dissolution of calcium phosphate in vitro and in vivo. Calcif. Tissue Res., 2(Suppl.), 10-10A Russell, R. G. G., Bisaz, S., Donath, A., Morgan, D. B., and Fleisch, H. (1971). Inorganic pyrophosphate in plasma in normal persons and in patients with hypophosphatasia, osteogenesis imperfecta and other disorders of bone. J. Clin. Invest., 50, 961-969 Schibler, D., Russell, R. G. G., and Fleisch, H. (1968). Inhibition by pyrophosphate and polyphosphate of aortic calcification induced by vitamin D3 in rats. Clin. Sci., 35, 363372

29

2.2. CHEMISTRY Bisphosphonates, formerly called diphosphonates, are compounds characterized by two CroP bonds. If the two bonds are located on the same carbon atom, resulting in a P - - C ~ P structure, the compounds are called geminal bisphosphonates. They are therefore analogs of pyrophosphate that contain a carbon instead of an oxygen atom. For the sake of simplicity, and since so far only P ~ C - - P bisphosphonates have been found to exert strong activity on the skeleton, the geminal bisphosphonates will simply be called bisphosphonates in this book. This simplification is usually also made in the literature. Fig. 2.2-1 Chemical structure of pyrophosphate and bisphosphonates.

Chemical structure OI

OI

O=P-O-P=O I O-

I O-

Pyrophosphate

OI

R' I

0I

O=P-C-P=O I O-

I I R" O-

Geminal bisphosphonate

Geminal bisphosphonates, simply called bisphosphonates in this book and the literature, are synthetic compounds characterized by a PmC--P bond.

Commercial index pp. 182-206

The geminal bisphosphonates have been known for a long time, the first synthesis by German chemists dating back to 1865. Etidronate was synthesized as early as 1897. They were used for a variety of industrial applications, among them as antiscaling agents. The P--CroP structure allows a great number of possible variations, either by changing the two lateral chains on the carbon atom, or by esterilying the phosphate groups. Many bisphosphonates have been investigated in animals and humans with respect to their effect on bone. Alendronate, clodronate, etidronate, ibandronate, pamidronate, risedronate, and tiludronate are commercially available in some countries for use in human bone disease (Fig. 2.2-2). Each bisphosphonate has its own physicochemical and biological characteristics. This variability in effect makes it impossible to extrapolate with certainty from data for one compound to others, so that each compound has to be considered on its own, with respect to both its use and its toxicology.

30

2.2. Chemistry Bisphosphonates used in humans NH 2 OI

I O - (CH2)3 O-

I

I

O--P

I

O:P--C--P:O

I

I

I

O - OH

O-

CI

I

O-

I

-- C - - P - - O

I

I

I

O-

CI

o-

(Dichloromethylene)bis-phosphonate

(4-Amino-1-hydroxybutylidene)bis-phosphonate

clodronate* Abiogen; Astra Leiras; Rhdne-Poulenc Rorer; Roche

alendronate* Abiogen; Merck Sharp & Dohme

9 I O - (CH2)2 0 -

I

I

O--P

I

-- C - - P - - O

I

I

O-

t

O--P

O-

!

-- C - - P - - O

I

OH

CH 3 O-

O-

I

I

I

t

O-

OH

o-

[1-Hydroxy-3-(1-pyrrolidinyl)propylidene]bis-phosphonate

(1-Hydroxyethylidene)bis-phosphonate

EB-1053

etidronate* Abiogen; Procter & Gamble

Leo /CH3 C~H3N / (0H2)4

I

I

O - (CH2)2 O -

O-

NH

I

I

I

I

O--'P

I

-- C-- P-'-O

O'-P

OI

-- C - - P - - O

I

1

I

I

I

1

O-

OH

O-

O-

H

o-

[1-Hydroxy-3-(methylpentylamino) propylidene]bis-phosphonate

[(Cycloheptylamino)methylene]bis-phosphonate

ibandronate* Roche

incadronate Yamanouchi NH 2

I Ol O--P--

I O-

I

CH2 O -

I

O - (CH2)s O -

t

I

C--P--O

O--P

1

I

--C--P--O

I

I

i

I

1

OH

O-

O-

OH

o-

[1-Hydroxy-2-imidazo-(1, 2-a) pyridin3-ylethylidene]bis-phosphonate

(6-Amino-l-hydroxyhexylidene)bis-phosphonate

minodronate Yamanouchi- Hoechst

neridronate Abiogen

Fig. 2.2-2 Chemical structure of bisphosphonates investigated for their effect on bone in humans. *Commercially available.

31

2. Bisphosphonatesmpreclinical H3C\ N / CH3

Y

NH 2

I

I

O- (CH2)2 O-

O - (CH2)2 O-

I

I

O--P

I

I

O--P

--C--P--O

I

I

I

O-

OH

o-

I

I

--C--P--O

I O-

I

I

OH

o-

[3-(Dimethylamino)-l-hydroxypropylidene]bis-phosphonate

(3-Amino-l-hydroxypropylidene)bis-phosphonate

olpadronate Gador

pamidronate* Gador; Novartis

cI I

9

9

1

I

O- CH2 OI I I O-----P -- C - - P - - O

I

I

I

O-

OH

o-

o-

s

o-

I

t

I

O--P

--C--P=O

I

I

I

O-

H

o-

[1-Hydroxy-2-(3-pyridinyl)ethylidene]bis-phosphonate

[[(4-Chlorophenyl)thio]methylene]bis-phosphonate

risedronate* Procter & Gamble

tiludronate* Sanofi

I O- CH 2 OI I I O - - P -- C - - P = O

I

t

O-

OH

I O-

[1-Hydroxy-2-(1H-imidazole-1-yl) ethylidene]bis-phosphonat zoledronate Novartis

Fig. 2.2-2 (continued)

Many bisphosphonates have been investigated. Each has its characteristic profile of activity.

own

The P ~ C ~ P bonds of the bisphosphonates are stable to heat and most *chemical reagents, and completely resistant to enzymatic hydrolysis, but can be hydrolyzed in solution by ultraviolet light. These compounds have a strong affinity for metal ions, among them calcium, with which they can form both soluble and insoluble complexes and aggregates, depending on *-

32

2.2. Chemistry the pH of the solution and the metal present. This can occur in vivo when large amounts are infused rapidly, so that great care has to be taken when these compounds are given intravenously. Some uncertainty still exists as to the state of bisphosphonates when in solution. In plasma they are only partially ultrafilterable, because of binding to proteins and possibly of other causes. The ultrafilterability varies greatly between compounds. This is of importance when renal clearance is calculated.

Bisphosphonates are resistant to chemical and enzymatic hydrolysis. They can l:orm insoluble complexes with metal ions.

Recommended selected reading Reviews Blomen, L. J. M. J. (1995). History of bisphosphonates: Discovery and history of the nonmedical uses of bisphosphonates. In Bijvoet, O. L. M., Fleisch, H. A., Canfield, R. E., and Russell, R. G. G. (eds.) Bisphosphonate on Bones, pp. 111-124. (Amsterdam: Elsevier)

Original articles Menschutkin, N. (1865). Ueber die Einwirkung des Chloracetyls auf phosphorige SZiure. Ann. Chem. Pharm., 133,317-320

33

Adverse events p. 168 Ultrafilterability p. 57 Renal clearance p. 59

2.3. ACTIONS 2.3.1.

Physicochemical effects

The physicochemical effects of many of the bisphosphonates are very similar to those of pyrophosphate. Thus, they inhibit the formation, delay the aggregation, and also slow down the dissolution of calcium phosphate crystals. All these effects are related to the marked affinity of these compounds for solid-phase calcium phosphate, on the surface of which they bind strongly. This property is of great importance, because it is the basis for the use of these compounds as skeletal markers in nuclear medicine and the basis for their selective localization in bone when used as drugs.

Effects on calcium phosphate 9Bind stronglyto crystals 9Inhibit crystal formation 9Inhibit crystal aggregation 9Inhibit crystal dissolution

Fig. 2.3-1 Physicochemical effects of bisphosphonates on calcium phosphate.

Bisphosphonates also inhibit the formation and the aggregation of calcium oxalate crystals.

Bisphosphonates bind avidly to calcium phosphate crystals and inhibit their growth, aggregation, and dissolution. The affinity for bone mineral is the basis for their use as skeletal markers and as inhibitors of ectopic calcification and of bone resorption. 2.3.2.

Biological effects

Inhibition o f bone resorption The main effect of the pharmacologically active bisphosphonates is to inhibit bone resorption. Indeed these compounds proved to be extremely powerful inhibitors of resorption when tested in a variety of conditions, both in vitro and in vivo.

In vitro Bisphosphonates block bone resorption induced by various means in cell and organ culture. In the former, they inhibit the formation of pits by isolated osteoclasts cultured on mineralized substrata. In organ culture they

34

2.3. A c t i o n s

decrease the destruction of bone in embryonic long bones and in neonatal calvaria. This inhibition is present whether resorption is stimulated or not. Up to now, the effect of all the stimulators of bone resorption, such as parathyroid hormone, 1,25(OH)2 D, and prostaglandins, as well as the products of tumor cells, has been inhibited. Fig. 2.3-2 Effectof bisphosphonates on resorption of rat calvaria in culture, assayed by 4sCa release. Open circles, etidronate; filled circles, clodronate. [Adapted from Reynolds, J. J., et al. (1972). Reproduced from Calcified Tissue Res., 10, 302-313, with copyright permission from the author and SpringerVerlag, Heidelberg.]

Resorption of calvaria Bisphosphonate ~tg P/ml 0.007 l

0.12 I

I

2 I

8 16

a

I

i

v 9 4-~

90

~

80

~

70

~

60

v

o " onate

Clodronate

In the past, the correlation between the results obtained in calvaria in vitro and those obtained in v i v o was rather poor. However, a more recent study performed with nine compounds varying in their activity by 5 - 6 orders of magnitude showed a satisfactory correlation using the 4 - 7 day-old mouse calvaria assay. Fig. 2.3-3 Inhibitory activity of various bisphosphonates in vitro in mouse calvariae and in vivo in the TPTX rat. [Adapted from Green, J. R., et al. (1994), J. Bone Miner. Res. 9, 745751, with permission from the author and publisher.]

Bisphosphonates in o

. ,~

1

0

Neri ronate. Pamidrona~.~" Alendronat~Olpadronate Risedronate~_ _ ~ 9Ibandronate

~ ~

--1"

0 ~

--2"

0

~

vitro and in vivo

-3- 7

~

-2

mClodronate

9Zoledronate 0 2 Log EDs0 TPTX assay in vivo

35

4

2. Bisphosphonatesmpreclinical Bisphosphonates inhibit bone resorption in cell and organ culture. Intact animals In growing rats, bisphosphonates can block the degradation of both primary and secondary trabeculae, thus arresting the modeling and remodeling of the metaphysis. The latter therefore becomes club-shaped and radiologically more dense than normal, leading to a picture similar to that seen in congenital osteopetrotic animals. This effect is often used as an experimental assay to estimate the potency of new compounds.

Inhibition of bone resorption Control

With clodronate

Bone resorption

Bone resorption blocked

Fig. 2.3-4 Inhibition of metaphyseal modeling and remodeling by a bisphosphonate in the growing rat. Upper panel: Diagram of the locations of bone resorption in the rat tibia during longitudinal growth (left): osteoclasts resorb calcified cartilage (1), subperiosteal bone (2), and primary spongiosa (3), therefore enlarging the marrow cavity. Effect of clodronate (right). Lower panel: Microradiograph of a normal tibia (left) and of a bone from an animal treated with clodronate (right). [Adapted from Schenk, R. K., et al. (1973). Reproduced from Calcified Tissue Res., 11, 196-214, with copyright permission from the author and Springer-Verlag, Heidelberg.] 36

2.3. Actions The inhibition of bone r e s o r p t i o n by b i s p h o s p h o n a t e s has also been d o c u m e n t e d using 4s Ca kinetic studies and h y d r o x y p r o l i n e excretion, as well as by other means. The effect occurs within 2 4 - 4 8 h and is therefore slower than that of calcitonin. Fig. 2.3-5 Inhibition of bone resorption by subcutaneous (sc) administration of clodronate in the rat, as assessed by 45Ca kinetics.

Effect of clodronate on bone resorption 60+1

40-

k)

0

"= 200

r 0

0.01

0.1

1

1.0

Clodronate (mg P/kg/day sc)

Fig. 2.3-6 Effect of one injection of 0.1 mg P/kg subcutaneously of alendronate on bone resorption, assessed by monitoring the urinary excretion of radioactive tetracycline from prelabeled rats. [Adapted from Miihlbauer, R. C., and Fleisch, H. (1990). A method for continuing monitoring of bone resorption in rats: Evidence for a diurnal rhythm. Am. J. Physiol., 259, R679-R689, with permission from the authors and publisher.]

Inhibition of bone resorption Alendronate

+i o 1-o

o 100 o

.,..~

.,.~

~

O-

I

2

I

I

4

I

I

6 Days

I

I

8

Bisphosphonates inhibit bone resorption in intact animals.

37

' 1'0

2. Bisphosphonates--preclinical

Calcium balance pp. 15-16

Coupling p. 15

Bisphosphonates in osteoporosis models pp. 133-136

The decrease in resorption is accompanied, at least in the growing animal, by a positive calcium balance, and an increase in the mineral content of bone and in bone mass. This is possible because of an increase in intestinal calcium absorption, consequent to an elevation of 1,25(OH)2 D. The increase is, however, smaller than expected considering the dramatic decrease in bone resorption. This is due to the fact that, after a certain § time, bone formation also decreases, because of the so-called coupling between formation and resorption characteristic of a decrease in remodeling and therefore of turnover. The main effect of bisphosphonates is therefore a reduction in bone turnover. It is not known how long the increase in balance lasts after discontinuation of the bisphosphonate. This increase is the basis for the administration of these compounds to prevent and treat osteoporosis in humans. Fig. 2.3-7 Effect of 1 Ftg P/kg sc daily of ibandronate on calcium metabolism in the rat. Bone resorption was decreased and calcium balance increased. [Data from Fleisch, H. (1996)].

Calcium metabolism I-7 Controls ~! Ibandronate l~tgP/kg sc daily 50 40 +1

-I-

30

"

eq

20

210

Bone resorption

Intestinal absorption

Balance

Bisphosphonates increase calcium balance in normal growing animals.

Mechanostat pp. 1 9 - 2 0

Less is known about the effect in the normal adult animal. In dogs and minipigs, the long-term administration of alendronate did not lead to an increase in bone mass. This might be explained by the physiological biomechanical homeostasis of bone structure, which would eliminate a biomechanically unnecessary excess of bone. This fact suggests that the fear of the dangers of long-term use of therapeutic doses may not be warranted. Some studies have addressed the question of the effect of the bisphosphonates on the mechanical properties of the skeleton. This issue is of importance, since it is known that a long-lasting, strong inhibition of bone 38

2.3. Actions

resorption can lead to increased bone fragility both in animals and in humans. This is well illustrated in the human osteopetrosis described by Albers-Sch6nberg. Many studies have shown that clinically relevant doses of bisphosphonates have a positive effect on mechanical characteristics in various experimental osteoporosis models. In normal animals there is either a positive effect or no effect, but not negative. Bisphosphonates proven to have this action include alendronate, clodronate, etidronate, ibandronate, incadronate, olpadronate, pamidronate, tiludronate, minodronate, and zoledronate. However, under very high and long-term, clinically not relevant dosage, increased fractures have been reported in the dog given clodronate. Etidronate at high doses may also induce an opposite effect, probably because of an inhibition of mineralization.

Effect in experiment~ osteoporosi~ p. 134 Chemistry c bisphosphonates pp. 31-32

Animals with experimentally increased bone resorption Bisphosphonates can also prevent an experimentally induced increase in bone resorption. Thus, they impair resorption induced by many bone resorbing agents such as parathyroid hormone, 1,25(OH)2 D and retinoids, the latter effect having been used to develop a powerful and rapid screening assay for new compounds. --~ Fig. 2.3-8 Assessmentof the action of inhibitors of bone resorption by means of retinoid-induced hypercalcemia in thyroparathyroidectomized rats.

Retinoid

E 10-

. . ~

O O

E

assay

No effect

8-

.- 100% ~

6-

~ ~

Maximal

4I

0

I

I

Days

- - B..Retinoidalone

t

Inhibition

I

3 ~ Retinoid + bisphosphonate

They also inhibit bone loss induced by different procedures such as immobilization, ovariectomy, corticosteroids, or lactation combined with a low calcium diet. This aspect will be discussed in the chapter on osteoporosis in Section 3.5. Bisphosphonates also inhibit bone resorption induced experimentally by implantation of various t u m o r cells. They reduce both the local destruction of bone near the invading tumor cells, as well as the resorption induced by systemically circulating factors. These effects lead to a partial 39

Osteoporos models pp. 133-13

Effect on experiment: tumors pp. 95-97

2. Bisphosphonatesmpreclinical Tumor bone disease p. 95

or total prevention of hypercalcemia and hypercalciuria. This effect is the basis for their use in tumor-related bone disease and is discussed in Section 3.3. Of interest in the dental field is the fact that they also slow down periodontal bone destruction in rats susceptible to periodontal disease and in experimental periodontitis in monkeys. Furthermore, they inhibit tooth movement induced by orthodontic procedures, and these effects can be achieved even when the compounds are administered topically.

Bisphosphonates very efficiently prevent experimentally induced bone resorption. Relative activity of bisphosphonates The activity of bisphosphonates on bone resorption varies greatly from compound to compound. For etidronate, the dose required to inhibit resorption is relatively high, in the rat above 1 mg/kg parenterally per day. This dose is very near that which impairs normal mineralization. One of the aims of bisphosphonate research has therefore been to develop compounds with a more powerful antiresorptive activity, without a stronger inhibition of mineralization. This has proven to be possible. Clodronate was already more potent than etidronate, and pamidronate was found to be even more active. In more recent years, compounds have been developed that are up to 10,000 times more powerful than etidronate in the inhibition of bone resorption in experimental animals without being more active in inhibiting mineralization.

Potency to inhibit bone resorption

Chemistry of

-lx

--lOx

Etidronate

Clodronate Neridronate Alendronate Tiludronate Pamidronate EB-1053 Incadronate Olpadronate

-100 x

>100 1000 10 000 •

Ibandronate Minodronate Risedronate Zoledronate

bisphosphonates pp. 31-32

Fig. 2.3-9 Potencyof some bisphosphonates to inhibit bone resorption in the rat. The compounds in each column are listed in alphabetical order. For the development of future compounds it is of relevance that, so far, the potency evaluated in the rat corresponds quite well with that found in humans, at least with respect to their relative place in the scale of potency. However, the difference of activity between the least and the most potent compound is less in humans and depends on the disease for which the compounds are used. It is much smaller for osteoporosis, less so for Paget's disease and tumor-induced hypercalcemia.

40

2.3. Actions The potency of different bisphosphonates on bone resorption varies from 1 for etidronate to approximately 10,000 for zoledronate in the rat, about 10 or more times less in humans. There is quite a good correlation between the potencies found in the rat and those in humans as to their place in the scale. At present, the structural requirements for activity are only partially defined. The length of the aliphatic carbon chain is important, the effect on bone resorption increasing and then decreasing again with increasing chain length. Adding a hydroxyl group to the carbon atom at position 1 increases potency, and compounds with a nitrogen atom in the side chains are more active. The first compound of the latter kind to be described, pamidronate, has an amino group at the end of the alkyl chain. When the chain is altered in its length, the highest activity is present with a backbone of four carbons, as seen in alendronate. A primary amine is not necessary for this activity, as dimethylation of the amino nitrogen of pamidronate, as seen in olpadronate, increases potency. The latter can still be further increased when other groups are added to the nitrogen, as is the case, for example, in ibandronate, [1-hydroxy-3(methylpentylamino)propylidene]bisphosphonate, which is extremely potent. Geminal bisphosphonates containing cyclic substituents are also very potent, especially those containing a nitrogen atom in the ring, such as risedronate. The most active compounds described so far, zoledronate and minodronate, belong to this class and contain an imidazole ring. It must be noted that at present, all effective compounds have a P - - C - - P structure, which appears to be a prerequisite for activity. The intensity of the effect is, however, also dependent on the side chain. A threedimensional structural requirement appears to be involved. Indeed stereoisomers of the same chemical structure have shown 10-fold differences in activity. This opens the possibility of binding onto some kind of receptor.

No clear structure-effect relationship has yet emerged. The binding to the mineral appears to be due to the P m C m P structure, while the antiresorptive activity is influenced both by the P m C ~ P part and by the structure of the side chains, therefore by the threedimensional structure.

Mechanisms of action Our understanding of the mode of action of the bisphosphonates has made great progress in the last few years. There is no doubt that the action in vivo is mediated mostly, if not completely, through mechanisms other than the physicochemical inhibition of crystal dissolution, as was initially

41

Chemistry of bisphosphonates pp. 31-32

2. Bisphosphonates--preclinical postulated. However, the exact nature of these mechanisms is still not entirely unraveled. It may well be that several mechanisms are operating simultaneously.

The action of bisphosphonates on b o n e resorption is not mediated, as thought earlier, by their pbysicochemical effect on crystal dissolution, but mostly if not entirely through cellular mechanisms.

Osteoporosis p. 127

The mechanism of action of the bisphosphonates can be considered at three levels which are, however, tightly linked one to another: At the tissue level their main effect is a decrease in bone turnover, which is secondary to the inhibition of bone resorption. This effect is due to a decrease in the number and the activity of osteoclasts destroying bone, which leads to a decrease in the number of new BMUs. Since bone loss is intimately linked to turnover in diseases like tumor-induced bone disease and osteoporosis, this loss will be reduced by the bisphosphonates. Furthermore the bisphosphonates act to a certain extent at the individual BMU level by decreasing the depth of the resorption site. Since the amount of new bone formed in the BMU is not decreased, but possibly even increased, the local and consequently the whole body bone balance will be less negative or possibly sometimes even positive.

Bisphosphonates decrease bone turnover and therefore bone loss. At the cellular level four mechanisms appear to be possibly involved: (1) inhibition of osteoclast recruitment; (2) possibly inhibition of osteoclastic adhesion; (3) shortening of the life span of osteoclasts due to earlier apoptosis; (4) inhibition of osteoclast activity. The former three will lead to a decrease in the number of osteoclasts which is usually seen after treatment. The fourth will lead to inactive osteoclasts.

Bisphosphonates decrease the number of osteoclasts by inhibiting the recruitment and activating apoptosis. Furthermore they inhibit osteoclast activity.

Effect on mevalonate pathway p. 45

At the molecular level, the low concentrations necessary for activity suggests some sort of "pocket" which induces a cellular transduction mechanism. This site could be either on the cell membrane or within the cell and might be an enzyme, a pump, or some other intracellular protein involved in the signaling cascade. As discussed below, one of them could be farnesyl pyrophosphate synthase, which has recently been found to be inhibited by N-containing bisphosphonates and responsible for at least some of their effects.

42

2.3. Actions Direct effect The bisphosphonates can influence osteoclasts either directly as a result of their cellular binding or intracellular uptake, as well as indirectly via other cells. The direct effects are made possible by the uptake of these compounds by the osteoclasts during the resorption process, a process favored by the fact that the bisphosphonates also deposit preferentially under the osteoclasts where they can attain very high concentrations, in the range of 10 -4 M or higher. Fig. 2.3-10 Directmechanism of action. The osteoclast is inhibited directly after having taken up bisphosphonate from bone.

Direct effect on osteoclast

Active

Osteoclast

Inactive

Bisphosphonate Bone

Bisphosphonates may directly affect the osteoclast when it dissolves bisphosphonate-coated mineral, or when they deposit under the osteoclasts. The direct effects of the bisphosphonates are many. Thus it has been known for a long time that they alter the morphology of osteoclasts both in vitro and in vivo. The changes are numerous and include alterations in the cytoskeleton, among others in actin and vinculin as well as disruption of the ruffled border (Fig. 2.3-11).

Bisphosphonates induce morphological changes in the osteoclasts both in vitro and in vivo. A great number of different biochemical effects on various cell types have been described in vitro, but only few data exist on the osteoclasts themselves. Some of the changes which are possibly relevant to bone resorption, are reduction in lactic acid production, proton secretion, lysosomal enzyme activity, and prostaglandin synthesis. Experiments on osteoclasts themselves have shown an inhibition of the acid extrusion performed by a sodium-independent mechanism, and of the vacuolar-type proton ATPase present in the ruffled border. An inhibition of certain protein tyrosine phosphatases (PTPase), namely PTPase e and o, has also been described. But unfortunately, in many cases there is no structure-effect

43

Deposition i bone pp. 57-58

2. Bisphosphonates--preclinical Fig. 2.3-11 An osteoclast of a normal rat (above) and of an animal treated with a bisphosphonate (below). (Courtesy of Dr. R. K. Schenk.)

Effect on osteoclasts

,.,~' , ...~

D

correlation between these effects in vitro and those on bone resorption in vivo.

Bisphosphonates induce in vitro many biochemical and enzymatic changes, some of them decreasing acid production. This might be one of the causes o1:decreased bone destruction. Very recently it was found that nitrogen containing bisphosphonates *-. can inhibit the mevalonate pathway, by inhibiting farnesyl pyrophosphate synthase. This inhibition is due to the docking of the bisphosphonates into the pyrophosphate Mg 2+ binding site of the enzyme, while the charged side chains act as inhibiting transition state analogs. This leads to a decrease of the formation of isoprenoid lipids such as farnesyl- and geranylgeranylpyrophosphates. These are required for the post-translational prenylation (transfer of fatty acid chains) of proteins, including the GTP-binding proteins Ras, Rho, Rac, and Rab. These proteins are important for many cell functions, including cytoskeletal assembly and intracellular signaling. Therefore, disruption of their activity will induce a series of changes lead44

2.3. Actions ing to decreased activity, probably the main effect, and to earlier apoptosis in several cell types, including osteoclasts. In osteoclasts the lack of geranylgeranylpyrophosphate is probably responsible for the effects. Fig. 2.3-1.2 Effectof bisphosphonates on the mevalonate pathway.

The mevalonate pathway HMG-CoA ~ ~ mevalonate

Statins

C10 geranylpyrophosphate ,,FarnesylPP ....SYnthase I ~ ~ ~ i"Bisph~176 squalene ~ C15 farnesylpyrophosphate~Farnesylated proteins ~ Ras, Rho, Rac, Rab, Lamins cholestrol C20 geranylgeranylpyrophosphate--~ geranylgeranylated proteins

Some bisphosphonates inhibit the mevalonate pathway which can lead to altered cell activity and apoptosis. It was also shown recently that some non-nitrogen-containing bisphosphonates that closely resemble pyrophosphate, such as etidronate, tiludronate, and clodronate, can be incorporated into the phosphate chain of ATP-containing compounds so that they become nonhydrolyzable. The new P m C - - P containing ATP analogs inhibit cell function and may lead to apoptosis and cell death. Thus, the bisphosphonates can be classified into two major groups with different modes of action. The latter may explain the various cellular changes described above (Fig. 2.3-13).

The bisphosphonates can be divided into two groups. The nitrogencontaining compounds can inhibit the mevalonate pathway and hence inhibit protein prenylation. Others, like etidronate, clodronate, and tiludronate, can be incorporated in ATP-containing compounds. Both may act by inhibiting cell function and inducing apoptosis.

Indirect effect through other cells It is likely that bisphosphonates act, at least in part, also through other cells. One candidate is the osteoblast. It is now generally accepted that 45

2. Bisphosphonates--preclinical Molecular mechanisms of action of bisphosphonates Risedronate Zoledronate Ibandronate Alendronate Pamidronate

Clodronate Etidronate Tiludronate

Incorporated into intracellular analogues of A TP NH2 0 C1 0 0 " o,p,~/l'._..P, .

6_ o 6 _ o

C1

~

'{)',,,__.~_

HO OH N

o

o

0-6.

Inhibit the prenylationand function of GTP-bindingproteins required for osteoclastformation, function and survival

NNI~~q < O' N ~ .I

NH2 N

o

o O 6_o OH

Fig. 2.3-13

HO OH The two classes of bisphosphonates. Courtesy of Dr. M. Rogers.

cells of osteoblastic lineage control the recruitment and activity of osteoclasts. One of the modulators involved in this mechanism appear to be the bisphosphonates. Indeed these compounds induce the osteoblasts to synthesize inhibitor(s) for osteoclast recruitment and therefore of bone resorption. Fig. 2.3-14 Indirect mechanism of action. The inhibition of the osteoclast is secondary to an increase in production by osteoblast lineage cells of an inhibitor(s) of osteoclast recruitment. The primary target is therefore the osteoblastic cell type.

Effect t h r o u g h o s t e o b l a s t

~ Recruitment

Osteoclast

Osteoblasts

Bone

Bisphosphonates also inhibit osteoclasts by stimulating the secretion of an inhibitor(s) of osteoclast recruitment by osteoblast lineage cells. Therefore the target cells may include members of the osteoblastic cell family.

46

2.3. Actions Another candidate target cell population are the macrophages which release many cytokines which are able to modulate the osteoclasts, and which are influenced by the bisphosphonates. Thus, under certain conditions, bisphosphonates inhibit their release of IL-113, IL-6, and TNFa in vitro. Alternatively, at high concentrations such as after intravenous administration, the release of these cytokines can be stimulated, producing an acute phase reaction. It is not known at present to which extent these mechanisms, the direct effect on the osteoclast or indirect action through osteoblastic or other cells, are operating in vivo and, if both do, which of the two is more important.

Acute phase reaction p. 173

It is not yet known which of the mechanisms, the direct effect on the osteoclast or indirect action through the osteoblastic cells, is more important in vivo.

Fig. 2.3-15 Summary of the effects of bisphosphonates on the osteoclast.

Mode of action of bisphosphonates Binding to apatite crystals Preferential accumulation under osteoclasts ~b Local release during bone resorption .

.

.

[ '~ osteoclast activity ! Decreased ruffled border Altered cytoskeleton Decreased acid production Decreased enzyme activity Decreased prenylation Incorporation into nucleotides

.

.

] ~osteoclast nu, number[ Increased apoptosis Decreased recruitment Osteoblast lineage cells

In view of the large array of their effects on cells, it is surprising that the bisphosphonates act almost exclusively on calcified tissues. This selectivity is explained by the strong affinity of these compounds for calcium phosphate, which allows them to be cleared very rapidly from blood and to be incorporated into calcified tissues, especially bone.

Bisphosphonates act specifically on bone, because of their affinity for bone mineral.

47

Pharmacokinetics p. 57

2. Bisphosphonates--preclinical I n h i b i t i o n o/r m i n e r a l i z a t i o n Ectopic

mineralization

Like pyrophosphate, bisphosphonates inhibit calcification in vivo very efficiently. Thus, they prevent experimentally induced calcification of many soft tissues such as arteries, kidneys, and skin. In contrast to pyrophosphate, which acts only when given parenterally, they are also active when administered orally. In the arteries they decrease not only mineral deposition, but also the accumulation of cholesterol, elastin, and collagen.

Experimental aortic calcification ~-80+l e 60-

fl

>-. r

40-

Fig. 2.3-16 Effectof 10 mg P/kg body weight of bisphosphonates on vitamin D3-induced aortic calcification in the rat. Subcutaneous (sc) compared with oral (po)administration. [Adapted from Fleisch, H., et al. (1970). Reproduced with permission from the author and the publisher.]

era =I. o

=oa 2 0 ka

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

Control Vit.D3 Vit.D3 Vit.D3 Etidronate Clodronate sc po sc po

Clinical use in ectopic calcification and ossification pp. 162-164 Human urolithiasis p. 162

A possibly interesting future use of bisphosphonates could arise from the finding that some of them, such as etidronate, can also inhibit the calcification of bioprosthetic heart valves, either when administered subcutaneously or when released locally from various matrices. Investigations are in progress to bind bisphosphonates covalently to the valves. Etidronate also inhibits ectopic ossification, when given either systemically or locally. This effect has led to the clinical use of etidronate in ectopic ossification, but unfortunately normal mineralization is inhibited as well. Similarly, certain bisphosphonates such as etidronate decrease the formation of experimental urinary stones. However the active dose also leads to the inhibition of normal mineralization of bone. Lastly, topical administration of etidronate induces a decrease in the formation of dental calculus, a property which is made use of in some toothpastes.

48

2.3. Actions Bisphosphonates in vivo inhibit experimentally induced soft tissue calcification and ossification, urinary stones, and dental calculus. Normal mineralization "+ The dose, at least of etidronate, which inhibits experimental ectopic mineralization, also impairs the mineralization of normal calcified tissues such as bone, cartilage, dentine, and enamel. The amount required to have this effect varies according to the animal species and the length of treatment. In contrast to bone resorption, where the different compounds vary greatly in their activity, this does not seem to be so much the case for the inhibition of mineralization. For most species, and most compounds, the effective daily parenteral dose is in the order of 1-10 mg of compound phosphorus per kilogram. Interestingly, clodronate inhibits normal mineralization somewhat less than does etidronate, despite the fact that it is more active on bone resorption. This may be due to the fact that clodronate has no hydroxyl side chain and is therefore less bound to the mineral. The inhibition of calcification with high doses can lead to fractures and to an impairment of fracture healing. The mineralization defect is eventually reversed after discontinuation of the drug. Nevertheless, the propensity to inhibit the mineralization of normal bone has hampered the therapeutic use of bisphosphonates in ectopic calcification. This is not the case for their use in bone resorption, since compounds have been developed that inhibit this process at doses at least 1000 times lower than those that inhibit mineralization. .~ 9 Fig. 2.3-17 Inhibitionof mineralization of bone and cartilage in the growing rat by 10 mg P/kg subcutaneously of etidronate, but not clodronate, daily for 7 days. (Courtesy of Dr. R. K. Schenk.)

Effect on growth plate

Bisphosphonates, if given at high doses, inhibit the mineralization of normal calcified tissues inducing rickets and osteomalacia. While this is a problem in humans when it is used to prevent ectopic calcification or ossification, it is not the case for most bisphosphonates when they are used to decrease bone resorption.

49

Adverse eve pp. 171-17

Heterotopic calcification and ossific, pp. 162-16

2. Bisphosphonates--preclinical Mechanisms of action in the inhibition of calcification Physicochemical effects p. 34

There is a close relationship between the ability of an individual bisphosphonate to inhibit the formation of calcium phosphate in vitro and its effectiveness on calcification in vivo, strongly suggesting that the latter can be explained in terms of a physicochemical mechanism. The inhibition of calcium phosphate formation is closely related to the affinity of the bisphosphonate to the solid-phase calcium phosphate. The binding can be bidentate through the two phosphates, as is the case for clodronate. Or it can be tridentate through a third moiety, such as a hydroxyl or a nitrogen attached to the carbon atom. This is the case for most bisphosphonates used clinically today. The third binding site increases the affinity and hence the inhibitory effect on calcification. However, additional effects on matrix formation, involving changes in glycosaminoglycan and collagen synthesis, may occur. These may be direct via cellular effects, or mediated indirectly by effects on crystals.

The inhibition of mineralization in vivo, both on normal and ectopic calcification, is most probably explained by a physicochemical mechanism involving the binding of the bisphosphonate on the surface of the mineral. Other effects

Use in osteoporosis p. 136

Another interesting finding is that several bisphosphonates, including clodronate, risedronate, and zoledronate, inhibit local bone and cartilage resorption, preserve the joint architecture, and decrease the inflammatory reaction in various types of experimental arthritis, such as that induced by Freund's adjuvant or by carrageenan. This effect is especially pronounced when the bisphosphonates are encapsulated in liposomes. Similar results were obtained with a new bisphosphonate, TRK-530, which furthermore inhibits splenomegaly and inflammation. Furthermore, clodronate and newer bisphosphonates also inhibit the delayed-type hypersensitivity granuloma response. Moreover, bisphosphonates or phosphonosulfonates linked to an isoprene chain are potent inhibitors of squalene synthase and hence are cholesterol-lowering agents in the animal. These results may open some interesting new therapeutic possibilities for these drugs. It is worth mentioning that very low concentrations of bisphosphonates were found to increase colony formation, nodule formation, mineralization, and osteocalcin synthesis in bone cell cultures in vitro. Furthermore, when administered in vivo, the amount of bone formed at each individual BMU is not decreased, but may possibly be somewhat increased. Lastly, since statins, which are inhibitors of the mevalonate pathway, increase bone formation at least in vitro, probably through an elevation of

50

2.3. A c t i o n s

BMP-2, it could be conceivable that the bisphosphonates have a similar action. Thus, it could be that bisphosphonates might, under certain conditions, increase bone formation in vivo. However, this still needs to be verified. Under some conditions, bisphosphonates may increase bone formation, at least in vitro. Whether this is true in vivo, needs confirmation.

Recommended selected reading Reviews Ebetino, F. H., Francis, M.D., Rogers, M. J., and Russell, R. G. G. (1998). Mechanisms of action of etidronate and other bisphosphonates. Rev. Contemp. Pharmacother., 9, 233243 Fleisch, H. (1998). Bisphosphonates: Mechanisms of action. Endocr. Rev., 19, 80-100 Rodan, G. A. (1998). Mechanisms of action of bisphosphonates. Annu. Rev. Pharmacol. Toxicol., 38, 375-388 Rodan, G. A. and Fleisch, H. A. (1996). Bisphosphonates: Mechanisms of action. J. Clin. Invest., 97, 2692-2696 Roldan, E. J. A., P6rez-Llore, A., and Ferretti, J. L. R. (1998). Olpadronate: A new aminobisphosphonate for the treatment of medical osteopathies. Exp. Opin. Invest. Drugs, 7, 1521-1538 Russell, R. G. G., Rogers, M. J., Frith, J. C., Luckman, S. P., Coxon, F. P., Benford, H. L., Croucher, P. I., Shipman, C., and Fleisch, H. A. (1999). The pharmacology of bisphosphonates and new insights into their mechanisms of action. J. Bone Miner. Res., 14(Suppl. 2), 53-65 O r i g i n a l articles Physical

chemistry

Fleisch, H., Russell, R. G. G., Bisaz, S., Miihlbauer, R. C., and Williams, D. A. (1970). The inhibitory effect of phosphonates on the formation of calcium phosphate crystals in vitro and on aortic and kidney calcification in vivo. Eur. J. Clin. Invest., 1, 12-18 Francis, M. D. (1969). The inhibition of calcium hydroxyapatite crystal growth by polyphosphonates and polyphosphates. Calcif. Tissue Res., 3, 151-162 Jung, A., Bisaz, S., and Fleisch, H. (1973). The binding of pyrophosphate and two diphosphonates by hydroxyapatite crystals. Calcif. Tissue Res., 11,269-280 Russell, R. G. G., M/ihlbauer, R. C., Bisaz, S., Williams, D. A., and Fleisch, H. (1970). The influence of pyrophosphate, condensed phosphates, phosphonates and other phosphate compounds on the dissolution of hydroxyapatite in vitro and on bone resorption induced by parathyroid hormone in tissue culture and in thyroparathyroidectomised rats. Calcif. Tissue Res., 6, 183-196

Bone resorption Adachi, H., Igarashi, K., Mitani, H., and Shinoda, H. (1994). Effects of topical administration of a bisphosphonate (risedronate) on orthodontic tooth movements in rats. J. Dent. Res., 73, 148-184 Balena, R., Markatos, A., Seedor, J. G., Gentile, M., Stark, C., Peter, C. P., and Rodan, G. A. (1996). Long-term safety of the aminobisphosphonate alendronate in adult dogs. II. 51

2. B i s p h o s p h o n a t e s ~ p r e c l i n i c a l Histomorphometric analysis of the L5 vertebrae. J. Pharmacol. Exp. Tber., 276,277-283 Ferretti, J. L., Cointry, G., Capozza, R., Montuori, E., Roldfin, E., and P~rez Lloret, A. (1990). Biomechanical effects of the full range of useful doses of (3-amino-l-hydroxy propylidene)-l,l-bisphosphonate (APD) on femur diaphyses and cortical bone tissue in rats. Bone Miner., 11,111-122 Ferretti, J. L., Mondelo, N., Capozza, R. F., Cointry, G. R., Zanchetta, J. R., and Montuori, E. (1995). Effects of large doses of olpadronate (dimethyl-pamidronate) on mineral density, cross-sectional architecture, and mechanical properties of rat femurs. Bone, 16 (Suppl. 4), 285S-293S Fleisch, H. (1996). The bisphosphonate ibandronate, given daily as well as discontinuously, decreases bone resorption and increases calcium retention as assessed by 4s Ca kinetics in the intact rat. Osteoporosis Int., 6, 166-170 Fleisch, H., Russell, R. G. G., and Francis, M. D. (1969). Diphosphonates inhibit hydroxy apatite dissolution in vitro and bone resorption in tissue culture and in vivo. Science, 165, 1262-1264 Gasser, A. B., Morgan, D. B., Fleisch, H. A., and Richelle, L. J. (1972). The influence of two diphosphonates on calcium metabolism in the rat. Clin. Sci., 43, 31-45 Geusens, P., Nijs, J., van der Perre, G., van Audekercke, R., Lowet, G., Goovaerts, S., Barbier, A., Lacheretz, F., Remandet, B., Jiang, Y., and Dequeker, J. (1992). Longitudinal effect of tiludronate on bone mineral density, resonant frequency, and strength in monkeys. J. Bone Miner. Res., 7, 599-609 Green, J. R., Miiller, K., and Jaeggi, K. A. (1994). Preclinical pharmacology of CGP 42'446, a new, potent, heterocyclic bisphosphonate compound. J. Bone Miner. Res., 9, 745-751 Guy, J. A., Shea, M., Peter, C. P., Morrissey, R., and Hayes, W. C. (1993). Continuous alendronate treatment throughout growth, maturation, and aging in the rat results in increases in bone mass and mechanical properties. Calcif. Tissue Int., 53,283-288 Igarashi, K., Adachi, H., Mitani, H., and Shinoda, H. (1996). Inhibitory effect of the topical administration of a bisphosphonate (risedronate) on root resorption incident to orthodontic tooth movements in rats. J. Dent. Res., 75, 1644-1659 Lafage, M. H., Balena, R., Battle, M. A., Shea, M., Seedor, J. G., Klein, H., Hayes, W. C., and Rodan, G. A. (1995). Comparison of alendronate and sodium fluoride effects on cancellous and cortical bones in minipigs. J. Clin. Invest., 95, 2127-2133 Miihlbauer, R. C., Bauss, F., Schenk, R., Janner, M., Bosies, E., Strein, K., and Fleisch, H. (1991). BM 21.0955, a potent new bisphosphonate to inhibit bone resorption. J. Bone Miner. Res., 6, 1003-1011 Peter, C. P., Guy, J., Shea, M., Bagdon, W., Kline, W. F., and Hayes, W. C. (1996). Longterm safety of the aminobisphosphonate alendronate in adult dogs. I. General safety and biomechanical properties of bone. J. Pharmacol. Exp. Tber., 276, 271-276 Reitsma, P. H., Bijvoet, O. L. M., Verlinden-Ooms, H., and van der Wee-Pals, L. J. A. (1980). Kinetic studies of bone and mineral metabolism during treatment with (3amino-l-hydroxypropylidene)-l,l-bisphosphonate (ADP) in rats. Calcif. Tissue Int., 32, 145-157 Reynolds, J. J., Minkin, C., Morgan, D. B., Spycher, D., and Fleisch, H. (1972). The effect of two diphosphonates on the resorption of mouse calvaria in vitro. Calcif. Tissue Res., 10, 302-313 Reynolds, J. J., Murphy, H., Miihlbauer, R. C., Morgan, D. B., and Fleisch, H. (1973). Inhibition by diphosphonates of bone resorption in mice and comparison with grey-lethal osteopetrosis. Calcif. Tissue Res., 12, 59-71 Russell, R. G. G., Miihlbauer, R. C., Bisaz, S., Williams, D. A., and Fleisch, H. (1970). The influence of pyrophosphate, condensed phosphates, phosphonates and other phosphate compounds on the dissolution of hydroxyapatite in vitro and on bone resorption induced by parathyroid hormone in tissue culture and in thyroparathyroidectomised rats. Calcif. Tissue Res., 6, 183-196 Schenk, R., Eggli, P., Fleisch, H., and Rosini, S. (1986). Quantitative morphometric evaluation of the inhibitory activity of new aminobisphosphonates on bone resorption in the rat. Calcif. Tissue Int., 38,342-349 Shoji, K., Horiuchi, H., and Shinoda, H. (1995). Inhibitory effects of a bisphosphonate (risedronate) on experimental periodontitis in rats. J. Periodont. Res., 30, 27-84 Trechsel, U., Stutzer, A., and Fleisch, H. (1987). Hypercalcemia induced with an arotinoid

52

2.3. Actions in thyroparathyroidectomized rats. New model to study bone resorption in vivo. J. Clin. Invest., 80, 1679-1686

van der Pluijm, G., Binderup, L., Bramm, E., van der Wee-Pals, L., de Groot, H., Binderup, E., L6wik, C., and Papapoulos, S. (1992). Disodium 1-hydroxy-3-(1-pyrrolidinyl)propylidene-l,l-bisphosphonate (EB-1053) is a potent inhibitor of bone resorption in vitro and in vivo. J. Bone Miner. Res., 7, 981-986

Mechanisms of resorption van Beek, E. R., L6wik, C. W. G. M., Ebetino, F. H., and Papapoulos, S. E. (1998). Binding and antiresorptive properties of heterocycle-containing bisphosphonate analogs: Structure-activity relationships. Bone, 23, 437-442 van Beek, E., L6wik, C. van der Pluijm, G., and Papapoulos, S. (1999). The role of geranylgeranylation in bone resorption and its suppression by bisphosphonates in fetal bone explants in vitro: A clue to the mechanism of action of nitrogen-containing bisphosphonates. J. Bone Miner. Res., 14, 722-729 van Beek, E., Pieterman, E., Cohen, L., L6wik, C., and Papapoulos, S. (1999). Nitrogencontaining bisphosphonates inhibit isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase activity with relative potencies corresponding to their antiresorptive potencies in vitro and in vivo. Biochem. Biophys. Res. Commun., 255,491-494 van Beek, E., Pieterman, E., Cohen, L., L6wik, C., and Papapoulos, S. (1999). Farnesyl pyrophosphate synthase is the molecular target of nitrogen-containing bisphosphonates. Biochem. Biophys. Res. Commun., 264, 108-111 Boonekamp, P. M., van der Wee-Pals, L. J. A., van Wijk-van Lennep, M. M. L., Thesing, C. W., and Bijvoet, O. L. M. (1986). Two modes of action of bisphosphonates on osteoclastic resorption of mineralized matrix. Bone Miner., 1, 27-39 Carano, A., Teitelbaum, S. L., Konsek, J. D., Schlesinger, P. H., and Blair, H. C. (1990). Bisphosphonates directly inhibit the bone resorption activity of isolated avian osteoclasts in vitro. J. Clin. Invest., 85,456-461 Cecchini, M. G., Felix, R., Fleisch, H., and Cooper, P. H. (1987). Effects of bisphosphonates on proliferation and viability of mouse bone marrow-derived macrophages. J. Bone Miner. Res., 2, 135-142 Colucci, S., Minielli, V., Zambonin, G., Cirulli, N., Mori, G., Serra, M., Patella, V., Zambonin Zallone, A., and Grano, M. (1998). Alendronate reduces adhesion of human osteoclast-like cells to bone and bone protein-coated surfaces. Calc. Tissue Int., 63, 230-235 David, P., Nguyen, H., Barbier, A., and Baron, R. (1996) The bisphosphonate tiludronate is a potent inhibitor of the osteoclast vacuolar H+-ATPase. J. Bone Miner. Res., 11, 1498-1507 Endo, N., Rutledge, S. J., Opas, E. E., Vogel, R., Rodan, G. A., and Schmidt, A. (1996). Human protein tyrosine phosphatase-o: Alternative splicing and inhibition by bisphosphonates. J. Bone Miner. Res., 11,535-543 Fast, D. K., Felix, R., Dowse, C., Neuman, W. F., and Fleisch, H. (1978). The effects of diphosphonates on the growth and glycolysis of connective-tissue cells in culture. Biochem. J., 172, 97-107 Felix, R., Russell, R. G. G., and Fleisch, H. (1976). The effect of several diphosphonates on acid phosphohydrolases and other lysosomal enzymes. Biochim. Biophys. Acta, 429, 429-438 Felix, R., Bettex, J. D., and Fleisch, H. (1981). Effect of diphosphonates on the synthesis of prostaglandins in cultured calvaria cells. Calcif. Tissue Int., 33,549-552 Fisher, J. E., Rogers, M. J., Halasy, J. M., Luckman, S. P., Hughes, D. E., Masarachia, P. J., Wesolowski, G., Russell, R. G. G., Rodan, G. A., and Reszka, A. A. (1999). Alendronate mechanism of action: Gernylgeraniol, an intermediate in the mevalonate pathway, prevents inhibition of osteoclast formation, bone resorption, and kinase activation in vitro. Proc. Natl. Acad. Sci. U.S.A., 96, 133-138 Flanagan, A. M., and Chambers, T. J. (1989). Dichloromethylenebisphosphonate (C12MBP) inhibits bone resorption through injury to osteoclasts that resorb C12MBP-coated bone. Bone Miner., 6, 33-43

53

2. B i s p h o s p h o n a t e s - - p r e c l i n i c a l

Frith, J. C., M6nkk6nen, J., Blackburn, G. M., Russell, R. G. G., and Rogers, M. J. (1997). Clodronate and liposome-encapsulated clodronate are metabolised to a toxic ATP analog, adenosine5'([3,3/-dichloromethylene)triphosphate, by mammalian cells in vitro. J. Bone Miner. Res., 12, 1358-1367 Hughes, D. E., Wright, K. R., Uy, H. L., Sasaki, A., Yoneda, T., Roodman, G. D., Mundy, G. R., and Boyce, B. F. (1995). Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo. J. Bone Miner. Res., 10, 1478-1487 Luckman, S. P., Coxon, F. P., Ebetino, F. H., Russell, R. G. G., and Rogers, M. J. (1998). Heterocycle-containing bisphosphonates cause apoptosis and inhibit bone resorption by preventing protein prenylation: Evidence from structure-activity relationships in J774 macrophages. J. Bone Miner. Res., 13, 1668-1678 Luckman, S. P., Hughes, D. E., Coxon, F. P., Russell, R. G. G., and Rogers, M. J. (1998). Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent posttranslational prenylation of GTP-binding proteins, including Ras. J. Bone Miner. Res., 13,581-589 Martin, M. B., Arnold, W., Heath III, H. T., Urbina, J. A., and Oldfield, E. (1999). Nitrogencontaining bisphosphonates as carbocation transition state analogs for isoprenoid biosynthesis. Biochem. Biophys. Res. Commun., 263,754-758 Masarachia, P., Weinreb, M., Balena, R., and Rodan, G. A. (1996). Comparison of the distribution of 3H-alendronate and 3H-etidronate in rat and mouse bones. Bone, 19, 281-290 Murakami, H., Takahashi, N., Sasaki, T., Udagawa, N., Tanaka, S., Nakamura, I., Zhang, D., Barbier, A., and Suda, T. (1995). A possible mechanism of the specific action of bisphosphonates on osteoclasts: Tiludronate preferentially affects polarized osteoclasts having ruffled borders. Bone, 17, 137-144 Nishikawa, M., Akatsu, T., Katayama, Y., Yasutomo, Y., Kado, S., Kugai, N., Yamamoto, M., and Nagata, N. (1996). Bisphosphonates act on osteoblastic cells and inhibit osteoclast formation in mouse marrow cultures. Bone, 18, 9-14 Sahni, M., Guenther, H. L., Fleisch, H., Collin, P., and Martin, T. J. (1993). Bisphosphonates act on rat bone resorption through the mediation of osteoblasts. J. Clin. Invest., 91, 2004-2011 Sato, M., and Grasser, W. (1990). Effects of bisphosphonates on isolated rat osteoclasts as examined by reflected light microscopy. J. Bone Miner. Res., 5, 31-40 Sato, M., Grasser, W., Endo, N., Akins, R., Simmons, H., Thompson, D. D., Golub, E., and Rodan, G. A. (1991). Bisphosponate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure. J. Clin. Invest., 88, 2095-2105 Schmidt, A., Rutledge, S. J., Endo, N., Opas, E. E., Tanaka, H., Wesolowski, G., Leu, C. T., Huang, Z., Ramachandaran, C., Rodan, S. B., and Rodan, G. A. (1996). Protein-tyrosine phosphatase activity regulates osteoclast formation and function: Inhibition by alendronate. Proc. Natl. Acad. Sci. U.S.A., 93, 3068-3073 Vitt~, C., Fleisch, H., and Guenther, H. L. (1996). Bisphosphonates induce osteoblasts to secrete an inhibitor of osteoclast-mediated resorption. Endocrinology, 137, 2324-2333 Zimolo, Z., Wesolowski, G., and Rodan, G. A. (1995). Acid extrusion is induced by osteoclast attachment to bone: Inhibition by alendronate and calcitonin. J. Clin. Invest., 96, 2277-2283

Mineralization Briner, W. W., Francis, M. D., and Widder, J. S. (1971). The control of dental calculus in experimental animals. Int. Dent. J., 21, 61-73 Fleisch, H., Russell, R. G. G., Bisaz, S., Miihlbauer, R. C., and Williams, D. A. (1970). The inhibitory effect of phosphonates on the formation of calcium phosphate crystals in vitro and on aortic and kidney calcification in vivo. Eur. J. Clin. Invest., 1, 12-18 Francis, M. D., Russell, R. G. G., and Fleisch, H. (1969). Diphosphonates inhibit formation of calcium phosphate crystals in vitro and pathological calcification in vivo. Science, 165, 1264-1266 King, W. R., Francis, M. D., and Michael, W. R. (1971). Effect of disodium ethane-l-hydroxy-l,l-diphosphonate on bone formation. Clin. Orthop., 78, 251-270

54

2.3. A c t i o n s

Schenk, R., Merz, W. A., Miihlbauer, R., Russell, R. G. G., and Fleisch, H. (1973). Effect of ethane-l-hydro• (EHDP) and dichloromethylene diphosphonate (C12MDP) on the calcification and resorption of cartilage and bone in the tibial epiphysis and metaphysis of rats. Calcif. Tissue Res., 11,196-214 Shinoda, H., Adamek, G., Felix, R., Fleisch, H., Schenk, R., and Hagan, P. (1983). Structure-activity relationships of various bisphosphonates. Calcif. Tissue Int., 35, 8799 O t h e r effects Boyce, R. W., Paddock, C. L., Gleason, J. R., Sietsema, W. K., and Eriksen, E. F. (1995). The effects of risedronate on canine cancellous bone remodeling: Three-dimensional kinetic reconstruction of the remodeling site. J. Bone Miner. Res., 10, 211-221 Ciosek, C. P., Magnin, D. R., Harrity, T. W., Logan, J. V. H., Dickson, J. K., Gordon, E. M., Hamilton, K. A., Jolibois, K. G., Kunselman, L. K., Lawrence, R. M., Mookhtiar, K. A., Rich, L. C., Slusarchyk, D. A., Sulsky, R. B., and Biller, S. A. (1993). Lipophilic 1, 1-bisphosphonates are potent squalene synthase inhibitors and orally active cholesterol lowering agents in vivo. J. Biol. Chem., 268, 24832-24837 Dunn, C. J., Galinet, L. A., Wu, H., Nugent, R. A., Schlachter, S. T., Staite, N. D., Aspar, D. G., Elliott, G. A., Essani, N. A., Rohloff, N. A., and Smith, R. J. (1993). Demonstration of novel anti-arthritic and anti-inflammatory effects of diphosphonates. J. Pharmacol. Exp. Ther., 266, 1691-1698 Endo, Y., Nakamura, M., Kikuchi, T., Shinoda, H., Takeda, Y., Nitta, Y., and Kumagai, K. (1993). Aminoalkylbisphosphonates, potent inhibitors of bone resorption, induce a prolonged stimulation of histamine synthesis and increase machrophages, granulocytes, and osteoclasts in vivo. Calcif. Tissue Int., 52, 248-254 Francis, M. D., Hovancik, K., and Boyce, R. W. (1989). NE-58095: A diphosphonate which prevents bone erosion and preserves joint architecture in experimental arthritis. Int. J. Tissue React., 11,239-252 Giuliani, N., Pedrazzoni, M., Negri, G., Passeri, G., Impicciatore, M., and Girasole, G. (1998). Bisphosphonates stimulate formation of osteoblast precursors and mineralized nodules in murine and human bone marrow cultures in vitro and promote early osteoblastogenesis in young and aged mice in vivo. Bone, 22, 455-461 Goziotis, A., Sukhu, B., Torontali, M., Dowhaniuk, M., and Tenenbaum, H. C. (1995). Effects of bisphosphonates APD and HEBP on bone metabolism in vitro. Bone, 16(Suppl.), 317S-327S Guenther, H. L., Guenther, H. E., and Fleisch, H. (1981). The effects of 1-hydroxyethane1, 1-diphosphonate and dichloromethanedisphosphonate on collagen synthesis by rabbit articular chondrocytes and rat bone cells. Biochem. J., 196, 293-301 Kinne, R. W., Schmidt-Weber, C. B., Hoppe, R., Buchner, E., Palombo-Kinne, E., Niirnberg, E., and Emmrich, F. (1995). Long-term amelioration of rat adjuvant arthritis following systemic elimination of macrophages by clodronate-containing liposomes. Arthritis Rheum., 38, 1777-1790 Osterman, T., Kippo, K., Laur8n, L., Hannuniemi, R., and Sellman, R. (1994). Effect of clodronate on established adjuvant arthritis. Rheumatol. Int., 14, 139-147 Tanahashi, M., Funaba, Y., Itoh, M., Kawabe, N., and Nakadate-Matsushita, T. (1998). Inhibitory effects of TRK-530 on rat adjuvant arthritis. Pharmacology, 56, 242-251 Tsuchimoto, M., Azuma, Y., Higuchi, O., Sugimoto, I., Hirata, N., Kiyoki, M., and Yamamoto, I. (1994). Alendronate modulates osteogenesis of human osteoblastic cells in vitro. Jpn. J. Pharmacol., 66, 25-33

55

2.4. PHARMACOKINETICS

dechanism of action p. 45

Bisphosphonates are synthetic compounds, which have not yet been found to occur naturally in animals or humans. No enzymes able to cleave the P - - C - - P bonds have been described. The bisphosphonates on which data have been published so far, appear to be absorbed, stored, and excreted unaltered from the body. Therefore, these bisphosphonates seem to be nonbiodegradable, in solution and in animals. However, it cannot be excluded that some bisphosphonates may be metabolized, especially in their side chains. Some of them may be incorporated in ATP-containing compounds. Bisphosphonates are not biodegradable, at least not at their P - - C m P bond.

Data from relatively few pharmacokinetic studies are available. Most of the published data have been obtained with alendronate, clodronate, etidronate, pamidronate, and tiludronate. 2.4.1.

Inhibition of lineralization pp. 171-172

Intestinal absorption

The bioavailability of an oral dose of a bisphosphonate in animals as well as in humans is low, probably because of their low lipophilicity which prevents transcellular transport, and their high negative charge which prevents paracellular transport. It lies between less than 1 and 10%. Absorption is proportionally greater when large doses are given, such as with etidronate and clodronate. This is possibly the reason why it is generally lower for the more potent bisphosphonates which are administered in lower amounts. It is in general higher in the young and shows great inter- and intraspecies variation. This variability can present a problem in humans, especially for compounds such as etidronate where the dose which shows an adverse event, such as an inhibition of mineralization, is close to that which inhibits bone resorption. The location of the absorption in the gastrointestinal tract is not yet elucidated, although it can occur in the stomach as well as in the small intestine. It appears to occur by passive diffusion, probably through a paracellular pathway. Absorption is substantially diminished when the drug is given with meals, especially in the presence of calcium and iron. The mechanism of this reduction may be due to the conversion of the bisphosphonate into a nonabsorbable form, or to a decrease of the absorption process itself. Therefore, bisphosphonates should never be given at mealtimes and never together with milk or dairy products or with iron supplements. For some unknown reasons, orange juice and coffee also decrease absorption.

56

2.4. Pharmacokinetics Bisphosphonates are poorly absorbed, especially in the presence of food and of calcium.

2.4.2.

Distribution

In the blood, only part of the bisphosphonates are ultrafilterable. The values vary between about two-thirds to only a few percent and are strongly species-dependent, being low for the rat, higher for larger animals and humans. The nonfilterable fraction is either bound to proteins, especially albumin, or present in very small calcium-containing aggregates. Some 2 0 - 8 0 % of the absorbed bisphosphonate is then taken up by bone, the remainder being rapidly excreted in the urine. The skeletal uptake varies with species, sex, and age and with the dose and nature of the compound. In humans receiving clinical doses, the values are about 20% for clodronate, 50% for etidronate, and more for alendronate and pamidronate. Sometimes bisphosphonates, especially pamidronate, can deposit in other organs, mostly the liver and the spleen. The deposition is proportionally greater when large amounts of the compounds are given. At least part of this extraosseous deposition appears to be due to the formation of complexes with metals or to aggregates after too high or too rapid intravenous injection. These complexes are then phagocytosed by the macrophages of the reticuloendothelial system. Therefore, data obtained from studies using large amounts of labeled bisphosphonate given rapidly intravenously should be interpreted with caution. The formation of aggregates in the blood is thought to occur in humans following rapid intravenous injections of large quantities, possibly explaining the renal failure that can ensue.

Bisphosphonates should not be infused rapidly in large quantities, as this can cause the formation of insoluble aggregates or complexes which may impair kidney function. The half-life of circulating bisphosphonates is short, in the order of minutes in the rat. In humans it is somewhat longer, 0.5-2 h. The rate of entry into bone is very fast, similar to that of calcium and phosphate. Bone clearance is compatible with a complete extraction by the skeleton after the first passage, so that skeletal uptake might be determined to a large extent by skeletal vascularization and blood flow. The areas of deposition were generally thought to be mostly those of bone formation. This property is used to measure areas of high bone turnover in nuclear medicine by means of 99mTc-linked bisphosphonates. However, alendronate, when

57

Adverse eve p. 168

2. Bisphosphonates~preclinical

Deposition in bone p. 43

given in therapeutic doses, has been found to accumulate preferentially under the osteoclasts. This is also the case, although to a lesser extent, for etidronate when given in the same amount. When given at a therapeutic dose, the latter, however, accumulates equally under both cells. This suggests that when a bisphosphonate is given in small doses, which is the case for all newer compounds, it is likely to deposit preferentially in locations of bone resorption. The rapid uptake by bone means that the soft tissues are exposed to bisphosphonates for only short periods, explaining why practically only bone is affected in vivo.

Bisphosphonates deposit rapidly into bone, in areas of both bone formation and bone destruction. The half-life in plasma is therefore very short.

Effect in humans p. 143

When bisphosphonates are given in clinically effective doses, there seems to be no saturation in their total skeletal uptake in humans, at least within periods as long as years or decades. In contrast, with continuous administration, the antiresorbing effect reaches a maximum relatively rapidly, both in animals and in man. The level of this maximal effect depends on the dose administered, as does the duration of the effect after discontinuation of the drug. The fact that a plateau of activity is reached, despite the fact that the bisphosphonate continues to be incorporated, suggests that the compounds are buried in the bone and become inactive (Fig. 2.4-1).

The accumulation in the skeleton reaches a plateau with chronic administration only after a very long time, possibly decades. In contrast, the plateau of biological response on resorption is attained more rapidly and is dose-dependent. Once deposited in the skeleton, part of the bisphosphonate is liberated again by physicochemical mechanisms. Once buried under new layers of bone, they will be released to a large extent only when the bone in which they were deposited is resorbed. Thus the half-life in the body depends on the rate of bone turnover itself. As the bisphosphonates slow down the resorption of the bone in which they are deposited, their half-life may be even longer than the normal half-life of the skeleton. The half-life of various bisphosphonates is between 3 months and up to a year in mice or rats, clodronate being cleared somewhat faster than etidronate and pamidronate. For humans it is much longer, for some bisphosphonates over 10 years, and it is possible that a portion of the administered compounds remains in the body for life. However, this is also true for other bone seekers such as tetracyclines, heavy metals, and fluoride. There is no indication that the bisphosphonate buried in the skeleton has any pharmacological activity. On the contrary, in the rat, bone formed

58

2.4. Pharmacokinetics --~ Fig. 2.4-1 Effectof various doses of pamidronate administered daily subcutaneously on urinary hydroxyproline excretion in the rat. The maximal effect is obtained rapidly and depends on the dose given. [Adapted and reproduced from Reitsma, P. H., et al. (1980). Calcif. Tissue Int., 32, 145-157, with copyright permission from the author and SpringerVerlag, Heidelberg.]

Effect of dose on bone resorption 100 80 0.16 ~moles/day +1

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under administration of even high doses of alendronate can be resorbed normally. However, at the sites where the bisphosphonate is deposited in large amounts, such as in high turnover locations of patients with bone metastases or with Paget's disease, the long skeletal retention may explain why one single administration of a bisphosphonate can be active for long periods of time, both in animals and in humans.

Tumor hypercalcen p. 100 Paget's dise~ p. 76

The skeletal retention of bisphosphonates is very long, possibly life-long.

2.4.3.

Renal clearance

The renal clearance of bisphosphonates is high. When taking into account their only partial ultrafilterability, it can be, at least in animals, higher than that of inulin, indicating active secretion. The secretory pathway involved is not yet characterized. Urinary excretion is decreased in renal failure and the removal by peritoneal dialysis is poor, which has to be accounted for when the compounds are administered in patients with kidney disease.

59

Administrat:

in renal faih p. 178

2. Bisphosphonates--preclinical Fig. 2.4-2 Pharmacokinetics of bisphosphonates.

Bisphosphonate kinetics Gastrointestinal Tract ~ : ~/(

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Effect on fractures Etidronate study p. 137

The question of whether bisphosphonates will also decrease fracture incidence has been definitively established over the past few years. Some decrease in vertebral fractures was found earlier in the above mentioned etidronate study, but significance was obtained only on patients with lower bone density chosen during the analysis of the data. Other studies with etidronate show a trend to a reduction of fractures but have been performed on too few patients to give significant results. A recent epidemiological study also shows a similar effect. To establish that etidronate decreases significantly fracture risk, which is probable, a new study on a large scale would be necessary (Fig. 3.5-20).

TheeffeciofetidronateonfractureratewasassessedonsmaUstud..... ies and is nbr pet Clear. Itneeds furtberinvestigaaon~

Alendronate on BMD p. 139

More recently it was reported in the above-mentioned study on alendronate on 994 women that, when the various groups were pooled, the daily administration of alendronate led to a decrease of 489'0 in vertebral fractures. This was accompanied by an improvement of the spine deformity index, and a decrease in the loss of height. These results were confirmed in another study on 2027 women between 144

3.5. Osteoporosis Fig. 3.5-20 Effect of etidronate administered for 2 weeks every 3 months for 3 years on vertebral fractures. In the left panel all patients were included, while in the right only those with a low bone mineral density at the start were analyzed. [Adapted from Harris, S. T., et al. (1993). Reproduced from J. Med., 95,557567, with permission from the author and publisher.]

Placebo ......................

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the ages of 55 and 81, with low bone mineral density and with at least one vertebral fracture. The oral administration of 5 mg daily for 2 years followed by approximately 1 year at 10 mg reduced the risk of vertebral fractures by 47% and the risk of sustaining more than one fracture by 89%. Furthermore, both hip and wrist fractures were significantly decreased by 51 and 44 %, respectively. The incidence of any clinical (symptomatic)fracture was reduced by 28%, and that of multiple new clinical fractures by 48 %. The effect was independent of age, initial bone mineral density, a n d the n u m b e r of pre-existing fractures. The patients w h o h a d an increase of B M D above 3 % in the first year of t r e a t m e n t , h a d a lower incidence of n e w vertebral fractures during the w h o l e study. A decrease in n o n v e r t e b r a l fractures of 4 7 % was also f o u n d in a n o t h e r study on 1908 patients. Fig. 3.5-21 Effect of the daily oral administration of either 5 or 10 mg for 3 years or 20 mg for 2 years, followed by 5 mg for 1 year of alendronate (all data pooled) on new vertebral fractures in postmenopausal osteoporotic women with or without fractures. [Data from Liberman et al., (1995).]

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145

_>2 fractures

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3. Bisphosphonates--clinical

Effect of alendronate on all fractures Nlultiple S.vmptomatic Any Vertebral vertebral vertebral symptomatic Hip Wrist fracture fracture fracture fracture fracture fracture ,-

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Fig. 3.5-22 Effect of 5 mg of alendronate orally daily for 2 years followed by 10 mg for approximately 1 year in postmenopausal osteoporotic women with at least one vertebral fracture. All data are expressed in percentage of patients who received placebo. All differences are statistically significant. [Data from Black et al. (1996).]

Alendronate given orally for 3 years decreases significantly by about one-half the occurrence of vertebral and nonvertebral fractures in patients with low bone mineral density and at least one fracture. In a further study on 4432 w o m e n with a bone mineral density below - 1 . 6 SD, but without a fracture, the treatment mentioned previously with alendronate over 4 years reduced significantly radiographic vertebral fractures by 44 %, as well as height loss, but induced only a trend towards a reduction in clinical fractures. However, if the w o m e n with a T-score below - 2 . 5 were analyzed, the risk of clinical fractures significantly decreased by 3 6 % , nonvertebral fractures by 3 5 % , and hip fractures by 56%. Thus, the effect of the bisphosphonate was dependent upon the severity of the initial osteoporosis. In an additional study on 1908 postmenopausal w o m e n at 153 centers with a T-score of the lumbar spine lower than - 2 SD, 10 mg of alendronate daily for one year also decreased significantly the risk of nonvertebral fractures by 4 7 % .

In patients without any fracture the results were similar for new vertebral fractures but significant/:or nonvertebral fractures only when initial BMD was low.

146

3.5. Osteoporosis

Very recently, risedronate has been shown to reduce the incidence of vertebral and nonvertebral fractures in osteoporotic women with vertebral fractures based upon two 3-year double-blind, placebo-controlled studies. In the multinational study conducted in Europe and Australia, patients receiving 5 mg therapy daily (n=407) had a reduction in the risk of new vertebral fractures by 49% (p