Drug Discovery and Development for Alzheimer's Disease (2000)

p:

um

/fo r

4a ll. vn

.c he m

w

w

//w

s/ in de

x

p x. ph

s/ in de

DRUG DISCOVERY AND DEVELOPMENT FOR

ht

tp

://

w

w

w

.c he

m

4a ll.

vn

/fo

ru

m

ALZHEIMER'S DISEASE

2000

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Howard Martin Fillit, MD is the founding Executive Director of the Institute for the Study of Aging, Inc., a private venture philanthropy based in New York City. The Institute's mission is to catalyze and fund research on drug discovery and drug development for cognitive aging and Alzheimer's disease. The Institute supports research nationally and internationally, in both academia and in the biotechnology industry. In addition, Dr. Fillit serves as a consultant in gerontology and geriatric medicine to individuals, managed care organizations, health care systems, pharmaceutical and biotechnology companies. He is also a clinical professor of geriatrics and medicine and a professor of neurobiology at The Mount SinaiNYU Medical Center. Dr. Fillit previously served as Corporate Medical Director for Medicare for New York Life's managed health plans (NYLCare), now a subsidiary of Aetna US Healthcare. During his tenure, NYLCare served more than 100,000 elderly individuals in several regional Medicare managed care programs and a total of more than 2.5 million individuals in 50 states. He received a BA cum laude from Cornell University and earned his Medical Degree at the State University of New YorkUpstate Medical Center at Syracuse. From 1976 to 1987, he received postgraduate training and held academic positions at The Rockefeller University and The New York Hospital-Cornell Medical Center. Dr. Fillit has received numerous awards and honors, including the Rita Hayworth Award for lifetime achievement in research and practice in Alzheimer's disease from the Alzheimer's Association (1999). He is also a fellow of the American Geriatrics Society, the American College of Physicians, the Gerontological Society of America, and the New York Academy of Medicine. He has published more than 200 scientific and clinical articles, abstracts, and books and is an editor of the leading international Textbook of Geriatric Medicine and Gerontology.

ht

tp

://

w

w

w

.c he

m

Alan O'Connell, PhD is Scientific Affairs Officer at the Institute for the Study of Aging, Inc. He is a neuroscientist by training and has extensive professional experience primarily in the biotechnology and pharmaceutical industries. During his career, Dr. O'Connell has held positions in pharmaceutical product development, licensing and technology evaluation. Before joining the institute, Dr. O'Connell was Manager of Neurobiology Product Development at American Biogenetic Sciences, Inc., a New York-based biotechnology company. Dr. O'Connell completed his undergraduate studies at the University of London and subsequently obtained a PhD in neurobiology from the National University of Ireland. He then completed a two-year postdoctoral fellowship in aging and Alzheimer's disease research at University College, Dublin, before joining Elan Corporation Plc., as Manager of Preclinical Pharmacology within the Pharmaceutical Technology Division. Dr. O'Connell has also worked as a Scientific Officer with the Medicines Control Agency (MCA) in London. During his time at the MCA he coordinated contacts between the Pharmacoviligance Section of the agency and the U.K. pharmaceutical industry. Dr. O'Connell has published numerous scientific abstracts and papers.

INSTITUTE FOR THE STUDY OF AGING

x. ph

p

DRUG DISCOVERY AND

s/ in de

DEVELOPMENT FOR

2000

w

w

.c he

m

4a ll.

vn

/fo

ru

m

ALZHEIMER'S DISEASE

ht

tp

://

w

Howard M. Fillit, MD Alan W. O'Connell,PhD Editors

Springer Publishing Company

p s/ in de

All rights reserved

x. ph

Copyright © 2002 by Springer Publishing Company, Inc.

/fo

m

4a ll.

Acquisitions Editor: Sheri W. Sussman Production Editor: Sara Yoo Cover design by Susan Hauley

vn

Springer Publishing Company, Inc. 536 Broadway New York, NY 10012-3955

ru

m

No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior permission of Springer Publishing Company, Inc.

.c he

02 03 04 05 06 / 5 4 3 2 1

w

Library of Congress Cataloging-in-Publication Data

ht

tp

://

w

w

Drug discovery and development for Alzheimer's disease 2000 / Howard Fillit, Alan O'Connell, editors. p.; cm. Includes bibliographical references and index. ISBN 0-8261 -1542-X 1. Alzheimer's disease—Chemotherapy—Congresses. 2. Alzheimer's disease—Chemotherapy—Evaluation—Congresses. 3. Drugs—Testing— Congresses. I. Fillit, Howard. II. O'Connell, Alan. III. Institute for the Study of Aging. [DNLM: 1. Alzheimer Disease—diagnosis—Congresses. 2. Alzheimer Disease—drug therapy—Congresses. 3. Dementia—drug therapy—Congresses. WT 155 A24383 2002] RC523 .A329 2002 616.831061—dc21 2001049760

Printed in the United States of America by Sheridan Books.

x. ph

p

Contents

s/ in de

Contributors Acknowledgments

m

The Challenges of Drug Discovery and Drug Development for Cognitive Aging and Alzheimer's Disease Howard Fillit and Alan O'Connell

xvi 1

vn

/fo

ru

1

ix

4a ll.

Part I. Early Detection Genetic Correlates of Successful Cognitive Aging Thomas Perls

3

Genetic and Environmental Risk Factors for Alzheimer's Disease in Arabs Residing in Israel Robert P. Friedland, Amos Korczyn, and Lindsay Farrer

4

Telephonic Screening of Mental Status: A Feasibility Study in a Managed Care Clinic David Knopman, Thomas Vonsternberg, and Jim Haefemeyer

17

Use of Brain MRI Volumetric Analysis in a Mild Cognitive Impairment Trial to Delay the Diagnosis of Alzheimer's Disease Michael Grundman, Drahomira Sencakova, Clifford R. Jack, Jr., Ronald C. Petersen, Hyun T. Kim, Arlan Schultz, Ronald G. Thomas, Leon J. Thal, for the Alzheimer's Disease Cooperative Study

24

Hippocampal Volume Assessment in Mild Cognitive Impairment Michael S. Mega, Paul M. Thompson, Arthur W. Toga, and Jeffrey L. Cummings

33

7 13

ht

tp

5

://

w

w

w

.c

he m

2

6

v

vi

Contents

7

Brain Imaging Surrogate Markers for Detection and Prevention of Cognitive Aging and Alzheimer's Disease Gary W. Small

42

8

Novel -Amyloid Probes 50 Mei-Ping Kung, Zhi-Ping Zhuang, Catherine Hou, Daniel Skovronsky, Tamar Gur, Sumalee Chumpradit, Virginia M.-Y. Lee, John Q. Trojanowski, and Hank F. Kung

9

Early Clinical and Biological Manifestations of Alzheimer's Disease: Implications for Screening and Treatment Richard C. Mohs, Deborah Marin, and Vahram Haroutunian

s/ in de

x. ph

p

57

Part II. Drug Discovery

Identification of Brain-Expressed ABC Transporters That May 67 Mediate Detachment of ß-Amyloid From Biological Membranes Peter B. Reiner and Stephane Le Bihan

11

Targeting an RNA Structure in the Amyloid Precursor Protein Gene as a New Therapeutic Strategy for Alzheimer's Disease Principal Investigator: Jack T. Rogers

74

12

Novel Tricyclic Pyrone Compounds Prevent Intracellular APP C99-Induced Cell Death Lee-Way Jin, Duy H. Hua, Feng-Shiun Shie, Izumi Maezawa, Bryce Sopher, and George M. Martin

89

13

Novel Glycosaminoglycans as Antiamyloid Agents Robert Kisilevsky and Walter A. Szarek

98

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

10

106

tp

://

14 Strategies for Inhibiting Alzheimer's y-Secretase Michael S. Wolfe

Phosphorylations of Tau and APP as Targets for Drug 116 Discovery John DeBernardis, Dan Kerkman, Cintia Vianna, and Peter Davies

16

Estrogen and ApoE: Drug Discovery Implications Jonathan D. Smith and Justine A. Levin

129

17

Structure-Based Functional Design of Agonists and Antagonists for AMPA-Subtype Glutamate Receptors Ming-Ming Zhou, Harel Weinstein, Diomedes E. Logothetis, and Eric Gouaux

137

ht

15

Contents

18

vii

Alzheimer's Therapeutics: Neurotrophin Small Molecule Mimetics Stephen M. Massa, You-Mei Xie, and Frank M. Longo

147

Part HI. Drug Development—Preclinical Antioxidants in the Treatment of Alzheimer's Disease: 159 Breakthrough Potential of Indole-3-Propionic Acid Daniel G. Chain, Douglas Galasko, Eyal Neria, Miguel Pappolla, Paul E. Bendheim, and Burkhard Poeggeler

20

AF150(S) and AF267B: Ml Muscarinic Agonists for Treatment and as Disease-Modifying Agents in Alzheimer's and Related Diseases A. Fisher, Z. Pittel, R. Bmndeis, N. Bar-Ner, H. Sonego, I. Marcovitch, N. Natan, and R. Haring

21

ABS-205: Effects on Cognition in Mature and Aged Rats Ciaran M. Regan

184

22

Calpain Inhibitors: A Treatment of Alzheimer's Disease Ottavio Arancio

194

23

Femtomolar-Acting Neuroprotective Peptides: Application for Inhibition of Alzheimer's Disease Illana Gozes, Sharon Furman, Ruth A. Steingart, Albert Pinhasov, Inna Vulih, Jacob Romano, Roy Zaltzman, Rachel Zamostiano, Eliezer Giladi, Sara Rubinraut, Mati Fridkin, Janet Hauser, and Douglas E. Brenneman

204

171

Part IV. Drug Development—Clinical

tp

://

w

w

w

.c he

m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

19

A Phase I Clinical Study of Ex Vivo Nerve Growth Factor Gene Therapy for Alzheimer's Disease Mark H. Tuszynski

217

25

Lowering Homocysteine Levels in Patients With Alzheimer's Disease Paul Aisen, Mary Sano, Ramon Diaz-Arrastia, and William Jagust

225

26

Therapeutic Potential of Positive AMPA Receptor Modulators 234 in Mild Cognitive Impairment and Alzheimer's Disease Steven A. Johnson, Gary Lynch, Gary A. Rogers, and Ursula V. Staubli

ht

24

viii

Contents The Alzheimer's Disease Atorvastatin Treatment Trial: Scientific Basis and Position on the Use of HMG-CoA Reductase Inhibitors (Statins) That Do or Do Not Cross the Blood-Brain Barrier D. Larry Sparks, Donald J. Connor, Patrick J. Browne, Dawn R. Wasser, Jean E. Lopez, and Marwan N. Sabbagh

244

28

A Double-Blind, Randomized Investigation of the Effects of Testosterone or Placebo in Male Patients With Mild-to-Moderate Alzheimer's Disease Bruce Miller

253

29

Estrogen and Cognitive Functioning in Men With Mild Cognitive Impairment Barbara B. Sherwin and Howard Chertkow

s/ in de

x. ph

p

27

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

Index

263

271

s/ in de

x. ph

p

Contributors

Paul Aisen, MD Department of Psychiatry Georgetown University Medical College Washington, DC

m

vn

/fo

ru

Howard Chertkow, MD Lady Davis Institute Jewish General Hospital Bloomfield Center for Research in Aging Montreal, Canada

4a ll.

Ottavio Arancio, MD, PhD Nathan Kline Institute Orangeburg, NY

Daniel G. Chain, PhD President and Chief Scientist Mindset BioPharmaceuticals Jerusalem, Israel

N. Bar-Ner, PhD Israel Institute for Biological Research Ness-Ziona, Israel

he m

Sumalee Chumpradit, PhD Department of Radiology University of Pennsylvania Philadelphia, PA

w

.c

Paul E. Bendheim, MD Mindset BioPharmaceuticals Jerusalem, Israel

w

Donald J. Connor, PhD Sun Health Center for Clinical Research Sun Health Research Institute Sun City, AZ

ht

tp

://

w

R. Brandeis, PhD Israel Institute for Biological Research Ness-Ziona, Israel

Jeffrey L. Cummings, MD Director UCLA School of Medicine Los Angeles, CA

Douglas E. Brenneman, PhD Laboratory of Developmental Neurobiology National Institutes of Health Bethesda, MD

Peter Davies, PhD Professor Albert Einstein College of Medicine Bronx, NY

Patrick J. Browne, MD Department of Cardiology Boswell Hospital Baltimore, MD IX

Contributors

X

s/ in de

Lindsay Farrer, PhD Boston University School of Medicine Boston, MA

ru

m

Illana Gozes, PhD Professor of Clinical Biochemistry Sackler Medical School Tel Aviv University Tel Aviv, Israel

/fo

Michael Grundman, MD, MPH Associate Director Department of Neurosciences University of California, San Diego La Jolla, CA

vn

Abraham Fisher, PhD Senior scientist, Head of Alzheimer's Disease R&D Israel Institute for Biological Research Ness-Ziona, Israel

Eric Gouaux, PhD Department of Biochemistry and Molecular Biophysics Columbia University New York, NY

x. ph

Ramon Diaz-Arrastia, MD, PhD Department of Neurology University of Texas Dallas, TX

Eliezer Giladi, PhD Department of Clinical Biochemistry Sackler Medical School Tel Aviv University Tel Aviv, Israel

p

John DeBernardis, PhD President and Chief Operating Officer Molecular Geriatrics Corporation Vernon Hills, IL

he m

4a ll.

Mati Fridkin, PhD Department of Organic Chemistry The Weizmann Institute of Science Rehovot, Israel

ht

tp

://

w

w

w

.c

Robert P. Friedland, MD, PhD Professor of Neurology and Chief, Laboratory of Neurogeriatrics Case Western Reserve University School of Medicine Cleveland, OH Sharon Furman, MSc Department of Clinical Biochemistry Sackler Medical School Tel Aviv University Tel Aviv, Israel Douglas Galasko, MD University of California San Diego, CA

Tamar Gur, PhD Department of Pathology and Laboratory Medicine University of Pennsylvania Philadelphia, PA Jim Haefemeyer, MD Department of Family Practice HealthPartners Minneapolis, MN R. Haring, PhD Israel Institute for Biological Research Ness-Ziona, Israel Vahram Haroutunian, PhD Department of Psychiatry Mount Sinai School of Medicine New York, NY

Contributors

XI

Janet Hauser, MSc Laboratory of Developmental Neurobiology National Institutes of Health Bethesda, MD

Robert Kisilevsky, MD, PhD Department of Pathology Queen's University Ontario, Canada David Knopman, MD Professor of Neurology Mayo Clinic Rochester, MN

p

Catherine Hou, BS Department of Radiology University of Pennsylvania Philadelphia, PA

x. ph

Amos Korczyn, MD, MSc Tel Aviv University Faculty of Medicine Tel Aviv, Israel

s/ in de

Duy H. Hua, PhD Department of Chemistry Kansas State University Manhattan, KS

Hank F. Kung, PhD Department of Radiology, and Pharmacology University of Pennsylvania Philadelphia, PA

ru

/fo vn

Mei-Ping Kung, PhD Department of Radiology University of Pennsylvania Philadelphia, PA

he m

4a ll.

William Jagust, MD Department of Neurology UC Davis Sacramento, CA

m

Clifford R. Jack, Jr., MD Department of Diagnostic Radiology Mayo Clinic and Foundation Rochester, MN

w

w

w

.c

Lee-Way Jin, MD, PhD Assistant Professor Department of Pathology University of Washington Seattle, WA

ht

tp

://

Steven A. Johnson, PhD Director Cortex Pharmaceuticals Irvine, CA Dan Kerkman, PhD Molecular Geriatrics Corporation Vernon Hills, IL Hyun T. Kim, MS Department of Neurosciences University of California, San Diego La Jolla, CA

Stephane Le Bihan, PhD Active Pass Pharmaceuticals Vancouver, Canada Virginia M.-Y. Lee, PhD Department of Pathology and Laboratory Medicine, and Pharmacology University of Pennsylvania Philadelphia, PA Justine A. Levin, BA The Rockefeller University New York, NY Diomedes E. Logothetis, PhD Department of Physiology and Biophysics Mount Sinai School of Medicine New York, NY

Contributors

Xll

p

x. ph

s/ in de

Miguel Pappolla, MD University of South Alabama Mobile, AL Thomas Perls, MD, MPH Principal Investigator Harvard Medical School Boston, MA

he m

4a ll.

I. Marcovitch, BSc Israel Institute for Biological Research Ness-Ziona, Israel

Eyal Neria, PhD Mindset BioPharmaceuticals Jerusalem, Israel

m

Izumi Maezawa, DPharm Department of Pathology University of Washington Seattle, WA

N. Natan, BSc Israel Institute for Biological Research Ness-Ziona, Israel

ru

Gary Lynch, PhD Department of Psychiatry and Human Behavior University of California Irvine, CA

Richard C. Mohs, PhD Associate Chief of Staff for Research VA Medical Center Bronx, NY

/fo

Jean E. Lopez, RN, MSN Sun Health Center for Clinical Research Sun Health Research Institute Sun City, AZ

Bruce Miller, MD University of California San Francisco, CA

vn

Frank M. Longo, MD, PhD Associate Chief of Staff for R&D University of California San Francisco, CA

w

w

w

.c

Deborah Marin, MD Department of Psychiatry Mount Sinai School of Medicine New York, NY

ht

tp

://

George M. Martin, MD Department of Pathology University of Washington Seattle, WA Stephen M. Massa, MD Department of Neurology UCSF School of Medicine San Francisco, CA Michael S. Mega, MD, PhD Director UCLA School of Medicine Los Angeles, CA

Ronald C. Petersen, PhD, MD Department of Neurology Mayo Clinic and Foundation Rochester, MN Albert Pinhasov, MSc Department of Clinical Biochemistry Sackler Medical School Tel Aviv University Tel Aviv, Israel Z. Pittel, PhD Israel Institute for Biological Research Ness-Ziona, Israel Burkhard Poeggeler, PhD University of Goettingen Goettingen, Germany

Contributors Xlll

xiii

Ciaran M. Regan, PhD Associate Professor University College Dublin Dublin, Ireland Peter B. Reiner, PhD President and CEO Active Pass Pharmaceuticals Vancouver, Canada

Barbara B. Sherwin, PhD Professor McGill University Montreal, Canada

x. ph

Gary A. Rogers, PhD Cortex Pharmaceuticals Irvine, CA

p

Drahomira Sencakova, MD Department of Diagnostic Radiology Mayo Clinic and Foundation Rochester, MN

s/ in de

Feng-Shiun Shie, PhD Department of Pathology University of Washington Seattle, WA

Daniel Skovronsky, PhD Department of Pathology and Laboratory Medicine University of Pennsylvania Philadelphia, PA

/fo

vn

Jacob Romano, BSc Department of Clinical Biochemistry Sackler Medical School Tel Aviv University Tel Aviv, Israel

ru

m

Jack T. Rogers, PhD Assistant Professor Massachusetts General Hospital Boston, MA

4a ll.

Gary W. Small, MD Professor of Psychiatry University of California Los Angeles, CA

.c he

m

Sara Rubinraut, MSc Department of Organic Chemistry The Weizmann Institute of Science Rehovot, Israel

ht

tp

://

w

w

w

Marwan N. Sabbagh, MD Sun Health Center for Clinical Research Sun Health Research Institute Sun City, AZ Mary Sano, PhD Associate Professor of Clinical Neuropsychology Columbia University New York, NY Arlan Schultz, MA Department of Neurosciences University of California, San Diego La Jolla, CA

Jonathan D. Smith, PhD Associate Professor The Rockefeller University New York, NY H. Sonego, MSc Israel Institute for Biological Research Ness-Ziona, Israel Bryce Sopher, PhD Department of Laboratory Medicine University of Washington Seattle, WA D. Larry Sparks, PhD Director Sun Health Research Institute Sun City, AZ

Contributors

xiv

Cintia Vianna, BS Departments of Pathology and Neuroscience Albert Einstein College of Medicine Bronx, NY

Ursula V. Staubli, PhD Cortex Pharmaceuticals Irvine, CA Ruth A. Steingart, PhD Department of Clinical Biochemistry Sackler Medical School Tel Aviv University Tel Aviv, Israel

x. ph

p

Thomas Vonsternberg, MD Department of Family Practice HealthPartners Minneapolis, MN

s/ in de

m

Dawn R. Wasser, PharmD Pharmacy Department Del Webb Hospital Sun City West, AZ

4a ll.

vn

Ronald G. Thomas, PhD Department of Neurosciences University of California, San Diego La Jolla, CA

ru

Leon J. Thal, MD Department of Neurosciences University of California, San Diego La Jolla, CA

Inna Vulih, BSc Department of Clinical Biochemistry Sackler Medical School Tel Aviv University Tel Aviv, Israel

/fo

Walter A. Szarek Department of Chemistry Queen's University Ontario, Canada

w

.c he

m

Paul M. Thompson, PhD Department of Neurology UCLA School of Medicine Los Angeles, CA

tp

://

w

w

Arthur W. Toga, PhD Department of Neurology UCLA School of Medicine Los Angeles, CA

ht

John Q. Trojanowski, MD, PhD Department of Pathology and Laboratory Medicine University of Pennsylvania Philadelphia, PA Mark H. Tuszynski, MD, PhD Associate Professor University of California San Diego, CA

Harel Weinstein, PhD Department of Physiology and Biophysics Mount Sinai School of Medicine New York, NY Michael S. Wolfe, PhD Associate Professor of Neurology Harvard Medical School Boston, MA You-Mei Xie, MD, PhD Department of Neurology UCSF School of Medicine San Francisco, CA Roy Zaltzman, MD Department of Clinical Biochemistry Sackler Medical School Tel Aviv University Tel Aviv, Israel

Contributors

xv

Rachel Zamostiano, PhD Department of Clinical Biochemistry Sackler Medical School Tel Aviv University Tel Aviv, Israel

Zhi-Ping Zhuang, PhD Department of Radiology University of Pennsylvania Philadelphia, PA

ht

tp

://

w

w

w

.c he

m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Ming-Ming Zhou, PhD Assistant Professor Mount Sinai School of Medicine New York, NY

x. ph

p

Acknowledgments

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

Our thanks are due to all of our funded investigators who so generously gave of their time to prepare papers and attend the meeting. Special thanks go to Tonya Lee and Sue Reynolds-Foley for their help in planning and coordinating the meeting and this publication. We would also like to thank Elan Pharmaceuticals (formerly Athena Neurosciences, Inc.), Memory Pharmaceuticals, and Pfizer, Inc. for their generous support of this meeting and publication, as well as the staff at Springer Publishing, NY for their cooperation and technical assistance in publishing this volume.

xvi

s/ in de

x. ph

The Challenges of Drug Discovery and Drug Development for Cognitive Aging and

p

1

ru

m

Alzheimer's Disease

m

4a ll.

vn

/fo

Howard Fillit and Alan O'Connell

.c he

INTRODUCTION

ht

tp

://

w

w

w

Alzheimer's disease and cognitive aging are major health care concerns. Alzheimer's disease is the most common cause of dementia in the elderly, affecting about 5% to 10% of individuals over the age of 65, and up to 40% of individuals over the age of 80, the most rapidly growing population segment in the developed world. Cognitive aging is more difficult to quantify, but reflects a decline in cognitive function with age and affects most of the population over the age of 65. Some success has been achieved in the treatment of Alzheimer's disease and there are now four drugs, all inhibitors of acetylcholinesterase, that have been approved for the treatment of mild to moderate Alzheimer's disease in the United States. These drugs are safe and moderately effective in relieving cognitive symptoms in some patients. However, these drugs are far from ideal, and there are currently no drugs with diseasemodifying or preventive properties. Therefore, there is a clear need to develop newer, more effective drugs to treat and/or modify the progression of Alzheimer's disease and related dementias. Development of safe and effective medications represents a considerable market opportunity. 1

Introductiiomn

2Introduction

x. ph

p

This market for Alzheimer's disease therapeutics will grow rapidly in the coming decades, as most of the developed world will experience a marked increase in its elderly population. The market for drugs to treat Alzheimer's disease and cognitive aging is likely to expand to include individuals with mild cognitive impairment (MCI), which is believed by many health care professionals to be a precursor to Alzheimer's disease, with about 80% converting within 7 to 8 years. Drug discovery and drug development is a long and expensive process with many challenges. This is particularly true for Alzheimer's disease. The challenges specifically relevant to Alzheimer's disease include:

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

1. A lack of validated therapeutic targets: Alzheimer's disease is a complex disorder and there is no clear understanding regarding the etiology of the disease. A number of genetic risk factors, such as apolipoprotein E, and environmental risk factors, such as head trauma, have been implicated. Comorbidities such as high cholesterol, homocysteine, diabetes, and hypertension may also play a role. Other potential targets include the p-amyloid and tau pathology, inflammation, and oxidative stress. These and many other targets are currently the subject of research, as well as drug-discovery and drug-development efforts, but have yet to be validated in human clinical trials. 2. Lack of adequate animal models: The available animal models of Alzheimer's disease do not provide the full spectrum of neuropathological and clinical aspects of the disease. Progress has been made and there are now transgenic mouse models of (3-amyloid production and deposition in which to study the (3-amyloid hypothesis. Models of tau pathology are also in development and some progress has been made. An important consideration in developing new drugs for Alzheimer's disease is access to the available models for drug screening. There are access restrictions on many of the currently available models and, when available, the models are costly. 3. Drug-discovery efforts for Alzheimer's disease in academia: Drugdiscovery programs in academia are underfunded and do not have the infrastructure and multidisciplinary collaboration necessary for successful drug-discovery research. Most funding organizations do not fund these programs in academia believing them to be primarily the role of the biotechnology and pharmaceutical industries. National Institutes of Health review panels typically lack expertise and mission in drug discovery and drug development and turn down many such programs. Academia is the source of much neurobiological research that leads to innovative therapeutic targets for Alzheimer's disease. Adequate funding can help advance these efforts and "translate" them into potential new drugs.

Introduction 3

3

4. Drug discovery and drug development for Alzheimer's disease in the biotechnology industry: These programs also suffer from a lack of investment. Developing new drugs for Alzheimer's disease is considered high risk by business managers and the investment community. For small biotechnology companies, attracting capital, especially for early-stage, high-risk projects, is difficult. The cost of conducting clinical trials is also difficult for small companies.

ht

tp

://

w

w

w

.c he

m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

For these and many other reasons, it is clear that the drug-discovery and drug-development process for Alzheimer's disease is in need of help. The Institute for the Study of Aging (Institute) was founded in 1998 to catalyze and fund the discovery and development of new drugs for Alzheimer's disease and cognitive aging. The Institute funds the drugdiscovery and drug-development continuum from the earliest phases in which therapeutic targets are identified and validated to the phases in which new drugs are discovered and optimized. The Institute also funds the later stages of preclinical and human clinical testing through phases I, II and off-label trials. The Institute's scientific portfolio is broad and reflects the multifactorial nature of the disease. The Institute's funding market includes the biotechnology industry and academic centers. In the biotechnology industry, the Institute is committed to helping small biotechnology companies focused on Alzheimer's disease realize their potential, and has funded many drug-discovery and clinical-trial programs at U.S. and international companies. Most of the funding is directed to earlier stage programs that do not attract funding from other sources. These investments give these companies the needed resources to validate their "proof of concept" programs. The Institute also faculties the formation of new biotechnology "start-up" companies by providing seed investment to acquire intellectual property and develop a scientific program and business plan. In academia, the Institute primarily funds early-stage target identification and drug-discovery programs, and encourages the formation of collaborations between the multidisciplinary teams necessary for successful drug discovery in academia. The Institute has developed an extensive network of funded investigators in the Untied States, Canada, Ireland, Israel, and the United Kingdom. In the coming years, it is hoped to expand this network to other regions of the world. As part of its mission to facilitate drug discovery and drug development for Alzheimer's disease, the Institute organized its First Annual Investigator's meeting, which brought together Institute-funded academic and biotechnology investigators from the United States and internationally. The meeting was held at Tarrytown House, NY, on October 29 to 31, 2000, and provided a forum for this unique group of researchers

4

In troduction

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

focused on drug discovery and development for Alzheimer's disease to present their findings. In addition, the meeting encouraged the exchange of ideas and facilitated networking and collaboration to facilitate Alzheimer's disease drug-discovery and drug-development efforts. Twenty-seven of the Institute's investigators attended the meeting, and topics ranged from early detection and drug discovery to preclinical and clinical drug development. The early detection program focused on genetic factors, novel probes for detecting |3-amyloicl in the living brain, and the use of telephonic screening and magnetic resonance imaging as a tool for identifying those at risk of developing Alzheimer's disease. A diverse range of therapeutic areas were covered in the drug-discovery and drug-development sessions, such as antioxidants, estrogen agonists, various anti-|3-amyloid and anti-tangle approaches, MI agonists, calpain inhibitors, neuroprotective peptides, nerve growth factor agonists, and novel cognitive enhancers. In addition, a number of off-label clinical approaches were discussed, such as the use of Lipitor® and hormonal supplements to treat Alzheimer's disease. A clinical trial to determine the potential role of elevated homocysteine in Alzheimer's disease was also discussed. The present volume includes the papers presented at the meeting, the first in a planned series entitled, "Advances in Drug Discovery and Drug Development for Cognitive Aging and Alzheimer's Disease." It is hoped that this volume will provide an overview of the most recent developments in drug-discovery and drug-development research, as well as potential treatment and early detection strategies. It is our hope that the scientists reporting in this volume and others within our network will help us achieve our vision: "That one clay, in the not-too-distant future, old age will no longer be accompanied by the dreadful fear and the indignity of the loss of memory and mind."

p

Early Detection x. ph

s/ in de

m

ru

/fo

vn

4a ll.

m

.c he

w

w

w

://

tp

ht I

p

x. ph

s/ in de

m

ru

/fo

vn

4a ll.

m

.c he

w

w

w

://

tp

ht This page intentionally left blank

2

p

Genetic Correlates of

x. ph

Successful Cognitive Aging

/fo

ru

m

s/ in de

Thomas perls*

4a ll.

vn

IDENTIFYING GENES PREDISPOSING TO SUCCESSFUL COGNITIVE AGING

ht

tp

://

w

w

w

.c

he m

The hypothesis driving this study is that centenarians are a select group of people who have a history of aging relatively slowly and who have either markedly delayed or entirely escaped diseases normally associated with aging, such as Alzheimer's disease (AD), cancer, stroke, and heart disease. The ultimate challenge in this area of research is to identify the genes that are associated with such a survival advantage and the ability to age so well for such a long time without cognitive impairment. We have very exciting preliminary results revealing that centenarians can be the key to discovering these genes. Primarily because of funding by the Institute for the Study of Aging, we continue careful annual neuropsychological testing and eventual neuropathological study of the centenarian subjects. Approximately 20% of our participants wish to be postmortem brain donors, but we anticipate increasing this rate to 50%, again, in part, due to the generosity of the Institute. These neuropsychological-neuropathological correlations will help us better understand what disease-free aging of the brain means and what it looks like; if causes of dementia are different in the extremely old compared with younger individuals; and the type and quantity of changes in the brain that correlate with different levels of cognitive impairment. * See chapter appendix for collaborators. 7

8

Early Detection

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

At the same time, we are studying the centenarians for genes that may play pivotal roles in determining how centenarians markedly delay or, in some cases, escape cognitive impairment. We have obtained the largest collection of centenarian sibships in the world (N ~ 200). Genome-wide scans from these individuals were performed and the data was given to our statistics colleagues at the Whitehead Institute and Rutgers University for linkage analyses. Based upon scans of 308 individuals making up 137 families, currently we have noted statistically significant linkage to a 20 cM region on chromosome 4. A manuscript reporting these results is currently in the review process. Discovery of genes that powerfully affect processes as broad as rates of aging and/or susceptibility to diseases, such as AD and stroke, would have dramatic impact upon understanding the underpinnings of aging and could, ultimately, lead to promising targets for drug discovery. In our attempts to locate and recruit sibships, we have enrolled five families with many members achieving extreme old age. Such clustering lends itself to linkage studies similar to those performed in families with clustering for rare diseases (such as Tay-Sachs, cystic fibrosis, and sickle-cell anemia). In this instance, we are not looking for the cause of a disease but rather a fantastic advantage. Four of these families are described in a recent article published in the Journal of the American Geriatrics Society. Unfortunately, because there is likely to be more than one gene (or the lack of a specific mutation) increasing the probability of achieving exceptional old age, we believe that even 10 members of one family being included in a linkage study is unlikely to produce statistically significant results. However, as we include cousins and perhaps children, such studies might prove to be feasible. In another approach to gene discovery, we have established a collaboration with gene-expression expert Steven Gullens, PhD, from the Brigham and Women's Hospital. Because we are able to obtain brain tissue at postmortem autopsy within 4 hours of death, we will provide Dr. Gullens with ideal brain tissue samples from regions of specific interest regarding AD, as well as regions that play critical roles in cognition (e.g., frontal lobe and its influence upon executive function) for differential gene expression studies. Determining which genes are active in cognitively intact participants versus those with various causes of cognitive impairment versus other younger controls should be an efficient approach to discovering genes critical to AD pathogenesis.

DEMENTIA PREVALENCE IN EXTREME OLD AGE Numerous researchers have extrapolated from dementia and AD prevalence rates in younger people that all centenarians should have at least

9

Genetic Correlates of Cognitive Aging 9

w

w

.c he

m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

some degree of cognitive impairment. Yet our studies, as well as those of other centenarian studies, indicate that approximately 20% to 30% are cognitively intact. Dr. Silver, our lead neuropsychologist, examined a series of 74 centenarians during a 3-year period. Some of their characteristics are noted in Table 2.1, and Table 2.2 lists the neuropsychological tests performed. We are collaborating with other centenarian studies to establish norms for components of the Mattis Dementia Rating Scale and other tests. The neuropsychological examination, as well as other phenotyping, is performed where the person lives. Specific maneuvers are performed to accommodate for hearing and vision deficits. Multiple visits are performed to prevent fatigue from factoring into the testing, and family is often invited to participate in order to help language barriers. Table 2.3 summarizes Dr. Silver's neuropsychological test results of 74 subjects. The neuropathological findings for 14 of these participants are summarized in Table 2.4. It is noteworthy that evidence of microvascular disease was conspicuously absent in the 14 cases found in Table 2.4. We have noted the absence of diagnoses and blood pressure measurements for many of our participants, which would predispose to vascular disease. We are now in the process of recruiting individuals who are willing to eventually proceed to postmortem autopsy, following them longitudinally with annual detailed neuropsychological examinations. Our aim is to recruit 33 centenarians per year, who live in close enough proximity to the Massachusetts ADRC to ensure that the brain can be obtained within 4 hours of death.

://

w

TABLE 2.1 Centenarians Who Have Undergone Neuropsychological Testing

tp

Age range

100-110 years 86% women 14% men

Living situation

7% live alone 26% with family 67% in nursing homes

Country of origin

50% foreign born

Most frequent birthplaces

Italy, Ireland, and Canada

Education

Mean = 11 years Range = 1-20 years

ht

Gender

10

Early Detection

TABLE 2.2 Neuropsychological Test Battery Geriatric Depression Scale

• Mattis Dementia Rating Scale

• Telephone Interview for Cognitive Status

• Boston Naming Test (CERAD)

• Test for Severe Impairment

• Trail Making Tests AandB

• Tactile Naming

• Clock Drawing

• Cognition and Health History (Informant)

• Drilled Word Span

• Psychiatry History (Informant)

• Cowboy Story (Boston-Rochester)

• Clinical Dementia Rating Scale

• Presidents since FDR

• NEO-Five Factor Personality Inventory: Self report and Observer report

s/ in de

x. ph

p

Mini-Mental State Exam

/fo

ru

m

• Spiers' Calculations

vn

GENETIC STUDIES

w

w

.c he

m

4a ll.

We have previously published findings supporting the hypothesis that centenarians live the vast majority of their lives in exceptional health. At an average of 92 years, 90% of the participants were living independently and in very good health (Hitt, Young-Xu, & Perls, 1999). These findings are consistent with the idea that centenarians markedly delay age-related diseases and, specifically, cognitive impairment relative to the general

tp

://

w

TABLE 2.3 Summary of Results of Neuropsychological Examinations—74 Participants

ht

CDR Score

Description

Number of participants

0

No dementia

0.5

Uncertain or deferred diagnosis

1

Mild dementia

10 (14%)

2

Moderate dementia

22 (30%)

3

Severe dementia

14 (19%)

4

Profound dementia

4 (5%)

5

Terminal dementia

6 (8%)

12 (16%) 6 (8%)

Genetic Correlates of Cognitive Aging

11

TABLE 2.4 Neuropathological Correlations—14 Participants Participants Age /Sex

CDR Score

Neuritic Plaques

Braak & Braak Stage

100/F

0

Sparse-moderate

II

AM

101/F

0

Sparse

I

FF

102/M

0

Sparse

I

LBL

104/F

0

Sparse

II-III

AB

104/M

0

Sparse

JL

103/F

0

Moderate

p

JD

x. ph

So: subjects with CDR = 0 (no dementia)

II

s/ in de

V

Two participants with CDR. = 1 or 2 with depression as an etiological factor

104/F

2

Sparse to moderate

LB

109/F

1

Sparse

104/F

MK

102/F

GG

103/F

JB

102/M

MN

100/F

.c

w

ru

/fo 2

Sparse

0

5

Frequent

V

2

Moderate

V

I

Sparse to moderate

3

Moderate

Sparse

4a ll.

MM

IV

II

I

he m

101/F

II

vn

Six participants with CDR = 1-5

KJ

m

MV

III-IV V

tp

://

w

w

Of particular note are participants AM and FF who have CDR scores of "0" and who demonstrated negligible neurofibrillary tangles or neuritic plaques despite their ages of 101 and 102 (Silver et al., 1998).

ht

population, and, in some cases, avoid the diseases entirely. Our analyses of pedigrees (Perls, Wager, Bubrick, Vijg, & Kruglyak, 1998) and our linkage and association studies thus far highly suggest to us that much of this survival advantage is genetically based. Several lines of evidence support the possibility that genetic polymorphisms (SNPs) that impart such a survival advantage are likely to also affect susceptibility to AD. First, there is a clear association between age and AD prevalence and incidence, and there is something about the aging process that predisposes to AD. Second, we have not encountered centenarians with a long history of dementia perhaps because it is eventually a lethal disease. As a result, centenarians must either lack SNPs that

12

Early Detection

ru

m

s/ in de

x. ph

p

predispose to AD and/or have SNPs that protect against it. It is possible that such polymorphisms will be detected by our methods. At this point, we have noted one chromosomal region that is nearly significant in our association studies of approximately 160 sibships. Probable association has been noted for an additional three regions. We are awaiting additional results status post the addition of 60 more sibships to the genotype data. We anticipate publishing this data soon and, in the meantime, we are in the midst of establishing a collaboration with a major biotech firm to quicken the determination of candidate genes in these regions. In perhaps a more direct means of addressing the centenarians' decreased susceptibility to AD, we are collaborating with Dr. Gullens at the Brigham and Women's Hospital to determine gene-expression profiles for regions of the brain specifically affected by AD. We plan to compare cognitively intact centenarians against those with AD, other forms of dementia, and various controls.

/fo

REFERENCES

tp

://

w

w

w

.c he

m

4a ll.

vn

Hitt, R., Young-Xu, Y, & Perls, T. (1999). Centenarians: The older you get, the healthier you've been. Lancet, 354, 652. Perls, T., Shea-Drinkwater, M., Bowen-Flynn, ]., Ridge, S. B., Kang, S., Joyce, E., Daly, M., Brewster, S.}., Kunkel, L., & Puca, A. A. (2000). Exceptional familial clustering for extreme longevity in humans. Journal of the American Geriatric Society, 48,1483-1485. Perls, T., Wager, C, Bubrick, E., Vijg, J., & Kruglyak, L. (1998). Siblings of centenarians live longer. Lancet, 351,1560. Silver, M., Newell, K., Growdon, J., Hyman, B. T., Hedley-Whyte, E. G., & Perls, T. (1998). Unraveling the mystery of cognitive changes in extreme old age: Correlation of neuropsychological evaluation with neuropathological findings in centenarians. International Psychogeriatrics, 10,

ht

APPENDIX Following is a list of collaborators. Louis Kunkel, PhD, and Annibale Puca; Division of Genetics, Children's Hospital, Boston, MA Eric Lander, PhD, and Mark Daly, PhD; The Whitehead Institute, MIT, Boston, MA Kathy Newell, PhD, and E. Tessa Hedley-Whyte, PhD; Alzheimer's Disease Research Center, Massachusetts General Hospital, Boston, MA Bruce Price, PhD; McLean Hospital, Boston, MA Mathew Frosch, PhD, and Steven Gullens, PhD Cynthia Lemere, PhD; Brigham and Women's Hospital, Boston, MA

3

Environmental

p

Genetic and

x. ph

Risk Factors for Alzheimer's

s/ in de

Disease in Arabs Residing

m

in Israel

4a ll.

vn

/fo

ru

Robert P. Friedland, Amos Korczyn, and Lindsay Farrer

ht

tp

://

w

w

w

.c

he m

There is relatively little known about the distribution of Alzheimer's disease (AD) around the world. Knowledge of high- or low-prevalence foci of disease may aid our understanding of disease mechanisms. Alzheimer's disease occurs less frequently in Asia than in Europe and North America, probably because of lower allele frequency of the apolipoprotein E (apo E) e4 allele. The disease appears to be also less common in India and Africa. There are few studies of the disease in Arabs, and few in populations with a high level of consanguinity. We have found a remarkably high prevalence of the disease in Arabs residing in Israel. In a study in Tunisia, unspecified dementia was found to be 3 times less prevalent than in the United States. Treves, Chandra, and Korczyn (1993) found a lower prevalence of presenile AD in Sephardi, as compared with Ashkenazi Jews, but more late-onset disease in Sephardi than Ashkenazi, perhaps because of lower levels of education in Sephardi. The state of Israel is comprised of about 5 million Jews and 1 million non-Jews, mostly Moslem Arabs. This Arab population of Israel is valuable for medical research because of a high level of inbreeding, large family size, high level of smoking exposure, high level of medical care available through the Israeli Health System, excellent demographic databases available from the government, absence of alcohol use because of the religious proscription, and high participation rate (>98%). 13

24

Early Detection

ht

tp

://

w

w

w

.c he

m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Consanguinity increases the prevalence in a population of autosomal recessive genes that may have undesirable characteristics. For example, an additional 1/16 of the variation of DNA is made homozygous by the inbreeding of a marriage of first-cousins. Conversely, the probability of identifying recessive or quasirecessive susceptibility factors is enhanced by inbreeding. Childhood mortality is increased in offspring of first-cousin marriages by a factor of 1.4 to 1.7, and children born to consanguineous unions have poorer health than the offspring of nonconsanguineous unions (including malignancies, congenital abnormalities, mental retardation, and physical handicap). Curiously, there are few studies of the effects of inbreeding on the health of adults. We have hypothesized that consanguinity in the Wadi Ara community has increased the prevalence of autosomal recessive genes that are responsible, in part, for the increased prevalence of the disease. It has been reported that 44% of all Arab marriages in Israel are consanguineous, with a mean inbreeding coefficient of .0192 (Jaber, Shohat, Rotter, & Shohat, 1997). The inbreeding rates for Israeli Arabs may be particularly high (as compared with Egyptians or Syrians) because mobility was reduced for centuries by the Turks. At times in Arab communities, it has been required for children to marry into their own family in part to avoid sharing resources with competing family groups. Linkage studies by the Boston University group and others focused on inbred Arab families have helped to locate genes for several inherited, neurological diseases, including Wilson's disease, spinal muscular atrophy, sensorineural deafness, and autosomal recessive Duchenne-like muscular dystrophy. Of critical relevance, successes in mapping genes for Wilson's disease and deafness using inbred Arab kindreds facilitated the cloning of these genes, which were subsequently shown to have diseasecausing mutations prevalent in outbred populations from other parts of the world, including the United States. All of the known Alzheimer-related genes (on chromosomes 21, 14, 1, and 19) are dominant (chromosomes 21,14,1) or codominant (chromosome 19). There are no known genes affecting the development of AD that are recessive, perhaps because there have been few studies of AD in populations with high levels of inbreeding. De Braekeleer et al. (1989) reported an association between inbreeding and the development of AD (inbreeding coefficient 9 times higher in AD cases than in controls) in a rural population in Quebec. Studies in the Old Order Amish have found a low prevalence of disease in this inbred community, also having a low frequency of the apo E-e4 allele (.037). Inbreeding could be linked to AD through a confounding effect of education, but the association of inbreeding and education is controversial (found for a Saudi population but not for Israeli Arabs). We expect that there are recessive factors for AD

Genetic and Environmental Risk Factors

15

ht

tp

://

w

w

w

.c he

m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

because of indirect evidence: genetic modeling studies of AD in apo E-e4 negative families are unable to reject a recessive model (Rao et al., 1996). However, it is difficult to evaluate recessive models in outbred populations. The high prevalence we have seen in our highly inbred Arab population in Israel might be due to the inculcation of recessive AD susceptibility alleles or to a dominant gene that became frequent by founder effect. In a population-based study of AD, we have screened all elderly residents of Wadi Ara, an Arab community in northern Israel, and observed an unusually high prevalence (20.5% of those >60 years, 60.5% of those >85 years). This prevalence is higher than that found in other populations in Israel, China, Europe, or the United States, even after adjustment for age, education, and gender, and is not due to increased frequency of the apo E-e4 allele (Corder, Saunders, Strittmatter, et al., 1993; Saunders, Strittmatter, Schmechel, et al., 1993) which is actually reduced in this community (0.035 for nondemented elders) as compared with other Caucasians. DNA samples were collected randomly from 256 participants of the Wadi Ara study, aged 75 ± 9, 118 male, and their apo E genotype was determined by a PCR-based method (Chapman, Estupinan, Asherov, & Goldfarb, 1996). Of the 256 cases examined, 22 carried an apo E-e4 allele (all heterozygous), including 3 of 34 with AD (apo E-e4 allele frequency 0.04), 8 of 128 nondemented elderly participants (apo E-e4 allele frequency 0.03), 7 of 56 with age-associated memory impairment (AAMI; apo E-e4 allele frequency 0.06), and 4 of 38 with other types of dementia and pseudodementia (apo E-e4 allele frequency 0.05). These data suggest that the apo E-e4 allele is relatively uncommon in Arabs in Wadi Ara. In fact, this is the lowest frequency of apo E-e4 ever recorded. Although the possibility that it is associated with dementia was not excluded, it cannot explain the high AD prevalence in this population. Pedigree studies showed that more than one of three of the 168 prevalent cases were from one hamula (extended family) of the 14 found in Wadi Ara. A lOcM genome scan showed significant association with a site that has been narrowed to 1.6 cM. Evidence for linkage stemmed primarily from excess homozygosity of one of the alleles for a marker in this region in the total sample of cases (15%) compared with controls (3%). The crude odds ratio of AD associated with this genotype was 5.7 (95% CI = 1.5,21.7). The odds increased to 10.0 (1.3-75.9) after adjustment for age, sex, and systolic blood pressure. The observation of significant but different patterns of association within multiple hamulas suggests the existence of multiple recombination between the marker and the AD susceptibility locus. This genetic location notably overlaps a region showing evidence for linkage to AD in a genome scan (Kehoe et al., 1999). This location includes several genes, including a gene strongly related to lipid metabolism. We are currently working on the precise identification of the linked gene.

16

Early Detection

We are also investigating the possibility that the high prevalence of AD in Wadi Ara is related to environmental risk factors and medical illness, including a high-fat diet, altered lipid metabolism, thyroid disease, smoking, hypertension, heart disease, and stroke.

REFERENCES

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Chapman, ]., Estupinan, }., Asherov, A., & Goldfarb, L. G. (1996). A simple and efficient method for apolipoprotein E genotype determination, Neurology, 46(5), 1484-1485. Corder, E. H., Saunders, A. M., Strittmatter, W. }., Schmechel, D. E., Gaskell, P. C, Small, G. W., Roses, A. D., & Pericak-Vance, M. A. (1993). Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late-onset families. Science, 261, 921-923. De Braekeleer, M., Cholette, A., Mathieu, J., Boily, C., Robitaille, Y, & Gauvreau, D. (1989). Familial factors in Alzheimer's disease (IMAGE project). European Neurology, 29(suppl 3), 2-8. Jaber, L., Shohat, T., Rotter, J. L, & Shohat, M. (1997). Consanguinity and common adult disease in Israeli Arab communities. American Journal of Medical Genetics, 70, 346-348. Kehoe, P., Wavrant-De Vrieze, E, Crook, R., Wu, W. S., Holmans, P., Fenton, I., Spurlock, G., Norton, N., Williams, H., Williams, N., Lovestone, S., Perez-Tur, J., Hutton, M., Chartier-Harlin, M C., Shears, S., Roehl, K., Booth, J., Van Voorst, W., Ramie, D., Williams, J., Goate, A., & Hardy, J. (1999). A full genome scan for late-onset Alzheimer's disease. Human Molecular Genetics, 8(2), 237-245. Rao, V. S., Cupples, L. A., van Duijn, C. M., Kurz, A., Green, R. C., Chui,, H., Duara, R., Auerbach, S. A., Volicer, L., Wells, J., van Broeckhoven, C., Growdon, J. H., Haines, J. L., & Farrer, L A. (1996). Evidence for major gene inheritance of Alzheimer disease in families of patients with and without Apo e 4. American Journal of Human Genetics, 59, 664-675. Saunders, A. M., Strittmatter, W. J., Schmechel, D., George-Hyslop, P. H., PeriackVance, M. A., Rosi, B. L., Gusella, J. F, & Alberts, M. J. (1993). Association of apolipoprotein E allele with late-onset familial and sporadic Alzheimer's disease. Neurology, 43,1467-1472. Treves, T. A., Chandra, V, & Korczyn, A. D. (1993). Parkinson's and Alzheimer's diseases: Epidemiological comparison. 2. Persons at risk. Neuroepidemiology, 12, 345-349.

ACKNOWLEDGMENTS This report was supported by the Institute for the Study of Aging, New York, NY, the National Institute on Aging, the National Institutes of Health, and the Joseph and Florence Mandel Foundation.

4

p

Telephonic Screening

x. ph

of Mental Status:

m

s/ in de

A Feasibility Study in a Managed Care Clinic

4a ll.

vn

/fo

ru

David Knopman, Thomas Vonsternberg, and Jim Haefemeyer

he m

ABSTRACT

ht

tp

://

w

w

w

.c

Background and rationale: Despite the presumed benefits, many patients with early dementia are not recognized. One strategy to improve the rate of diagnosis is routine mental status assessments for elders. Telephonic assessment of mental status offers a solution to some of the problems of cognitive assessment, and therefore, perhaps, to timely diagnosis of dementia. We were interested in learning about the acceptance rate and yield of cognitively impaired participants with telephonic screening. Methods: We contacted by mail 447 individuals from the roster of a large clinic who were over the age of 75 years without diagnoses of dementia or synonymous illnesses. We then called to invite each participant to undergo a telephonic mental status examination. Either a shorter or longer mental status examination was used. Participants were questioned about their responses to the interview and the process. Results: Under conditions that might be present in many clinical practices, the acceptance/completion rate for telephonic mental status assessment among participants 75 years and older was 55% (190/348) among those actually contacted. Of those who underwent cognitive assessment, the yield of cognitively impaired participants was low (3%). Among the interviewees, willingness to consult 17

18

Early Detection

x. ph

p

with a physician regarding memory problems detected during a telephonic screen was 87%. Conclusions: The findings of the present stud}^ suggested that telephonic screening is a useful and efficient strategy to identify those elders who do not need face-to-face cognitive assessments. Telephonic screening of mental status, by the approach we used, was less satisfactory for detection of previously unrecognized, cognitively impaired participants. Whether the deficiency lies with the telephonic approach or with "uninvited" mental status testing is not clear.

s/ in de

INTRODUCTION

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

The timely detection of early dementia should offer advantages to the individuals involved, as well as to society as a whole. In a properly developed health care system, detection of early dementia would enable the patients and their families to institute safeguards against misadventures that occur in dementia patients. Despite the presumed benefits, many patients with early dementia are not recognized (Callahan, Hendrie, & Tierney, 1995; Eefsting, Boersma, Van den Brink, & Van Tilburg, 1996; Perkins, Annegers, Doody, et al., 1997; Knopman, Donohue, & Gutterman, 2000). Because patients and families are quite tardy in bringing dementia patients to medical attention (Knopman et al., 2000), mental status assessments that are part of yearly physicals for all elders would be a more proactive approach. Strategies that circumvent the barriers to mental status testing need to be explored. Telephonic assessment of mental status offers a solution to some of the problems of cognitive assessment, and therefore, perhaps, to timely diagnosis of dementia. The actual telephonic interviews can be carried out by trained nonphysicians. Telephonic screening has been used successfully in research settings (Grodstein, Chen, Pollen, et al., 2000), but to our knowledge, it has not been used in the context of routine care.

METHODS

Approximately 830 individuals over age 75 years were on the computer roster of a large outpatient clinic affiliated with a managed care organization in Minneapolis, Minnesota. We selected those individuals from this roster without diagnoses of dementia, Alzheimer's disease (AD), and their synonymous diagnostic codes in any patient encounter in the prior 2 years. Of the 611 individuals who met these criteria, we sent an introductory letter to 447 potential participants, informing them that they would be receiving a phone call as part of this study.

Telephonic Screening of Mental Status

19

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Upon reaching a participant on the telephone, the examiner (an RN experienced in geriatrics and formally trained to administer the mental status examinations) informed the individual of who she was and the nature of the study. For those who agreed to participate, they were given either the Orientation-Memory-Concentration (OMC) test (Katzman, Brown, Fuld, et al., 1983) or the Minnesota Cognitive Acuity Screen (MCAS) (Knopman, Knudson, Yoes, & Weiss, 2000). Administration of the two generally alternated, except in a few circumstances in which a participant would agree only to the shorter of the two tests. After completion of the mental status examination, the participants were questioned on four issues. The first question asked how they thought they performed on the mental status examination ("How do you think that you did on the tests we just finished?"), and the responses were good, fair, poor, or very poor. The second question was, "Apart from this interview, in the past few months, have you felt that you have had memory problems?", with the rating scheme as follows: "my memory is excellent," "my memory is as good as anyone else my age," "my memory isn't as good as it used to be, but it doesn't cause me any problems," "my memory has definitely deteriorated in the past few years and interferes with my daily activities," or "my memory is severely impaired." The third question was whether they would follow-up with their physician if they were told that they had done poorly ("Suppose that the present interview showed that you had memory problems, would you be willing to have a medical assessment for memory problems at your clinic?", with yes, no, or possibly as the responses. The final question was open-ended, asking what they thought of the process of telephonic examinations. The study was approved by the HealthPartners Institutional Review Board. Oral consent was obtained to conduct the phone interview.

tp

://

RESULTS

ht

Out of 447 participants who were sent letters announcing the possibility that they would be contacted, 190 (122 women; mean age, 81.3 years; range, 75 to 93 years) underwent a mental status examination and completed the questionnaire about the experience. There were 158 (45%) elders who were contacted by phone who declined to participate. The remaining 88 participants (20% of the 447) drawn from the clinic roster could not be contacted, either because of phone number inaccuracies or repeated no-response on attempted contacts. The interviewer required 79 hours to complete the 190 interviews and deal with the 158 refusals, which translated to approximately 25 minutes per completed interview.

20

Early Detection

ht

tp

://

w

w

w

.c he

m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Hearing difficulties were reported by a number of participants (39%) who completed interviews, but in these individuals, the examiner did not believe that performance was adversely affected. Of participants who refused, hearing problems were occasionally cited as a reason for refusal, but we were not able to collect this data in a systematic fashion (because people hung up without responding to any questions of the examiner). There were only two reported instances where the examiner believed that the participant was being coached, and only one instance where the background noise was sufficient to be a distraction by the perceptions of the examiner. There were 95 participants who were given the OMC test. All but seven (93%) scored >24 (fewer than four errors). Only three participants scored in the impaired range of 16, LMII 24)

UCLA

(n = 188) age = 68.8 yr. edu = 15.9 yr. 63% female

vn

/fo

ru

Only 10 per group were enrolled from each center.

m

Imaging Protocol Magnet Voxel dimension

Mayo Clinic

ht

tp

://

w

w

w

.c

he m

4a ll.

of the patients provided to them for hippocampal assessment. The 3-D SPGR sequence was chosen because the near isotropic voxel in a 3-D acquisition is capable of being reformatted perpendicular to the long axis of the hippocampus without significant loss of resolution, can accommodate 3-D surface analysis, and is easy to acquire with most 1.5 T scanners across the world. Table 6.1 records the number of patients capable of being recruited from each site with the details of their scanning acquisition. Assessment methods. Four assessment techniques were implemented at each center. The first was a traditional volumetric assessment technique with manual outline of left and right hippocampi according to the methods of Jack et al. (1990,1989). Contours were made using a Java-based outlining package distributed to all centers over the Internet, which can be compiled on all current personal computers, laptop computers, or unixbased workstations. The digital output of these contours were electronically sent to UCLA for processing. The second procedure was to reformat the scans axially, parallel to the long axis of the hippocampus, according to the methods of de Leon et al. (1993) and to provide an individual slice through each volume to obtain qualitative assessments of medial temporal atrophy using a 4-point scale. The images of each slice were accessed via the Internet, and scores were entered electronically over the web. The third method was a linear measure of medial temporal lobe width that used the centimeter-distance scale of images that have the midpoint

Hippocampal Volume Assessment

37

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

of the rostral caudal extent of the hippocampal formation at each side. This linear measure of the narrowest point of the medial temporal lobes adjacent to the brain stem used the procedure described by Jobst et al. (1998,1992). Images of each slice were accessed via the Internet, and measures were entered electronically over the web. The fourth method produced population-based, average 3-D hippocampal surface maps for both the control and MCI groups, derived from the manual outlines produced across centers. The methodology for constructing a surface map was described (Thompson, Schwartz, Lin, Khan, & Toga, 1996) and was initially applied to document the variability of the elderly control, MCI, and AD hippocampus in the Talairach atlas space, arguing for a refinement of this commonly used space in the functional imaging assessment of the elderly and demented population (Figure 6.1) (Mega, Thompson, Toga, & Cummings, 2000). Hippocampal contours were traced using a track ball on each relevant slice. The points that made up a traced contour, after smoothing the effects of irregular hand movements, were connected across slices to create regularly ordered 3-D meshes corresponding to the hippocampal surface. To quantify an

FIGURE 6.1 The average 3-D surface models of 10 control, 10 MCI, and 10 AD patients' hippocampi derived from manual outlines according to the methods of Jack et al. (1990,1992). Note the mismatch of these averages in the Taliarach coordinate space.

Early Detection

38

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

individual patient's anatomy an average hippocampal 3-D model was resolved by averaging the vectorial displacements, on a point-by-point basis from the parametric mesh, for each patient's hippocampus. Hippocampal variability, expressed as a 3-D distance in the common coordinate space, was computed by taking the mean of the square roots of a 3-D displacement vector necessary to align each node along a patient's mesh onto the average representation of the population's hippocampus. This computation of the root mean square (RMS) allows production of a variability map is shown in Figure 6.2. Standardization of the output from each center in their volumetric analysis permitted the construction of outliner and morphological variability maps, as well as displacement maps of the MCI group from the average control hippocampus. In addition, a fully automated hippocampal volume extraction method was tested based upon the manually outlined data that generate an average hippocampal location in a fixed coordinate space. Knowing the average location of the hippocampus in the atlas space permitted a refinement of the resolution of the atlas template, constructed via continuum-mechanical surface-based warping (Thompson, Woods, Mega, & Toga, 2000), which can then serve as the new target for a fully automated hippocampal volume extractor.

FIGURE 6.2 Variability maps such as this can be constructed to detail the intercenter outliner variability of the same scans, as well as resolving the variability of the hippocampus within a population.

Hippocampal Volume Assessment

39

s/ in de

x. ph

p

By using the inverse of a nonlinear high-order intensity based warp (e.g., 8th order AIR, Roger Woods' software package that is freely available on the web) that brings a new participant into the atlas template, the hippocampal surface was brought from the atlas to segment the new participant's native data for volumetric assessment. By jack-knifing each participant tested so that their contour does not make up the average atlas, an estimate of the validity of this assessment was achieved based on their manual outlines. Given the known accuracy of 8th order AIR into a nonlinear target (see Figure 6.3), this procedure would require little manual interaction with the data. UCLA made the hippocampal atlas template derived from this proposal freely available on the web to any institution, as well as the Java-based tools to extract hippocampi in new participants for use in the clinical and scientific community.

ru

m

DATA ANALYSIS

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

Statistical analysis. Volumetric assessment included normalization for interpatient variation of head size by dividing hippocampal volume by

FIGURE 6.3 Using the fully automated AIR package, freely available on the Internet, near-perfect hippocampal registration accuracy to an atlas template can be achieved with nonlinear high-order warpings, as shown in these variability maps constructed from 10 AD patients. Inverting the warping field to carry back the template's hippocampal surface onto the individual's native scan will allow fully automated hippocampal extraction. The template atlas and Java-based tools for this procedure will be freely available on the Internet as a result of this proposal.

40

Early Detection

he m

REFERENCES

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

the total intracranial volume of that particular patient. Percentiles for normalized hippocampal volume had been previously reported. Percentile scores were then converted to a W score, which is a value from a standard normal distribution corresponding to the observed percentile (in a standard normal distribution, the 50th, 5th, and 2.5th percentiles are given by W scores of 0, -1.645, and -1.96, respectively). A W score of less than or equal to -2.5 would be associated with the 0.6th percentile of hippocampal volume. Sixty-seven of 80 MCI patients in a recent study (Jack et al., 1999) had W scores less than 0 (13 had W scores less than or equal to -2.5, which is 16.25% of the population of MCI patients studied). Assuming outliner variability as previously reported across studies of errors less than 1.9% coefficient of variation, then the number of MCI patients needed to produce sufficient power to find a significant effect would be 30. This would provide 80% power to detect a difference of 16% as a deviation from .5, with alpha equal to .01. If 10.5% of the 30 MCI patients fall below a W score of -2.5, rather than the expected 16.25%, we would have 80% power to detect an alpha at .01 (h = .45 at 0.6th percentile of the control's standard normal distribution) (Cohen, 1988). Sensitivity (true positives / (true positives + false negatives) and specificity (true negatives / (false positives + true negatives) calculations were also computed for each method's ability to correctly identify incipient AD, MCI, and controls.

ht

tp

://

w

w

w

.c

Berg, L. (1988). Clinical dementia rating. Psychopharmacological Bulletin, 24, 637-639. Cohen, J. (1988). Statistical power analysis for the behavioral sciences. New York: Academic Press. de Leon, M. J., George, A. E., Golomb, J., Tarshish, C, Convit, A., Kluger, A., De Santi, S., McRae, T., Ferris, S. H., Reisberg, B., Ince, C., Rusinek, H., Bobinski, M., Quinn, B., Miller, D. C., & Wisniewski, H. M. (1997). Frequency of hippocampus atrophy in normal elderly and Alzheimer's disease patients. Neurobiology of Aging, 18,1-11. de Leon, M. J., George, A. E., Stylopoulos, L. A., Smith, G., & Miller, D. C. (1989). Early marker for Alzheimer's disease: The atrophic hippocampus. Lancet, 2, 672-673. de Leon, M. J., Golomb, J., George, A. E., Convit, A., Tarshish, C. Y, McRae, T, De Santi, S., Smith, G., Ferris, S. H., Noz, M., & Rusinek, H. (1993). The radiologic prediction of Alzheimer disease: The atrophic hippocampal formation. American Journal of Neuroradiology, 14, 897-906. Jack, C. R., Bently, M., Twomey, C. K., & Zinsmeister, A. R. (1990). MR-based volume measurements of the hippocampal formation and anterior temporal lobe: Validation studies. Radiology, 176, 205-209.

Hippocampal Volume Assessment

41

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Jack, C. R v Petersen, R. C., O'Brien, P. C, & Tangalos, E. G. (1992). MR-based hippocampal volumetry in the diagnosis of Alzheimer's disease. Neurology, 42, 183-188. Jack, C. R., Petersen, R. C., Xu, Y. C., O'Brien, P. C., Smith, G. E., Ivnik, R. J., Boeve, B. R, Waring, S. C., Tangalos, E. G., & Kokmen, E. (1999). Prediction of AD with MRI-based hippocampal volume in mild cognitive impairment. Neurology, 52,1397-1403. Jack, C. R., Twomey, C. K., Zinsmeister, A. R., Sharbrough, F. W., Petersen, R. C., & Cascino, G. D. (1989). Anterior temporal lobes and hippocampal formations: Normative volumetric measurements from MR images in young adults. Radiology, 172, 549-554. Jobst, K. A., Barnetson, L. P. D., Shepstone, B. J., & members of OPTIMA. (1998). Accurate prediction of histologically confirmed Alzheimer's disease and the differential diagnosis of dementia: The use of NINCDS-ADRDA and DSMIII-R criteria, SPECT, X-ray CT, and Apo E4 in medial temporal lobe dementias. International Psychogeriatrics, 10, 271-302. Jobst, K. A., Smith, A. D., Barker, C. S., Wear, A., King, E. M., Smith, A., Anslow, P. A., Molyneux, A. J., Shepstone, B. J., Holmes, K. A., Robinson, J. R., Hope, R. A., Oppenheimer, C., Brockbank, K., & McDonald, B. (1992). Association of atrophy of the medial temporal lobe with reduced blood flow in the posterior parietotemporal cortex in patients with a clinical and pathological diagnosis of Alzheimer's disease. Journal of Neurology Neurosurgery Psychiatry, 55,190-194. Mega, M. S., Thompson, P. M., Toga, A. W., & Cummings, J. L. (2000). Neuroimaging in dementia. In J. C. Mazziotta, A. W. Toga, & R. Frackowiak (Eds.), Brain mapping: The disorders (pp. 217-293). San Diego: Academic Press. Petersen, R. C., Smith, G. E., Waring, S. C., Ivnik, R. J., Tangalos, E. G., & Kokmen, E. (1999). Mild cognitive impairment: Clinical characterization and outcome. Archives of Neurology, 56, 303-308. Sled, J. G., Zijdenbos, A. P., & Evans, A. C. (1998). A nonparametric method for automatic correction of intensity nonuniformity in MRI data. IEEE Transactions on Medical Imaging, 17, 87-97. Thompson, P. M., Schwartz, C., Lin, R. T., Khan, A. A., & Toga, A. W. (1996). Threedimensional statistical analysis of sulcal variability in the human brain. Journal ofNeuroscience, 16, 4261-4274. Thompson, P. M., Schwartz, C., & Toga, A. W. (1996). High-resolution random mesh algorithms for creating a probabilistic 3-D surface atlas of the human brain. Neuroimage, 3,19-34. Thompson, P. M., & Toga, A. W. (1996). A surface-based technique for warping three dimensional images of the brain. IEEE Transactions on Medical Imaging, 15,1-16. Thompson, P. M., Woods, R. P., Mega, M. S., & Toga, A. W. (2000). Mathematical and computational challenges in creating deformable and probabilistic atlases of the human brain. Human Brain Mapping, 9, 81-92.

7

x. ph

s/ in de

Markers for Detection and Prevention of

p

Brain Imaging Surrogate

Cognitive Aging and

ru

m

Alzheimer's Disease

ABSTRACT

.c he

m

4a ll.

vn

/fo

Gary W. Small

ht

tp

://

w

w

w

With increasing knowledge of the neuropathological and cognitive changes preceding Alzheimer's disease (AD), clinical trials have begun to focus on preventive treatments aimed at slowing age-related cognitive decline and delaying onset of AD. Studying participants with minimal deficits leads to diagnostic heterogeneity and a need for larger samples in order to detect active drug effects. This chapter summarizes results of several studies designed to address such issues. Middle-aged and older adults with mild memory complaints were studied using brain imaging and measures of the major known genetic risk for AD, the apolipoprotein E-4 (apo E-e4) allele. In a study of positron emission tomography during mental rest, glucose metabolic rates were significantly lower in apo E-e4 carriers in brain regions affected by AD. Another study using functional magnetic resonance imaging showed increased brain activation during memory tasks in apo E-e4 carriers in similar brain regions. Longitudinal follow-up after 2 years indicated the potential utility of such brain-imaging measures, combined with genetic-risk information, as surrogate markers 42

Brain Imaging Surrogate Markers

43

in prevention-treatment trials for cognitive aging and AD. Additional development of novel approaches using positron emission tomography to directly measure the neuritic plaques and neurofibrillary tangles of AD offers promise of more specific measures of disease progression in future clinical trials.

p

INTRODUCTION

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

Medical technological advances have led to an increased proportion of older persons and a greater number afflicted with the major cause of latelife cognitive decline, Alzheimer's disease (AD). Gradual progressive memory loss precedes clinically diagnosed AD. Findings of neuritic plaques (NPs) (Price & Morris, 1999) and neurofibrillary tangles (NFTs) (Braak & Braak, 1991), the neuropathological hallmarks of AD, in adults without dementia suggest that the neuronal deficits leading to AD begin years before any clinical changes. New and developing antidementia treatments focus on slowing disease progression rather than reversing neuronal death, emphasizing the importance of identifying early markers of future cognitive decline. Genetic studies have identified an association between the apolipoprotein E-4 (apo E-e4) allele on chromosome 19 and the common form of AD that begins after age 60 years (Saunders, Strittmatter, Schmechel, et al., 1993). Apo E has three allelic variants (apo E-2, apo E-3, and apo E-e4) and five common genotypes (2/3, 3/3, 2/4, 3/4, 4/4). The apo E-e4 allele has a dose-related effect on increasing risk and lowering the age of onset of late-onset familial and sporadic AD (Saunders et al., 1993; Corder et al., 1993), while apo E-2 appears to confer protection (Corder et al., 1994). Although the apo E-e4 allele may have a modest effect in predicting cognitive decline in older persons, Apo E genotype alone is not considered a useful predictor in nondemented people (Relkin, Tanzi, Breitner, et al., 1996). Structural magnetic resonance imaging (MRI) in normal older persons may show medial temporal atrophy and predict future cognitive decline (Golomb, Kluger, de Leon, et al., 1996); cerebral atrophy, however, is seen only after substantial cell death. Positron emission tomography (PET) studies of glucose metabolism during mental rest have identified parietal, temporal, and prefrontal deficits in glucose metabolism in normal, middle-aged, apo E-e4 carriers (Small et al., 1995; Reiman, Caselli, Yun, et al., 1996) who are not likely to develop the disease for decades. Activation imaging, which compares brain activity while participants perform a task relative to a control or resting state, may reveal more subtle alterations in brain function, perhaps before the emergence of mild memory impairments. Activation PET studies, using cognitive and passive

44

Early Detection

ru

m

s/ in de

x. ph

p

stimuli, have revealed more widespread brain activity among patients with AD, compared with age-matched normal participants (Grady, Haxby, Horwitz, et al., 1993; Mentis, Horwitz, Grady, et al., 1996; Backman et al., 1999). Like PET, functional MRI provides measures of signal intensity associated with relative cerebral blood flow during memory or other cognitive tasks (Gabrieli, Brewer, Desmond, & Glover, 1997), but has the advantages of high resolution in space and time and lack of radiation exposure. The MRI signal intensity associated with a particular task, in comparison to the control condition, reflects relative blood flow and, consequently, neural activity, although indirectly (Fox & Raichle, 1986; Ogawa, Tank, Menon, et al., 1992; Kwong, Belliveau, Chesler, et al., 1992). In this chapter, I will summarize recent findings from PET and functional MRI in middle-aged and older adults with mild memory complaints. These studies show a pattern of brain activity that differs according to genetic risk, which may be useful in future clinical trials of drugs designed to prevent age-related cognitive decline.

vn

/fo

Cerebral Metabolic and Cognitive Decline in Apo E-e4 Carriers

ht

tp

://

w

w

w

.c

he m

4a ll.

To determine cognitive and metabolic decline patterns according to genetic risk, Small et al. (2000) investigated cerebral metabolic rates using PET in middle-aged and older nondemented persons with normal memory performance. Participants were right-handed and in the 50- to 84-year-age range. Of the 54 participants with mild memory complaints, 27 were apo E-e4 carriers and 27 were noncarriers. A single copy of the apo E-e4 allele was associated with lowered inferior parietal, lateral temporal, and posterior cingulate metabolism, which predicted cognitive decline after 2 years of longitudinal follow-up. For the 20 nondemented participants followed longitudinally, memory-performance scores did not decline significantly, but cortical metabolic rates did. In apo E-e4 carriers, a 4% left posterior cingulate metabolic decline was observed, and inferior parietal and lateral temporal regions demonstrated the greatest magnitude (5%) of metabolic decline after 2 years. These results have practical implications for clinical trials of dementiaprevention treatments. The right lateral temporal metabolism for apo E-e4 carriers at baseline and 2-year follow-up yielded an estimated power under the most conservative scenario (i.e., assuming that the points are connected exactly in reverse order) of 0.9, to detect a 1-unit decline from baseline to follow-up using a one-tailed test. A sample size of only 20 participants, therefore, would be needed in each treatment arm (i.e., active drug or placebo) to detect a drug-effect size of 0.8 (a = 0.05, power = 0.8). Thus, a clinical trial of a novel intervention to prevent cerebral metabolic

Brain Imaging Surrogate Markers

45

s/ in de

Brain Activation During Memory Tasks in People at Genetic Risk

x. ph

p

decline would require only 40 participants over a 2-year treatment period. Such findings are consistent with previous PET studies showing stable and replicable results (Andreasen, Arndt, Cizadlo, et al., 1996), and suggest that combining PET and AD genetic-risk measures will allow investigators to use relatively small sample sizes when testing antidementia treatments in preclinical AD stages. These results indicate that the combination of cerebral metabolic rates and genetic-risk factors provides a means for preclinical AD detection that will assist in response monitoring during experimental treatments.

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

To determine the relationship between brain responses to memory tasks and genetic risk for AD, Bookheimer et al. (2000) performed apo E genotyping and functional MRI while cognitively intact older persons performed memory tasks. The study included 30 participants, aged 47 to 82 years, with mild memory complaints but normal memory performance, of whom 16 were apo E-e4 carriers and 14 were not. The age and prior educational achievement in the two groups were similar. Brain-activation patterns were determined from functional MRI scanning while participants memorized and recalled unrelated word pairs. Memory performance was reassessed on 14 participants 2 years later. The magnitude and spatial extent of brain activation during memory performance in regions affected by AD, including left hippocarnpal, parietal, and prefrontal regions, was greater in the participants with apo E-e4 alleles, as compared with those with no apo E-e4 alleles. During memory performance tasks, the apo E-e4 carriers demonstrated a greater percentage increase in hippocarnpal MRIsignal intensity and a greater number of activated regions throughout the brain than did participants without apo E-e4. Longitudinal assessment after 2 years indicated that greater baseline brain activation correlated with verbal-memory decline. These results indicate that brain-activation patterns during memory tasks differ according to genetic risk for AD and may provide information that eventually predicts future cognitive decline. Preclinical Detection: Benefits and Strategies Although no cure exists for AD, preclinical disease detection has several benefits. When early detection assessments are negative, people with mild memory complaints can be reassured that their forgetfulness reflects a normal age-related change that probably will not progress. In addition, many people would like to know about a poor prognosis while still in a mildly impaired state in order to plan their futures while mental faculties

46

Early Detection

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

remain. Perhaps the most compelling argument for preclinical detection strategies is to identify candidates for novel antidementia treatments before the dementing process causes extensive neuronal death, since new antidementia treatments are more likely to delay the dementing process than to reverse neuronal death. Although current cholinergic treatments have been shown to result in symptomatic, rather than disease-altering or structural effects, it would certainly be of interest to initiate treatments very early when searching for a disease-modifying effect. Moreover, both the expense and potential risks of treatment make it reasonable to reserve treatment only for those people who are at the greatest risk for developing the disease. Several lines of research suggest that AD actually begins years before its clinical manifestations are obvious. The PET studies of glucose metabolism combined with genetic-risk assessment show regional glucose abnormalities in middle-aged persons with the apo E-e4 allele (Small et al., 1995, 2000; Reiman et al., 1996). Studies of structural images suggest that regional atrophy of hippocampus and other medial temporal regions may be an early predictor of future cognitive decline (Golomb et al., 1996). Brain autopsy studies of normal aging and older persons with mild cognitive impairment also indicate very early, preclinical accumulation of NPs and NFTs, the neuropathological hallmarks of AD, years before a clinical diagnosis can be confirmed (Price & Morris, 1999; Braak & Braak, 1991). Finally, findings from a study of 93 nuns also support the notion of subtle, preclinical functional abnormalities. In that study (Snowdon, Kemper, Mortimer, et al., 1996), a systemic assessment of these nuns' early autobiographies (mean age = 22 years) and their later (age 75-95) cognitive performances found that low idea density and lack of grammatical complexity in early life predicted low cognitive test scores in late life (Snowdon et al., 1996).

ht

tp

Positron Emission Tomography Imaging of Amyloid Senile Plaques and Neurofibrillary Tangles Several groups have been developing new, small-molecule probes to image the amyloid NPs and NFTs. Current methods for measuring brain amyloid, such as histochemical stains, require tissue fixation on postmortem or biopsy material. Available in vivo methods for measuring NPs or NFTs are indirect (e.g., cerebrospinal fluid measures) (Motter, Vigo-Pelfrey, Kholodenko, et al., 1995). Studies that may lead to direct in vivo, human AP imaging include various radiolabeled probes using small organic and organometallic molecules capable of detecting differences in amyloid-fibril structure or amyloid-protein sequences (Ashburn, Han, McGuinness, & Lansbury, 1996). Investigators also have used chrysamine-G, a carboxylic

Brain Imaging Surrogate Markers 47

47

vn

/fo

ru

m

s/ in de

x. ph

p

acid analogue of Congo red, an amyloid-staining histologic dye (Klunk, Debnath, & Pettegrew, 1995), serum amyloid-P component, a normal plasma glycoprotein that binds to amyloid-deposit fibrils (Lovat, O'Brien, Armstrong, et al., 1998), or monoclonal antibodies (Majocha et al, 1992). Methodological difficulties that hinder progress with these techniques include poor blood-brain barrier crossing and limited specificity and sensitivity. In addition, most approaches do not measure both NPs and NFTs. Recently, Barrio et al. (1999) reported using a hydrophobic, radiofluorinated derivative of l,l-dicyano-2-[6-(dimethylamino)naphthalen-2yllpropene (FDDNP) (Jacobson, Petric, Hogenkamp, Sinur, & Barrio, 1996) with PET to measure the cerebral localization and load of NFTs and SPs in AD patients. The probe showed visualization of NFTs, NPs, and diffuse amyloid in AD brain specimens using in vitro fluorescence microscropy, which matched results using conventional stains (e.g., thioflavin S) in the same tissue specimens. Such approaches may ultimately aid in the early detection of AD and brain-function monitoring during antidementia treatment trials, particularly those designed to interrupt accumulation of NPs and NFTs.

4a ll.

REFERENCES

ht

tp

://

w

w

w

.c

he m

Andreasen, N. C, Arndt, S., Cizadlo, T., O'Leary, D., & Gold, S. (1996). Sample size and statistical power in [15O]H2O studies of human cognition. Journal of Cerebral Blood Flow and Metabolism, 16, 804-816. Ashburn, T. T., Han, H., McGuinness, B. R, & Lansbury, P. T. (1996). Amyloid probes based on Congo Red distinguish between fibrils comprising different peptides. Chemistry and Biology, 3, 351-358. Backman, L., Andersson, J. L. R., Nyberg, L., Winblad, B., Nordberg, A., & Almkvist, O. (1999). Brain regions associated with episodic retrieval in normal aging and Alzheimer's disease. Neurology, 52,1861-1870. Barrio, J. R., Huang, S-C, Cole, G. M, Satyamurthy, N., Petric, A., & Small, G. W. (1999). PET imaging of tangles and plaques in Alzheimer disease. Journal of Nuclear Medicine, 40(Suppl), 70P-71P. Bookheimer, S. Y., Strojwas, M. H., Cohen, M. S., Saunders, A. M., Pericak-Vance, M. A., Mazziotta, J. C., & Small, G. W. (2000). Brain activation in people at genetic risk for Alzheimer's disease. New England Journal of Medicine, 343, 450-456. Braak, H., & Braak, E. (1991). Neuropathological staging of Alzheimer-related changes. Acta Neuropathology, 82, 239-259. Corder, E. H., Saunders, A. M., Strittmatter, W. J., Schmechel, D., Gaskell, P., Small, G. W., Roses, A. D., Haines, J. L., & Pericak-Vance, M. A. (1993). Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late-onset families. Science, 261, 921-923. Corder, E. H., Saunders, A. M., Risch, N. J., Strittmatter, W. J., Schmechel, D. E.,

48

Early Detection

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Gaskell, P. C, Rimmler, J. B., Locke, P. A., Conneally, P. M., Schmader, K. E., Small, G. W., Roses, A. D., Haines, J. L., & Pericak-Vance, M. A. (1994). Protective effect of apolipoprotein E type 2 allele for late-onset Alzheimer disease. Nature Genetics, 7,180-183. Fox, P. T., & Raichle, M. E. (1986). Focal physiological uncoupling of cerebral blood flow and oxidative metabolism during somatosensory stimulation in human subjects. Proceedings of the National Academy of Sciences (USA), 83,1140-1144. Gabrieli, J. D. E., Brewer, J. B., Desmond, J. E., & Glover, G. H. (1997). Separate neural bases of two fundamental memory processes in the human medial temporal lobe. Science, 276, 264-266. Golomb, J., Kluger, A., de Leon, M. J., Ferris, S. H., Mittleman, M., Cohen, J., & George, A. F. (1996). Hippocampal formation size predicts declining memory performance in normal aging. Neurology, 47, 810-813. Grady, C. L., Haxby, J. V., Horwitz, B., Gillette, J., Salerno, J. A., Gonzalez-Aviles, A., & Rappaport, S. I. (1993). Activation of cerebral blood flow during a visuoperceptual task in patients with Alzheimer-type dementia. Neurobiology of Aging, 14, 35-44. Jacobson, A., Petric, A., Hogenkamp, D., Sinur, A., & Barrio, J. R. (1996). 1,1dicyano-2-(6- dimethylamino)naphthalen-2-yl)propene (DDNP): A solvent polarity and viscosity sensitive fluorophore for fluorescence microscopy. Journal of the American Chemical Society, 118, 5572-5579. Klunk, W. E., Debnath, M. L., & Pettegrew, J. W. (1995). Chrysamine-G binding to Alzheimer and control brain: Autopsy study of a new amyloid probe. Neurobiology of Aging, 16, 541-548. Kwong, K. K., Belliveau, J. W., Chesler, D. A., Goldberg, I. E., Weisskoff, R. M., Poncelet, B. P., & Tarner, R. (1992). Dynamic magnetic resonance imaging of human brain activity during primary sensory stimulation. Proceedings of the National Academy of Sciences (USA), 89, 5675-5679. Lovat, L. B., O'Brien, A. A., Armstrong, S. R, et al. (1998). Scintigraphy with 1231serum amyloid P component in Alzheimer disease. Alzheimer's Disease and Associated Disorders, 12, 208-210. Majocha, R. E., Reno, J. M., Friedland, R. P., VanHaight, C., Lyle, L. R., & Marotta, C. A. (1992). Development of a monoclonal antibody specific for beta/A4 amyloid in Alzheimer's disease brain for application to in vivo imaging of amyloid angiopathy. Journal of Nuclear Medicine, 33, 2184-2189. Mentis, M. J., Horwitz, B., Grady, C. L., et al (1996). Visual cortical dysfunction in Alzheimer's disease evaluated with a temporally graded "stress test" during PET. American Journal of Psychiatry, 153, 32-40. Motter, R., Vigo-Pelfrey, C., Kholodenko, D., Barbour, R., Johnson-Wood, K., Galasko, D., & Chang, L. (1995). Reduction of beta-amyloid peptide42 in the cerebrospinal fluid of patients with Alzheimer's disease. Annals of Neurology, 38, 643-648. Ogawa, S., Tank, D. W., Menon, R., Ellermann, J. M., Merkle, H., Kim, S. G., & Ugurbil, K. (1992). Intrinsic signal changes accompanying sensory stimulation: Functional brain mapping with magnetic resonance imaging. Proceedings of the National Academy of Sciences (USA), 89, 5951-5955.

Brain Imaging Surrogate Markers 49

49

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Price, J. L., & Morris, J. C. (1999). Tangles and plaques in nondemented aging and "preclinical" Alzheimer's disease. Annals of Neurology, 45, 358-368. Reiman, E. M., Caselli, R. J., Yun, L. S., Chen, K., Bandy, D., de Leon, M. J., & De Santi, S. (1996). Preclinical evidence of Alzheimer's disease in persons homozygous for the e4 allele for apolipoprotein E. New England Journal of Medicine, 334, 752-758. Relkin, N. R., Tanzi, R., Breitner, J., et al. (1996). Apolipoprotein E genotyping in Alzheimer's disease: Position statement of the National Institute on Aging/Alzheimer's Association Working Group. Lancet, 347,1091-1095. Saunders, A. M., Strittmatter, W. J., Schmechel, D., et al. (1993). Association of apolipoprotein E allele epsilon 4 with late-onset familial and sporadic Alzheimer's disease. Neurology, 43,1467-1472. Small, G. W., Ercoli, L. M., Silverman, D. H. S., Huang, S-C, Komo, S., Bookheimer, S. Y., Lavretsky, H., Miller, K., Siddarth, P., Mazziotta, J. C., Saxena, S., Wu, H. M., Mega, M. S., Cummings, J. L., Saunders, A. M., PericakVance, M. A., Roses, A. D., Barrio, J. R., & Phelps, M. E. (2000). Cerebral metabolic and cognitive decline in persons at genetic risk for Alzheimer's disease. Proceedings of the National Academy of Sciences (USA), 97, 6037-6042. Small, G. W., Mazziotta, J. C., Collins, M. T., Baxter, L. R., Phelps, M. E., Mandelkern, M. A., Kaplan, A., La Rue, A., Adamson, C. E, Chang, L., Guze, B. H., Corder, E. H., Saunders, A. M., Haines, J. L., Pericak-Vance, M. A., & Roses, A. D. (1995). Apolipoprotein E type 4 allele and cerebral glucose metabolism in relatives at risk for familial Alzheimer disease. Journal of the American Medical Association, 273, 942-947. Snowdon, D. A., Kemper, S. ]., Mortimer, J. A., Greiner, L. H., Wekstein, D. R., & Markesbery, W. R. (1996). Linguistic ability in early life and cognitive function and Alzheimer's disease in late life. Findings from the Nun Study. Journal of the American Medical Association, 275, 528-532.

w

w

ACKNOWLEDGMENTS

ht

tp

://

Supported by the Montgomery Street Foundation, San Francisco, CA; the Fran and Ray Stark Foundation Fund for Alzheimer's Disease Research, Los Angeles, C A; The Institute for the Study of Aging, Inc.; and NIH grants MH52453, AG10123, AG13308, and the Alzheimer's Association grant IIRG94101. The views expressed are those of the authors and do not necessarily represent those of the Department of Veterans Affairs. Presented in part at The Institute for the Study of Aging First Annual Investigator's Meeting, October 29-31, 2000.

8

p

Novel (3-Amyloid Probes

vn

/fo

ru

m

s/ in de

x. ph

Mei-Ping Kung, Zhi-Ping Zhuang, Catherine Hou, Daniel Skovronsky, Tamar Gur, Sumalee Chumpradit, Virginia M.-Y. Lee, John Q. Trojanowski, and Hank F. Kung

4a ll.

ABSTRACT

ht

tp

://

w

w

w

.c he

m

This chapter proposes to develop novel probes, which may be useful as in vivo imaging agents, for studying (3-amyloid aggregates in living human brains. These novel probes are based on highly conjugated styrene derivatives labeled with I-125/I-123. An initial binding study showed that these agents displayed high-binding affinity and distinctively labeled (3-amyloid aggregates in the postmortem brain sections of patients with Alzheimer's disease (AD). The proposed agents may provide simple and effective tools to diagnose and monitor patients with Alzheimer's disease (AD). It is well known that AD is a neurodegenerative disease associated with regional neuronal loss (Selkoe, 1998; Selkoe, 1999; Sinha & Lieberburg, 1999). Clinical symptoms of AD include but are not limited to cognitive decline, irreversible memory loss, disorientation, and language impairment (Selkoe, 1997). Postmortem brain tissue examinations show neuropathology observations—the presence of senile plaques (Sps), neurofibrillary tangles, and neurophil threads containing (3-amyloid aggregates and highly phosphorylated tau proteins (Lee, 1996; Trojanowski & Lee, 1994). Several genomic factors have been linked to AD. Familial Alzheimer's disease has been reported to have mutations encoding (3-amyloid precursor protein (APP), apolipoprotein E4 (apo E-e4), presenilin 1 (PS1), and presenilin 2 (PS2) genes (Selkoe, 1997). The exact mechanisms of these four mutations, which lead to the development of AD, are not fully understood; however, 50

Novel f3-Amyloid Probes

51

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

the hallmark of AD is the formation of P-amyloid aggregates. It is likely that P-amyloid precursor proteins are degraded by several proteases, among which the catabolism reactions of P- and y-secretases on APP lead to the production of excess A[3 (P-amyloid). The excessive burden of Ap, produced by various normal or abnormal mechanisms, may represent the starting point of neurodegenerative events. Formation of P-amyloid aggregates in the brain may be the pivotal event, which produces various toxic effects in neuronal cells, leading to the formation of neuritic plaques. The plaques consist of extracellular masses of Ap filaments intimately associated with dystrophic dendrites and axons, activated microglia, and reactive astrocytes (Selkoe, 1997). It is, therefore, of great scientific interest to develop ligands that can specifically bind to the P-amyloid aggregates (Selkoe, 2000; Styren, Hamilton, Styren, & Klunk, 2000; Wengenack, Curran, & Poduslo, 2000). The new ligands will not only be useful as in vivo diagnostic tools, but may also provide in vitro labeling agents for studying the P-amyloid aggregates. They can be further modified as potential agents for inhibiting the formation of the P-amyloid aggregates. The proposed new ligands in this chapter may be useful as in vivo diagnostic tools for monitoring the formation of P-amyloid aggregates. Advances in early diagnosis of P-amyloid formation and aggregation may also lead to development of inhibitors for treatment of AD. In the past few years, several interesting reports on developing P-amyloid aggregate-specific imaging agents have appeared in the literature (Ashburn, Han, McGuinness, & Lansbury, 1996; Han, Cho, & Lansbury, 1996; Klunk, Debnath, & Pettegrew, 1995; Klunk et al, 1997; Ma this, Mahmood, Debnath, & Klunk, 1997; Styren et al., 2000; Wengenack et al., 2000). By far, the most attractive approach is based on highly conjugated chrysamine-G (CG) and congo red (CR), normally used for fluorescent staining of the plaques and tangles in postmortem brain sections of AD patients. It was demonstrated that the binding of CG and CR (also 3'-bromo and 3'-iodo derivatives; see Table 8.1) appeared to be selective toward the P-amyloid aggregates in vitro (AP 140 peptide aggregated) or in AD brain tissues with confirmed P-amyloid aggregates. At equilibrium, the binding of [I4C]CG toward P-amyloid aggregates is a saturable and reversible process similar to that observed for receptor-ligand binding. The unique observation of the interaction between P-amyloid aggregates (normally existing as an antiparallel p-sheet structure) and small, negatively charged and highly conjugated ligands may represent a novel opportunity to design specific ligands for single photon emission computed tomography (SPECT) imaging (Klunk, Debnath, & Pettegrew, 1994; Klunk et al., 1995). Recently, a significant advance has been demonstrated by Klunk and Mathis, who reported the used of a

Early Detection

52

s/ in de

x. ph

p

TABLE 8.1 In Vitro Binding Affinity (Ki) and Partition Coefficients of Congo Red, Chrysamine G (CG), 3'-Bromo-CG, and 3'-Ioclo-CG

0.7 60 — 200

/fo

ru

m

2800 370 300 250

CR CG 3'-Br-CG 3'-I-CG

4a ll.

vn

CR, congo red. CG, Chrysamine G Note. From "Synthesis of a Lipophilic, Radioiodinated Ligand with High Affinity to Amyloid Protein in Alzheimer's Disease Brain," by C. A. Mathis, K. Mahmood, M. L. Debnath, and W. E. Klunk, 1997, Proceedings of the Xllth International Symposium on Radiopharmaceutical

he m

Chemistry, pp. 94-95. Reprinted with permission.

ht

tp

://

w

w

w

.c

compound called "X-34" (Styren et al., 2000). Replacing the diazo group with a simple vinyl group (both CG and CR are diazo dyes) appears to preserve the binding affinity (Mathis et al., 1997). This substitution has several advantages over the diazo derivatives: (a) the molecular weight decreases, smaller molecules may show preferable ability to penetrate intact blood-brain barrier (BBB); (b) the vinyl group improve the in vitro and in vivo stabilities; (c) X-34 showed a similar separation of two negative charges by highly conjugated aromatic rings; and (d) derivatives can be prepared via a versatile reaction scheme, which is amenable to additional substitution. Results of a preliminary study suggest that the initial synthesis of a 3'bromo-derivative of X-34 (BSB) showed excellent binding affinity to |3-amyloid aggregates comparable to CG (Ki = 300 to 500 nM). It is postulated that the basic requirements for designing a |3-amyloid aggregate-specific ligand are: (a) a highly conjugated back bone; (b) two negative charges that are highly conjugated; (c) the negative charges are associated with salicylate moieties; (d) it is likely that there is bulk tolerance

Novel ft-Amyloid Probes

53

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

at 3-,4- and 3'-positions on the CG or X-34; and (e) the molecular weight of the tracer will be smaller than 750 for penetrating the intact bloodbrain barrier. Based on the requirements listed above, a novel fluorescent probe, (E,E)-l-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styryl-benzene (BSB), and two iodinated ligands, (E,E)-l-iodo-2,5-bis-(3-hydroxycarbonyl4-hydroxy)styryl-benzene (ISB) and (E,E)-l-iodo-2, 5-bis-(3-hydroxycarbonyl-4-methoxy)styryl-benzene (1MSB), were synthesized (see Figure 8.1). Preliminary results on the new fluorescent compound, BSB, were reported recently (Skovronsky et al., 2000). Basic conclusions of initial studies in cell lines and in transgenic mice suggest that: (a) BSB sensitively labels SPs in AD brain sections; (b) BSB permeates living cells in culture and binds specifically to intracellular A(3 aggregates; (c) following intracerebral injection in living transgenic mouse models of AD amyloidosis, BSB labels SPs composed of human A(3 with high sensitivity and specificity; and (d) BSB crosses the BBB and labels numerous AD-like SPs throughout the brain of the transgenic mice following intravenous injection. Under the sponsorship of this project, we have developed two radioiodinated ligands ([123/12SI]ISB and ([123/125I]IMSB). These "prototype" compounds were tested for their in vitro and in vivo binding characteristics to (3-amyloid. The proposed initial studies are designed to test and assess the parameters required for a radioiodinated ligand as a suitable biological marker for detecting senile plaques in living human brain. It is hypothesized that when labeled with 1-123 (T |/2 = 13 hr, 159 KeV), the (3 amyloidselective ligands with the proper sensitivity and in vivo pharmacokinetics will be useful as imaging agents in conjunction with SPECT. These ligands, if successfully developed, will be useful for the detection of A[3 aggregates prior to the onset of the disease and they ultimately may be applicable for quantitation of senile plaques in AD patients. We believe that early detection of the formation of plaques may be beneficial for the older population in which AD is relatively prevalent. Aggregated Af5(140) or AP 42) in solution was used as the model system for initial screening and characterization of ligand binding to amyloid fibrils (Figure 8.2). High-binding affinities were observed with both [125I]ISB and [125I]IMSB for Ap (140) aggregates. Similarly, [125I]IMSB displayed specific binding to AP 4,} aggregates, but with a lower affinity as compared with its binding to A|3(1 _40) aggregates (0.70 nM vs. 0.13 nM; see Figure 8.2).

FIGURE 8.1 Chemical structures of BSB, ISB, and IMSB.

Early Detection

s/ in de

x. ph

p

54

ru

m

FIGURE 8.2 The representative saturation and Scatchard plots of [125I]ISB and [125]IMSB for A(3(l-40) aggregates.

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

Postmortem brain sections from confirmed AD patients, as well as brain sections of normal brain were labeled with [125I]ISB and [125I]IMSB. Both ligands labeled plaques well, but [125I]IMSB displayed a more clear and distinct labeling with a minimal background signal (see Figure 8.3). In summary, AD is a brain disorder showing progressive memory loss and other cognitive loss. However, a definitive diagnosis of AD can only

FIGURE 8.3

Novel (3-Amyloid Probes

55

vn

/fo

ru

m

s/ in de

x. ph

p

be established by demonstrating the presence of abundant SPs and neurofibrillary tangles (NFTs) in the postmortem brain. The plaques are extracellular deposits of amyloid fibrils formed by |3-amyloid peptides (A(3), which can serve as suitable targets for early detection and monitoring the progression of this disease. This project proposes to develop new inhibitors of amyloid fibrils containing (3-amyloid aggregates, which may potentially be useful for diagnosis and treatment of AD. In this project, a novel series of compounds based on CG analogs showing specific binding to the A(3 aggregates in vitro and in vivo will be developed and characterized. The proposed agents, if successfully developed, may detect and monitor the changes of (3-amyloid deposition in living brain as a potential marker for this disease. They themselves may also serve as lead compounds for potential drugs for slowing or even reversing the formation of (3-amyloid aggregates in patients with AD. The new compounds will be synthesized. Radiolabeling and binding studies will be tested in in vitro and in vivo. Currently, there is no specific imaging agent available for direct mapping of (3-amyloid aggregates in the living brain; therefore, it is important to develop such an agent for the early diagnosis of AD, which could potentially benefit these patients.

4a ll.

REFERENCES

ht

tp

://

w

w

w

.c

he m

Ashburn, T. T., Han, H., McGuinness, B. E, & Lansbury, P. T., Jr. (1996). Amyloid probes based on Congo red distinguish between fibrils comprising different peptides. Chemical Biology, 3, 351-358. Han, G., Cho, C.-G., & Lansbury, P. T., Jr. (1996). Technetium complexes for the quantitation of brain amyloid. Journal of the American Chemical Society, 118, 4506-4507. Klunk, W. E., Debnath, M. L., & Pettegrew, J. W. (1994). Small-molecule beta-amyloid probes which distinguish homogenates of Alzheimer's and control brains. Biological Psychiatry, 35, 627. Klunk, W. E., Debnath, M. L., & Pettegrew, J. W. (1995). Chrysamine-G binding to Alzheimer and control brain: Autopsy study of a new amyloid probe. Neiirobiology of Aging, 16, 541-548. Klunk, W. E., Hamilton, R. L., Styren, S. D., Styren, G., Debnath, M. L., Mathis, C. A., Mahmood, A., & Hsiao, K. K. (1997). Staining of AD and Tg2576 mouse brain with X-34, a highly fluorescent derivative of chrysamine G and a potential in vivo probe for (3-sheet fibrils, [Abstract 636.12]. Society for Neuwscience Abstract, 23,1638. Lee, V. M. (1996). Regulation of tau phosphorylation in Alzheimer's disease. Annals of the New York Academy of Sciences, 777,107-113. Mathis, C. A!, Mahmood, K., Debnath, M. L., & Klunk, W. E. (1997). Synthesis of a lipophilic, radioiodinated ligand with high affinity to amyloid protein in Alzheimer's disease brain tissue, [Abstract II-9]. Proceedings of the Xllth International Symposium on Radiophannaceutical Chemistry, Uppsala, Sweden, 94-95.

56

Early Detection

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Selkoe, D. J. (1997). Alzheimer's disease: Genotypes, phenotypes, and treatments. Science,275, 630-631. Selkoe, D. J. (1998). The cell biology of beta-amyloid precursor protein and presenilin in Alzheimer's disease. Trends in Cell Biology, 8, 447-53. Selkoe, D. J. (1999). Biology of (3-amyloid precursor protein and the mechanism of Alzheimer's disease. In R. D. Terry, R. Katzman, K. L. Bick, & S. S. Sisodia (Eds.), Alzheimer's disease (pp. 293-310). Philadelphia: Lippincott Williams & Wilkins. Selkoe, D. J. (2000). Imaging Alzheimer's amyloid. Nature Biotechnology, 18, 823-824. Sinha, S., & Lieberburg, I. (1999). Cellular mechanisms of beta-amyloid production and secretion. Proceedings of the National Academy of Sciences (USA), 96, 11049-11053. Skovronsky, D., Zhang, B., Kung, M.-R, Kung, H. E, Trojanowski, J. Q., & Lee, V. M.-Y. (2000). In vivo detection of amyloid plaques in a mouse model of Alzheimer's disease. Proceedings of the National Academy of Sciences (USA), 97, 7609-7614. Styren, S. D., Hamilton, R. L., Styren, G. C, & Klunk, W. E. (2000). X-34, a fluorescent derivative of Congo red. A novel histochemical stain for Alzheimer's disease pathology. Journal of Histochemistry and Cytochemisty, 48,1223-1232. Trojanowski, J. Q., & Lee, V. M. (1994). Paired helical filament tau in Alzheimer's disease: The kinase connection. American Journal of Pathology, 144, 449-453. Wengenack, T. M., Curran, G. L., & Poduslo, J. E (2000). Targeting Alzheimer amyloid plaques in vivo. Nature Biotechnology, 18, 868-872.

x. ph

Early Clinical and Biological Manifestations

p

9

s/ in de

of Alzheimer's Disease:

Implications for Screening

ru

m

and Treatment

w

w

ABSTRACT

.c

he m

4a ll.

vn

/fo

Richard C. Mohs, Deborah Marin, and Vahram Haroutunian

ht

tp

://

w

Alzheimer's disease (AD) is a slowly progressive disease with an average of more than 10 years from first manifestations to death. Both the underlying biology of AD and its clinical manifestations change substantially over the course of the illness. This chapter reviews briefly some recent studies investigating the neurobiologic changes in the brains of AD patients over the course of the illness, with an emphasis on the earliest changes. Cognitive deficits also change over the course of illness and an understanding of the earliest cognitive changes may enable the development of screening instruments for AD. An ongoing set of studies designed to determine the reliability and validity of telephonic screening instruments is described. An instrument of this type could be useful in clinical trials of agents for the primary prevention of AD, as well as in health service-delivery organizations where there is a need to identify cases of AD that have not yet been diagnosed. 57

58

Early Detection

INTRODUCTION

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Alzheimer's disease (AD) is associated with a wide range of neurobiologic changes in the brain. Among them are the development of neuritic plaques and neurofibrillary tangles, neuronal loss, diminished cholinergic neurotransmission, increased oxidative stress, increased neuroinflammation, and loss of synapses. A major goal of AD neurobiologic research has been to determine which of these many changes are most responsible for the cognitive and other deficits observed clinically in patients with AD. A topic of particular interest has been the study of factors that are implicated in initiating the cascade of neurodegenerative processes involved in AD. Understanding the neurobiologic events that occur at the start of AD might lead to the development of therapies that could delay or even prevent the onset of this disease. Research conducted in our center has attempted to examine this question by looking at brain tissue from patients who die without dementia, with very mild dementia of the Alzheimer's type, or with definite AD of either mild, moderate, or severe impairment (Haroutunian et al., 1998). While these studies are primarily concerned with identifying the earliest biologic changes in AD patients, they also enable us to examine neurobiologic events at each stage in the longitudinal development of AD symptoms. Since the neuropathologic events underlying AD symptomatology may change over the entire course of illness, these studies may provide insight into the most appropriate therapeutic interventions for each stage of disease. Clinical manifestations of AD over the entire course of illness have been studied extensively (Morris et al., 1993; Stern et al., 1994). Changes in cognition, neuropsychiatric symptomatology (Marin et al., 1997), and in ability to perform activities of daily living (Galasko et al., 1997; Green et al., 1999) have been documented in longitudinal studies. As investigators have become more interested in early detection of disease and in the possibility of intervening early in the course of disease, there has been increased emphasis on changes that are the earliest indicators of AD (Petersen et al., 1999). In the absence of any clear biological test for early AD that can be used clinically, most efforts to improve screening and early detection have focused on neuropsychological tests of cognitive function. Studies have documented some of the earliest cognitive changes in AD, and studies are being conducted to determine the utility of some very brief screening tests for cognitive impairment. One study outlined below is being conducted in collaboration with the Institute for the Study of Aging. The study seeks to determine whether a telephone screen, lasting less than 5 minutes and administered by a lay person, can help identify persons likely to have AD.

Early Clinical and Biological Manifestations of AD 59

59

NEUROBIOLOGY OF EARLY ALZHEIMER'S DISEASE

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Details of the studies on early AD conducted at our center have been published (Haroutunian et al., 1998, 1999). The population for these studies are the residents of the Jewish Home and Hospital (JHH), a long-term care facility affiliated with the Mount Sinai School of Medicine in New York. The two main campuses of the JHH have approximately 1,600 residents, with an average age of over 85. Cognitive screening is a routine part of clinical care in the JHH, and Mini-Mental State Examination (MMSE) scores are available for nearly all residents. For the early AD study, all consenting residents with MMSE scores of 15 or greater are given a thorough diagnostic evaluation and are assigned a score on the Clinical Dementia Rating (CDR; Morris et al., 1993). Those with CDR scores of 0 (no dementia), 0.5 (questionable dementia), or 1.0 (mild dementia) are administered a battery of neuropsychological tests. The JHH routinely requests autopsy permission when a resident dies and, over the period of the early AD project, more than 50 autopsies have been obtained from residents who died with CDR scores of 0 to 1.0, and evidenced either no significant neuropathology or had neuropathologic lesions associated with AD only. As might be expected with cases from a long-term care facility, many more autopsies have been obtained from residents who died with more severe dementia or with comorbid neuropathologic lesions. Neuropathologic studies of cases dying with no dementia, questionable dementia, or mild dementia have shown that senile plaques are more abundant in most areas of the neocortex in patients with CDR 0.5 than in persons dying without any evidence of dementia (CDR 0) (Haroutunian et al., 1998). Examination of specific amyloid fragments has shown that the Afi peptides, especially the A^ 42 peptide, are markedly elevated even in the CDR 0.5 cases, compared with the CDR 0 cases (Naslund et al., 2000). By contrast, neurofibrillary tangles were evident in the entorhinal cortex and hippocampus of virtually all brains from persons over age 80, even those from nondemented individuals; extensive neurofibrillary tangles in the neocortex were evident only in patients with CDR 2.0 (moderate dementia) or greater (Haroutunian et al., 1999). These data support the view that overproduction and accumulation of amyloid protein, particularly the ^ 42 fragment, is a very early manifestation of AD. Neurofibrillary tangles in entorhinal cortex and hippocampus are age-related phenomena that are further influenced by AD as dementia progresses from severe to terminal, while the development of tangles in the neocortex is associated with progression to moderate and severe dementia. Other biologic manifestations of AD have also been examined in this series. Cholinergic markers, including choline acetyltransferase (ChAT)

60

Early Detection

/fo

ru

m

s/ in de

x. ph

p

and acetylcholinesterase, were not diminished in early AD cases, but were reduced substantially in those patients dying with moderate-to-severe dementia (Davis et al., 1999a). Quantitative studies of somatostatin and corticotropin-releasing factor levels in these brain tissues showed significant deficits of both neuropeptides in patients with severe dementia, but only CRF was significantly reduced in specimens obtained from cases with mild dementia (Davis et al., 1999b). Compared with age-matched controls, subjects dying with severe dementia but not subjects at earlier disease stages had higher levels of the inflammatory cytokines IL-6 and TGF-pl mRNA expression in the entorhinal cortex and superior temporal gyrus (Luterman et al., 2000). These studies of neurochemical markers and inflammatory cytokines indicate that neither is affected in the very earliest patients (those with CDR 0.5) who already have markedly increased concentrations of the (3-amyloid protein. Taken together, these studies provide a comprehensive picture of the development of neurobiologic changes over the course of AD, and may serve to guide the development and testing of therapies for each stage of disease.

4a ll.

vn

NEUROPSYCHOLOGY OF EARLY ALZHEIMER'S DISEASE

ht

tp

://

w

w

w

.c

he m

Cross-sectional studies comparing patients with very mild AD to agematched, nondemented control subjects have demonstrated that a deficit in memory is the earliest and most prominent neuropsychological deficit in patients with diagnosed AD (Welsh, Butters, Hughes, Mohs, & Heyman, 1991). This deficit is most pronounced on tasks for which the patients are asked to recall previously learned information (such as a short list of words) after a brief delay during which the patient engages in other cognitive activity. Longitudinal studies of nondemented persons who are at risk for dementia have examined the question of whether there are neuropsychological deficits that are measurable before patients are impaired enough to warrant a diagnosis of AD. In these studies, baseline neuropsychological data are used to compare performance of patients who subsequently were diagnosed with AD with those who remain dementia free. Results of these studies indicate that there are measurable deficits in memory and, to a lesser extent, in language and cognitive-processing speed at least 1 year before patients meet diagnostic criteria for dementia (Masur, Sliwinski, Lipton, Blau, & Crystal, 1994; Petersen et al., 1999). Our own results from the JHH study confirm that patients with a CDR score of 0.0 who convert to CDR 0.5 1 year later have baseline memory scores that are poorer than the baseline memory scores of patients who remain CDR 0.0 on follow-up. Thus, data from a variety of sources indicates that poor

Early Clinical and Biological Manifestations of AD 61

61

scores on a test of memory, particularly delayed-recall memory, are a sensitive indicator of early dementia.

TELEPHONE SCREENING FOR ALZHEIMER'S DISEASE

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

A complete diagnostic evaluation for AD is time-consuming, expensive, and requires substantial expertise on the part of the examining physician. In clinical settings and in research studies, there is a need for brief screening tools that could help to identify those individuals who should receive a full diagnostic evaluation. Effective screening tools could help to ensure that scarce diagnostic services are focused on those individuals who are most likely to have dementia. Screening can be conducted in a variety of settings and, potentially, using a variety of different tools. As examples, screening could be done in clinics, doctors' offices, door-to-door surveys, by mail, or by telephone. Several mental-status interviews have been developed or adapted for use over the telephone (e.g., Brandt, Spencer, & Folstein, 1988; Roccaforte, Burke, Bayer, & Wengel, 1992). Most of these instruments are designed as telephonic versions of brief mental-status examinations, such as the MMSE and, therefore, have enough questions to measure the severity, as well as detect the presence of dementia. Recently, we have initiated a study to test the reliability and validity of a very brief telephonic-screening interview that could be given by nonprofessionals in a few minutes. The interview is not designed to enable a specific dementia diagnosis or to evaluate the severity of dementia, but simply to identify persons with a high probability of being demented.

w

Materials and Methods

ht

tp

://

The interview was designed to require no more than 5 minutes to administer and to be standardized so that a person with no clinical training could conduct the interview over the phone. The interview contains one item testing for the delayed recall of a list of three objects. The interview also contains some simple questions about cognitively demanding activities of daily living. Initially, the interview was given to a group of AD patients and agematched controls to determine its acceptability and whether there were obvious problems with telephonic administration and scoring. This pilot evaluation lead to rewording of some items and to clarifications in the administration procedure. We are now conducting a reliability and concurrent validity study. There are 90 participants in this study, all of whom have received a cognitive and diagnostic evaluation by a team of dementia

62

Early Detection

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

experts in the past 6 months. Some of the participants have AD and others are elderly, nondemented controls. The telephone interviewers are blind to the participant's diagnosis and cognitive evaluation data. To assess reliability, each participant is being interviewed twice during a 1-month period. Concurrent validity will be assessed by determining the correspondence between scores on the screening interview and the participant's most recent clinical diagnosis. More detailed analyses will look at the ability of the screening instrument to identify AD patients with very mild disease. Although the sample size is too small to draw definitive conclusions about the possible influence of confounding variables, we will also examine the relationship of screening test scores to factors such as education, age, and medical comorbidities. These analyses will determine the reliability of the screening tool and provide a first look at its ability to identify cases of AD. Further validity studies will be conducted, subsequently, provided that the reliability and validity data in the current study are acceptable. One additional study will involve elderly patients enrolled in a managed care plan. None of the participants in this study will have received a full diagnostic evaluation for dementia. Participants will be screened over the telephone and determined to be cognitively normal or possibly impaired. Subsets from each group will then be examined clinically to determine the extent to which the screening classification agrees with the results of a diagnostic evaluation.

Discussion

ht

tp

://

w

w

w

.c

The neurobiologic and clinical events leading to the development of clear-cut AD are now being clarified. Emphasis in therapeutic trials is increasingly focused on strategies for preventing the development and progression of AD. Screening tools that can be used both in routine clinical practice and in research studies may play an important role in the development and effective use of these new therapies.

REFERENCES

Brandt, J., Spencer, M., & Folstein, M. (1988). The telephone interview of cognitive status. Neuropsychiatry, Neuropsychology, and Behavioral Neurology, 1,111-117. Davis, K. L., Mohs, R. C., Marin, D. B., Purohit, D. P., Perl, D. P., Lantz, M., Austin, G., & Haroutunian, V. (1999a). Cholinergic markers are not decreased in early Alzheimer's disease. Journal of the American Medical Association, 281,1401-1406. Davis, K. L., Mohs, R. C., Marin, D. B., Purohit, D. P., Perl, D. P., Lantz, M., Austin, G., & Haroutunian, V. (1999b). Neuropeptide abnormalities in patients with early Alzheimer's disease. Archives of Geriatric Psychiatry, 56, 981-987.

Early Clinical and Biological Manifestations of AD 63

63

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Galasko, D., Bennett, D., Sano, M., Ernesto, C., Thomas, R., Grundman, M., Ferris, S., and the Alzheimer's Disease Cooperative Study. (1997). An inventory to assess activities of daily living for clinical trials in Alzheimer's disease. Alzheimers Disease and Associated Disorders, 11, S33-S39. Green, C. R., Marin, D. B., Mohs, R. C., Schmeidler, J., Aryan, M., Fine, E., & Davis, K. L. (1999). The impact of behavioral impairment on functional ability in Alzheimer's disease. International Journal of Geriatric Psychiatry, 14, 307-316. Haroutunian, V, Perl, D. P., Purohit, D. P., Marin, D. B., Khan, K., Lantz, M., Davis, K. L., & Mohs, R. C. (1998). Regional distribution of senile plaques in nondemented elderly and cases of very mild Alzheimer's disease. Archives of Neurology, 55,1185-1191. Haroutunian, V., Purohit, D. P., Perl, D. P., Marin, D., Khan, K., Lantz, M., Davis, K. L., & Mohs, R. C. (1999). Neurofibrillary tangles in nondemented elderly and very mild Alzheimer's disease. Archives of Neurology, 56, 713-718. Luterman, J. D., Haroutunian, V., Yemul, S., Ho, L., Purohit, D., Aisen, P. S., Mohs, R., & Pasinetti, G. M. (2000). Cytokine gene expression as a function of the clinical progression of Alzheimer disease dementia. Archives of Neurology, 57, 1153-1160. Marin, D. B., Green, C. R., Schmeidler, J., Harvey, P. D., Lawlor, B. A., Ryan, T., Aryan, M., Davis, K. L., & Mohs, R. C. (1997). Noncognitive disturbances in Alzheimer's disease: Frequency, longitudinal course and relationship to cognitive symptoms. Journal of the American Geriatric Society, 45,1331-1338. Masur, D. M., Sliwinski, M., Lipton, R. B., Blau, A. D., & Crystal, H. A. (1994). Neuropsychological prediction of dementia and the absence of dementia in healthy elderly persons. Neurology, 44,1427-1432. Morris, J. C., Edland, S., Clark, C., Galasko, D., Koss, E., Mohs, R., van Belle, G., Fillenbaum, G., & Heyman, A. (1993). The Consortium to establish a registry for Alzheimer's disease (CERAD). Part IV. Rates of cognitive change in the longitudinal assessment of probable Alzheimer's disease. Neurology, 43, 2457-2465. Naslund, J., Haroutunian, V., Mohs, R., Davis, K. L., Davies, P., Greengard, P., & Buxbaum, J. D. (2000). Elevated amyloid p-peptides in brain: Correlation with cognitive decline. Journal of the American Medical Association, 283,1571-1577. Petersen, R. C., Smith, G. E., Waring, S. C., Ivnik, R. J., Tangalos, E. G., & Kokmen, E. (1999). Mild cognitive impairment: Clinical characterization and outcome. Archives of Neurology, 56, 303-308. Roccaforte, W. H., Burke, W. J., Bayer, B. L., & Wengel, S. P. (1992). Validation of a telephone version of the Mini-Mental State Examination. Journal of the American Geriatric Society, 40, 697-702. Stern, R. G., Mohs, R. C., Davidson, M., Schmeidler, J., Silverman, J. M., KramerGinzberg, E., Searcey, T., Bierer, L. M., & Davis, K. L. (1994). A longitudinal study of Alzheimer's disease: Measurement, rate and predictors of cognitive deterioration. American Journal of Psychiatry, 151, 390-396. Welsh, K. A., Butters, N., Hughes, J., Mohs, R. C., & Heyman, A. (1991). Detection of abnormal memory decline in mild cases of Alzheimer's disease using CERAD neuropsychological measures. Archives of Neurology, 48, 278-281.

p

x. ph

s/ in de

m

ru

/fo

vn

4a ll.

m

.c he

w

w

w

://

tp

ht This page intentionally left blank

he m

.c

w

w

w

://

tp

ht

m

ru

/fo

vn

4a ll.

p

Drug Discovery x. ph

s/ in de

II

p

x. ph

s/ in de

m

ru

/fo

vn

4a ll.

m

.c he

w

w

w

://

tp

ht This page intentionally left blank

10

x. ph

s/ in de

Brain-Expressed ABC Transporters That May

p

Identification of

Mediate Detachment of

/fo vn

Membranes

ru

m

p-Amyloid From Biological

.c he

m

4a ll.

Peter B. Reiner and Stephane Le Bihan

w

w

INTRODUCTION

ht

tp

://

w

Alzheimer's disease is characterized by neurofibrillary tangles, senile plaques, and neuronal death. The neurofibrillary tangles contain paired helical filaments composed of hyperphosphorylated tau, while the senile plaques are comprised of an array of proteins deposited around a core of insoluble A(3 peptide. The cause of neuronal death remains unknown, but considerable evidence suggests that it is secondary to an increase in the brain A(3 load (Selkoe, 1999). The molecular events involved in the constitutive production of Ap are increasingly being understood. The first step appears to be cleavage of the amyloid precursor protein (APP) by (3-secretase (Vassar et al., 1999; Lin et al., 2000), yielding an extracellular fragment known as sAPP(3, which is shed into the extracellular space (Mills & Reiner, 1999). The remaining 99 amino acid COOH-terminal fragments (C99) consist of 28 charged amino acids on the extracellular side of the membrane, 23 hydrophobic amino acids that 67

68

Drug Discovery

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

presumably traverse the membrane as an a-helix, and 52 charged amino acids on the intracellular side of the membrane. Cleavage of C99 within the membrane by an enzyme known as y-secretase, which appears to be identical to the presenilins (Wolfe et al., 1999; Lin et al., 2000), liberates intact A{3. Both the 40 and 42 amino acid versions of A(3 are amphipathic, consisting of 28 charged amino acids and either 12 or 14 hydrophobic amino acids (for A(3t_40 and A(3142, respectively). It has been known for nearly 10 years that the A|3 peptides are rapidly released from cells (Haass et al., 1992; Seubert et al., 1992; Shoji et al., 1992; Busciglio, Gabuzda, Matsudaira, & Yankner, 1993), but the hydrophobic amino acids at the COOH-terminus make it likely that the peptide will remain associated with the membrane following y-secretase cleavage. Thus, we hypothesized that an active process was required in order for A(3 to detach from the membrane. Selected members of the ATP-binding cassette (ABC) superfamily of transporters are responsible for the energydependent efflux of a variety of lipophilic and amphipathic molecules from cells, and the process bears a striking similarity to that which occurs with the AP peptide (van Veen & Konings, 1998; Croop, 1998; Kuchler & Thorner, 1992; Ambudkar et al., 1999; Yakushi, Masuda, Narita, & Tokuda, 2000). Using a variety of techniques, we recently demonstrated that the ABC transporter known as MDR1 is an Ap efflux pump (Lam et al., 2001). We have identified a single ABC transporter, MDR1, as an AP efflux pump. Cells throughout the body constitutively produce and release Ap, yet the MDR1 protein is only expressed in a limited number of tissues, and is essentially undetectable in neurons (Fojo et al., 1987; Thiebaut et al., 1987; Cordon-Cardo et al., 1989; Pardridge, Golden, Kang, & Bickel, 1997). This suggests that other ABC transporters are responsible for detachment of Ap from the membranes of neurons and/or glial cells. The goal of our current research program is to identify such brain-expressed ABC transporters.

ht

tp

METHODS AND RESULTS Allikmets, Gerrard, Hutchinson, and Dean (1996) characterized a large number of human ABC transporter genes by searching the human EST database using sequences derived from ABC (Walker A, ABC signature, and Walker B) of MDR1, as well as the entire sequence of cystic fibrosis transmembrane conductane regulator (CFTR). Because many of the ESTs are derived from brain libraries, the data suggested that many of these ABC transporters were expressed in brain. Because the number of ESTs deposited in public databases has increased substantially in the past 4 years, we recently repeated this analysis in order to ensure that the full complement of brain-expressed ABC transporters was represented in the

Identification of Brain-Expressed ABC Transporters 69

69

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

database. Moreover, to increase the power of our search, we developed consensus amino acid sequences for the Walker A motif, as well as the ABC signature and Walker B motifs, by comparing the sequences of the following human ABC transporters: gi4501849; gi4557876; gi4505769; gi9665248; gi4826838; gi6995996; gi4826958; and gi8051575. The consensus sequences that emerged from this analysis were (G(X)2G(X)GK(X)T(X)4L(X)2L(X)2PT(X)3G for the Walker A motif, and LSGG(X)4L(X)2A(X)AL(X)3PKV(X)2LDE(X)TS(X) for the ABC signature and Walker B motifs. The BLAST programs (Altschul, Gish, Miller, Myers, & Lipman, 1990) were used to search the human database EST using these consensus sequences, as well as the complete amino acid sequences of the human ABC-transporter proteins identified above. Human clones with the highest scores were retrieved and primers were designed using the program Mac Vector (Oxford Molecular, UK). Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using brain fetal and adult total RNA (Invitrogen) in order to confirm human brain expression, and PCR products were sequenced to authenticate the ESTs. Based on both the cDNA library source used to generate these ESTs, as well as the expression profile of RNA as seen in Northern blots (Allikmets et al. 1996, and unpublished data), we identified the 14 ABC transporters listed in Table 10.1 as being brain-expressed.

he m

DISCUSSION

ht

tp

://

w

w

w

.c

Using bioinformatic tools, we have identified 14 ABC transporters that are expressed in the brain. It is apparent that there are no brain-specific ABC transporters: all ABC transporters that have been identified as being expressed in brain are also expressed in at least one other tissue, and several are expressed quite widely. While Table 10.1 provides data about general tissue distribution, further analyses will be required to determine whether these transporters are localized to neurons, glia, or even to cerebral vasculature. The latter point is important, as ABC transporters such as MDR1 and MRP1 represent key elements of the blood-brain barrier and are prominently expressed in cerebrovascular endothelial cells (CordonCardo et al., 1989). We have put forth the hypothesis that ABC transporters are involved in the process of detachment of AP from cellular membranes (Lam et al., 2001). A(3 is unlikely to aggregate while attached to the membrane, as the hydrophobic amino acids in the peptide COOH tail would be shielded by their association with the lipid bilayer. On the other hand, the likelihood of Ap aggregation increases substantially following membrane detachment. Thus, detachment of Ap} from the membrane may represent a critical change in the biophysical properties of the peptide, and may very well

Drug Discovery

70

TABLE 10.1 Brain-Expressed ABC Transporters Chromosome

ABCA1

U18236

9q31

Adipose, aorta, brain, ear, foreskin, kidney, lung, ovary, pancreas, placenta, prostate, smooth muscle, stomach, thyroid, tonsil, colon, lung

ABCA2

U18235

9q34

Brain, spinal cord, thyroid, kidney, spleen, brain, spinal cord

ABCA3

111653

16pl3.3

Aorta, bone, brain, germ cell, heart, kidney, lung, pancreas, prostate, brain, colon, eye, lung

ABCA5

90625

17q21-24

Aorta, brain, breast, kidney, lung, pancreas, parathyroid, spleen, testis, tonsil, uterus, lung

ABCA6

155051

17q21

Brain, germ cell, kidney, lymph, pancreas, uterus

ABCA8

AL041916

17

Aorta, brain, colon, ear, germ cell, heart, kidney, lung, prostate, stomach, testis, brain

ABCB5

422562

7pl4

ABCB6

45597

/fo

vn 4a ll.

w

.c

2q33

w w

122234

Foreskin, brain

Bone, brain, breast, colon, germ cell, heart, kidney, lung, marrow, ovary, pancreas, prostate, stomach, testis, uterus, bone marrow, brain, eye, genitourinary tract, lung, lymph, placenta, whole blood

12q24

Brain, germ cell, testis, tonsil, uterus

157481

4q22-23

Brain, germ cell, kidney, placenta, prostate, small intestine, yestis, uterus, brain

ABCC5

277145

3q25-27

Brain, breast, colon, esophagus, eye, foreskin, kidney, lung, pancreas, parathyroid, prostate, stomach, synovial membrane, tonsil, uterus, brain, breast, cervix, colon, genitourinary tract, kidney, lung, skin, uterus

ABCC10

182763

6p21

Brain, kidney, lung, pancreas, prostate, uterus, brain, breast, kidney, lung

://

ABCB9

ru

m

s/ in de

x. ph

p

EST

he m

Expression information (cDNA and mRNA source)

HUGO

ht

tp

ABCB10

Identification of Brain-Expressed ABC Transporters

TABLE 10.1

71

(Continued) Expression information (cDNA and mRNA source)

EST

Chromosome

ABCD4

352188

14q23.3

Blood, brain, colon, eye, germ cell, heart, kidney, lung, lymph, muscle, pancreas, parathyroid, stomach, testis, tonsil, uterus, breast, connective tissue, eye, muscle, ovary, skin

ABCF1

123147

6p21.33

Adrenal gland, aorta, blood, brain, breast, colon, esophagus, eye, germ cell, heart, kidney, lung, lymph, muscle, ovary, pancreas, placenta, prostate, skin, spleen, testis, tonsil, umbilical cord vein, uterus, brain, colon, eye, kidney, lung, muscle, ovary, placenta, stomach, uterus

m

s/ in de

x. ph

p

HUGO

4a ll.

vn

/fo

ru

Note. The table lists the 14 brain-expressed ABC transporters according to the HUGO nomenclature (http://www.gene.ucl.ac.uk/cgi-bin/nomenclature/searchgenes.pl), as well as via the ESTs, which identify the ABC transporter as being expressed in brain. In addition, tissue-expression information as available in the public EST database is included (in alphabetical order), with adult tissues identified in normal type and fetal tissues in italics.

tp

://

w

w

w

.c

he m

be a prerequisite to the aggregation events that are thought to be at the core of the pathology of Alzheimer's disease. In summary, we have identified the suite of ABC transporters that are expressed in the brain. Such bioinformatic analysis is a prerequisite to the functional expression of each of these transporters in model systems. With such information in hand, we will be able to determine which brainexpressed ABC transporters function as (3-amyloid efflux pumps. Such proteins represent novel targets for the development of drugs that can regulate p-amyloid levels in the brain.

ht

REFERENCES

Altschul, S. R, Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403-410. Allikmets, R., Gerrard, B., Hutchinson, A., & Dean, M. (1996). Characterization of the human ABC superfamily: Isolation and mapping of 21 new genes using the expressed sequence tags database. Human Molecular Genetics, 5,1649-1655. Ambudkar, S. V., Dey, S., Hrycyna, C. A., Ramachandra, M., Pastan, I., & Gottesman, M. M. (1999). Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annual Review of Pharmacology and Toxicology, 39, 361-398.

72

Drug Discovery

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Busciglio, }., Gabuzda, D. H., Matsudaira, P., & Yankner, B. A. (1993). Generation of P-amyloid in the secretory pathway in neuronal and nonneuronal cells. Proceedings of the National Academy of Sciences (USA), 90, 2092-2096. Croop, J. M. (1998). Evolutionary relationships among ABC transporters. Methods in Enzymology, 292,101-116. Cordon-Cardo, C, O'Brien, J. P., Casals, D., Rittman-Grauer, L., Biedler, J. L., Melamed, M. R., & Bertino, J. R. (1989). Multidrug resistance gene (p-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites. Proceedings of the National Academy of Sciences (USA), 86, 695-698. Fojo, A. T., Ueda, K., Slamon, D. J., Poplack, D. G., Gottesman, M. M., & Pastan, I. (1987). Expression of a multidrug-resistance gene in human tumors and tissues. Proceedings of the National Academy of Sciences (USA), 84, 265-269. Haass, C., Schlossmacher, M. G., Hung, A. Y, Vigo-Pelfrey, C., Mellon, A., Ostaszewski, B. L., Lieberburg, I., Koo, E. H., Schenk, D., Teplow, D. B., et al. (1992). Amyloid-(3 peptide is produced by cultured cells during normal metabolism. Nature, 359, 322-325. Kuchler, K., & Thorner, J. (1992). Secretion of peptides and proteins lacking hydrophobic signal sequences: The role of adenosine triphosphate-driven membrane translocators. Endocrine Review, 13, 499-514. Lam, F. C.-L., Liu, R., Lu, P., Shapiro, A. B., Renoir, J.-M., Sharom, E J., & Reiner, P. B. (2001). P-amyloid efflux mediated by P-glycoprotein. Journal ofNeurochemistry, 76,1121-1128. Lin, X., Koelsch, G., Wu, S., Downs, D., Dashti, A., & Tang, J. (2000). Human aspartic protease memapsin 2 cleaves the p-secretase site of P-amyloid precursor protein. Proceedings of the National Academy of Sciences (USA), 97,1456-1460. Mills, J. A., & Reiner, P. B. (1999). Regulation of amyloid precursor protein cleavage. Journal of Neurochemistry, 72, 443-460. Pardridge, W. M., Golden, P. L., Kang, Y.-S., & Bickel, U. (1997). Brain microvascular and astrocyte localization of p-glycoprotein. Journal of Neurochemistry, 68, 1278-1285. Selkoe, D. J. (1999). Translating cell biology into therapeutic advances in Alzheimer's disease. Nature, 399, A23-31. Seubert, P., Vigo-Pelfrey, C., Esch, E, Lee, M., Dovey, H., Davis, D., Sinha, S., Schlossmacher, M., Whaley, J., Swindlehurst, C., et al. (1992). Isolation and quantification of soluble Alzheimer's p-peptide from biological fluids. Nature, 359, 325-327. Shoji, M., Golde, T. E., Ghiso, J., Cheung, T. T., Estus, S., Shaffer, L. M., Cai, X. D., McKay, D. M., Tintner, R., Frangione, B., et al. (1992). Production of the Alzheimer amyloid-p protein by normal proteolytic processing. Science, 258, 126-129. Thiebaut, E, Tsuruo, T., Hamada, H., Gottesman, M. M., Pastan, L, & Willingham, M. C. (1987). Cellular localization of the multidrug-resistance gene product Pglycoprotein in normal human tissues. Proceedings of the National Academy of Sciences, USA, 84, 7735-7738. van Veen, H. W., & Konings, W. N. (1998). The ABC family of multidrug transporters in microorganisms. Biochimica et Biophysica Acta, 1365, 31-36. Vassar, R., Bennett, B. D., Babu-Khan, S., Khan S., Mendiaz, E. A., Davis, P.,

Identification of Brain-Expressed ABC Transporters 73

73

s/ in de

x. ph

p

Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., Lou, Y., Fisher, S., Fuller,}., Edemson, S., Lile, J., Jarosinski, M. A., Biere, A. L., Curran, E., Burgess, T., Louis, J. C., Collins, E, Treanor, J., Rogers, G., & Citron, M. (1999). p-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science, 286, 735-741. Wolfe, M. S., Zia, W., Ostaszewski, B. L., Diehl, T. S., & Kimberly, W. T. (1999). Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and y-secretase activity. Nature, 398, 513-517. Yakushi, T., Masuda, K., Narita, S.-L, & Tokuda, H. (2000). A new ABC transporter mediating the detachment of lipid-modified proteins from membranes. Nature Cell Biology, 2, 212-218.

ACKNOWLEDGMENTS

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

This research was supported by grants from the Institute for the Study of Aging, Inc., and the Science Council of British Columbia. We thank our colleagues at Active Pass Pharmaceuticals for advice and encouragement.

1 1

s/ in de

Protein Gene as a New

x. ph

p

Targeting an RNA Structure in the Amyloid Precursor Therapeutic Strategy for

ru

m

Alzheimer's Disease

he m

4a ll.

vn

/fo

Principal Investigator: Jack T. Rogers

ht

tp

://

w

w

w

.c

Recently, the possibility of developing drugs that knock out the unwanted genetic function associated with unique RNA structures has led laboratories in industry and academia to screen for medicinal compounds directed to disease-associated RNA targets. Small molecules that influence RNAdirected activity are to be screened from large combinatorial libraries of compounds. To date, most accumulated knowledge in this field centers around the development of drugs that offer therapies against infectious diseases. For example, drug discovery related to hepatitis C virus infection targets a unique translation control signal, the internal ribosome initiation site (IRES), which is essential for the viral life cycle (Anwar, Ali, Tanveer, & Siddiqui, 2000). We will discuss the RNA structure formed by the 5' untranslated region (5'UTR) of the endogenous amyloid precursor protein (APP) gene (APP 5'UTR) as a therapeutic drug target for Alzheimer's disease (AD) (Figure 11.1) (Rogers et al., 1999). Interleukin-1 and iron regulate APP gene expression at the translational level through the APP 5'UTR, similar to the iron-storage protein, ferritin (Rogers, 1996, 1999). This finding is consistent with the fact that the secreted ectodomain of the precursor (APPs) binds (and probably sequesters) the neurotoxic metals, copper, and iron (Multhaup et al.,1996; Bush et al., 1994). 74

p x. ph s/ in de m ru /fo vn 4a ll. m .c he w w w ://

ht

tp

FIGURE 11.1 RNA secondary structure formed by the 5'untranslated region of the Alzheimer's amyloid precursor protein (RNA Fold program, Zuker, 1989). The chemical structure of the small molecules, phenserine (anticholinesterase) and desferrioxamine (iron chelator), are provided. Phenserine and desferrioxamine exemplify medicinal compounds that suppress APP mRNA translation via 5'untranslated region (5'UTR) sequences. An assay to selectively limit APP mRNA translation through this target will provide an opportunity for the selection of medicinal compounds for treatment of Alzheimer's disease.

76

Drug Discovery

PROTEIN TARGETING

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Many existing drugs are designed to bind to the active sites and then suppress protein activities that promote the development of disease progress. For example, cocktails of protease inhibitors are used to therapeutically block hepatitis C virus infections (Fattori et al., 2000) by inactivating the viral NS3 protease (Barbato et al., 2000). Statins are low molecular-weight compounds that suppress HMG-CoA reductase activity and, thus, cholesterol synthesis, to therapeutically assist patients at risk for coronary heart disease (Penzak, Chuck, & Stajich, 2000). RNA aptmers are also used as protease inhibitors (Fukuda et al., 1997). There are some drawbacks to the use of viral-associated protein targets for drug selection. In the case of HIV-associated diseases, rapid genetic drift in protein sequences makes viral targets immunoevasive. Therefore, these protein sequences are less attractive targets for long-term therapeutic strategies (Condra et al., 1995; Bally, Martinez, Peters, Sudre, & Telenti, 2000; Bangsberg et al.,, 2000). Most AD patients are currently treated with acetylcholinesterase inhibitors, which act as cognitive enhancers. Acetylcholinesterase inhibitors (e.g., Aricept, Eisai Co Ltd, Tokyo, Japan; Pfizer Inc., New York, NY) are proteintargeted inhibitors of the esterase that cleaves acetylcholine, the cholinergic neurotransmitter. These drugs slow down the rate of cognitive decline by increasing acetylcholine neurotransmitter levels in the brain (al-Jafari, Kamal, Greig, Alhomida, & Perry, 1998). The anticholinesterases provide limited improvement of cognitive performance in AD patients, and thus are of only partial benefit to AD patients early in disease progression. One problem associated with the use of anticholinesterases is that they have not been shown to confer any therapeutic action on the neuropathological events that might cause AD (i.e., amyloid, apolipoprotein E, alpha-1 antichymotrypsin, heparan sulfate proteoglycans, and the microtubule-associated protein, tau). We will discuss our discovery that a new anticholinesterase, phenserine, directly suppresses APP mRNA translation through the 5'UTR, thus imparting a therapeutic impact on A(3-peptide buildup, in addition to being an anticholinesterase (Shaw, Utsuki, Rogers, Yu, Lahiri, & Grieg, 2001). Current protein-based therapeutic approaches for AD aim to arrest the accumulation of the major amyloid plaque-associated protein, the A(3 peptide. The exact mechanism by which amyloid becomes toxic is unknown, but the presence of copper and iron and oxidative stress is a critical event (Huang et al., 1999; Bush et al., 2000). At the same time, neurotoxic protofibrils deposit in the neuritic amyloid plaques as Ap peptide is converted from an open-coil structure into a beta-sheet conformation (Kimberly, Xia, Rahmati, Wolfe, & Selkoe, 2000; Teplow, 1998; Walsh et al., 1999). Haass et al. (1992) first showed that Ap peptide is secreted from all cells in the body after being cleaved from the transmembrane APP. The

Targeting an RNA Structure

77

he m

DNA TARGETING

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

elegant work of Wolfe et al. (1999) demonstrated that two transmembrane aspartates in presenilin-1 constitute the endoproteolytic peptidase conferring y-secretase activity. Thus, PS-1 mutations cause familial AD (LevyLahad, 1995; Scheuner et al., 1996), and PS-1 appears to function as the y-secretase that catalyzes the final cleavage of APP to Ap (Wolfe et al., 1999). Companies, including Bristol-Myers Squibb, have programs to screen, from large combinatorial libraries, for small molecules that can suppress the secretases (PS-1 and PS-2) that cleave APP to the 40-42 amino acid Ap peptide. One drawback of developing drugs that inhibit both (3 and y-secretase activity (and hence the generation of A(3 peptide) is that these secretases have other cellular targets. For example, the transcription factor notch is cleaved by PS-1, and it remains to be seen whether drug-induced inhibition of y-secretase will generate cytotoxic side effects associated with the disappearance of an essential downstream transcriptionally activated protein (Song et al., 1999). This concern was addressed by recent work showing that transfectants bearing PS-1 and PS-2 mutations maintain nuclear translocation of notch to the nucleus with relative preservation of notch-1 signaling (Berezovska et al., 2000). However, new protease inhibitors directed toward (3- and y-secretases may also affect other unrelated cellular targets, although this subject remains open to the development of a drug that proves the concept.

ht

tp

://

w

w

w

.c

Like the use of protease inhibitors to limit APP cleavage, drug-induced down-regulation of APP-gene transcriptional control has the intrinsic problem that DNA motifs are shared with several other genes. Certainly targeting the DNA promoter sites in front of the APP gene as a therapeutic strategy for AD has the disadvantage that several genes share the common enhancer sequences that control APP gene expression. As an example, NFkB is a well-characterized transcription enhancer that controls immunoglobulin gene expression, in addition to APP gene expression, in response to inflammatory signals (Grilli, Pizzi, Memo, & Pier Franco, 1996; Ghosh & Baltimore, 1990). AP-1 sites are palindromes in front of the APP gene, which bind the cJun/cFos proto-oncogenes during stress, but the presence of this site in the enhancers of several other stress-responsive genes precludes the use of this site as a therapeutic target for AD (Karin, 1995). These considerations imply that new DNA-targeting drugs will not only suppress the gene of interest (e.g., APP for AD), but will also interfere with the expression of related and essential housekeeping genes, resulting in unwanted metabolic side effects. Despite these difficulties, companies such as Abbott Laboratories (Pan, Monteggia, & Giordano, 1993) and

78

Drug Discovery

Hoechst Marion Roussel (Ringheim et al., 1998) have explored the potential to modulate APP gene transcription.

RNA TARGETING RNA Targets in HIV, Infectious Disease, and Cytokines

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

RNA structure has become the focus for developing therapeutic strategies to regulate the expression of many disease-associated genes because any given RNA structure is unique to the gene from which it is expressed. However, the concept that RNA-directed compounds can confer a therapeutic impact is not new. For decades, antibiotics, like erythromycin, have been characterized to be bactericidal based on their capacity to bind to unique ribosomal RNA sequences in the bacterial 23S ribosome subunit. The B component of streptogramins inhibits peptide elongation in vivo during the early rounds of protein synthesis in a manner similar to that of the smaller microlides, including erythromycin (Porse, Kirillov, Awayez, & Garrett, 1999). Ribotargets (Cambridge, UK) is a company that seeks to inhibit the essential TAT-Tar interaction to prevent HIV infection and AIDS (Hamy et al., 1997). In this case, the TAT transactivating protein binds to the TAR stemloop at the viral LTR to promote viral growth. Blocking this interaction with selected compounds will interfere with the viral life cycle and be of therapeutic benefit (Keen, Churcher, & Karn, 1997) (Figure 11.2). Scriptgen Inc. (Waltham, MA) has a drug-discovery program directed to a single RNA target, the replication origin of hepatitis C virus. Message Pharmaceuticals (Malvern, PA), is the only company interested in diseaseassociated RNA targets of endogenously expressed genes (Figure 11.2). A major project in their program is to modulate cytokine expression as a therapeutic strategy for arthritis and cancer. Tumor necrosis factor (TNF) and interleukin-1 (IL-1) gene expression is up-regulated in the joints aof arthritis patients (Rogers, 1996). Low molecular weight RNA-binding compounds can be screened for their capacity to inhibit binding of the AUF-1 to the AU-rich sequences in the 3'UTR of these cytokine mRNAs. Another class of drugs (CSAIDS) were previously shown to interfere with a kinase that phosphorylates the 3'UTR AUF-1, which controls cytokine mRNA stability in response to cellular signaling (Lee et al., 1994). RNA Targets Therapeutic for the Alzheimer's Disease Amyloid Precursor Protein Background

Our goal to suppress Alzheimer's APP gene expression at the translational level has the advantage that the APP 5'UTR RNA target is unique, thus

Targeting an RNA Structure

79

RNA TARGETS IN BIOLOGY AND MEDICINE HIV: DRUGS THAT SUPPRESS THE TAR STEMLOOP FROM INTERACTING WITH A TAT TRANS-ACTIVATOR.

x. ph

p

INFECTIOUS DISEASE Message Pharmaceuticals

m

CYTOKINE mRNAs: DRUGS THAT SUPPRESS AUF-1 BINDING TO THE 3'UTRS OF IL-1- AND TNF-mRNAs -STABILITY & TRANSLATION.

vn

/fo

ru

ARTHRITIS/CANCER Message Pharmaceuticals

s/ in de

ANTIBIOTICS: i.e: BACTERIAL RIBOSOME PEPTIDAL TRANSFERASE IS BLOCKED BY ERYTHROMYCIN.

he m

4a ll.

FIGURE 11.2 RNA targets that are currently being screened to obtain drugs for suppressing disease process.

ht

tp

://

w

w

w

.c

affording a higher degree of selectivity (Figures 11.1 and 11.3). There are regulatory sites in the 3'UTR of APP mRNA that can be utilized as targets to suppress APP gene expression (Mbella, Bertrand, Huez, & Octave, 2000). However, our finding that the 5'UTR of APP mRNA is a powerful, translational enhancer element makes this RNA structure (in front of the start codon) a very attractive target for regulating APP gene expression as a therapeutic strategy to slow AD progression. The vaccination strategy to target and suppress steady-state levels of A(3 peptide has been successfully developed by Elan Pharmaceuticals, who reduced later stage formation of (3-amyloid plaques, neuritic dystrophy, and astrogliosis, and other "Alzheimer's disease-like pathology" in the PDAPP transgenic mouse (Schenk et al., 1999). RNA targeting and vaccination approaches may ultimately be complementary approaches as therapeutic strategies to help AD patients. Werstuck and Green (1998) recently demonstrated a model system for the selection of RNA-binding compounds to inhibit translation of reporter proteins under the control of specific upstream RNA stemloops.

80

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Drug Discovery

ht

tp

://

w

w

w

.c

he m

FIGURE 11.3 Phenserine and desferrioxamine suppress translation through the 5'untranslated region (5'UTR) target in APP mRNA. Neuroblastoma cells (SY5Y) were transfected with (a) the pSV2(APP)LUC construct (APP-5'UTR construct designated as pAS-1) (b) pGL-3 (Parental vector that lacks APP sequences). After transfection, the cells were passaged equally into 6 well flasks and treated for 48 hours in-triplicate with either phenserine, tacrine, donapezil (25 ug/ml AChE), EDTA and desferrioxamine (5 uM chelators) . After drug treatment, derivative lysates were assessed for luciferase activity. As a control for specificity, compounds did not markedly suppress transfected luciferase gene expression in cells expressing the parental construct (pGL-3, Arbitrary value = 10) relative to pAS-1 (APP 5'UTR) transfectants. Using pAS-1 (APP 5'UTR-specific) transfected cells, drug treatment with EDTA only reduced APP 5'UTR-driven gene expression by 18% (n = 3), whereas desferrioxamine suppressed APP 5'UTR-driven luciferase gene expression by 80% to 90% relative to PGL-3 transfected controls. Drug treatment with phenserine reduced APP 5'UTR-driven luciferase gene expression by 77%, whereas tacrine and donapezil exhibited a lesser 55% suppression of luciferase relative to the PGL-3 control. Note the (3-galactosidase plasmid (1:20 ratio) was cotransfected to ensure equal transfection efficiency and representative lysates from the two sets of transfectants were assayed for p-galactosidase activity and found to display equal activity.

Targeting an RNA Structure 81

81

The APP mRNA 5'Untranslated Region

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

IL-1 and iron-dependent translation enhancement. We reported that the mRNA coding for the Alzheimer's APP is already abundant in astrocytes and neuronal cells. APP synthesis and A^-peptide secretion are increased in response to IL-1 in astrocytes, and the APP 5'UTR confers translational regulation to transfected hybrid APP/CAT mRN As in response to IL-1 a and IL-lp stimulation (Rogers et al., 1999). Astrocytic APP mRNA is activated to the polyribosomes after IL-1 (3 stimulus. Both (FMR-1) and the mRNA for the "Fragile x" genes have the APP mRNA 5' leaders have stable RNA secondary structures (Feng et al., 1995) (Figure 11.1). Unlike the case for FMR-1, the APP 5'UTR clearly up-regulates 43S small ribosomal subunit prior to the onset of protein synthesis (AA = -54.9 kcal/mol) (Zuker, 1989). Our model for APP mRNA translational regulation was extended when we discovered that the APP 5'UTR also contains functional iron-responsive sequences. Computer-generated alignments show that sequences in the APP 5'UTR are related to the iron-responsive element (IRE) found in the mRNAs encoding the light and heavy subunits of the ubiquitous iron storage protein, ferritin (Thomson, Rogers, & Leedman, 1999). Indeed, the iron-regulatory proteins (IRP-1 and IRP-2) trans-acting proteins capable of binding to APP mRNA sequences in front of the start codon. We showed that IRP-1 and IRP-2 control ferritin mRNA translation, not only in response to iron, but also hormonal activation from thyroid-releasing hormone in pituitary cells (Thomson, Rogers, & Leedman, 2000).

ht

tp

://

w

w

w

.c

Small molecules that suppress APP mRNA translation. Since the APP 5'UTR is a significant regulator of APP protein levels, we have begun to test whether the RNA secondary structure in this region can provide an attractive therapeutic target for small RNA-binding compounds to down-regulate APP synthesis and, thus, limit A(3-peptide output. Two sets of lead compounds appear to specifically suppress APP mRNA translation, through 5' UTR RNA secondary structure (Figures 11.1 and 11.3). The first compound is the anticholinesterase, phenserine (al-Jafari et al., 1998). In collaboration with Dr. Nigel Greig (NIA), we reported that phenserine derivatives suppressed APP mRNA translation by targeting 5'UTR-mediated regulation (Shaw, Utsuki, Rogers, Yu, Lahiri, & Grieg, 2001). The second compound is desferrioxamine (Df), which specifically chelates intracellular iron (Rogers & Munro, 1987) and is known to block ferritin mRNA translation through the iron-responsive element in the 5'UTR (Thomson, Rogers, & Leedman, 1999). Desferrioxamine. Autopsy samples from AD patients reveal elevated levels of iron, particularly in the neurons of the basal ganglia (Bartzokis et al.,

82

Drug Discovery

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

2000). Iron is relevant to neurodegenerative pathogenesis in the brains of AD patients, as evidenced by their disrupted brain-iron distribution (Connor, Menzies, St. Martin, & Mufson, 1992; Pinero, Hu, & Connor, 2000). Since we had discovered that the APP 5'UTR is a modified ironresponsive element, we decided to test whether iron chelation by Df can suppress translation conferred to a luciferase reporter by APP mRNA 5'UTR sequences. In the first step, a Luciferase-reporter construct, MS121, was prepared by inserting a PCR-generated APP-mRNA 5'UTR fragment (90nt, nt 58-147 according to Genebank accession number NM_000484). Neuroblastoma cells (SY5Y) were transfected with this APP-5'UTR-specific construct and cells were then exposed to Df (5uM), before preparation aof cell lysates and use of a luciferase assay to determine levels of reporter gene activity. As a negative control, neuroblastoma cells were transfected with a parental pGL-3 reporter plasmid. The results in Figure 11.3 showed that the APP 5'UTR was clearly a target for the action of Df to suppress APP-5'UTR translational regulation. Desferrioxine was shown to be beneficial for Alzheimer's patients in one study (Crapper McLachlan et al., 1991). However, the use of this iron chelator is currently restricted to the treatment of patients with sickle cell disease, thalassemias, arid to counteract iron poisoning (Hallaway & Hedlund, 1992). We reasoned that Df would be a strong positive control to screen for new compounds, including anticholinesterase derivatives (phenserine), which might suppress APP mRNA translation without chelating intracellular iron (Figure 11.3).

ht

tp

://

w

w

w

.c

Phenserine. Impairment of the cholinergic system is one of the most important clinical featured symptoms of AD. This process can be partially reversed by acetylcholinesterase inhibitors, including phenserine (al-Jafari et al., 1998). Coincidentally, we have discovered that phenserine is a member of a class of preexisting medicinal compounds that also block APP translation by targeting the unique stemloop formed from 5'UTR sequences in APP mRNA (Figure 11.3). Indeed, studies of rats with forebrain cholinergic lesions have shown that phenserine decreases the level of APP production, unlike some other acetylcholinesterase inhibitors that increase the production of APP (Haroutunian et al., 1997; Lahiri, Farlow, Nunberger, & Greig, 1997). Phenserine is an example of a small molecule (Mw = 487) that acts, at least in part, by blocking translation of APP mRNA through the regulation of the 5'UTR, to suppress APP synthesis, thus inhibiting A(3-peptide output (Dr. N. Greig, NIA, Baltimore, MD). We have tested the capacity of the lead compound phenserine (and other related RNA-targeting compounds) to block APP gene expression through 5'UTR sequences in APP mRNA. Figure 11.3 shows that phenserine blocks the capacity of the APP 5'UTR to drive translation to a greater extent than other anticholinesterases, donapezil and tacrine. The iron

Targeting an RNA Structure

83

chelator, Df, was used as a positive control, representing a small molecule that markedly suppressed APP mRNA translation via 5'UTR sequences (Molecular weight of Df = 656.8).

CONCLUSIONS

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Phenserine improves cognitive performance in rats and is undergoing phase II clinical trials as an acetylcholinesterase inhibitor (Yu, Holloway, Utsuki, Brossi, & Grieg, 1998; Patel et al., 1998). We found that phenserine imparts a double therapeutic action by targeting 5'UTR sequences in APP mRNA to suppress translation of the precursor, and reduces A(3-peptide secretion with a high degree of specificity (Shaw, Utsuki, Rogers, Yu, Lahiri, & Grieg, 2001). The finding that Df suppresses translational enhancement by 5'UTR sequences in APP mRNA is consistent with evidence for the presence of a modified IRE in the 5'UTR of APP mRNA (Figure 11.1). Neither Df or most anticholinesterases is considered to be completely desirable as agents for AD therapy. Desferrioxamine generates hypotension at high doses (Hallaway & Hedlund, 1992), and current anticholinesterases have been shown to be beneficial only for mild cases of AD over 1 year of usage with marked side effects to the liver (Qizilbash, Birks, Lopez Arrieta, Lewington, & Szeto, 2000). There is clearly a need to develop effective screens to generate more promising APP 5'UTR-directed drugs, such as phenserine, for future clinical use.

w

.c

FUTURE DIRECTIONS

ht

tp

://

w

w

Our ultimate goal is to use newly discovered compounds directed to APP-mRNA 5'UTR to limit A(3-peptide output in cell culture systems, and subsequently to therapeutically test these compounds in transgenic mouse models for APP and A(3 over-expression. We have developed a collaboration with Dr. Steve Gullans (Director of Renal Division Research, Harvard Institutes of Medicine). During the course of this project, we have started a screen for APP 5'UTR-binding compounds from a unique library of 1,500 FDA preapproved drugs arranged in a convenient format for transferring to cells growing in 96 well plates. Using a new transfection-based assay, in which APP-5'UTR sequences drive the expression of luciferase and green fluorescent protein (GFP) reporter genes, our two laboratories are conducting a screen to identify new "hits" of therapeutic compounds that already have FDA approval. Single lead drugs, or more potent combinations, will be tested for their capacity to suppress APP translation by RNA targeting. In conducting these

84

Drug Discovery

p

screens, great care has to be taken to ensure that lead compounds do not alter APLP-1 and APLP-2 and ferritin gene expression. This serves as a screening control designed to minimize side effects. New and powerful combinations of FDA-approved drugs will soon be available as therapy for AD patients to use at low doses. Their efficacy will be directed to suppress APP translation, an effect that subsequently would reduce neurotoxic production of A(3-peptide..

x. ph

REFERENCES

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

al-Jafari, A. A., Kamal, M. A., Greig, N. H., Alhomida, A. S., & Perry, E. R. (1998). Kinetics of human erythrocyte acetylcholinesterase inhibition by a novel derivative of physostigmine: Phenserine. Biochemistry Biophysics Research Communications, 248,180-185. Anwar, A., Ali, N., Tanveer, R., & Siddiqui, A. (2000). Demonstration of functional requirement of polypyrimidine tract-binding protein by SELEX RNA during hepatitis C virus internal ribosome entry site-mediated translation initiation. Journal of Biological Chemistry, 275, 34231-34235. Bally, E, Martinez, R., Peters, S., Sudre, P., & Telenti, A. (2000). Polymorphism of HIV Type 1 Gag p7/pl and pl/p6 cleavage sites: Clinical significance and implications for resistance to protease inhibitors. AIDS Research and Human Retroviruses, 16,1209-1213. Bangsberg, D. R., Hecht, F. M., Charlebois, E. D., Zolopa, A. R., Holodniy, M., Sheiner, L., Bamberger, J. D., Chesney, M. A., & Moss, A. (2000). Adherence to protease inhibitors, HIV-1 viral load, and development of drug resistance in an indigent population. AIDS, 14, 357-366. Barbato, G., Cicero, D. O., Cordier, F, Narjes, F, Gerlach, B., Sarnbucini, S., Grzesiek, S., Matassa, V. G., De Francesco, R., & Bazzo, R. (2000). Inhibitor binding induces active site stabilization of the HCV NS3 protein serine protease domain. EMBO Journal, 19,1195-1206. Bartzokis, G., Sultzer, D., Cummings, J., Holt, L. E., Hance, D. B., Henderson, V. W., & Mintz, J. (2000). In vivo evaluation of brain iron in Alzheimer disease using magnetic resonance imaging. Archives of General Psychiatry, 57, 47-53. Berezovska, O., Jack, C., McLean, P., Aster, J. C., Hicks, C, Xia, W., Wolfe, M. S., Kimberly, W. T, Weinmaster, G., Selkoe, D. J., & Hyman, B. T. (2000). Aspartate mutations in presenilin and gamma-secretase inhibitors both impair notchl proteolysis and nuclear translocation with relative preservation of notchl signaling. Journal of Neurochemistry, 75, 583-593. Bush, A. I., Atwood, C., Goldstein, L., Huang, X., & Rogers, J. T. (2000). Could A(3 and PPP be antioxidants? Journal of Alzheimer's Disease, 2, 83-84. Bush, A., Pettingell, W. H., Multhaup, G., Paradis, D., Vonsattel, J. P., Gusella, J. F, Beyreuther, K., Masters, C. L., & Tanzi, R. E. (1994). Rapid induction of Alzheimer A-beta amyloid formation by zinc. Science, 265,1464-1467. Condra, J. H., Schleif, W. A., Blahy, O. M., Gabryelski, L. J., Graham, D. J., Quintero, J. C., Rhodes, A., Robbins, H. L., Roth, E., & Shivaprakash, M.

Targeting an RNA Structure

85

ht

tp

://

w

w

w

.c he

m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

(1995). In vivo emergence of HIV-1 variants resistant to multiple protease inhibitors [see comments]. Nature, 374, 569-571. Connor, J. R., Menzies, S. L., St. Martin, S. M, & Mufson, E. J. (1992). A histochemical study of iron, transferrin, and ferritin in Alzheimer's diseased brains. Journal of Neuroscience Research, 31, 75-83. Crapper McLachlan, D., Dalton, A. J., Kruck, T. P. A., Bell, M. Y., Smith, W. L., Kalow, W., & Andrews, D. F. (1991). Intramuscular desferrioxamine in patients with Alzheimer's disease. Lancet, 337,1304-1308. Fattori, D., Urbani, A., Brunetti, M., Ingenito, R., Pessi, A., Prendergast, K., Narjes, F, Matassa, V. G., De Francesco, R., & Steinkuhler, C. (2000). Probing the active site of the hepatitis C virus serine protease by fluorescence resonance energy transfer. Journal of Biological Chemistry, 275,15106-15113. Feng, Y, Zhang, F, Lokey, L. K., Chastain, J. L., Lakkis, L., Eberhart, D., & Warren, S. T. (1995). Translational suppression by a trinucleotide repeat expansion at FMR1. Science, 268, 731-734. Fukuda, K., Vishinuvardhan, D., Sekiya, S., Kakiuchi, N., Shimotohno, K., Kumar, P. K., & Nishikawa, S. (1997). Specific RNA aptamers to NS3 protease domain of hepatitis C virus. Nucleic Acids Symposium Series, 37, 237-238. Ghosh, S., & Baltimore, D. (1990). Activation in vitro of NF-KB by phosphorylation of its inhibitor IkB. Nature, 344, 678-682. Grilli, M., Pizzi, M., Memo, M., & PierFranco, S. (1996). Neuroprotection by aspirin and sodium salicylate through blockade of NF-kB activation. Science, 274,1383-1385. Haass, C., Schlossmacher, M. G., Hung, A. Y, Vigo-Pelfrey, C., Mellon, A., Ostaszewski, B. L., Liebhaber, I., Koo, E. H., Schenk, D., Teplow, D. B., & Selkoe, D. J. (1992). Amyloid (3-peptide is produced by cultured cells during normal metabolism. Nature, 359, 322-325. Hallaway, P. E., & Hedlund, B. (1992). Therapeutic strategies to inhibit iron-catalyzed tissue damage. In R. Laufer (Ed.), Iron and Human Disease (pp. 477-500). Hamy, E, Felder, E. R., Heizmann, G., Lazdins, J., Aboul-ela, F., Varani, G., Karn, J., & Klimkait, T. (1997). An inhibitor of the TAT.TAR RNA interaction that effectively suppresses HIV-1 replication. Proceedings of the National Academy of Sciences (USA), 94, 3548-3553. Haroutunian, V., Greig, N., Pei, X. E, Utsaki, T., Gluck, R., Acevedo, L. D., Davis, K. L., & Wallace, W. C. (1997). Pharmacological modulation of Alzheimer's beta-amyloid precursor protein levels in the CDSF of rats with forebrain cholinergic system lesions. Molecular Brain Research, 46,161-168. Huang, X., Cuajungco, M. P., Atwood, C. S., Hartshorn, M. A., Tyndall, J. D., Hanson, G. R., Stokes, K. C., Leopold, M., Multhaup, G., Goldstein, L. E., Scarpa, R. C., Saunders, A. J., Lim, J., Moir, R. D., Glabe, C., Bowden, E. F, Masters, C. L., Fairlie, D. P., Tanzi, R. E., & Bush, A. I. (1999). Cu(II) potentiation of alzheimer abeta neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction. Journal of Biological Chemistry, 274, 37111-37116. Karin, M. (1995). The regulation of AP-1 activity by mitogen-activated protein kinases. Journal of Biological Chemistry, 270,16483-16486. Keen, N. J., Churcher, M. J., & Karn, J. (1997). Transfer of Tat and release of TAR

Drug Discovery

86

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

RNA during the activation of the human imunodeficiency virus type-1 transcription cells. EMBO Journal, 16, 5260-5272. Kimberly, W. T., Xia, W., Rahmati, T., Wolfe, M. S., & Selkoe, D. J. (2000). The transmembrane aspartates in presenilin 1 and 2 are obligatory for gamma-secretase activity and amyloid beta-protein generation. Journal of Biological Chemistry, 275, 3173-3178. Lahiri, D., Farlow, M. R., Nunberger, J. I., Jr., & Greig, N. H. (1997). Effects of cholinesterase inhibitors on the secretion of beta-amyloid precursor protein in cell cultures. Annals of the New York Academy of Sciences, 26, 416-421. Lee, J. C, Laydon, J. T., McDonnell, P. C, Gallagher, T. R, Kumar, S., Green, D., McNulty, D., Blumenthal, M. J., Heys, J. R., Landvatter, S. W., Stickler, J. E., Mclaughlin, M. M., Siemens, I. R., Fisher, S. M., Livi, G. P., White, J. R., Adams, J. L., & Young, P. R. (1994). A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature, 372, 739-745. Levy-Lahad, E., Wasco, W., Poorkaj, P., Romano, D. M, Oshima, J., & Tanzi, R. (1995). Candidate gene for the chromosome 1 familial Alzheimer's disease locus. Science, 269, 973-977. Mbella, E. G., Bertrand, S., Huez, G., & Octave, J. N. (2000). A GG nucleotide sequence of the 3' untranslated region of amyloid precursor protein mRNA plays a key role in the regulation of translation and the binding of proteins. Molecular and Cellular Biology, 20, 4572-4579. Multhaup, G., Schlicksupp, A., Hesse, L., Beher, D., Ruppert, T., Masters, C., & Beyreuther, K. (1996). The amyloid precursor protein of Alzheimer's disease in the reduction of copper(II) to copper(I). Science, 271,1406-1409. Pan, J. B., Monteggia, L. M., & Giordano, T. (1993). Altered levels and splicing of the amyloid precursor protein in the adult rat hippocampus after treatment with DMSO or retinoic acid. Molecular Brain Research, 18, 259-266. Patel, N., Spangler, E. L., Greig, N. H., Yu, Q. S., Ingram, D. K., & Meyer, R. C. (1998). Phenserine, a novel acetylcholinesterase inhibitor, attenuates impaired learning in rats in a 14-unit T-maze induced by blockade of the N-methyl-Daspartate receptor. Neuroreport, 9(1), 171-176. Penzak, S. R., Chuck, S. K., & Stajich, G. V. (2000). Safety and efficacy of HMGCoA reductase inhibitors for treatment of hyperlipidemia in patients with HIV. Pharmacotherapy, 20,1066-1071. Pinero, D. J., Hu, J., & Connor, J. R. (2000). Alterations in the interaction between iron regulatory proteins and their iron responsive element in normal and Alzheimer's diseased brains. Cellular and Molecular Biology (Noisy-le-Grand, France), 46, 761-776. Porse, B., Kirillov, S. V., Awayez, M. J., & Garrett, R. A. (1999). UV-induced modifications in the peptidyl transferase loop of 23S rRNA dependent on binding of the streptgramin B antibiotic, Pristinamycin 1 A. RNA, 5,586-595. Qizilbash, N., Birks, J., Lopez Arrieta, J., Lewington, S., & Szeto, S. (2000). Tacrine for Alzheimer's disease (Cochrane review). Cochrane Database Systems Reviews, (3):CD000202. Ringheim, G. E., Szczepanik, A. M., Petko, W., Burgher, K. L., Zhu, S. Z., & Chao, C. C. (1998). Enhancement of beta-amyloid precursor protein transcription and expression by the soluble interleukin-6 receptor/interleukin-6 complex. Molecular Brain Research, 55, 35-4432.

Targeting an RNA Structure

87

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Rogers, J. T. (1996). Ferritin translation by interleukin-1 and interleukin-6: The role of sequences upstream of the start codons of the heavy and light subunit genes. Blood, 87, 2525-2537. Rogers, J. T., Leiter, L. M, McPhee, J., Cahill, C. M., Zhan, S. S., Potter, H., & Nilsson, L. N. (1999). Translation of the alzheimer amyloid precursor protein mRNA is up-regulated by interleukin-1 through 5'-untranslated region sequences. Journal of Biological Chemistry, 274, 6421-6431. Rogers, J. T., & Munro, H. N. (1987). Translation of ferritin light and heavy subunit mRNAs is regulated by intracellular chelatable iron levels in rat hepatoma cells. Proceedings of the National Academy of Sciences (USA), 84, 2277-2281. Schenk, D., Barbour, R., Dunn, W., Gordon, G., Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Liao, Z., Lieberburg, L, Motter, R., Mutter, L., Soriano, R, Shopp, G., Vasquez, N., Vandevert, C., Walker, S., Wogulis, M., Yednock, T., Games, D., & Seubert, P. (1999). Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature, 400,173-177. Scheuner, D., Eckman, C., Jensen, M., Song, X., Citron, M., Suzuki, N., Bird, T. D., Hardy, J., Hutton, M., Kukull, W., Larson, E., Levy-Lahad, E., Viitanen, M., Peskind, E., Poorkaj, P., Schellenberg, G. D., Tanzi, R., Wasco, W., Lannfelt, L., Selkoe, D., & Younkin, S. (1996). Ab42(43) increased in vivo by PS1 /2 and APP mutations linked to familial Alzheimer's disease. Nature Medicine, 2, 864-870. Shaw, K. T. Y, LJtsuki, T., Rogers, J. T., Yu, Q.-S., Lahiri, D., & Grieg, N. H. (2001). Translational regulation of Alzheimer's [3-amyloid precursor protein mRNA by a putative interleukin-1 responsive element, a new target for drug development. Proceedings of the National Academy of Sciences, 98, 7605-7610. Song, W., Nadeu, P., Yuan, M., Yang, X., Shen, J., & Yankner, B. (1999). Proteolytic release and nuclear translocation of Notch-1 are induced by presenillin-1 and impaired by pathogeneic presenilin-mutations. Proceedings of the National Academy of Sciences (USA), 96, 6959-6963. Teplow, D. (1998). Structural and kinetic features of amyloid (3-protein fibrillogenesis. Amyloid, 5, 121-142. Thomson, A., Rogers, J. T., & Leedman, P. J. (1999). Iron-regulatory proteins, iron responsive elements and ferritin mRNA translation. International Journal of Biochemistry and Cell Biology, 10,1139-1152. Thomson, A., Rogers, J. T., & Leedman, P. J. (2000). Thyrotropin-releasing hormone and epidermal growth factor regulate iron regulatory protein binding in pituitary cells via protein kinase C-dependent and -independent signaling pathways. Journal of Biological Chemistry, 275, 31609-31615. Walsh, D., Hartley, D. M., Kusumoto, Y, Fezoui, Y, Conron, M. M., Lomakin, A., Benedek, G. M., & Teplow, D. B. (1999). Amyloid (3-protein fibrillogenesis. Journal of Biological Chemistry, 274, 25945-25952. Werstuck, G., & Green, M. R. (1998). Controlling gene expression in living cells through small molecule-RNA interactions. Science, 282, 296-298. Wolfe, M. S., Xia, W., Moore, C. L., Leatherwood, D. D., Ostaszewski, B., Rahmati, T., Donkor, I. O., & Selkoe, D. J. (1999). Peptidomimetic probes and molecular modeling suggest that Alzheimer's gamma-secretase is an intramembranecleaving aspartyl protease. Biochemistry, 38, 4720-4727.

88

Drug Discovery

p

Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T., & Selkoe, D. J. (1999). Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity. Nature, 398, 512-517. Yu, Q., Holloway, H. W, Utsuki, T., Brossi, A., & Grieg, N. H. (1999). Synthesis of novel phenserine-based-selective inhibitors of butyrylcholiriesterase for Alzheimmer's disease. Journal of Medicinal Chemistry, 42(10), 1855-1861. Zuker, M. (1989). On finding all suboptimal foldings of an RNA molecule. Science, 244, 48-52.

x. ph

ACKNOWLEDGMENTS

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

This work was partially supported by a phase I Small Business Innovation Grant (SBIR) from the National Institute on Aging (NIA), in association with Dr. Tony Giordano (Vice President, Message Pharmaceuticals, Malvern, PA) and Dr. Nigel Greig (Director of Drug Design and Development, NIA, Baltimore, MD). I am very grateful to Dr. Rudolph Tanzi and Dr. Ashley Bush for their scientific input and advice. Dr. Steve Gullans, Director of Renal Division Research (Department of Medicine, Brigham and Womens Hospital, Boston, MA), gave expert advice and provides his library of FDA-preapproved drugs to be screened against the APP 5'UTR target. Dr. Robert Noir kindly assisted in preparing the graphics for Figure 11.1. Many thanks are due to Dr. Catherine Cahill and Dr. Xudong Huang who provided very helpful suggestions.

s/ in de

Intracellular APP

x. ph

Novel Tricyclic Pyrone Compounds Prevent

p

12

m

C99-Induced Cell Death

.c

w

ABSTRACT

he m

4a ll.

vn

/fo

ru

Lee-Way Jin, Duy H. Una, Feng-Shiun Shie, humi Maezawa, Eryce Sopher, and George M. Martin

ht

tp

://

w

w

Alzheimer's disease (AD) is an age-related, neurodegenerative disorder characterized by the progressive and global loss of cognitive functions. Pathological features include a loss of neurons in vulnerable brain regions and the extracellular deposition of abnormal protein aggregates known as amyloid plaques. Amyloid-^ protein (A(3) is the major component of amyloid plaques and is derived from a larger transmembrane glycoprotein, termed amyloid-^ protein precursor (APP), by proteolysis. The AD research has focused on A(3 production and metabolism, its extracellular deposition, and its cellular toxicity. Recent evidence, however, suggests that A(3, as well as the C-terminal fragments (CTF) of APP, can accumulate intraneuronally. The neuronal loss and synaptic transmission deficit in AD may, therefore, depend upon intraneuronal accumulation of A(VCTF, rather than upon extracellular plaque formation. Accordingly, we propose that one of the primary targets of therapeutic intervention should be intracellular AJ3/CTF and its toxic cellular effect. 89

90

Drug Discovery

x. ph

p

We have established a cell-culture model in which the neurons degenerate upon induction of endogenous expression of A(3/CTF of APR These cultures have been used to test whether tricyclic pyrone (TP) compounds may prevent Ap/CTF-mediated neuronal death. The results to date have been encouraging. Lead compounds will now be selected for their abilities to ameliorate Ap/CTF-mediated pathology in transgenic mice. Our belief is that these compounds may eventually prove beneficial for the prevention and treatment of AD.

s/ in de

INTRODUCTION

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

Increasing evidence suggests that the modulation of amyloid-):* precursor protein (APP) metabolism and amyloid-(3 protein (Ap, a proteolytic product of APP) production and deposition may be early and central pathogenetic events leading to Alzheimer's disease (AD). Ap is a 3942-amino acid, self-assembling peptide that is normally secreted. It is largely deposited extracellularly as a component of senile plaques in AD. Based on the belief that aggregated, fibrillary AP in plaques is the source of toxicity causing local cell death, the usual approach to the evaluation of the toxicity of AP has been to modulate the extent of these extracellular depositions. Recently, "AP vaccination" has been used to diminish extracellular Ap aggregates of the transgenic mice as a potential therapeutic approach for AD (Schenk et al., 1999; Bard et al., 2000), and has been shown to prevent cognitive decline in mice (Janus, Pearson, McLaurin, et al., 2000; Morgan, Diamond, Gottschall, et al., 2000) On the other hand, numerous observations suggest that extracellular Ap aggregates may be a secondary and late event in AD. First,, the number of plaques does not correlate well with the degree of cognitive impairment. The distribution of plaques lacks a consistent relationship with the vulnerability of individual cytoarchitectural fields (Arriagada, Growdon, Hedley-Whyte, & Hyman, 1992; Hyman, Morzloff, & Arriagada, 1993). Second, cell death is not found in the vicinity of the plaques, either in the brains of humans or transgenic animals. Finally, the levels of secreted Ap, as reflected in cerebrospinal fluid, are not increased in sporadic AD. These levels are too low (picomolar to nanomolar range) to initiate fibril formation. Therefore, alternative hypotheses to account for Ap-related neurodegeneration is necessary. Work from our laboratory and others has pointed out that a physiologically relevant C-terminal fragment (CTF) of APP (called C100 or C99) may induce toxicity from within the cells (Yankner et al., 1989; Yoshikawa, Aizawa, & Hayashi, 1992; Fukuchi et al., 1993; Sopher et al., 1994; Jin et al., 1998; Neve, McPhie, & Chen, 2000). In addition, recent studies have

Novel Tricyclic Pyrone Compounds 91

91

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

established that A(3 can be produced at multiple locations within the cell. An example is the formation of a nonsecreted, intracellular pool that contains amyloidogenic A(342 (reviewed by Wilson, Doms, & Lee, 1999). Alternatively, extracellular A(342 can be selectively internalized within ADvulnerable CA1 neurons and induce a buildup of intracellular C99 (the carboxyl 99-amino acids of APP), an amyloidogenic precursor to A(3 (Bahr et al., 1998). Neurons in AD brains containing intracellular A(3 epitope exhibited TUNEL-positive DNA damage (LaFerla, Troncoso, Strickland, Kawas, & Jay, 1997), and frequently contained neurofibrillary tangles (NFT), another hallmark lesion of AD (Grundke-Iqbal et al., 1989). It was also demonstrated that neurons in AD-vulnerable brain regions specifically accumulate amyloidogenic A(342 and that this intraneuronal A(342 immunoreactivity appears to precede both NFT and A(3 plaque depositions (Gouras et al., 2000). Taken together, intracellular accumulation of A(3/CTF may occur early in the pathogenesis of AD and may induce toxicity, leading to cell dysfunction, synaptic loss, and eventually cell death. Therefore, one of the primary therapeutic concerns should be to prevent intracellular accumulation of A[VCTF and/or to ameliorate its toxic intracellular effects. We have established a neuronal culture model for conditional expression of intracellular C99 (Sopher et al., 1994). We report that analogs of tricyclic pyrone compounds (TP) rescue the cultured C99-expressing neurons from cell death.

.c

MATERIALS AND METHODS

ht

tp

://

w

w

w

The TP analogs were synthesized using a simple one-pot coupling reaction of pyrones and enals as described (Hua, Chen, Sin, et al., 1997), followed by various functional group interconversions. The MC65 cells were cultured as described (Sopher et al., 1994; Sopher, Fukuchi, Kavanagh, Furlong, & Martin, 1996). The cytotoxicity in each experimental condition was estimated using a colorimetric MTT (tetrazolium salt)-based assay (Hansen, Nielsen, & Berg, 1989). Between control and test cultures (cultures with and without tetracycline and cultures with and without TP compounds), the MTT-based estimates of viable cells were comparable to estimates obtained using counts of viable cells based on trypan blue exclusion. The bicine/Tris SDS-PAGE system in the presence of 8 M urea for the separation of A(340 and A(342 was performed as described (Klafki, Wiltfang, & Staufenbiel, 1996). The monoclonal antibody 6E10 against Ap, 17 was purchased from Senetek Inc. (Maryland Heights, MI). The polyclonal antibody B994 against the C-terminal 39 amino acids of APP was a gift from Dr. Thomas Hinds at University of Washington (Jin et al., 1998).

92

Drug Discovery

RESULTS

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

MC65 is a stably transformed, human neuroblastoma cell line that conditionally expresses a partial APP-fusion protein (amino-17 residues + carboxyl-99 residues) (Sopher et al., 1994). The conditional expression was achieved using the tetracycline-responsive promoter system (Gossen & Bujard, 1992). MC65 cells maintained in the presence of tetracycline did not express any C99 and proteolytic fragments of C99. Days after tetracycline withdrawal, MC65 cells demonstrated pronounced nuclear DNA fragmentation and gradually degenerated (Sopher et al., 1996). One day after induction by the withdrawal of tetracycline from the medium, both C99 and AP40 were present in the cell extracts (Figure 12.1 A), but not in the conditioned medium (data not shown). In addition, there were several higher molecular weight bands that were recognized by both 6E10, an antibody against Ap t 17 (Figure 12.1A), and B994, an antibody against C-terminal 39 amino acids of APP (data not shown), representing possible A(3-CTF complexes. At 24 hours after induction, the levels of A(3, C99, and the possible AfJ-CTF complexes were markedly decreased in the presence of 2 uM CP2, a lead TP compound (Figure 12.1 A). This effect was not due to an interference with the induction of C99 expression since at 12 hours after induction, the levels of C99 and A{3 in the treated and control cultures showed no significant difference. Obvious neuronal death was observed only on the third day after C99 induction, and significant numbers of cells were dead on the fourth day after C99 induction. We have tested 12 analogs of TP to determine their effects on this model of cell death and their cellular toxicity. The goal was to obtain data to guide the future design of new analogs, those that achieve higher effects with minimal toxicity (high EC50/IC50). CP2 is one of the best TP compounds so far. CP2, when added to the cultures at the same time as tetracycline withdrawal, completely protected the cells from death (Figure 12.IB). Its EC50 was around 0.1 to 0.2 uM.

DISCUSSION

The TP compounds were first synthesized based on the structures of pyripyropenes and arisugacin (Hua et al., 1997). These compounds have a variety of activities. Pyripyropenes are potent ACAT (acyl-CoA: cholesterol O-acyltransferase) inhibitors and arisugacin is a potent acetylcholinesterase inhibitor. (Unlike the acetylcholinesterase inhibitor tacrine, which is a charged molecule, arisugacin is a neutral molecule.) Both are tetracyclic pyrones. Novel TP compounds were used to screen a variety of activities. They were applied to models of AD since they have tacrine-like

Novel Tricyclic Pyrone Compounds

s/ in de

x. ph

p

93

4a ll.

vn

/fo

ru

m

FIGURE 12.1 CP2 reduces Ap/CTF levels and prevents MC65 cell death. A. Changes of induced Ap/CTF in MC65 cells after CP2 (2 uM) treatment. MC65 cells were lysed at indicated hours after C99 induction. The lysates were separated by Bicine SDS-PAGE and the western blot probed with 6E10. Ap40, C99, and proteins indicated by arrowheads are decreased upon CP2 treatment. These bands were not seen in lysates from tetracycline (TC)-treated controls. B. MC65 cell survival 4 days after withdrawal of TC in the presence of different concentrations of CP2. The cell survival was measured by the MTT method.

ht

tp

://

w

w

w

.c

he m

activity. Relevant to our MC65 model, C100 was shown to induce increases in acetylcholinesterase (Sberna et al., 1998). Surprisingly, TP compounds were effective in preventing MC65 death at much lower concentrations than those required for acetylcholinesterase-inhibitor activity. Therefore, this effect is the result of other actions of the compounds. MC65 cell death has been found to be mediated by an intracellular process (Sopher et al., 1996). This process appears to be modulated and/or mediated by a reactive oxygen specie(s). Unpublished data from our laboratory indicate that TP compounds do not inhibit the common pathway of apoptosis, since they did not prevent PC 12 cells from death due to serum withdrawal and did not prevent differentiated PCI2 cells from death due to nerve growth factor withdrawal (Greene, 1978). They are not substrates of caspase 3, as determined by measuring the release of p-nitroaniline after incubating TP with caspase 3. They did not show radical scavenger activity at nanomolar concentrations (capable of preventing MC65 death), as reflected in the measurement of lipid peroxidation using rat brain membranes. Finally, at nanomolar concentrations, they did not inhibit in vitro A(340 fibrillogenesis, and they did not prevent the death of primary neurons after A(3 treatment (Yankner, Duffy, & Kirschner, 1990).

94

Drug Discovery

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Our data suggested that TP may reduce the accumulation of A(3 and C99 after they are produced, and may prevent the formation of complexes that contain both A(3 epitope and the epitope contained in the most C-terminal portion of C99. The effect of TP on the levels of A|3, C99, and the possible A(3-CTF complexes occurs early in the course of C99 induction, indicating that this effect is not secondary to the prevention of cell death. While the mechanism of action of TP compounds remains to be elucidated, it is intriguing to speculate that they may bind directly to C99. Experiments addressing this possibility are underway in our laboratory. Furthermore, TP compounds may be used as a probe for the study of mechanism of cell toxicity rendered by intracellular Ap/CTF. Several reports have indicated the existence of cytotoxic domains in the CTF outside the A(3 domain (e.g., Lu et al., 2000). It will be interesting to determine whether TP can block the neuronal death resulting from the accumulation of these domains. It will also be important to determine if they can ameliorate the physiological, morphological, and behavioral deficits in transgenic mice overexpressing APP. Several groups of investigators have created such transgenic mice. They exhibit some of the prominent behavioral and pathological features of AD. We have studied two lines of such mice using sensitive immunocytochemical staining for A(3. The first line, APPsw, was obtained from Dr. Karen Hsiao at the University of Minnesota (Hsiao et al., 1996). We found punctate, apparently intracellular deposits containing an A(3-epitope in the hippocampal CA1 neurons as early as 4 months of age (Shie, Jin, Leverenz, & LeBoeuf, 2000). This precedes the extracellular, amyloid plaque formation and behavioral deficits (e.g., impaired spatialreference memory), phenotypes seen only at 9 to 10 months of age (Hsiao et al., 1996). It is much earlier than the impaired induction of long-term potentiation at synapses in the CA1 and dentate gyrus hippocampal regions, which occurs by 15 to 17 months of age (Chapman, White, Jones, et al., 1999). A second line, SpCkv, was produced in our laboratory, and showed muscle pathology similar to that of human inclusion body myositis, the most common degenerative muscle disorder of the elderly (Jin et al., 1998). We found punctate intracellular deposits containing an A(3 epitope in the morphologically normal muscle fibers at 9 months of age, preceding the amyloid fibril formation and muscle fiber degeneration that was only seen at 24 months of age (Jin et al., 1999). These two lines of evidence suggest that, in addition to the detrimental effect from amyloid fibrils, which occurs at later stages in both cases, the intracellular accumulation of A(3 or Ap-containing CTF (Neve et al., 2000) may inflict an early and probably more significant toxic effect. Our TP compounds may have a unique effect on this early intracellular Ap/CTF toxicity. We are in the process of designing lead compounds to test their ameliorating effect on these animal models.

Novel Tricyclic Pi/rone Compounds 95

95

REFERENCES

ht

tp

://

w

w

w

.c he

m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Arriagada, P. V., Growdon, J. H., Hedley-Whyte, E. T., & Hyman, B.T. (1992). Neurofibrillary tangles but not senile plaques parallel duration and severity of Alzheimer's disease. Neurology, 42, 631-639. Bahr, B. A., Hoffman, K. B., Yang, A. J., Hess, U. S., Glabe, C. G., & Lynch, G. (1998). Amyloid (3 protein is internalized selectively by hippocampal field CA1 and causes neurons to accumulate amyloidogenic carboxyterminaal fragments of the amyloid precursor protein. Journal of Comparative Neurology, 397,139-147. Bard, R, Cannon, C., Barbour, R., Burke, R. L., Games, D., Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Lieberburg, I., Motter, R., Nguyen, M., Soriano, R, Vasquez, N., Weiss, K., Welch, B., Seubert, P., Schenk, D., & Yednock, T. (2000). Peripherally administered antibodies against amyloid f3-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nature Medicine, 6, 916-919. Chapman, P. R, White, G. L., Jones, M. W., Cooper-Blacketer, D., Marshall, V. J., Irizarry, M., Younkin, L., Good, M. A., Bliss, T. W., Hyman, B. T., Younkin, S. G., & Hsiao, K. K. (1999). Impaired synaptic plasticity and learning in aged amyloid precursor protein transgenic mice. Nature Neuroscience, 2, 271-276. Fukuchi, K., Sopher, B., Furlong, C. E., Smith, A. C., Dang, N., & Martin, G. M. (1993). Selective neurotoxicity of COOH-terminal fragments of the (3-amyloid precursor protein. Neuroscience Letters, 154,145-148. Gossen, M., & Bujard, H. (1992). Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proceedings of the National Academy of Sciences (USA), 89, 5547-5551. Gouras, G. K., Tsai, J., Naslund, J., Vincent, B., Edgar, M., Checler, R, Greenfield, J. P., Haroutunian, V., Buxbaum, J. D., Xu, H., Greengard, P., & Relkin, N. R. (2000). Intraneuronal A[342 accumulation in human brain. American Journal of Pathology, 156,15-20. Greene, L. A. (1978). Nerve growth factor prevents the death and stimulates the neuronal differentiation of clonal PC12 pheochromocytoma cells in serumfree medium. Journal of Cell Biology, 78, 747-755. Grundke-Iqbal, I., Iqbal, K., George, L., Tung, Y. C, Kim, K. S., & Wisniewski, H. M. (1989). Amyloid protein and neurofibrillary tangles coexist in the same neuron in Alzheimer disease. Proceedings of the National Academy of Sciences (USA), 86, 2853-2857. Hansen, M. B., Nielsen, S. E., & Berg, K. (1989). Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. Journal of hnmunological Methods, 119, 203-210. Hsiao, K., Chapman, P., Nilsen, S., Eckman, C., Harigaya, Y, Younkin, S., Yang, R, & Cole, G. (1996). Correlative memory deficits, A(3 elevation, and amyloid plaques in transgenic mice. Science, 274, 99-102. Hua, D. H., Chen, Y, Sin, H.-S., Newell, S. W., Ladesich, J. B., Perchellet, E. M., Kraft, S. L., Omura, S., & Perchellet, J. B. (1997). A one-pot condensation of pyrones and enals. Synthesis of lH,7H-5a,6,8,9-tetrahydrol-l-oxopyrano[4,3b][l]benzopyrans. Journal of Organic Chemistry, 62, 6888-6896.

96

Drug Discovery

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Hyman, B. T., Marzloff, K., & Arriagada, P. V. (1993). The lack of accumulation of senile plaques or amyloid burden in Alzheimer's disease suggests a dynamic balance between amyloid deposition and resolution. Journal of Neuropathology and Experimental Neurology, 52, 594-600. Janus, C, Pearson, J., McLaurin, }., et al. (2000). A beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer's disease. Nature, 408, 979-982. Jin, L. W., Hearn, M., Ogburn, C. E., Dang, N., Nochlin, D., Ladiges, W. C., & Martin, G. M. (1998). Transgenic mice overexpressing the C-99 fragment of (3PP with an a-secretase site mutation develop a myopathy similar to human inclusion body myositis. American Journal of Pathology, 153,1679-1686. Jin, L. W., Hearn, M. G., Marcu, O., Starbuck, M. Y, Sopher, B., & Martin, G. M. (1999). Evidence of oxidative DNA damage in the skeletal muscles of a transgenie model of inclusion body myositis. Society for Neurosciences Abstracts, 25,1840. Klafki, H. W., Wiltfang, J., & Staufenbiel, M. (1996). Electrophoretic separation of betaA4 peptides (1-40) and (1-42). Analytical Biochemistry, 237, 24-29. LaFerla, F. M., Troncoso, J. C., Strickland, D. K., Kawas, C. H., & Jay, G. (1997). Neuronal cell death in Alzheimer's disease correlates with apoE uptake and intracellular A(3 stabilization. Journal of Clinical Investigations, 100, 310-320. Lu, D. C., Rabizadeh, S., Chandra, S., Shayya, R. F., Ellerby, L. M., Ye, X., Salvesen, G. S., Koo, E. H., & Bredesen, D. E. (2000). A second cytotoxic proteolytic peptide derived from amyloid beta-protein precursor. Nature Medicine, 6, 397-404. Morgan, D., Diamond, D. M., Gottschall, P. E., Ugen, K. E., Dickey, C., Hardy, J., Duff, K., Jantzen, P., DiCarlo, G., Wilcock, D., Connor, K., Hatcher, J., Hope, G., Gordon, M., & Arendash, G. W. (2000). A beta peptide vaccination prevents memory loss in an animal model of Alzheimer's disease. Nature, 408, 982-985. Neve, R. L., McPhie, D. L., & Chen, Y. (2000). Alzheimer's disease: A dysfunction of the amyloid precursor protein. Brain Research, 886, 54-66. Sberna, G., Saez-Valero, J., Li, Q. X., Czech, C., Beyreuther, K., Masters, C. L., McLean, C. A., & Small, D. H. (1998). Acetylcholinesterase is increased in the brains of transgenic mice expressing the C-terminal fragment (CT100) of the beta-amyloid protein precursor of Alzheimer's disease. Journal of Neurochemistry, 71, 723-731. Schenk, D., Barbour, R., Dunn, W., Gordon, G., Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Liao, Z., Lieberburg, I., Motter, R., Mutter, L., Soriano, F., Shopp, G., Vasquez, N., Vandevert, C., Walker, S., Wogulis, M., Yednock, T., Games, D., & Seubert, P. (1999). Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature, 400,173-177. Shie, F. S., Jin, L. W., Leverenz, J., & LeBoeuf, R. (2000). Early p-amyloid deposition in hippocampal neurons in p-amyloid precursor protein 695 (APP695) transgenic mice. Neurobiology of Aging, 21, S225. Sopher, B. L., Fukuchi, K., Smith, A. C., Leppig, K. A., Furlong, C. E., & Martin, G. M. (1994). Cytotoxicity mediated by conditional expression of a carboxyl-

97

Novel Tricyclic Pyrone Compounds 97

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

terminal derivative of the beta-amyloid precursor protein. Molecular Brain Research, 26, 207-217. Sopher, B. L., Fukuchi, K., Kavanagh, T.}., Furlong C. E., & Martin, G. M. (1996). Neurodegenerative mechanisms in Alzheimer disease. A role for oxidative damage in amyloid (3 protein precursor-mediated cell death. Molecular Chemical Neuropathology, 29,153-168. Wilson, C. A., Doms, R. W., & Lee, V. M.-Y. (1999). Intracellular APP processing and A(3 production in Alzheimer's disease. Journal of Neuropathology and Experimental Neurology, 58, 787-794. Yankner, B. A., Dawes, L. R., Fisher, S., Villa-Komaroff, L., Oster-Granite, M. L., & Neve, R. L. (1989). Neurotoxicity of a fragment of the amyloid precursor associated with Alzheimer's disease. Science, 245, 417-420. Yankner, B. A., Duffy, L. K., & Kirschner, D. A. (1990). Neurotrophic and neurotoxic effects of amyloid beta protein: Reversal by tachykinin neuropeptides. Science, 250, 279-282. Yoshikawa, K., Aizawa, T., & Hayashi, Y. (1992). Degeneration in vitro of postmitotic neurons overexpressing the Alzheimer amyloid protein precursor. Nature, 359, 64-67.

13

/fo

ru

m

s/ in de

Robert Kisilevsky and Walter A. Szarek

x. ph

p

Novel Glycosaminoglycans as Antiamyloid Agents

4a ll.

vn

ABSTRACT

ht

tp

://

w

w

w

.c

he m

In vivo amyloids consist of two classes of constituents. The first is the disease-defining protein (e.g., A(3 in Alzheimer's disease). The second is a set of common structural components that usually are the building blocks of basement membrane (BM), a tissue structure that serves as a scaffold onto which cells normally adhere. In vitro binding interactions between one of these BM components and amyloidogenic proteins rapidly change the conformation of the amyloidogenic protein into amyloid fibrils. The offending BM component is a heparan sulfate (HS) proteoglycan (HSPG), part of which is protein and the remaining part a specific linear polysaccharide that is the portion responsible for binding to and imparting the typical amyloid structure to the amyloid precursor protein/peptide. Our past work has demonstrated that agents that inhibit the binding between HS and the amyloid precursor are effective antiamyloid compounds both in vitro and in vivo. This chapter is concerned with the design and synthesis of modified sugar precursors of HS, which when incorporated into the polysaccharide will alter its structure so that it loses its amyloid-precursor protein/peptide binding and fibril-inducing properties. As part of a continuing study, four compounds have been designed and synthesized based on the known steps involved in HS biosynthesis. Using primary hepatocyte cultures, we have shown that one of these is incorporated into HS and terminates its elongation. Three others have been constructed so as to be incorporated into HS and allow elongation to 98

Novel Gh/cosciminoglycans as Antiamyloid Agents

99

occur, but also alter the sulfation pattern of HS, which is thought to be the critical aspect of the HS structure necessary for amyloid-precursor protein/peptide binding.

INTRODUCTION Nature of Amyloid

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Amyloid is a term for extracellular fibrillar protein deposits that have a specific set of staining and structural characteristics. Amyloid depaosits are involved in the pathogenesis of disorders, such as Alzheimer's disease, adult-onset diabetes, joint destruction during prolonged hemodialysis, and several other rarer disorders. Although in tissues, all amyloids look and stain the same, more than 20 different protein types have been identified (Westermark, 1997). In vivo, each amyloid is composed of two classes of components (Kisilevsky & Fraser, 1997). The first is the defining protein and the second is a set of common, structural constituents. These include serum amyloid P, proteoglycans (usually perlecan, the basement membrane form of HSPG), laminin, collagen IV, and apolipoprotein E. Significant evidence has accumulated indicating that interactions between the common components on the one hand, and the amyloidogenic protein on the other, play a role in amyloidogenesis (Kisilevsky & Fraser, 1997). Of particular importance are the glycosaminoglycans (GAGs), HS, and/or heparin. In the cases of amyloid associated with inflammatory diseases (AA amyloid) and Alzheimer's disease (A(3), such amyloid-associated GAGs have been shown to have subtle changes in structure (Lindahl et al., 1995,1996,1997, 1999), and, when interacting with their respective amyloidogenic proteins, have the ability to alter their conformations so that they take on the secondary and fibrillar-structural characteristics typical of an amyloid (McCubbin, Kay, Narindrasorasak, & Kisilvesky, 1988; Mclaurin et al., 1999a, 1999b; Kisilevsky & Fraser, 1997; Castillo et al., 1998).

ht

Amyloidogenic Protein and Heparin/Heparan Sulfate—Complementary Binding Domains

The structural features of one HS-binding domain of serum amyloid A (SAA; the precursor to AA amyloid) necessary for HS binding are indicated below. The critical amino acid residues have been identified (Ancsin & Kisilevsky, 1999) and are shown enlarged.

ADQEANRHGRSCKDPNYYRPPGLPAKY An analogous domain has been identified in A(3(l-42) (Davidson & Kisilevsky, unpublished results) and is shown below. DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

Drug Discovery

200

x. ph

p

The general structural features of HS and heparin are indicated in Figure 13.1. In HS, X may be H or SO3H, and R may be H, acetyl, or SO3H. Unpublished experimental work (Ancsin, Davidson, & Kisilevsky) has shown that the 6-OSO3H group on the glucosamine residue of heparin/HS is not necessary for binding to the HS-binding domain of SAA. More critical are the 2-OSO3~ on the uronate residue and the N-SO3~ of the glucosamine residue, both of which appear to be necessary for binding to SAA and AS.

s/ in de

RATIONALE

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

The rationale for the synthesis of novel GAG-sugar precursors follows from the structure of heparin/HS, and the biosynthetic steps that are responsible for their production (Lindahl, Kushke, Lindholt, & Oscarsson, 1989). Altering the structure at the 4-position of either the glucosamine or the uronate residues would interfere with the growth of the GAG chain, since this position is necessary for the ocl-4 linkages in the polysaccharide. Alternatively, altering the structure at the 2-position of the uronate or of the amino group of the glucosamine residue would likely change the manner in which the growing polysaccharide chain is sulfated. In addition, because N-sulfonation is a required step in the proper epimerization and sulfation of the uronate, modification of glucosamine precursors at the amino group will likely result in an undersulfated HS product, and, therefore, one that will probably not interact with amyloidogenic proteins/peptides.

FIGURE 13.1 Structural features of heparan sulfate/heparin.

Novel Glycosaminoglycans as Antiamyloid Agents

101

MATERIALS AND METHODS Chemical Syntheses

s/ in de

x. ph

p

Four novel sugars have been prepared. The first (compound 1) has a change in structure introduced at the 4-position of glucosamine (X in Figure 13.2), and a variation of compound 1 was prepared in radioactive form to determine if this compound is incorporated into the GAG chain. The remaining three compounds (2, 3, and 4) had substituents introduced into the amino group of glucosamine (Rs in Figure 13.2). Compound 4 was also acetylated at its remaining hydroxyl groups to facilitate entry into cells. Glycosaminoglycan Synthesis

4a ll.

vn

/fo

ru

m

Mouse hepatocytes were isolated and placed in culture as described previously (Berkin et al, 20003, 2000b; Subrahmanyan & Kisilevsky, 1988; Thomas et alv 1995). These were then cultured for 24 hours in the absence and presence of varying concentrations of the novel sugar analogues. [3H] Glucosamine and [35S]SO4 were used to monitor the synthesis and sulfation of the newly made HS as described previously (Berkin et al., 2000a, 2000b; Subrahmanyan & Kisilevsky, 1988; Thomas et al., 1995).

he m

RESULTS

ht

tp

://

w

w

w

.c

Figure 13.3 demonstrates the time course of [3H]glucosamine (panel A) and [35S]SO4 (panel B) incorporation into HS of 2x106 liver cells at various times during the incubation in the absence of novel sugars. Note an almost linear incorporation of the two isotopes for the first 20 hours, after which each of their levels reach a plateau. The values are the mean ± SD of triplicate assays. Figure 13.4 demonstrates the time course of [3H]glucosamine (panel A) and [35S]SO4 (panel B) incorporation into HS of 2x106 liver cells at various

FIGURE 13.2 Generic structure of glucosamine analogues.

Drug Discovery

x. ph

p

102

s/ in de

FIGURE 13.3 Time course of [3H]glucosamine (panel A) and and [35S]SO4 (panel B) incorporation into HS of 2 x 106 liver cells at various times during incubation in the absence of novel sugars.

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

times during their incubation in the presence of 1 mM of compound 1. Note that there is an abrupt termination of incorporation of the isotopes within 2 hours of exposure of the cells to compound 1. Experiments with [3H] leucine indicated that compound 1 did not inhibit protein synthesis. The values are the mean ± SD of triplicate assays. Figure 13.5 demonstrates the effect of increasing concentration of the tritiated variant of compound 1 and its incorporation into cellular hepatocyte HS over a 24-hour incubation period. The values are the mean ± SD of triplicate assays. The effects of compounds 2, 3, and 4 on [3H]glucosamine arid [35S]SO4 incorporation into hepatocyte HS are illustrated in Table 13.1.

FIGURE 13.4 Time course of [3H]glucosamine (panel A) and [35S]SO4 (panel B) incorporation into HS of 2 x 106 liver cells at various times during their incubation in the presence of 1 mM of compound 1.

Novel Gli/cosaminogh/cans as Antiamyloid Agents

s/ in de

x. ph

p

103

ru

m

FIGURE 13.5 The effect of increasing concentration of the tritiated variant of compound 1 and its incorporation into cellular hepatocyte HS over a 24-hour incubation period.

/fo

DISCUSSION

.c he

m

4a ll.

vn

The chemical design and synthetic procedures adopted produced the desired compounds in good yield, the minimum being 60% to 65% based on the starting material. The structures of the compounds were validated by elemental analysis, nuclear magnetic resonance (NMR) spectroscopy and Mass Spectrometry.

[3H]Glucosamine

5

S]S0 4

w

w

TABLE 13.1 Percent Incorporation Relative to Control*

95 ±4 119 ±11 114 ±4 113 ±5

87 + 4 122 + 18 114 ±5 115+9

Compound 3

10 1.0 0.1 0.01

23 ±1 48 ±1 101 ± 4 111 ±10

88+7 105 + 7 138 ±9 123 + 12

Compound 4

10 1.0 0.1 0.01

51 ± 1 69 ±4 85 ±4

w

mM 10 1.0 0.1 0.01

ht

tp

://

Compound 2

Toxic to cells

The values are the means ± SEM of triplicate cultures.

73 ±2 80 + 1 90+6

204

Drug Discovery

he m

REFERENCES

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

The effect of compound 1 on the biosynthesis of liver HS can best be interpreted as terminating elongation of the growing GAG chain. This conclusion is supported by the abrupt termination of [3H]glucosamine and [35S]SO4 incorporation into HS in the presence of compound 1, the incorporation of the radiolabelled analogue of compound 1 into the growing GAG chain, the modification of the novel sugar at the 4-position that precludes the further addition of uronate, and the dramatically shortened HS length (data not shown). The effects of compounds 2, 3, and 4 on HS synthesis were interesting and somewhat unexpected. Accurate interpretation will require additional information on the metabolic handling of these compounds. Our preliminary results are in keeping with the chemical design and synthetic objectives set out in our original proposal. They indicate the feasibility of preparing the desired compounds in good yield by the relatively inexpensive synthetic pathways chosen for this purpose. Some of the synthesized compounds examined to date exert the desired effects on the HS biosynthetic process. They will be examined for their effects on the metabolic processes that activate the precursor sugars (i.e., the critical metabolic pathways involved in HS synthesis), which will help explain the mechanisms by which they exert their effect. The compounds will then be tested in animal models of anti-amyloid properties.

ht

tp

://

w

w

w

.c

Ancsin, J. B., & Kisilevsky, R. (1999). The heparin/heparan sulfate-binding site on apo-serum amyloid A: Implications for the therapeutic intervention of amyloidosis. Journal of Biological Chemistry, 274, 7172-7181. Berkin, A., Szarek, M. A., Plenkiewicz, J., Szarek, W. A., & Kisilevsky, R. (2000a). Synthesis of 4-deoxy analogues of 2-acetamido2-deoxy-D-glucose and 2acetamido-2-deoxy-D-xylose and their effects on glycoconjugate biosynthesis. Carbohydrate Research, 325, 30-45. Berkin, A., Szarek, W. A., & Kisilevsky, R. (2000b). Synthesis of 4-deoxy-4-fluoro analogues of 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy~Dgalactose and their effects on cellular glycosaminoglycan biosynthesis. Carbohydrate Research, 326, 250-263. Castillo, G. M., Cummings, J. A., Yang, W. H., Judge, M. E., Sheardown, M. J., Rimvall, K., Hansen, J. B., & Snow, A. D. (1998). Sulfate content and specific glycosaminoglycan backbone of perlecan are critical for perlecan's enhancement of islet amyloid polypeptide (amylin) fibril formation. Diabetes, 47, 612-620. Kisilevsky, R., & Fraser, P. E. (1997). Ap amyloidogenesis: Unique or variation on a systemic theme? Reviews in Biochemistry and Molecular Biology, 32, 361-404. Lindahl, B., Eriksson, L., & Lindahl, U. (1995). Structure of heparan sulphate from human brain, with special regard to Alzheimer's disease. Biochemical Journal, 306,177-184.

Novel Glycosaminoglycans as Antiamyloid Agents

105

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Lindahl, B., Eriksson, L., Spillmann, D., Caterson, B., & Lindahl, U. (1996). Selective loss of cerebral keratan sulfate in Alzheimer's disease. Journal of Biological Chemistry, 271,16991-16994. Lindahl, B., & Lindahl, U. (1997). Amyloid-specific heparan sulfate from human liver and spleen. Journal of Biological Chemistry, 272, 26091-26094. Lindahl, B., Westling, C, Gimenez-Gallego, G., Lindahl, U., & Salmivirta, M. (1999). Common binding sites for p-amyloid fibrils and fibroblast growth factor-2 in heparan sulfate from human cerebral cortex. Journal of Biological Chemistry, 274, 30631-30635. Lindahl, U., Kushke, M., Lindholt, K., & Oscarsson, L. G. (1989). Biosynthesis of heparin and heparan sulfate. Annals of the New York Academy of Sciences, 556, 36-50. McCubbin, W. D., Kay, C. M., Narindrasorasak, S., & Kisilevsky, R. (1988). Circular dichroism and fluorescence studies on two murine serum amyloid A proteins. Biochemical Journal, 256, 775-783. Mclaurin, J., Franklin, T., Kuhns, W. J., & Fraser, P. E. (1999). A sulfated proteoglycan aggregation factor mediates amyloid-(3 peptide fibril formation and neurotoxicity. Amyloid, 6, 233-243. Mclaurin, J., Franklin, T., Zhang, X. Q., Deng, J. P., & Fraser, P. E. (1999). Interactions of Alzheimer amyloid-p peptides with glycosaminoglycans— Effects on fibril nucleation and growth. European Journal of Biochemistry, 266, 1101-1110. Subrahmanyan, L., & Kisilevsky, R. (1988). Effects of culture substrates and normal hepatic sinusoidal cells on in-vitro hepatocyte synthesis of apo-SAA. Scandanavian Journal of Immunology, 27, 251-260. Thomas, S. S., Plenkiewicz, J., Ison, E. R., Bols, M., Zou, W., Szarek, W. A., & Kisilevsky, R. (1995). Influence of monosaccharide derivatives on liver cell glycosaminoglycan synthesis: 3-deoxy-D-ry/o-hexose (3-deoxy-D-galactose) and methyl (methyl 4-chloro-4-deoxy-p-D- galactopyranosid) uronate. Biochimica Biophysica Acta, 1272, 37^48. Westermark, P. (1997). Classification of amyloid fibril proteins and their precursors: An ongoing discussion. Amyloid, 4, 216-218.

ht

tp

ACKNOWLEDGMENTS This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (WAS), the Medical Research Council of Canada, grant MT-3153 (RK), and the Institute for the Study of Aging (RK and WAS). We also express our appreciation to Drs. Ali Berkin and Shridhar Bhat, as well as Mrs. Ruth Tan and Carol Hegadoorn for their assistance with various aspects of this project.

x. ph

Strategies for Inhibiting Alzheimer's y-Secretase

p

14

ru

m

s/ in de

Michael S. Wolfe

vn

/fo

INTRODUCTION

ht

tp

://

w

w

w

.c

he m

4a ll.

Postmortem analysis invariably reveals that the Alzheimer's disease (AD) brain is littered with neuritic plaques (Selkoe, 1999). The principle protein component of these plaques is the 4 kDa amyloid-(3 protein (AP), derived from the amyloid-p precursor protein (APP) via sequential proteolysis by P- and y-secretases (Figure 14.1). Cleavage by p-secretase occurs just outside the single transmembrane domain of APP to release the large ectodomain, and subsequent processing of the membrane-associated C-terminal fragment by y-secretase takes place in the middle of the transmembrane region to produce 40- and 42-amino acid C-terminal A|3 variants, AP40 and AP42. While Ap is prone to self-association into fibrils that are neurotoxic (Lorenzo & Yankner, 1994), the longer and more hydrophobic AP42 is particularly fibrillogenic (Jarrett, Berger, & Lansbury, 1993). Moreover, mutations in APP and in the polytopic presenilins that cause autosomal-dominant familial Alzheimer's disease (FAD) all increase AP42 production (Hardy, 1997), and Ap42 is the principal Ap isoform found presymptomatically in early, diffuse plaques (Iwatsubo, Manri, Odaka, Suzuki, & Ihara, 1995). Thus, AP42 is particularly implicated in the pathogenesis of AD. Both P- and y-secretases are considered important targets for the development of new therapeutic agents to treat AD. p-Secretase has recently been identified as a new membrane-tethered aspartyl protease (Vassar et al., 1999; Sinha et al., 1999; Van et al., 1999; Hussain et al., 1999; Lin et al., 2000), a major step toward developing effective inhibitors as drug candidates. 106

Strategies for Inhibiting y-Secretase

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

107

ht

tp

://

w

w

w

.c

FIGURE 14.1 (A) Proteolytic processing of APR Ectodomain shedding by a- or (3-secretase generates 83- or 99-residue, membrane-associated, C-terminal fragments (C83 or C99, respectively). C83 and C99 are further processed by y-secretase, which hydrolyzes these substrates in the middle of their transmembrane regions to form p3 from C83 and A(3 from C99. (B) Topology of presenilins. Two conserved aspartates, one in TM6 and one in TM7, are required for y-secretase cleavage of APP, leading to the hypothesis that presenilins are novel intramembrane-cleaving aspartyl proteases.

While y-secretase has not been definitively identified, evidence strongly suggests that presenilin is the catalytic component of a larger y-secretase complex (Selkoe & Wolfe, 2000). We have shown through inhibitor studies that y-secretases have properties of aspartyl proteases (Wolfe et al., 1999), and modeling and mutagenesis support a helical conformation of the APP transmembrane region for its initial interaction with y-secretase (Wolfe et al., 1999; Lichtenthaler et al., 1999). Based on these results, we

108

Drug Discovery

ru

m

s/ in de

x. ph

p

found that two conserved, transmembrane aspartates in presenilin-1 are each required for y-secretase cleavage of APP (Wolfe et al., 1999). Similar results have been obtained with presenilin-2 (Steiner et al., 1999). These findings are consistent with the hypothesis that presenilins are y-secretases, novel intramembrane-cleaving aspartyl proteases (Wolfe, De Los Angeles, Miller, Xia, & Selkoe, 1999). While (3-secretase has clear sequence homology with other known aspartyl proteases, the eight-transmembrane presenilins bear no obvious resemblance to them. Moreover, presenilins apparently do not facilitate y-secretase proteolysis by themselves, but require other limiting cofactors (Thinakaran et al., 1997). Understanding the biochemistry of y-secretase should facilitate the development of therapeutic agents that inhibit this activity. Our approach has been to develop new y-secretase inhibitors that can serve both as prototypes for drug development and as biological tools for characterizing y-secretase and understanding the normal and pathological roles of this protease.

/fo

TRANSITION-STATE ANALOGUE INHIBITORS

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

We previously reported that substrate-based difluoro ketone peptidomimetic transition-state analogue 1 inhibits both Ap40 and Ap42 production at the level of y-secretase; levels of the soluble ectodomains, a- and f5-APPs/ are unaltered, demonstrating that (3- and y-secretase activities are not affected (Wolfe et al., 1998). We found that a number of changes in the flanking residues of the inhibitor still allowed y-secretase inhibition (Wolfe et al., 1999). Such loose sequence specificity in the inhibitors mirrored similar observations with substrates (Lichtenthaler et al., 1999; Maruyama et al., 1996; Tischer & Cordell, 1996; Lichtenthaler, Ida, Multhaup, Masters, & Beyreuther, 1997). Moreover, we have observed that difluoro alcohol analogues, while less effective, also block A|3 production (Wolfe et al., 1999). Difluoro ketones are readily hydrated to closely mimic the transition state of aspartyl protease catalysis, but certain analogues of this type can also inhibit serine and cysteine proteases (Parisi & Abeles, 1992; Angliker, Anagli, & Shaw, 1992). In contrast, difluoro alcohols are only known to inhibit aspartyl proteases (Thaisrivongs et al., 1986; Doherty et al., 1992). These results therefore suggest that y-secretases are aspartyl proteases.

Strategies for Inhibiting y-Secretase

109

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

To further explore the issue of loose sequence specificity, we synthesized a series of difluoro ketone transition-state mimics with alterations in the PI position (Figure 14.2). All of the compounds 2a-e effectively blocked total A(3 and A(342 production. The rank order of potency within a given experiment consistently showed 2e>2d>2c>2b>2a; thus, the preference for PI substituent was cyclohexylmethyl>sec-butyl>benzyl>zso-propyl>methyl, suggesting a relatively large SI pocket for y-secretase (Moore, Leatherwood, Diehl, Selkoe, & Wolfe, 2000). These findings are consistent with other observations, noted above, showing that y-secretase has loose sequence specificity. Installation of the cyclohexylmethyl group in PI also led to the identification of a difluoro alcohol (the immediate synthetic precursor to 2e) equipotent to its difluoro ketone counterpart toward inhibiting total A(3 production (IC50 ~ 5 uM). This difluoro alcohol was an order of magnitude more potent than other difluoro alcohols we have previously reported (Wolfe et al., 1999), providing strong additional evidence that y-secretase is an aspartyl protease. To identify the target of these y-secretase inhibitors, we designed derivatives of these peptide analogues that could bind covalently and irreversibly. The N-terminal Boc group of difluoro alcohol 3 was replaced with a bromoacetyl group, a functionality susceptible to nucleophilic attack that has been extensively employed as a means of creating affinity labels (Pongs & Lanka, 1975; Chang, Lobl, Rowley, & Tindall, 1984; Bateman, Kim,

FIGURE 14.2 Definition of S sites in proteases and P sites in their substrates. Inhibitors with large, hydrophobic PI substituents, such as 2e, retain y-secretase inhibitory potency, suggesting a large SI pocket in this protease.

110

Drug Discovery

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Slaughter, & Hersh, 1990; Smar et al., 1992; Safran, Farwell, Rokos, & Leonard, 1993; Kuliopulos et al., 1994), and biotin was added to the C-terminus. Thus, the difluoro ketone transition-state mimic should interact with the two active site aspartates of y-secretase (James, Sielecki, Hayakawa, & Gelb, 1992), while the bromoacetamide should react with any proximal nucleophilic residues (e.g., Ser, Cys, Thr) to form a stable covalent bond and irreversibly inactivate the enzyme, and the biotin acts as a handle for detection. This compound, BrA-3-Bt, retained y-secretase inhibitory activity in living cells and specifically bound to presenilin-1 in cell lysates, isolated microsomes, and whole cells, in strong support of presenilins being the catalytic component of y-secretase (Esler et al., 2000).

ht

More recently, we have identified a new class of transition-state analogue inhibitors, (hydroxyethyl)urea peptidomimetics, which blocks y-secretase much more effectively than any of the difluoro ketones or difluoro alcohols described above. In this class of peptidomimetic protease inhibitor (general structure 4), the hydroxyethyl moiety mimics the transition-state of aspartyl protease catalysis. The incorporation of this transition-state mimicking substructure has led to highly potent and selective inhibitors of other aspartyl proteases, such as renin, and HIV protease (Greenlee, 1990; Huff, 1991). In fact, all five HIV protease inhibitors currently marketed for the treatment of AIDS contain the hydroxyethyl isostere (Flexner, 1998). The replacement of the PI' a-carbon with nitrogen creates a urea

Strategies for Inhibiting j-Secretase III

111

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

functionality that greatly simplifies synthesis (Getman et al., 1993). Although we have only just begun examining this type of compound, we have already identified analogues that inhibit y-secretase activity with IC50 values as low as 100 nM in whole cells, nearly two orders of magnitude more potent than our best fluorinated peptide analogues. We also found that a- and (3-APP^ levels were unaltered by these compounds, indicating that a- and (3-secretase activities were not inhibited. Thus, despite the recent cloning and identification of (3-secretase as a membrane-tethered aspartyl protease (Vassar et al., 1999; Sinha et al., 1999; Van et al., 1999; Hussain et al, 1999; Lin et al., 2000), the (hydroxyethyl)ureas are apparently selective for y-secretase. These compounds are drug prototypes that should also serve as important tools for basic research on y-secretase.

he m

TOWARD HELICAL PEPTIDOMIMETIC Y-SECRETASE INHIBITORS

ht

tp

://

w

w

w

.c

y-Secretase is unusual in that it cleaves within the transmembrane region of its substrate APP, and this cleavage appears to actually take place within the lipid bilayer. Transmembrane regions typically assume an a-helical conformation. Indeed, molecular modeling (Wolfe et al., 1999) and mutagenesis (Lichtenthaler et al., 1999) experiments support a helical model for the APP transmembrane region upon initial binding to y-secretase. To take advantage of this unique characteristic of y-secretase, we have designed small, helical peptides, both to better understand the nature of the APP/y-secretase interaction and to obtain new prototype inhibitors for drug development. To design helical peptides for interaction with y-secretase, we considered the y-secretase cleavage site within the transmembrane regiaon of APP (Figure 14.3). 2-Aminoisobutyric acid (Aib) is a known helix-inducing amino acid, favoring helical conformations even in pentapeptides. Judicious replacement of selected residues of short APP-derived peptides with Aib could provide peptides that mimic the y-secretase cleavage site on one face of the helix, with Aib residues on the other face. Thus, the Aib residues could elicit conformational restraint in the peptide, presenting an APP-like face for interaction with y-secretase.

122

Drug Discovery

AFP sequence: —Val-Gly-Gly-Val-Val*Ile-Ala*ThrVal-Ile-Val-Ile— Designed peptidomimetics:

12 6 6 3 3

x. ph

s/ in de

Boc-Val*Ile-Aib-Thr(OBz)-Val-Aib-OMe Boc-Val-Val*Ile-Aib-Thr(OBz)-Val-Aib-OMe Boc-Aib-Val-Val*Ile-Aib-Thr(OBz)-Val-Aib-OMe Boc-Gly-Aib-Val-Val*Ile-Aib-Thr(OBz)-Val-Aib-OMe Boc-Val-Gly-Aib-Val-Val*Ile-Aib-Thr(OBz)-Val-Aib-OMe

25 15 10 2 5

p

Boc-Ala*Thr(OBz)-Aib-Ile-Val-Aib-OMe Boc-Ile-Ala*Thr(OBz)-Aib-Ile-Val-Aib-OMe Boc-Aib-Ile-Ala*Thr(OBz)-Aib-Ile-Val-Aib-OMe Boc-Val-Aib-Ile-Ala*Thr(OBz)-Aib-Ile-Val-Aib-OMe Boc-Gly-Val-Aib-Ile-Ala*Thr(OBz)-Aib-Ile-Val-Aib-OMe

5 6 7 8 9 10 11 12 13 14

IC50 (jaM)

4a ll.

vn

/fo

ru

m

FIGURE 14.3 Sequence of the y-secretase cleavage site within the APP transmembrane region and designed peptidomimetic inhibitors. Asterisks in APP denote sites of cleavage resulting in A(340 and A[342 production. The top set of peptidomimetics (5-9) was designed considering the cleavage site leading to A(342 and the bottom set (10-14) was designed considering the cleavage site leading to A(340. IC50 is the concentration of compound that reduced production of total Ap by 50% in a cellbased assay using CHO cells stably transfected with human APP. Removal of the threonine-protecting group substantially reduces activity.

ht

tp

://

w

w

w

.c

he m

We have identified Aib-containing peptides that inhibit A(3 production in APP-transfected cells with IC50 values as low as 2 uM (Figure 14.3). This inhibition occurs at the y-secretase level: y-secretase substrates C99 and C83 are substantially increased in the presence of the Aib-containing peptides. The compounds do not affect a- or p-secretase activity, as a- and P-APPs levels are unaltered. Conformational studies by circular dichroism (CD) show that these peptides display helical character in methanol: characteristic negative peaks were observed at 204 and 225 nm. However, the 225 nm trough was of much lower intensity than the 205 nm trough, suggesting that these peptides spend only a small portion of their time in a helical conformation. Moreover, we found that all D-amino acid counterparts of compounds 5-9 inhibited y-secretase activity just as well as the L-peptides. We conclude that these Aib-containing peptides are probably not inhibiting y-secretase due to their partial helical character, but likely because of their length and hydrophobicity. Therefore, simple incorporation of Aib into these peptides apparently cannot address the question of whether helical peptides can block y-secretase activity. We are currently working toward rigidly constrained peptides that possess clear helical character and plan on combining optimal features of helical peptide y-secretase inhibitors with those of transition-state analogue inhibitors as a strategy for realizing highly potent and selective compounds.

Strategies for Inhibiting y-Secretase

113

REFERENCES

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Angliker, H., Anagli,}., & Shaw, E. (1992). Inactivation of calpain by peptidyl fluoromethyl ketones. Journal of Medicinal Chemistry, 35, 216-220. Bateman, R. C, Jr., Kim, Y. A., Slaughter, C, & Hersh, L. B. (1990). N-bromoacetylD-leucylglycine. An affinity label for neutral endopeptidase 24.11. Journal of Biological Chemistry, 265, 8365-8368. Chang, C. H., Lobl, T.}., Rowley, D. R., & Tindall, D. J. (1984). Affinity labelinga of the androgen receptor in rat prostate cytosol with 17 beta-[(bromoacetyl)oxy]5 alpha-androstan-3-one. Biochemistry, 23, 2527-2533. Doherty, A. M., Sircar, I., Kornberg B. E., Quin, J. D., Winters, R. T., Kaltenbronn, J. S., Taylor, M. D., Batley, B. L., Rapundalo, S. R., Ryan, M. J., et al. (1992). Design and synthesis of potent, selective, and orally active fluorine-containing renin inhibitors. Journal of Medicinal Chemistry, 35, 2-14. Esler, W. P., Kimberly, W. T., Ostaszewski, B. L., Diehl, T. S., Moore, C. L., Tsai, J.-Y., Rahmati, T., Xia, W., Selkoe, D. J., & Wolfe, M. S. (2000). Transition-state analogue inhibitors of ysecretase bind directly to presenilin-1. Nature Cell Biology, 2, 428-434. Flexner, C. (1998). HIV-protease inhibitors. New England Journal of Medicine, 338, 1281-1292. Getman, D. P., DeCrescenzo, G. A., Heintz, R. M., Reed, K. L., Talley, J. J., Bryant, M. L., Clare, M., Houseman, K. A., Marr, J. J., Mueller, R. A., et al. (1993). Discovery of a novel class of potent HIV-1 protease inhibitors containing the (R)-(hydroxyethyDurea isostere. Journal of Medicinal Chemistry, 36, 288-291. Greenlee, W. J. (1990). Renin inhibitors. Medical Research Review, 10,173-236. Hardy, J. (1997). The Alzheimer family of diseases: Many etiologies, one pathogenesis? Proceedings of the National Academy of Sciences (USA), 94, 2095-2097. Huff, J. R. (1991). HIV protease: A novel chemotherapeutic target for AIDS. Journal of Medicinal Chemistry, 34, 2305-2314. Hussain, L, Powell, D., Hewlett, D. R., Tew, D. G., Meek, T. D., Chapman, C., Gloger, I. S., Murphy, K. E., Southan, C. D., Ryan, D. M., Smith, T. S., Simmons, D. L., Walsh, R S., Dingwall, C., & Christie, G. (1999). Identification of a novel aspartic protease (Asp 2) as beta-secretase. Molecular Cellular Neurosciences, 14, 419-427. Iwatsubo, T., Mann, D. M., Odaka, A., Suzuki, N., & Ihara, Y. (1995). Amyloid beta protein (A beta) deposition: A beta 42(43) precedes A beta 40 in Down syndrome. Annals of Neurology, 37, 294-299. James, M. N., Sielecki, A. R., Hayakawa, K., & Gelb, M. H. (1992). Crystallographic analysis of transition state mimics bound to penicillopepsin: Difluorostatineand difluorostatone-containing peptides. Biochemistry, 31, 4693-4697. Jarrett, J. T., Berger, E. P., & Lansbury, P. T., Jr. (1993). The carboxy terminus of the beta amyloid protein is critical for the seeding of amyloid formation: Implications for the pathogenesis of Alzheimer's disease. Biochemistry, 32, 4693-4697. Kuliopulos, A., Nelson, N. P., Yamada, M., Walsh, C. T., Furie, B., Furie, B. C., & Roth, D. A. (1994). Localization of the affinity peptide-substrate inactivator

114

Drug Discovery

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

site on recombinant vitamin K-dependent carboxylase. Journal of Biological Chemistry, 269, 21364-21370. Lichtenthaler, S. R, Ida, N., Multhaup, G., Masters, C. L., & Beyreuther, K. (1997). Mutations in the transmembrane domain of APP altering gamma-secretase specificity. Biochemistry, 36,15396-15403. Lichtenthaler, S. R, Wang, R., Grimm, H., Uljon, S. N., Masters, C. L., & Beyreuther, K. (1999). Mechanism of the cleavage specificity of Alzheimer's disease gamma-secretase identified by phenylalanine-scanning mutagenesis of the transmembrane domain of the amyloid precursor protein. Proceedings of the National Academy of Sciences (USA), 96, 3053-3058. Lin, X., Koelsch, G., Wu, S., Downs, D., Dashti, A., & Tang, J. (2000). Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein. Proceedings of the National Academy of Sciences (USA), 97,1456-1460. Lorenzo, A., & Yankner, B. A. (1994). Beta-amyloid neurotoxicity requires fibril formation and is inhibited by Congo Red. Proceedings of the National Academy of Sciences (USA), 91,12243-12247. Maruyama, K., Tomita, T., Shinozaki, K., Kume, H., Asada, H., Saido, T. C., Ishiura, S., Iwatsubo, T., & Obata, K. (1996). Familial Alzheimer's diseaselinked mutations at Val717 of amyloid precursor protein are specific for the increased secretion of A beta 42(43). Biochemical Biophysical Research Communications, 227, 730-735. Moore, C. L., Leatherwood, D. D., Diehl, T. S., Selkoe, D. J., & Wolfe, M. S. (2000). Difluoro ketone peptidomimetics suggest a large SI pocket for Alzheimer's ySecretase: Implications for inhibitor design. Journal of Medicinal Chemistry, 43, 3434-3442. Parisi, M. R, & Abeles, R. H. (1992). Inhibition of chymotrypsin by fluorinated alpha-keto acid derivatives. Biochemistry, 31, 9429-9435. Pongs, O., & Lanka, R. (1975). Affinity labeling of the ribosomal decoding site with an AUG-substrate analog. Proceedings of the National Academy of Sciences (USA), 72,1505-1509. Safran, M., Farwell, A. P., Rokos, H., & Leonard, J. L. (1993). Structural requirements of iodothyronines for the rapid inactivation and internalization of type II iodothyronine 5'-deiodinase in glial cells. Journal of Biological Chemistry, 268, 14224-14229. Selkoe, D. J. (1999). Translating cell biology into therapeutic advances in Alzheimer's disease. Nature, 399, A23-31. Selkoe, D. J., & Wolfe, M. S. (2000). In search of gamma-secretase: Presenilin at the cutting edge. Proceedings of the National Academy of Sciences (USA), 97,5690-5692. Sinha, S., Anderson, J. P., Barbour, R., Basi, G. S., Caccavello, R., Davis, D., Doan, M., Dovey, H. R, Prigon, N., Hong, J., Jacobson-Croak, K., Jewett, N., Keim, P., Knops, J., Lieberburg, L, Power, M., Tan, H., Tatsuno, G., Tung, J., Schenk, D., Seubert, P., Suomensaari, S. M., Wang, S., Walker, D., & John, V. (1999). Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature, 402, 537-540. Smar, M. W., Ares, J. J., Nakayama, T., Itabe, H., Kador, P. R, & Miller, D. D. (1992). Selective irreversible inhibitors of aldose reductase. Journal of Medicinal Chemistry, 35,1117-1120.

Strategies for Inhibiting y-Secretase

115

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Steiner, H., Duff, K., Capell, A., Romig, H., Grim, M. G., Lincoln, S., Hardy, J., Yu, X., Picciano, M., Fechteler, K., Citron, M., Kopan, R., Pesold, B., Keck, S., Baader, M., Tomita, T., Iwatsubo, T., Baumeister, R., & Haass, C. (1999). A loss of function mutation of presenilin-2 interferes with amyloid beta-peptide production and notch signaling. Journal of Biological Chemistry, 274, 28669-28673. Thaisrivongs, S., Pals, D. T., Kati, W. M., Turner, S. R., Thomasco, L. M., & Watt, W. (1986). Design and synthesis of potent and specific renin inhibitors containing difluorostatine, difluorostatone, and related analogues. Journal of Medicinal Chemistry, 29, 2080-2087. Thinakaran, G., Harris, C. L., Ratovitski, T., Davenport, R, Slunt, H. H., Price, D. L., Borchelt, D. R., & Sisodia, S. S. (1997). Evidence that levels of presenilins (PS1 and PS2) are coordinately regulated by competition for limiting cellular factors. Journal of Biological Chemistry, 272, 28415-28422. Tischer, E., & Cordell, B. (1996). Beta-amyloid precursor protein. Location of transmembrane domain and specificity of gamma-secretase cleavage. Journal of Biological Chemistry, 271, 21914-21919. Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., Fisher, S., Fuller, J., Edenson, S., Lile, J., Jarosinski, M. A., Biere, A. L., Curran, E., Burgess, T., Louis, J. C., Collins, F., Treanor, J., Rogers, G., & Citron, M. (1999). Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science, 286, 735-741. Wolfe, M. S., Citron, M., Diehl, T. S., Xia, W., Donkor, I. O., & Selkoe, D. J. (1998). A substrate-based difluoro ketone selectively inhibits Alzheimer's y-secretase activity. Journal of Medicinal Chemistry, 41, 6-9. Wolfe, M. S., De Los Angeles, J., Miller, D. D., Xia, W., & Selkoe, D. J. (1999a). Are presenilins intramembrane-cleaving proteases? Implications for the molecular mechanism of Alzheimer's disease. Biochemistry, 38,11223-11230. Wolfe, M. S., Xia, W., Moore, C. L., Leatherwood, D. D., Ostaszewski, B., Donkor, I. O., & Selkoe, D. J. (1999b). Peptidomimetic probes and molecular modeling suggest Alzheimer's y-secretases are intramembrane-cleaving aspartyl proteases. Biochemistry, 38, 4720-4727. Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T., & Selkoe, D. J. (1999c). Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and y-secretase activity. Nature, 398, 513-517. Yan, R., Bienkowski, M. J., Shuck, M. E., Miao, H., Tory, M. C., Pauley, A. M., Brashier, J. R., Stratman, N. C., Mathews, W. R., Buhl, A. E., Carter, D. B., Tomasselli, A. G., Parodi, L. A., Heinrikson, R. L., & Gurney, M. E. (1999). Membrane-anchored aspartyl protease with Alzheimer's disease beta-secretase activity. Nature, 402, 533-537.

s/ in de

x. ph

Phosphorylations of Tau and APP as Targets for Drug Discovery

p

15

4a ll.

vn

/fo

ru

m

John DeBernardis, Dan Kerkman, Cintia Vianna, and Peter Davies

he m

TAU ABNORMALITIES IN ALZHEIMER'S DISEASE

ht

tp

://

w

w

w

.c

Hyperphosphorylation of the microtubule-associated protein tau is a well-established feature in the brains of patients with Alzheimer's disease (AD) (Johnson & Hartigan, 1998). At least 21 sites in tau have been reported to be phosphorylated in Alzheimer brain, and while many of these sites are phosphorylated in the normal brain, the level of phosphorylation at any given site is probably higher in the AD brain (Hasegawa et al., 1992). There also appear to be a few of these amino acids that are not phosphorylated at detectable levels in the normal brain (Jicha, O'Donnell, Weaver, Angeletti, & Davies, 1999; Jicha et al., 1999). Hyperphosphorylated tau is also different in conformation from tau in the normal brain, and aggregates to form paired helical filament (PHF) structures that are the major protein components of the neurofibrillary tangles of AD (Terry & Wisniewski, 1970). Despite very extensive work over the last decade, there is still no agreement on two major points: 1. The nature of the process responsible for the accumulation of hyperphosphorylated tau is unknown. Changes in the activity of a number of different protein kinases have been suggested as a possible cause of the increased level of phosphorylation (Trojanowski & Lee, 1994; Drewes et al., 216

Phosphorylations of Tau and APP 117

117

ru

m

s/ in de

x. ph

p

1995; Mulot, Hughes, Woodgett, Anderton, & Hanger, 1994). Considering the different amino-acid sequences that are hyperphosphorylated in the AD brain, it is very unlikely that one protein kinase could be responsible for all the phosphorylations. This has led to the suggestion that the basic abnormality might be a deficit in protein-phosphatase activity, although no such defect has yet been identified in the AD brain (Carver et al, 1994,1995). 2. The significance of the phosphorylations for the conformational changes in tau and its aggregation into PHF remains unclear. It has been possible in vitro to induce recombinant tau to aggregate into PHF-like structures (Goedert, 1993). Although this process remains very inefficient, PHF formation in this manner does not require any phosphorylation of tau (Schweers, Schonbrunn-Hanebeck, Marx, & Mandelkow, 1994; Schweers, Mandelkow, Bierrat, & Mandelkow, 1995). Phosphorylation of recombinant tau actually inhibits the assembly into PHF-like structures, although it may be significant that the conformation of recombinant tau is closer to that of tau in the normal brain, than that in AD (Schweers et al., 1994).

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

One approach to attempt to unravel the significance of changes in the conformation and phosphorylation of tau in AD has been to focus studies on cases of AD as early in the process of the disease as can be reliably identified. Clinical and neuropathologic staining systems have been developed in recent years that allow identification of such cases (Braak, Duyckaerts, Braak, & Prette, 1993; Bancher, Braak, Fischer, & Jellinger, 1993). Using a panel of monoclonal antibodies that are able to detect either conformational changes of tau or specific phosphorylations, it is possible to determine which of these events occurs prior to the development of PHF (and, hence, before neurofibrillary tangles and degenerative changes) in neurons. A number of studies of this type have been published in the last few years (Jicha et al., 1999; Vincent, Zheng, Dickson, Kress, & Davies, 1998), and these have revealed two important features of the early biochemical alterations of tau. First, it is clear that conformational changes in tau occur prior to PHF formation, and not as a result of filament formation. There is now good agreement that specific conformational changes of tau are among the earliest detectable changes within neurons of the AD brain (Hyman et al., 1988; Carmel, Mager, Binder, & Kuret, 1996; Jicha, Bowser, Kazam, & Davies, 1997; Jicha, Berenfeld, & Davies, 1999). One specific phosphorylation of tau, on threonine 231, also appears to occur very early in the process of AD, probably at or close to the time at which conformational changes are detectable. Specific monoclonal antibodies detecting phosphothreonine 231 of tau were discovered that are also sensitive to conformational changes in the phosphorylated tau, suggesting a link between this phosphorylation and conformation changes in the protein (Jicha et al., 1997).

118

Drug Discovery

THE "MITOTIC" HYPOTHESIS

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Among the major protein kinases, cdc2 (also called cdkl) is the most efficient at phosphorylating threonine 231 of tau (Jicha et al., 1997). There have been suggestions that cdc2 activity is up regulated in the brains of AD patients, and that cdc2 may be associated with neurofibrillary tangles, especially in the early stages of formation (Vincent, Jicha, Rosado, & Dickson, 1997). This kinase is better known as a critical regulator of the cell cycle, although its appearance in postmitotic cells, such as neurons, was unexpected. This work has led to the so-called "Mitotic Hypothesis" of AD, which proposes that aberrant activation of the cell cycle in postmitotic neurons is responsible for neurofibrillary tangle formation and cellular degeneration (McShea, Harris, Webster, Wahl, & Smith, 1997; Nagy, Esiri, Cato, & Smith, 1997; Nagy, Esiri, & Smith, 1997; Illenberger et al., 1998). Recent work has suggested that a second key regulator of the cell cycle may also participate in tangle formation and neurodegeneration. Pinl is a prolyl isomerase that binds to and isomerizes serine/threonine-proline bonds in proteins, only if the serine or threonine is phosphorylated. It is essential for normal mitosis in all eukaryotic cells (Yaffe et al., 1997; Lu, Hanes, & Hunter, 1996). Sequences recognized by Pinl in peptide substrates were strikingly similar to sequences in tau, and it was discovered that Pinl binds strongly to the tau sequence surrounding threonine 231, only if the threonine was phosphorylated (Lu, Wolf, Zhou, Davies, & Lu, 1999). Binding of Pinl to cdc2-phosphorylated tau was demonstrated to alter the conformation of the protein. Further work showed that Pinl was tightly associated with phosphorylated tau isolated from the AD brain, and was colocalized with neurofibrillary tangles in sections of AD brain tissue (Lu et al., 1999). It is possible to propose that phosphorylation of tau by cdc2 at threonine 231 leads to the binding of Pinl, and that this protein alters the conformation of tau, such that it forms PHFs, which then aggregate into neurofibrillary tangles. Cell death could result either from the abnormalities of tau or from depletion and sequestration of Pinl into the tangles. This is an attractive scheme for tangle formation, as it provides an explanation linking both phosphorylation and conformational changes of tau, both of which are established as early events in the neuronal abnormalities of AD.

Amyloid Deposition in Alzheimer's Disease One of the most frustrating aspects of work on AD in recent years has been the difficulty in finding clear links between the neurofibrillary tangle pathology and the deposition of beta-amyloid, the major peptide component of the neuritic or senile plaque. Although the "Amyloid Cascade

Phosphorylations of Tau and APP 119

119

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Hypothesis," dominant in this field for several years, has proposed that deposition of beta-amyloid in the brain leads to neuronal degeneration and tangle formation, direct experimental evidence for this has been difficult to obtain (Selkoe, 1996; Da vies, 1994). Most notably, transgenic mice, which develop huge numbers of beta-amyloid deposits in the brain at a young age, fail to develop evidence of significant tau pathology, and no evidence of neurofibrillary tangle formation, even when aged (Holcomb et al., 1998). Because of these findings, there has recently been a variety of modifications to the amyloid cascade hypothesis. It has been suggested that intracellular accumulation of beta-amyloid (rather than extracellular deposition in plaque) (Skovronsky, Doms, & Lee, 1998; Yang, Chandswangbhuvana, Shu, Henschen, & Glabe, 1999) or formation of soluble beta-amyloid "protomers" (Selkoe, 1999) may be responsible for neurofibrillary degeneration, but again, direct, experimental evidence for this is lacking at this time. Beta-amyloid is formed by the cleavage of a larger precursor, the amyloid precursor protein or APP. This protein is abundant in neurons, and much of what is synthesized within neurons is cleaved near the center of the beta-amyloid peptide region, with secretion of the larger N-terminal fragment of the molecule, and presumably intracellular retention of the smaller C-terminal fragment (reviewed in Selkoe, 1996,1999). This cleavage is catalyzed by a currently unidentified protease called alpha-secretase. As alpha-secretase cleavage occurs within the beta-amyloid domain, production and deposition of this peptide is impossible following this secretory processing. Two different cleavages of the APP are required to liberate the beta-amyloid peptide, a beta-secretase cleavage to generate the N-terminus of the beta-amyloid peptide, and one or more gamma secretase cleavages to generate the C-terminus. At least some of the beta-amyloid peptide generated in normal cells is secreted; the fate of the rest of the APP molecule in this situation is unclear. Much attention has been focused on the beta and gamma secretases in recent years, and the beta-secretase was recently cloned (Vassar et al., 1999). Mechanisms that might control the production of the beta-amyloid from APP have attracted a great deal of interest, and a novel, potential control mechanism was recently identified.

Linking Tau and Amyloid Abnormalities The C-terminus of APP is known to be phosphorylated in the brains of animals, as well as in cell culture (Oishi et al., 1997; Suzuki et al., 1994). It has been suggested that phosphorylation of the C-terminus of APP controls the rate of cleavage, at least at the alpha-secretase site (Caporaso, Gandy, Buxbaum, Ramabhadran, & Greengard, 1992). There has been little work in this area, partly, perhaps, because unlike the situation with

220

Drug Discovery

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

tau, specific monoclonal antibodies to phosphorylation sites in APP were not available until recently. One site in the C-terminus of APP, at threonine 668, is reported to be a preferred site for cdc2 phosphorylation (Susuki et al., 1994). Because of interest in the cdc2 site on tau, as well as evidence of activation of cdc2 in early AD, two series of experiments were conducted. In the first series, monoclonal antibodies were raised to both the phosphothreonine 231 site of tau and the phosphothreonine 668 site of APP. Remarkably, antibodies were discovered that recognized both sites, but not several other phosphopeptides tested (Figure 15.1). This result would seem to argue that despite the lack of amino-acid sequence homology between these sites, these phosphoepitopes must share similarities in conformation. There is some additional experimental evidence on this point. NMR studies of peptide conformations from these regions of tau and APP are suggestive of an unusual reverse-beta turn in the structures of both peptides (Kroenke, Ziemnicka-Kotula, Xu, Kotula, & Palmer, 1997; Weaver, Cahill, Purpura, & Da vies, 1999). In addition to cross-reactive antibodies, specific antibodies to each site were prepared. Monoclonal antibodies to the APP 668 phosphoepitope show specific staining of brain tissues from cases of AD (Figure 15.2). Labeling is intraneuronal, and was found in the same regions that are known to show staining with the tau 231 phosphoepitope-specific antibodies. Double-labeling immunocytochemistry using tissues from early AD cases show that both phosphoepitopes accumulate in the same neurons of the hippocampus (Figure 15.3). This data strongly suggests that both tau and APP are phosphorylated by cdc2 (or a similar kinase) early in the course of AD. Because of the apparent similarity between the tau and APP phosphoepitopes, studies have been conducted to determine whether Pinl would bind to both proteins after phosphorylation by cdc2. This is indeed the case (Figure 15.4). Phosphopeptides derived from both the tau sequence around threonine 231 and around APP threonine 668 both bind Pinl with high affinity. Efforts are underway to determine the effects of Pinl binding on the conformation of APP and its processing by secretases. It is already established that APP interacts with other cellular proteins, such as Fe65, Go, and XI1, through binding of these proteins to the C-terminal region, in the same general region as Pinl (Russo et al., 1998; Borg, Ooi, Levy, & Margolis, 1996; Guenette, Chen, Jandro, & Tanzi, 1996; Nishimoto et al., 1993). It is very likely that phosphorylation with or without Pinl binding will disrupt these interactions. It thus appears highly probable that the normal cellular processing of APP will be disturbed by such binding. It appears that both tau and APP abnormalities will result from a similar mechanism, because both proteins have a site at which phosphorylation by cdc2 (or a similar kinase) will render them substrates for Pinl. The binding and action of Pinl will probably alter the conformation of both of

p x. ph s/ in de m ru /fo vn 4a ll. he m .c w w w :// tp ht FIGURE 15.1 Monoclonal antibodies were produced using mice immunized with phosphopeptides containing phosphothreonine 668 of APR GF3 and GF5 recognize both tau peptides containing phosphothreonine 231 (tau231P) and APP peptides containing phosphothreonine 668 (APP668P). The corresponding nonphosphopeptides (tau231 and APP668) are not recognized, nor are any other tau or APP phosphopeptides tested. Antibody GF7 recognizes only the phosphothreonine 668 of APP, and no other phosphopeptide tested to date. The remaining figures show some data for a few of the other phosphopeptides tested. 121

Drug Discovery

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

222

ht

tp

://

w

w

w

.c

he m

FIGURE 15.2 GF7 was used for immunocytochemical studies of Alzheimer and normal brain tissue. Representative results are shown, from examination of several dozen cases investigated. A: Normal brain tissue, CA2 region of hippocampus. No staining is visible. B: GF7 stains (brown) granular structures in the cytoplasm of CA1 and CA2 neurons in the Alzheimer cases. This is shown in low-power view in B, and in two higher-power views in C and D. Similar staining is seen in neurons of other affected cortical regions. Unaffected regions of the AD brain (primary visual cortex and the cerebellum) and the same regions from normal individuals show no staining.

these proteins, leading to PHF and tangle formation in the case of tail, and to changes in beta-amyloid peptide production and alteration of APP metabolism. Prevention of the conformational changes in these two proteins thus becomes an important potential therapeutic target for AD. Since Pinl is an essential regulator of mitosis in all eukaryotic cells, inhibition of the normal function of this protein is not desirable, and, thus, inhibitors directed toward the binding site or catalytic activity of this protein would not seem an appropriate therapy for AD. However, perhaps compounds can be discovered that selectively interact with phosphothreonine 231 of tau and/or phosphothreonine 668 of APP, as these will block binding of Pinl

Phosphorylations of Tau and APP

/fo

ru

m

s/ in de

x. ph

p

123

w

w

.c

he m

4a ll.

vn

FIGURE 15.3 Sections of hippocampus from an early stage case of Alzheimer disease were stained with both GF7 (a mouse IgGl) and TG3 (a mouse IgM), the latter antibody recognizing phosphothreonine 231 of tau. FITC-labeled goat antimouse IgGl was used to visualize the GF7 immunoreactivity and TRITC-labeled goat antimouse IgM was used to visualize TG3. Images of two representative neurons in the CA2 region were captured by confocal microscopy and merged. Similar results have been obtained in several additional cases with double labeling using nonfluorescent visualization techniques.

ht

tp

://

w

only to these proteins that have been phosphorylated in the AD brain. Normal functions of tau and APP will be undisturbed by these compounds, since neither site is phosphorylated to a significant degree in the normal brain. The discovery of such compounds is expected to be facilitated by the availability of monoclonal antibodies that recognize one or both of these sites. Discovery of compounds that bind to either phosphothreonine 231 of tau and/or phosphothreonine 668 of APP, will provide, at the very least, tools for examination of the functional roles of these phosphorylation sites in the parent proteins. It would be expected that such compounds would prevent the binding of Pinl to these sites, and presumably free this prolyl isomerase to perform its normal cellular function. The discovery of such compounds would greatly facilitate our ability to examine the possible interactions of tau and APP at the level of Pinl in suitable

Drug Discovery

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

224

w

w

.c

he m

FIGURE 15.4 Pinl binds both to tau peptides containing phosphothreonine 231 (tau231P) and to APP peptides containing phosphothreonine 668 (APP668P). The corresponding nonphosphopeptides (tau231 and APP668) do not bind Pinl, nor do any other tau or APP phosphopeptides tested.

ht

tp

://

w

cellular systems, as well as in appropriate transgenic mice. Ultimately, such compounds may provide novel therapeutics for AD, addressing mechanisms that may participate in both the tau and amyloid pathologies.

REFERENCES Bancher, C., Braak, H., Fischer, P., & Jellinger, K. A. (1993). Neuropathological staging of Alzheimer lesions and intellectual status in Alzheimer's and Parkinson's disease patients. Neuroscience Letters, 162,179-182. Borg, J.-P, Ooi, ]., Levy, E., & Margolis, B. (1996). The phosphotyrosine interaction domain of XI1 and Fe65 bind to distinct sites on the YENPTY motif of amyloid precursor protein. Molecular Biology of the Cell, 16, 6229-6241. Braak, K, Duyckaerts, E., Braak, E., & Piette, E (1993). Neuropathological staging of Alzheimer-related changes correlates with psychometrically assessed

Phosphorylations of Tau and APP 125

125

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

intellectual status. In B. Corain, K. Iqbal, M. Nicolini, B. Winblad, H. M. Wisniewski, & P. Zatta (Eds.), Alzheimer's disease: Advances in clinical and basic research (pp. 131-137). New York: John Wiley. Caporaso, G. L., Gandy, S. E., Buxbaum, J. D., Ramabhadran, T. V., & Greengard, P. (1992). Protein phosphorylation regulates secretion of Alzheimer/A4 amyloid precursor protein. Proceedings of the National Academy of Sciences (USA), 89, 3055-3059. Carmel, G., Mager, E. M., Binder, L. I., & Kuret, J. (1996). The structural basis of monoclonal antibody AlzSO's selectivity for Alzheimer's disease pathology. Journal of Biological Chemistry, 271, 32789-32795. Davies, P. (1994). Neuronal abnormalities, not amyloid, are the cause of dementia in Alzheimer's disease. In R. Katzman, R. D. Terry, & C. Bick (Eds.), Alzheimer's disease (pp. 327-333). New York: Raven Press. Drewes, G., Trinczek, B., Illenberger, S., Biernat, J., Schmitt-Ulms, G., Meyer, H. E., Mandelkow, E. M., & Mandelkow, E. (1995). Microtubule associated protein/microtubule affinity regulating kinase (p!10marc). Journal of Biological Chemistry, 270, 7679-7688. Carver, T. D. Harris, K. A., Lehman, R. A., Lee, V. M., Trojanowski, J. Q., & Billingsley, M. L. (1994). Tau phosphorylation in human, primate, and rat brain: Evidence that a pool of tau is highly phosphorylated in vivo and is rapidly dephosphorylated in vitro. Journal of Neurochemistry, 63, 2279-2287. Carver, T. D., Oyler, G. A., Harris, K. A., Polavarapu, R., Damuni, Z., Lehman, R. A., & Billingsley, M. L. (1995). Tau phosphorylation in brain slices: Pharmacological evidence for convergent effects of protein phosphatases on tau and mitogen-activated protein kinase. Molecular Pharmacology, 47,745-756. Goedert, M. (1993). Tau protein and the neurofibrillary pathology of Alzheimer's disease. Trends in Neurosciences, 16, 460-465. Guenette, S. Y, Chen, J., Jondro, P. D., & Tanzi, R. E. (1996). Association of a novel human FE65-like protein with the cytoplasmic domain of the amyloid precursor protein. Proceedings of the National Academy of Sciences (USA), 93,10832-10837. Hasegawa, M., Morishima-Kawashima, M., Takio, K., Suzuki, M., Titani, K., & Ihara, Y (1992). Protein sequence and mass spectrometric analyses of tau in the Alzheimer's disease brain. Journal of Biological Chemistry, 267,17047-17054. Holcomb, L., Gordon, M. N., McGowan, E., Yu, X., Benkovic, S., Jantzen, P., Wright, K., Saad, I., Mueller, R., Morgan, D., Sanders, S., Zehr, C., O'Campo, K., Hardy, J., Prada, C. M., Eckman, C., Younkin, S., Hsiao, K., & Duff, K. (1998). Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes. Nature Medicine, 4, 97-100. Hyman, B. T, Van Hoesen, G. T, Wolozin, B. L., Davies, P., Kromer, L. J., & Damasio, A. R. (1988). Alz-50 antibody recognizes Alzheimer-related neuronal changes. Annals of Neurology, 23, 371-379. Illenberger, S., Zheng-Fischhofer, Q., Preuss, U., Stamer, K., Baumann, K., Trinczek, J., Godemann, R., Mandelkow, E.-M., & Mandelkow, E. (1998). The endogenous and cell-cycle dependent phosphorylation of tau protein in living cell: Implications for Alzhiemer's disease. Molecular Biology of the Cell, 9, 1495-1512.

226

Drug Discovery

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Jicha, G. A., Berenfeld, B., & Davies, P. (1999). Sequence requirements for formation of conformational variants of tau similar to those found in Alzheimer's disease. Journal of Neuroscience Research, 55, 713-723. Jicha, G. A., Bowser, R., Kazam, I. G., & Davies, P. (1997). Alz-50 and MC-1, a new monoclonal antibody raised to paired helical filaments, recognize conformational epitopes on recombinant tau. Journal of Neuroscience Research, 48, 128-132. Jicha, G. A., Lane, E., Vincent, I. J., Otvos, L., Hoffmann, R., & Davies, P. A. (1997). Conformation and phosphorylation dependent antibody recognizing the paired helical filaments of Alzheimer's disease. Journal of Neurochemistry, 69, 2087-2095. Jicha, G. A., O'Donnell, A., Weaver, C, Angeletti, R., & Davies, P. (1999). Hierarchical phosphorylation of recombinant tau by the paired helical filament associated protein kinase is dependent on cAMP-dependent protein kinase. Journal ofNeurochemistry, 72, 214-224. Jicha, G. A., Weaver, C., Lane, E., Vianna, C., Kress, Y, Rockwood, J., & Davies, P. (1999). cAMP-dependent protein kinase phosphorylations on tau in Alzheimer's disease. Journal of Neuroscience, 19, 7486-7494. Johnson, G. V., & Hartigan, J. A. (1998). Tau protein in normal and Alzheimer's disease brain: An update. Alzheimer's Disease Review, 3,125-141. Kroenke, C. D., Ziemnicka-Kotula, D., Xu, J., Kotula, L., & Palmer, A. G. (1997). Solution conformations of a peptide containing the cytoplasmic domain sequence of the beta amyloid precursor protein. Biochemistry, 36, 8145-8152. Lu, K. P., Hanes, S. D., & Hunter, T. (1996). A human peptidyl-prolyl isomerase essential for regulation of mitosis. Nature, 380, 544-547. Lu, P.-J., Wolf, G., Zhou, X. Z., Davies, P., & Lu, K. P. (1999). The prolyl isomerase Pinl restores the biological function of Alzheimer-associated phosphorylated tau. Nature, 399, 784-788. McShea, A., Harris, P. L. R., Webster, K. R., Wahl, A. R, & Smith, M. A. (1997). Abnormal expression of the cell cycle regulators pi6 and cdk4 in Alzheimersdisease. American Journal of Pathology, 150,1933-1939. Mulot, S. R, Hughes, K., Woodgett, J. R., Anderton, B. H., & Hanger, D. P. (1994). PHP-tau from Alzheimer's brain comprises four species on SDS-PAGE which can be mimicked by in vitro phosphorylation of human brain tau by glycogen synthase kinase-3 beta. FEES Letters, 349, 359-364. Nagy, Z., Esiri, M. M., Cato, A. M., & Smith, A. D. (1997). Cell cycle markers in the hippocampus in Alzheimers-disease. Acta Neuropathologica, 94, 6-15. Nagy, Z., Esiri, M. M., & Smith, A. D. (1997). Expression of cell division markers in the hippocampus in Alzheimers disease and other neurodegenerative conditions. Acta Neuropathologica, 93, 294-300. Nishimoto, I., Okamoto, T., Matsuura, Y., Takahashi, S., Okamoto, T., Murayama, Y, & Ogata, E. (1993). Alzheimer amyloid protein precursor complexes with brain GTP-binding protein G(o). Nature, 362, 75-79. Oishi, M., Nairn, A. C., Czernik, A. J., Lim, G. S., Isohara, T., Gandy, S. E., Greengard, P., & Suzuki, T. (1997). The cytoplasmic domain of Alzheimer's amyloid precursor protein is phosphorylated at Thr654, Ser655, and Thr668 in adult rat brain and cultured cells. Molecular Medicine, 3,111-123.

Phosphori/lations of Tau and APP 127

127

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Russo, T., Faraonio, R v Minopoli, G., De Candia, P., De Renzis, S., & Zambrano, N. (1998). Fe65 and the protein network centered around the cytosolic domain of the Alzheimer's beta-amyloid precursor protein. FEES Letters, 434,1-7. Schweers, O., Mandelkow, E. M v Biernat, J., & Mandelkow, E. (1995). Oxidation of cysteine-322 in the repeat domain of microtubule-associated protein tau controls the in vitro assembly of paired helical filaments. Proceedings of the National Academy of Sciences (USA), 92, 8463-8467. Schweers, O., Schonbrunn-Hanebeck, E., Marx, A., & Mandelkow, E. (1994). Structural studies of tau protein and Alzheimer paired helical filaments show no evidence for beta structure. Journal of Biological Chemistry, 269,24290-24297. Selkoe, D. J. (1996). Cell biology of the beta-amyloid precursor protein and the genetics of Alzheimer's disease. Cold Spring Harbor Symposia on Quantitative Biology, 61, 587-596. Selkoe, D. J. (1999). Translating cell biology into therapeutic advances in Alzheimer's disease. Nature, 399, A23-31. Skovronsky, D. M., Doms, R. W., & Lee, V. M. (1998). Detection of a novel intraneuronal pool of insoluble amyloid beta protein that accumulates with time in culture, journal of Cell Biology, 141,1031-1039. Suzuki, T., Oishi, M., Marshak, D. M., Czernik, A. J., Nairn, A. C., & Greengard, P. (1994). Cell cycle-dependent regulation of the phosphorylation and metabolism of the Alzheimer amyloid precursor protein. European Molecular Biology Organization Journal, 13,1114-1122. Terry, R. D., & Wisniewski, H. M. (1970). The ultrastructure of the neurofibrillary tangle and the neuritic plaque. In G. E. W. Wolstenholme, M. J. O'Conner, & A. Churchill (Eds.), CIBA Foundation symposium on Alzheimer's disease and related conditions (pp. 145-155). London. Trojanowski, J. Q., & Lee, V. M. (1994). Paired helical filament tau in Alzheimer's disease. The kinase connection. American Journal of Pathology, U4, 449-453. Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., Fisher, S., Fuller, J., Edenson, S., Lile, J., Jarosinski, M. A., Biere, A. L., Curran, E., Burgess, T., Louis, J. C., Collins, F, Treanor, J., Rogers, G., & Citron, M. (1999). Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science, 286, 735-741. Vincent, I., Jicha, G., Rosado, M., & Dickson, D. W. (1997). Aberrant expression of mitotic cdc2/cyclin bl kinase in degenerating neurons of Alzheimers disease brain. Journal ofNeuroscience, 17, 3588-3598. Vincent, I. J., Zheng, J.-H., Dickson, D. W., Kress, Y. & Davies, P. (1998). Mitotic phosphoepitopes precede paired helical filaments in Alzheimer's disease. Neurobiology of Aging, 19, 287-296. Weaver, C. L., Cahill, S., Purpura, D. P., & Davies, P. (1999). Conformational requirements for the monoclonal antibody TG3 reactivity and specificity for Alzheimer's disease elucidated through NMR spectroscopy. Society for Neurosciences Abstract, 448,10. Yaffe, M. B., Schutkowski, M., Shen, M., Zhou, X. Z., Stukenberg, P. T., Rahfeld, J. U., Xu,)., Kuang, J., Kirschner, M. W., Fischer, G., Cantley, L. C., & Lu, K. P.

128

Drug Discovery

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

(1997). Sequence-specific and phosphorylation-dependent proline isomerization: A potential mitotic regulatory mechanism. Science, 278,1957-1960. Yang, A. J., Chandswangbhuvana, D., Shu, T., Henschen, A., & Glabe, C. G. (1999). Intracellular accumulation of insoluble, newly synthesized abetan-42 in amyloid precursor protein-transfected cells that have been treated with Abetal42. Journal of Biological Chemistry, 274, 20650-20656.

16

ru

m

s/ in de

Jonathan D. Smith and Justine A. Levin

x. ph

p

Estrogen and ApoE: Drug Discovery Implications

/fo

ABSTRACT

ht

tp

://

w

w

w

.c he

m

4a ll.

vn

Using the amyloid precursor protein (APP)-transgenic mouse model of Alzheimer's disease (AD), we have explored the effects of ovariectomy and estrogen treatment on cerebral levels of apolipoprotein E (apoE) and (5-amyloid peptide (A(3). Estrogen induces apoE in the mouse brain via different mechanisms in different brain regions. In the diencephalon, apoE induction is mediated by the estrogen-receptor a, but in the cortex, apoE induction is not mediated via this estrogen receptor. When young, APP-transgenic mice were ovariectomized, 58% mortality was observed by 8 months of age, which was not observed in wildtype mice. This lethality was associated with decreased A(5 levels. Ovariectomized, APP-transgenic mice treated with high-dose pellets of 17(3- or 17a-estradiol had 10% and 25% reductions in A(3 levels, respectively. In future studies, Afi levels and behavioral endpoints will be assessed in APP-transgenic mice after treatments with various estrogen-like compounds.

INTRODUCTION The single, strongest genetic susceptibility marker for AD is the E4 allele of the apoE gene. There are three common apoE alleles called E2, E3, and E4, with allele frequencies in a western European population of 8%, 77%, and 15%, respectively. About two-thirds of all AD subjects carry one or two copies of the E4 allele, while only 25% of the population as a whole carry the E4 allele, demonstrating that the E4 allele has a deleterious effect 229

130

Drug Discovery

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

in regard to susceptibility for AD (Farrer et al., 1997). One interesting observation from this study is that women with the E4/3 genotype are much more likely to develop AD than men with E4/3 genotype (Farrer et al., 1997). This gender effect of E4 on AD suggests a potential interaction between a gender specific factor (e.g., estrogen) and apoE. In general, drug therapy for AD has been limited and disappointing. One bright finding in regard to AD incidence has been the demonstration that postmenopausal estrogen-replacement therapy (ERT) is associated with dramatically decreased AD incidence in women as they age. A 1998 meta-analysis of 10 studies found a 29% decreased risk of dementia for postmenopausal estrogen users (Yaffe, Sawaya, Lieberburg, & Grady, 1998). Additionally, there appears to be an interaction between estrogen and apoE4, as a large, multicenter, longitudinal study of elderly women found that cognitive scores decreased less in estrogen users, but only among those who were not E4 carriers (Yaffe, Haan, Byers, Tangen, & Kuller, 2000). However, estrogen-therapy trials of women who already have AD have been disappointing (Mulnard et al., 2000; Wang et al., 2000; Henderson et al., 2000). Perhaps once AD is diagnosed, it is too late for estrogen to help, but even to prove that estrogens can decrease AD incidence will require a long-term, prospective, double-blind, placebo-controlled study. Cell-culture studies have shown that estrogen can decrease the processing of APP into A(3 (Xu et al., 1998), but it is not known whether this is the mechanism by which estrogen may decrease AD incidence in vivo. In mice, estrogen has been shown to lead to increased synaptic sprouting after brain injury, and remarkably, this effect of estrogen is dependent upon apoE, as it is not observed in apoE-deficient mice (Stone, Rozovsky, Morgan, Anderson, & Finch, 1998). In this chapter, we describe our findings using mouse models on the effects of estrogen on cerebral apoE levels, and the effects of ovariectomy and estrogen supplementation on mortality and A(3 levels in APP-transgenic mice.

ht

MATERIALS AND METHODS

Mice C57BL/6 mice were obtained from the Jackson Labs. APP-transgenic mice were obtained from Dave Borchelt of Johns Hopkins University; these mice utilize the murine prion promoter to drive an APP695swe construction (Borchelt et al., 1996). These mice have been bred 10 generations onto the C57BL/6 strain, and develop AD-like senile plaques by 14 to 18 months of age (Borchelt et al., 1997). Estrogen-receptor ex-deficient (ERKO) mice on the C57BL/6 background were obtained from Dennis Lubahn (University of Missouri-Columbia) (Lubahn et al., 1993).

Estrogen and ApoE

131

Ovariectomy and Estrogen Supplementation Mice were anesthetized with ketamine/xylazine and subjected to bilateral ovariectomy by cauterization. Estrogen supplementation was performed by implanting silastic pellets subcutaneously at the base of the dorsal side of the neck. The pellets were designed as 90-day release pellets (Innovative Research of America) containing 17p-estradiol (calculated to lead to plasma levels of 500 to 600 pg/mL), 17a-estradiol (500 pg/mL), or placebo.

/fo

ru

m

s/ in de

x. ph

p

Brain Homogenates Mice were anesthetized with ketamine/xylazine, and sacrificed by exsanguination and intracardiac saline perfusion. The brains were removed and either frozen whole or after dissection into the cortex, hippocampus, and diencephalon. The frozen brains or brain regions were homogenized with a polytron in a buffer containing 6M urea, 100 mM Tris, pH 8,0,1% SDS, 1 mM EDTA, 0.5 mM PMSF, and 10% of a protease inhibitor cocktail (Sigma). The samples were centrifuged at low speed to reduce foam and then at 100,000 x gm for 1 hour to pellet-insoluble debris.

tp

://

w

w

w

.c

he m

4a ll.

vn

Western Blot Analysis of Mouse apoE and GFAP Brain-region homogenates were assayed for protein concentration by an alkaline-modified Lowry assay (Markwell, Haas, Bieber, & Tolbert, 1978). Equal amounts of protein were run out on 10% Tris-Glycine gels and were subsequently transferred to nylon membrane. The blots were probed with two primary antibodies: sheep antimouse apoE antibody (gift from Charles Bisgaier) and goat antihuman GFAP antibody (Sigma), which we have demonstrated to cross-react with mouse GFAP. Antisheep and antigoat IgG-HRP conjugated secondary antibodies were incubated with the blots, and, after extensive washing in PBS/0.05% Tween-20, the blots were developed using ECL reagents (Amersham), followed by exposure to X-ray film. The band intensities were quantified using laser-scanning densitometry.

ht

Total A/3 ELISA Assay Ap40 standard was diluted in DMSO to 1 mg/mL, and then subsequently diluted in TBSE (PBS with 1% BSA, 0.05% Tween-20, and 0.02 % Na azide). All samples were diluted 5-fold in TBSE to dilute out the urea and SDS, and the standards were also prepared with 20% homogenization buffer so that they contained the same amount of urea and SDS. The monoclonal anti-Ap antibody 6E10 (Senetek) was diluted in 0.1 M sodium carbonate, pH 9.6, and coated onto 96-well MaxiSorp plates (Nunc) overnight. After blocking, samples and standards were added and allowed to bind overnight at 4°C. After washing, biotinylated anti-Ap

132

Drug Discovery

monoclonal 4G8 (Senetek) was added to detect the bound A(3. Strepavidinalkaline phosphatase and AttoPhos substrate were used to quantify A(3 fluorometrically.

RESULTS

p

Estrogen Regulation of Cerebral apoE in Mice

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

Four-week-old C547BL/6 female mice were ovariectomized, and at 6 weeks of age they were treated with subcutaneous pellets that released either a pharmacological dose of 17p-estradiol or placebo. Five days later, the mice were sacrificed, and brain region-specific dissection was performed to yield the cortex, hippocampus, and diencephalon. Homogenates of these regions were subjected to western blot analysis to determine the relative levels of apoE and another astrocyte-specific marker, glial acidic fibrillar protein (GFAP). ApoE levels were increased by the estrogen treatment, compared with the placebo, in the cortex, hippocampus, and diencephalon by 2.0, 1.5, and 2.3-fold, respectively (N > 6 for estrogen and placebo, p < 0.01 for all 3 brain regions by f-test). ApoE levels were also normalized to GFAP levels to examine whether the effects of estrogen were specific for apoE, or due to an increased number or activation state of astrocytes. The apoE/GFAP ratio was still significantly elevated in the cortex and diencephalon by 1.6 and 2.4-fold, respectively; while the 1.2-fold elevation of the apoE/GFAP ratio in the hippocampus failed to reach statistical significance. To determine whether the estrogen effects on apoE were mediated by classical estrogen receptors (ER), two additional experiments were performed. First, ovariectomized, C57BL/6 female mice were treated for 5 days with either 17oc-estradiol, which is not an ER agonist, or placebo. Second, ovariectomized, estrogen receptor-a knock out mice (ERKO) were treated for 5 days with either 17|3-estradiol or placebo. Interestingly, specific apoE induction in the cortex and diencephalon were mediated by completely different mechanisms. In the cortex, similar levels of specific apoE induction (apoE/GFAP ratio) were obtained with both 17oc- and 17(3-estradiol, and 17|3-estradiol was still fully functional to induce apoE specifically in the ERKO mice. However, in the diencephalon, the specific induction of apoE by 17p~estradiol was not recapitulated after treatment with 17oc-estradiol, nor was 17{3-estradiol effective in the ERKO mice. Thus, the specific induction of apoE by 17|3-estradiol in the diencephalon is completely mediated by the estrogen-receptor a, while in the cortex, the estrogen induction of apoE is not mediated by the estrogen-receptor a, and, in fact, may not be mediated by either of the classical estrogen receptors, as

Estrogen and ApoE

133

17a-estradiol, which is not an agonist for either ER-oc or ER-fS, also induced apoE in this brain region. Effects of Ovariectomy on APP-Transgenic Mice

x. ph

p

Out of 24 APP-transgenic mice ovariectomized at 4 weeks of age, none died before 3 months of age, but 14 (58%) died between 3 and 8 months of age. In contrast, 0 of 16 APP-transgenic mice subjected to sham ovariectomy at 4 weeks of age died by 8 months of age, and only 1 of 14 wildtype mice subjected to ovariectomy at 4 weeks of age died by 8 months of age.

s/ in de

Effects of Estrogen Supplementation on APP-Transgenic Mice

he m

DISCUSSION

4a ll.

vn

/fo

ru

m

The effects of estrogen treatment on AfJ levels were ascertained in APPtransgenic mice that were ovariectomized at 4 weeks of age. At 6 weeks of age, groups of six to seven mice each were treated with subcutaneous pellets releasing placebo, 17(5-estradiol, or 17a-estradiol. The mice were sacrificed 6 weeks later at 3 months of age, a time prior to the observed mortality of ovariectomized, APP-transgenic mice. Compared with placebo, both 17p- and 17a-estradiol led to significant 27% and 38% reductions in total A(3, respectively (p < 0.05).

ht

tp

://

w

w

w

.c

In this study, we demonstrated that estrogen induces apoE by different mechanisms in different brain regions. The estrogen effects were mediated by estrogen-receptor a in the diencephalon, consistent with the finding of the highest level of ER-oc mRNA in the hypothalmic region of rodents (Shughrue, Lane, & Merchenthaler, 1997). In contrast, our data with the ERKO mice demonstrate that ER-a does not mediate the estrogen induction of apoE in the cortex. The finding that 17a-estradiol induces apoE in the cortex is consistent with a nonestrogen receptor-mediated response. Ovariectomy of sexually immature, APP-transgenic mice led to 58% mortality by 8 months of age, while ovariectomy of sexually immature wildtype mice, or sexually mature APP-transgenic mice did not lead to increased mortality. We hypothesize that the increased mortality observed after ovariectomy of young, APP-transgenic mice is due to neurodegeneration-associated APP overexpression. This is an attractive hypothesis since it is known that APP overexpression in transgenic mice can lead to premature lethality associated with neurodegeneration, particularly in inbred mouse strains (Hsiao et al., 1995; Moechars et al., 1999; Mucke et al., 2000). Van Leuven's laboratory discovered that the early behavioral

134

Drug Discovery

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

and premature death phenotypes were best correlated to the levels of APP mRNA, irrespective of whether the APP was wildtype or mutant, whereas plaque load was associated with A(342 levels that were high only in the mutant APP transgenics (Moechars et al, 1999). Our finding that the ovariectomy must occur before sexual maturity to observe this mortality phenotype suggests that physiological estrogen is playing a developmental role, and that its absence during the period of normal sexual maturation allows for increased expression of this early death phenotype. Six-week treatments with pharmacological doses of 170- and 17a-estradiol led to significant decreases in A(3 levels, respectively, compared with placebo in ovariectomized, APP-transgenic mice. These findings suggest that the estrogen effect in decreasing A0 levels is not mediated by classical estrogen receptors, and may be mediated by alternate mechanisms, for example, via estrogen's antioxidant activity or activation of the MAPkinase pathway. These findings are in agreement with a guinea pig study, which also found that estrogen treatment lowered A(3 levels (Petanceska, Nagy, Frail, & Gandy, 2000), and with the in vitro studies, which found that estrogen decreased A|3 processing and secretion in cultured neurons (Xu et al., 1998). We are now performing additional studies to look at the long-term effects of pharmacological estrogens on nonovariectomized, APP-transgenic mice, as well as on APP-transgenic mice ovariectomized after sexual maturity when increased mortality is not observed. Once the best paradigm is discovered for observing the effects of estrogen on lowering A(3 levels in APP-transgenic mice, we will be in an excellent position to undertake preclinical drug development and screening studies, where we can examine the effects of various estrogen agonists, antagonists, and analogues to determine which have the most activity in lowering A|3 levels. Thus, these studies have the potential to suggest candidate compounds for investigation in human clinical trials.

ht

REFERENCES

Borchelt, D. R., Ratovitski, T., Van, L. J., Lee, M. K., Gonzales, V., Jenkins, N. A., Copeland, N. G., Price, D. L., & Sisodia, S. S. (1997). Accelerated amyloid deposition in the brains of transgenic mice coexpressing mutant presenilin 1 and amyloid precursor proteins. Neuron, 19, 939-945. Borchelt, D. R., Thinakaran, G., Eckman, C. B., Lee, M. K., Davenport, R, Ratovitsky, T., Prada, C. M., Kim, G., Seekins, S., Yager, D., Slunt, H. H., Wang, R., Seeger, M., Levey, A. I., Gandy, S. E., Copeland, N. G., Jenkins, N. A., Price, D. L., Younkin, S. G., & Sisodia, S. S. (1996). Familial Alzheimer's diseaselinked presenilin 1 variants elevate Abetal-42/1-40 ratio in vitro and in vivo. Neuron, 17,1005-1013.

Estrogen and ApoE

135

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Farrer, L. A., Cupples, L. A., Haines, J. L., Hyman, B., Kukull, W. A., Mayeux, R., Myers, R. H., Pericak-Vance, M. A., Risch, N., & Van, D. M. (1997). Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease. A meta-analysis. Journal of the American Medical Association, 278,1349-1356. Henderson, V. W., Paganini-Hill, A., Miller, B. L., Elble, R. J., Reyes, P. R, Shoupe, D., McCleary, C. A., Klein, R. A., Hake, A. M., & Farlow, M. R. (2000). Estrogen for Alzheimer's disease in women: Randomized, double-blind, placebo-controlled trial. Neurology, 54, 295-301. Hsiao, K. K., Borchelt, D. R., Olson, K., Johannsdottir, R., Kitt, C., Yunis, W., Xu, S., Eckman, C., Younkin, S., & Price, D. (1995). Age-related CNS disorder and early death in transgenic FVB/N mice overexpressing Alzheimer amyloid precursor proteins. Neuron, 15,1203-1218. Lubahn, D. B., Moyer, J. S., Golding, T. S., Couse, J. F., Korach, K. S., & Smithies, O. (1993). Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proceedings of the National Academy of Sciences (USA), 90,11162-11166. Markwell, M. A., Haas, S. M., Bieber, L. L., & Tolbert, N. E. (1978). A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Analytical Biochemistry, 87, 206-210. Moechars, D., Dewachter, I., Lorent, K., Reverse, D., Baekelandt, V, Naidu, A., Tesseur, L, Spittaels, K., Haute, C. V, Checler, R, Godaux, E., Cordell, B., & Van, L. R (1999). Early phenotypic changes in transgenic mice that overexpress different mutants of amyloid precursor protein in brain. Journal of Biological Chemistry, 274, 6483-6492. Mucke, L., Masliah, E., Yu, G. Q., Mallory, M., Rockenstein, E. M., Tatsuno, G., Hu, K., Kholodenko, D., Johnson-Wood, K., & McConlogue, L. (2000). High-level neuronal expression of abeta 1-42 in wild-type human amyloid protein precursor transgenic mice: Synaptotoxicity without plaque formation. Journal of Neuroscience, 20, 4050-4058. Mulnard, R. A., Cotman, C. W., Kawas, C., van Dyck, C. H., Sano, M., Doody, R., Koss, E., Pfeiffer, E., Jin, S., Gamst, A., Grundman, M., Thomas, R., & Thai, L. J. (2000). Estrogen replacement therapy for treatment of mild to moderate Alzheimer disease: A randomized controlled trial. Alzheimer's Disease Cooperative Study. Journal of the American Medical Association, 283, 1007-1015. Petanceska, S. S., Nagy, V, Frail, D., & Gandy, S. (2000). Ovariectomy and ITbetaestradiol modulate the levels of Alzheimer's amyloid beta peptides in brain. Neurology, 54, 2212-2217. Shughrue, P. J., Lane, M. V, & Merchenthaler, I. (1997). Comparative distribution of estrogen receptor-alpha and -beta mRNA in the rat central nervous system. Journal of Comparative Neurology, 388, 507-525. Stone, D. J., Rozovsky, I., Morgan, T. E., Anderson, C. P., & Finch, C. E. (1998). Increased synaptic sprouting in response to estrogen via an apolipoprotein Edependent mechanism: Implications for Alzheimer's disease. Journal of Neuroscience, 18, 3180-3185. Wang, P. N., Liao, S. Q., Liu, R. S., Liu, C. Y, Chao, H. T., Lu, S. R., Yu, H. Y, Wang,

236

Drug Discovery

s/ in de

x. ph

p

S. J., & Liu, H. C. (2000). Effects of estrogen on cognition, mood, and cerebral blood flow in AD: A controlled study. Neurology, 54, 2061-2066. Xu, H., Gouras, G. K., Greenfield, J. P., Vincent, B., Naslund, J., Mazzarelli, L, Fried, G., Jovanovic, J. N., Seeger, M., Relkin, N. R., Liao, R, Checler, R, Buxbaum, J. D., Chait, B. T., Thinakaran, G., Sisodia, S. S., Wang, R., Greengard, P., & Gandy, S. (1998). Estrogen reduces neuronal generation of Alzheimer beta-amyloid peptides. Nature Medicine, 4, 447-451. Yaffe, K., Haan, M., Byers, A., Tangen, C., & Kuller, L. (2000). Estrogen use, APOE, and cognitive decline: Evidence of gene-environment interaction. Neurology, 54,1949-1954. Yaffe, K., Sawaya, G., Lieberburg, L, & Grady, D. (1998). Estrogen therapy in postmenopausal women: Effects on cognitive function and dementia. Journal of the American Medical Association, 279, 688-695.

m

ACKNOWLEDGMENTS

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

This work was supported by a grant from the Institute for the Study of Aging. We would like to thank Bruce McEwen for his guidance and assistance in these studies.

17

s/ in de

x. ph

Design of Agonists and Antagonists for AMPASubtype Glutamate

p

Structure-Based Functional

ru

m

Receptors

he m

4a ll.

vn

/fo

Ming-Ming Zhou, Harel Weinstein, Diomedes E. Logothetis, and Eric Gouaux

w

.c

BACKGROUND

ht

tp

://

w

w

Glutamate is the major excitatory neurotransmitter in the central nervous system. Glutamate-receptor activity is central to cellular models of learning and memory (Seeburg, 1993; Hollmann & Heibemann, 1994; Nakanishi & Masu, 1994; Mori & Mishina, 1995; Wo & Oswald, 1995; Squire & Kendel, 1999), and receptor malfunction is implicated in both age-related memory impairment and Alzheimer's disease (AD), particularly in the context of aberrant calcium permeability (Morrison & Hot, 1997). Traditionally, ionotropic glutamate receptors (iGluRs) are classified into three subtypes, based upon their pharmacological and electrophysiological response to the agonists AMPA (alpha-amino-3-hydroxy-5-methy-4-isoxazole propionic acid), kainate (KA, a structural analogue of glutamate), or NMD A (N-methyl-D-aspartate) (Hollmann & Heibemann, 1994; Watkins, Krogsgaard-Larsen, & Honore, 1990) (Figure 17.1 A). Glutamate signaling through iGluRs regulates membrane excitability and intracellular Ca2+ levels (Hollmann & Heibemann, 1994). Like other 137

238

Drug Discovery

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

GluR families, the AMPA GluRs are complex oligomeric proteins, where the emergent functional attributes strongly reflect the relative subunit composition. Thus, the key to manipulating AMPA receptors pharmacologically lies in an understanding of the properties attributable to specific subunits, and the development of subunit-specific ligands. For example, AMPA receptors that contain GluR 1,3, and 4 provide a Ca2+ pathway into the cell, while others (GluR2 containing) do not directly flux calcium, but control membrane excitability by regulating indirectly Ca2+ influx through activation of another GluR class, the NMDA receptors (Burnashev, Monyer, Seeberg, & Sakmann, 1992; Pellegrini-Giampietro, Gorter, Bennett, & Zukin, 1997). Regulation of Ca2+ levels within narrow physiological limits is of paramount importance to neuronal survival, as low levels lead to apoptosis, while high levels lead to excitotoxicity and necrosis (Lee, Zipfel, & Choi, 1999). Increased Ca2+ influx within normal limits stimulates Ca2+-dependent enzymes to potentiate long-lasting synaptic activity, termed "long-term potentiation." Complex regulatory processes fine-tune intracellular Ca2* level with exquisite precision to produce neuronal plasticity, a hallmark of complex tasks, such as learning and memory. Failure of precise regulation of Ca2+ levels in AD leads to memory loss and a reduction in learning potential, and, potentially, neuron death. The complexity of regulating Ca2+ requires specific tools, such as those to be developed in this study that will block or stimulate selectively GluR subunits in order to fine-tune intracellular levels of Ca2+. Despite the significant role of the glutamate receptors in both the biology and pathology of the central nervous system, detailed understanding of molecular mechanisms by which iGluRs function in cells has been hampered by a lack of high-resolution structural information. Recently, Gouaux and colleagues reported the 3D-crystal structure of an iGluR ligand-binding region (GluR2 S1S2) in a complex with the neurotoxin (agonist) KA (Armstrong, Sun, Chen, & Gouaux, 1998). The iGluR ligandbinding core consists of polypeptide segments SI and S2 connected by a short linker (13 residues), where SI comprises residues N-terminal to the first transmembrane segment and S2 includes amino acids between membrane-associated segments 3 and 4 (Figure 17.IB). The GluR2 S1S2 protein has pharmacological properties similar to those of the wild-type membrane-bound receptor, which is believed to be a tetrameric complex where each subunit consists of a ligand-binding domain (SI and S2, Figure 17.IB) (O'Hara et al., 1993; Stern-Bach et al., 1994; Kuusinen, Arvola, & Keinanen, 1995) , three transmembrane segments (Ml, M3, and M4) (Hollmann, Maron, & Heinemann, 1994; Wo & Oswald, 1994; Bennett & Dingledine, 1995), and membrane-embedded re-entrant loop (M2) (Kuusinen et al., 1995; Chen, Sun, & Jin, 1998). The crystal structure (Figure 17.1C) revealed, for the first time, the determinants of receptor-agonist interactions, and

p x. ph s/ in de m ru /fo vn 4a ll. m .c he w w

ht

tp

://

w

Figure 17.1 A. Chemical structures of some iGluR agonists. B. Domain structure of iGluRs, consisting of the SI and S2 segments. "Cut" and "linker" denote the boundaries of the S1S2 construct. C. The X-ray structure of the GluR2 S1S2 in complex with the agonists kainate.

140

Drug Discovery

m

s/ in de

x. ph

p

provided the detailed structural explanation for the biochemical results obtained previously by mutagenesis (O'Hara et al., 1993; Kuryatov, Laube, Betz, & Kuhse, 1994; Mano, Lamed, & Teichberg, 1996; Paas, Devillers-Thoery, Changeux, Medevielle, & Teichberg, 1996; Wo & Oswald, 1996), by chimera (Stern-Bach et al., 1994), and by S1S2 fusion experiments (Kuusinen et al., 1995; Arvola & Keinanen, 1996). The structure further suggested how ligand-binding specificity is altered, by remote residues and redox state of a conserved disulphide bond. The new structural information of GluR2 lays the foundation for detailed study of the functional properties of the AMPA-subtype GluRs in a structural context. This new knowledge provides exciting opportunities to discover therapeutically useful receptor agonists and antagonists. The use of such structure-based rational approaches for this important family of glutamate receptors is the focus of this study.

ru

RESEARCH METHODS AND PROGRESS

/fo

Structure-Based Drug Design With NMR

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

High affinity and selectivity for biological targets are essential elements for the efficacy of potent therapeutic agents, which should also possess good bioavailability, metabolic stability, and low toxicity. Structure-based rational drug design offers great potentials in drug discovery (Hajduk, Meadows, & Fesik, 1997; Shuker, Hajduk, Meadows, & Fesik, 1996). Our approach of structure-based drug design utilizes nuclear magnetic resonance (NMR) spectroscopy and a linked-fragment method to identify and refine chemical leads for development of specific ligands for therapeutic targets. Specifically, the chemical building blocks identified from NMRbased screening and optimized for binding to a protein selected as a target for treatment of a specific human disease. The resulting linkecl-chemical compounds with high affinity and selectivity are then subjected to detailed, structure-based analysis of their interactions with the target protein, using a combination of NMR and computational modeling. Refinement, chemical diversification through various chemical linkages, and selectivity enhancement are achieved at this stage. Because this linked-fragment approach requires a library of small chemical molecules that are commercially available, this method is technically feasible for structure-based functional therapeutics design in an academic setting. The NMR-based screening of chemical compounds has significant advantages that make it preferable over the newest methods of highthroughput screening of natural products or combinatorial chemical libraries. These advantages include the ability to: (a) identify small and

Structure-Based Design of Agonists and Antagonists

141

vn

/fo

ru

m

s/ in de

x. ph

p

weakly binding chemical compounds to the target protein; (b) determine the location of the ligand-binding sites; (c) conduct reliable screening using heteronuclear-editing and saturation transfer-difference NMR spectroscopy techniques. The former method is clean and reliable because only signals of a stable isotope (13C or 15N)-labeled protein are detected without interference from other components of the assay. On the other hand, the latter approach is fast and effective for very large proteins and requires as little as 1 nmol of a protein; and (d) require a relatively small library of chemical compounds. Molecules in a relatively small library only represent fragments of the linked compounds in a very large virtual library. For example, for a chemical inhibitor with two independent, binding fragments assembled pairwise with 10 linkers, a compound collection of 10,000 fragments represents a virtual library of one billion compounds. Synthesis and analysis of a real library of this size would be an arduous and costly task, which is not required in this method. Thus, this NMRbased method overcomes two major hurdles in drug discovery by screening natural products or large chemical libraries (i.e., (a) limited diversity of chemical compounds in the libraries, and (b) enormous resources and time-associated high-throughput screening).

4a ll.

Protein Sample Preparation and NMR Assignment

ht

tp

://

w

w

w

.c

he m

To facilitate NMR-based compound screening and the structural-based drug design, we need the GluR2 S1S2 protein in quantities of tens of milligrams, which can be achieved using the bacterial expression system and the protein purification procedure developed by the Gouaux Lab (Chen et al., 1998; Chen & Gouaux, 1997). The proton NMR spectrum of the protein prepared using this method is excellent, suggesting that the protein behaves well in the phosphate buffer of pH 6.5 and is suitable for the NMR structural study (Figure 17.2A). This is supported by the observation of several well-resolved peaks with relative narrow linewidth between -1 and 0 ppm (methyl group protons) and between 9 and 11 ppm (amide or aromatic protons). These results suggested that the GluR2 S1S2 protein (-30 kDa) in this buffer condition contains a stable structural fold, and exists in a monomeric form suitable for the NMR study. In addition, we have also observed major differences in chemical shift changes of the protein residues upon binding to KA or NBQX, which are clearly visible in the less overlapping ranges of -1 to 0 ppm and 9 to 11 ppm (Figure 17.2B). These results suggest that the GluR2 S1S2 may bind in distinct modes to the agonist or the antagonist, respectively. Because the initial spectra looked so promising, we are preparing various stable isotope (2H, 13C, and 15N)-labeled protein samples, which are used for sequence-specific resonance assignment of the protein using

Drug Discovery

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

242

ht

FIGURE 17.2 Proton NMR spectra of the GluR2 S1S2. A. The free-form spectrum. B. Spectral comparison of the GluR2 protein in the free form, ancl complexed to kainate (agonist) or NBQX (antagonist). All the protein samples (~0.5mM) were prepared in a 50 mM phosphate buffer of pH 6.5. The spectra were collected on a Bruker RX600 NMR spectrometer at 25°C.

heteronuclear, multidimensional, NMR techniques (Zhou et al., 1995; Zhou et al., 1995; Zhou et al., 1996; Dhalluin et al., 1999). Information from the chemical shift assignment will greatly facilitate chemical compound screening in parallel. Specifically, to conduct sequence-specific resonance assignments for the structural studies of the protein, we have

Structure-Based Design of Agonists and Antagonists

143

prepared a uniformly 13C-, 15N-labeled and fractionally (75%) deuterated protein. This triple-labeled protein sample is essential for the NMR structural studies of a protein of this size, because of the favorable 1H, 13C, and 15 N relaxation rates caused by the partial deuteration of the protein (Zhou et alv 1995; Sattler & Fesik, 1996). This sample allows us to acquire constant-time triple-resonance NMR spectra with higher digital resolution and sensitivity.

x. ph

p

Chemical Compound Screening by NMR

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

We are building a library of small molecules using commercially available chemical compounds, which is used for NMR-based screening for the GluR2 S1S2 protein. Particularly, we conduct the NMR screening for the GluR2 in the presence of KA, which could enhance protein stability during the screening and could also be used as the first fragment to provide target selectivity. The resulting high-affinity compounds that would contain a KA moiety would bind to the KA-binding site and serve as effective antagonists for the GluR2 S1S2. Once the lead compounds that bind proximal to the KA subsite are identified, chemical-structural analogs will be tested to optimize binding to the protein, which will be evaluated by binding affinities. Dissociation constants will be obtained by monitoring the chemical shift changes of a specific amino acid residue at the ligandbinding site as a function of ligand concentration (Hajduk et al., 1997). Moreover, we will determine the 3D structure of the ternary complex of the new ligand and KA bound to the protein by NMR. Information from this NMR structural analysis will reveal location and orientation of the lead compound and KA in the protein/ligand complex, which could guide chemical synthesis to link together the lead compound with KA in order to obtain high-affinity ligands for the GluR2.

ht

tp

Chemical Synthesis and Structural Analysis of the Linked Ligands Based on the NMR structural information of the ternary complex of the GluR2 S1S2 bound to the new ligand and KA, we use molecular modeling methods to design suitable chemical linkers to assemble the two fragments by chemical synthesis (Weinstein & Osman, 1989; Lorber & Shoichet, 1998; Sandak, Wolfson, & Nussinov, 1998; Lybrand, 1995). Analysis of the protein/ligand complex by molecular modeling will enable the design of the chemical linkers by identifying sterically nonconflicting elements and optimal conformations that will not interfere with the binding of the linked fragments.

244

Drug Discovery

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Once the linked compounds are synthesized, we will test their binding to the protein by NMR and/or isothermal titration calorimetry (ITC) technique (Livingstone, 1996). ITC method should not only determine the binding constants of the linked ligand to the protein, but also reveal new insights into how the high-affinity binding is achieved in thermodynamic terms. In order to understand the structural basis of the GluR2 S1S2 and ligand recognition, we will determine the 3D structure of the protein in complex with the high-affinity linked compound by NMR. The new structure of the GluR2 in complex with the high affinity and specificity ligand provides the foundation for the use of computational molecular modeling methods (Honig & Nicholls, 1995; Leach & Kuntz, 1992) to analyze in great detail the protein/ligand complex in terms of the energetics of molecular conformations, the distribution of charges and electron densities, and the spatial distribution of reactivity properties, such as the molecular electrostatic potential that constitute determinants of molecular reactivity in protein-ligand interactions. Such detailed, computational analysis of the protein/ligand complex will establish a mechanistic relationship between the pharmacological properties and the structure-based binding models for GluR2. The chemical compounds designed in the NMR structural and molecular modeling studies that act as agonists or antagonists of glutamate receptors can be assayed in electrophysiological experiments. In the long term, this information of detailed, structural determinants for the specific molecular interactions will further support the design of subtype selective agonists and antagonists for different AMPAspecific GluRs based on sequence and structure homology.

w

w

REFERENCES

ht

tp

://

w

Armstrong, N., Sun, Y., Chen, G. Q., & Gouaux, E. (1998). Structure of a glutamatereceptor ligand-binding core in complex with kainate. Nature, 395, 913-917. Arvola, M., & Keinanen, K. (1996). Characterization of the ligand-binding domains of glutamate receptor (GluR)-B and GluR-D subunits expressed in Escherichia coli as periplasmic proteins. Journal of Biological Chemistry, 271, 15527-15532. Bennett, J. A., & Dingledine, R. (1995). Topology profile for a glutamate receptor: Three transmembrane domains and a channel-lining reentrant membrane loop. Neuron, 14, 373-384. Burnashev, N., Monyer, H., Seeberg, P. H., & Sakmann, B. (1992). Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit. Neuron, 8,189-198. Chen, G. Q., & Gouaux, E. (1997). Overexpression of a glutamate receptor (GluR2) ligand binding domain in Escherichia coli: Application of a novel protein folding screen. Proceedings of the National Academy of Sciences (USA), 94,13431-13436.

Structure-Based Design of Agonists and Antagonists

145

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Chen, G. Q., Sun, Y, Jin, R., & Gouaux, E. (1998). Probing the ligand binding domain of the GluR2 receptor by proteolysis and deletion mutagenesis defines domain boundaries and yields a crystallizable construct. Protein Science, 7, 2623-2630. Dhalluin, C, Carlson, J. R, Zeng, L., He, C, Aggarwal, A. K., & Zhow, M. M (1999). Structure and ligand of a histone acetyltransferase bromodomain. Nature, 399, 491-496. Hajduk, P. J., Meadows, R. P., & Fesik, S. W. (1997a). Discovering high-affinity ligands for proteins. Science, 278, 498-499. Hajduk, P. J., Meadows, R. P., & Fesik, S. W. (1997b). Discovery of potent nonpeptide inhibitors of stromelysin using SAR by NMR. Journal of the American Chemical Society, 119, 5818-5827. Hollmann, M., & Heinemann, S. (1994). Cloned glutamate receptors. (1994). Annual Review ofNeuroscience, 17, 31-108. Hollmann, M., Maron, C., & Heinemann, S. (1994). N-Glycosylation site tagging suggests a three transmembrane domain topology for the glutamate receptor GluRl. Neuron, 13,1331-1343. Honig, B., & Nicholls, A. (1995). Classical electrostatics in biology and chemistry. Science, 268,1144-1149. Kuryatov, A., Laube, B., Betz, H., & Kuhse, J. (1994). Mutational analysis of the glycine-binding site of the NMDA receptor: Structural similarity with bacterial amino acid-binding proteins. Neuron, 12,1291-1300. Kuusinen, A., Arvola, M., & Keinanen, K. (1995). Molecular dissection of the agonist binding site of an AMPA receptor. EMBO Journal, 14, 6327-6332. Leach, A. R., & Kuntz, I. D. (1992). Conformational analysis of flexible ligands in macromolecular receptor sites. Journal of Computational Chemistry, 13,370-379. Lee, J. M., Zipfel, G. J., & Choi, D. W. (1999). The changing landscape of ischaemic brain injury mechanisms. Nature, 399, A7-A14. Livingstone, J. R. (1996). Antibody characterization of isothermal titration calorimetry. Nature, 384, 491-492. Lorber, D. M., & Shoichet, B. K. (1998). Flexible ligand docking using conformational ensembles. Protein Science, 7, 938-950. Lybrand, T. P. (1995). Ligand-protein docking and rational drug design. Current Opinions in Structural Biology, 5, 224-228. Mano, I., Lamed, Y., & Teichberg, V. I. (1996). A Venus flytrap mechanism for activation and desensitization of a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors. Journal of Biological Chemistry, 271,15299-15302. Mori, H., & Mishina, M. (1995). Structure and function of the NMDA receptor channel. Neuropharmacology, 34,1219-1237. Morrison, J. H., & Hof, P. R. (1997). Life and death of neurons in the aging brain. Science, 278, 412-419. Nakanishi, S., & Masu, M. (1994). Molecular diversity and functions of glutamate receptors. Annual Review of Biophysics and Biomolecular Structure, 23, 319-348. O'Hara, P. J., Sheppard, P. O., Thogensen, H., Venezia, D., Haldeman, B. A., McGrane, V., Houamed, K. M., Thomsen, C., Gilbert, T. L., & Mulvihill, E. R. (1993). The ligand-binding domain in metabotropic glutamate receptors is related to bacterial periplasmic binding proteins. Neuron, 11, 41-52.

Drug Discovery

146

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Paas, Y., Devillers-Thoery, A., Changeux, J. P., Medevielle, E, & Teichberg, V. I. (1996). Identification of an extracellular motif involved in the binding of guanine nuclotides by a glutamate receptor. EMBO Journal, 15,1548-1556. Pellegrini-Giampietro, D. E., Gorter, J. A., Bennett, M. V. L., & Zukin, R. S. (1997). The GluR2 (GluR-B) hypothesis: Ca2+-permeable AMPA receptors in neurological disorders. Trends in Neuroscience, 20, 464-470. Sandak, B., Wolfson, H. J., & Nussinov, R. (1998). Flexible docking allowing induced fit in proteins: Insights from an open to closed conformational isomers. Proteins, 32,159-174. Sattler, M., & Fesik, S. W. (1996). Use of deuterium labeling in NMR: Overcoming a sizable problem. Structure, 4,1245-1249. Seeburg, P. H. (1993). The TINS/TiPS lecture. The molecular biology of mammalian glutamate receptor channels. Trends in Neurosciences, 16, 359-365. Shuker, S. B., Hajduk, P.}., Meadows, R. P., & Fesik, S. W. (1996). Discovering highaffinity ligands for proteins: SAR by NMR. Science, 274,1531-1534. Squire, L. R., & Kandel, E. R. (1999). Memory: From mind to molecules. New York: Scientific American Library, W.H. Freeman Company. Stern-Bach, Y, Bettler, B., Hartley, M., Sheppard, P. O., O'Hara, P. J., & Heinemann, S. F. (1994). Agonist selectively of glutamate receptors is specified by two domains structurally related to bacterial amino acid-binding proteins. Neuron, 13,1345-1357. Watkins, J. C., Krogsgaard-Larsen, P., & Honore, T. (1990). Structure-activity relationships in the development of excitatory amino acid receptor agonists and competitive antagonists. Trends in Pharmacological Sciences, 11, 25-33. Weinstein, H., & Osman, R. (1989). Interaction mechanisms at biological targets: Implications for design of serotonin receptor ligands. In W. G. Richards (Ed.), Computer-aided molecular design. London: IBC Technical Services. Wo, Z. G., & Oswald, R. E. (1994). Transmembrane topology of two kainate receptor subunits revealed by N-glycosylation. Proceedings of the National Academy of Sciences (USA), 91, 7154-7158. Wo, Z. G., & Oswald, R. E. (1995). Unraveling the modular design of glutamategated ion channels. Trends in Neuroscience, 18,161-168. Wo, Z. G., & Oswald, R. E. (1996). Ligand-binding characteristics and related structural features of the expressed goldfish kainate receptors: Identification of a conserved disulfide bond and three residues important for ligand binding. Molecular Pharmacology, 50, 770-780. Zhou, M.-M., Huang, B., Olejniczak, E. F, Meadows, R. P., Shuker, S. B., Miyazaki, M., & Fesik, S. W. (1996). Structural basis of IL-4 receptor phosphopeptide recognition by the IRS-1 PTB domain. Nature Structure Biology, 3, 388-393. Zhou, M.-M., Meadows, R. P., Logan, P. M., Yoon, H. S., Wade, W. S., Burakoff, S. J., & Fesnik, S. W. (1995). Solution structure of the She SH2 domain complexed with a tyrosine-phosphorylated peptide from the T-cell receptor. Proceedings of the National Academy of Sciences (USA), 92, 7784-7788. Zhou, M.-M., Ravichadran, K. S., Olejniczak, E. E, Petros, A. M., Meadows, R. P., & Fesik, S. W. (1995). Structure and ligand recognition of the phosphotyrosine binding domain of She. Nature, 378, 584-592.

Mimetics

s/ in de

Molecule

x. ph

Alzheimer's Therapeutics: Neurotrophin Small

p

18

vn

/fo

ru

m

Stephen M. Massa, You-Mei Xie, and Frank M. Longo

4a ll.

ABSTRACT

ht

tp

://

w

w

w

.c

he m

In Alzheimer's disease (AD), degeneration of basal forebrain, hippocampal and cortical neurons, and their related networks results in loss of cognitive function. The findings that neurotrophin proteins, such as nerve growth factor (NGF), prevent neuronal death and promote the maintenance of neuronal connections suggests that neurotrophins might be used to slow degenerative processes and optimize function of surviving networks. The demonstration that administration of NGF to aged primates leads to a reversal of age-related neuronal atrophy indicates that neurotrophins will be effective over a wide range of age-related neurodegenerative disorders. Like most proteins, neurotrophins have poor medicinal properties, including limited penetration of brain tissue and poor chemical stability. A major goal is to develop stable, small molecules that function as drugs to mimic the neurotrophin actions of preventing neuronal loss and maintaining neuronal connections. The development of small molecule mimetics of the insulin and erythropoietin (EPO) proteins has stimulated new approaches for the treatment of diabetes and anemia. Similar approaches for the neurotrophin proteins will introduce new strategies for creating a new class of AD and age-related neurodegenerative therapeutics. We have established that peptidomimetics (small, synthetic fragments of NGF) mimicking selected parts of the NGF protein can promote its 147

Drug Discovery

148

fundamental biological actions and can trigger NGF's normal signaling processes within its target cells. These peptidomimetics constitute a powerful intermediate step in developing NGF-like drug compounds. Peptidomimetics provide a means for searching small molecule chemical libraries for active lead compounds that will allow development of pharmaceutical compounds.

x. ph

p

INTRODUCTION

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

Preventing the onset and progression of AD and other age-related losses of cognitive function will require concomitant approaches (Cummings, Vinters, Cole, & Khachaturian, 1998). One key strategy will be to apply robust therapies capable of preventing neuronal degeneration and loss of neuritic networks in the setting of multiple toxic processes. The ability of neurotrophins to prevent atrophy, reduce vulnerability to a wide range of toxic factors, maintain neuritic integrity, and upregulate neuronal function encourages their application in the contexts of aging and neurodegenerative disease (Cummings et al., 1998; Hughes & O'Leary, 1999; Smith, Roberts, Gage, & Tuszynski, 1999). Development of small molecule mimetics for the EPO and insulin proteins (Wrighton et al., 1997; Zhang, Salituro, Szalkowski, et al., 1999) points to the importance of small molecule approaches in clinical therapeutics. NGF interacts with the p75 receptor primarily via residues in p-loop 1 and with TrkA primarily via residues in (3-loops 2 and 4 and in the N-terminus (McDonald & Chao, 1995) (Figure 18.1). We have created small peptide mimetics (peptidomimetics) corresponding to loop 1 that act via p75 (Longo, Vu, & Mobley, 1990; Longo, Manthorpe, Xie, & Varon, 1997) and mimetics corresponding to loop 4 that act via TrkA to prevent neuronal death (Xie & Longo, 1997; Xie, Tisi, Yeo, & Longo, 2000; Xie & Longo, 2000). Loop 4 mimetics activate ERK and AKT, key signaling intermediates activated by NGF-Trk binding (Xie et al., 2000). NGF peptidomimetics corresponding to loop 4 have also been reported by Maliartchouk et al. (2000a, 2000b). NGF peptidomimetics constitute an important reduction-to-practice of the theoretical possibility that active, NGF, small molecule mimetics can be created. Moreover, peptidomimetics are powerful intermediate-stage tools in the identification of nonpeptide, small molecule, pharmaceutical-lead compounds (Kieber-Emmons, Murali, & Greene, 1997). Our hypothesis is that the available crystal structure of NGF, along with quantitative structure activity relationship (QSAR) data derived from NGF peptidomimetics, can be used to identify nonpeptide, small molecule, NGF-mimetic lead compounds. Small molecule mimetics of the NGF

149

vn

/fo

ru

m

s/ in de

x. ph

p

+Alzheimer's Therapeutics

he m

4a ll.

FIGURE 18.1 NGF (3-turn loop 1 interacts with p75 receptors and NGF p-turn loop 4 interacts with TrkA receptors.

ht

tp

://

w

w

w

.c

loop 1 domain will modulate signal transduction pathways linked to the p75 NGF receptor. Small molecule mimetics of the NGF loop 4 domain will regulate signal-transduction pathways linked to the TrkA NGF receptor. Specific aims are as follows: (a) NGF peptidomimetic QSAR studies; (b) screening of small molecule library databases; (c) biological validation of small molecule "hits"; and (d) signal transduction validation of small molecule "hits."

MATERIALS AND METHODS Linear peptides containing various combinations of residues present in loop 1 or loop 4 of NGF are synthesized, purified, and analyzed using standard protocols as described in Longo et al. (1990,1997) and Xie et al. (2000). Peptide-conformational constraint is induced via cyclization of linear peptides into monomeric or dimeric forms. Following cyclization, peptides are purified by reversed-phase HPLC and submitted for analytical HPLC, quantitative amino-acid analysis, and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Peptidomimetics are

150

Drug Discovery

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

assessed for neurotrophic activity using standard, quantitative, dorsal root ganglia (DRG) bioassays (Xie et al., 2000). Control cultures performed with each assay include serial dilutions of NGF to generate NGF doseresponse curves and cultures without NGF or peptides to determine background survival. Neuronal survival and neurite outgrowth are assessed by a combination of morphological criteria and the MTT colorimetric assay. Peptidomimetic activity is expressed in terms of efficacy, compared with the NGF maximum effect and potency as indicated by concentrations required for a given effect. Dependence of peptidomimetic function via the p75 receptor is determined using function-blocking p75 antibody and DRG neurons isolated from p75 KO mice. Dependence of peptidomimetic function via Trk is determined using the Trk inhibitors K252a and AG879. The role of ERKpathway signaling in peptidomimetic activity is assessed using the UO126 ERK inhibitor and its inactive control, UO124. ERK, AKT, and JNK activation are assessed using phosphoactive antibodies and quantitative Western blot analysis (Xie et al., 2000). Small molecule libraries are being screened with queries based on the chemical features and potential structures of selected loop 1 or loop 4 residues using established pharmacophore approaches (Guner, 2000). Residues are selected based on several strategies, including assessment of relative bioactivities of peptidomimetics containing various combinations of residues, structural relationships between residues predicted by NGF crystal structure, and comparisons of residue conservation across different species and members of the neurotrophin gene family. Figure 18.2 demonstrates one example of small-molecule library screening with a NGF loop 4-based query (Figure 18.2a). In some cases, conformations of identified compounds demonstrate poor steric matches to the targeted NGF domain (Figure 18.2b). Addition of further steric constraints to the query (shape and excluded volumes) reduces the number of hits and increases the likelihood that the identified compound will better match the targeted NGF domain (Figure 18.2c). Limitations include the possibilities that crystal-derived structures do not match structures required for receptor activation and incomplete knowledge of which functional chemical groups are important. Chemical modifications and biologic testing of active loop 1 and 4 peptidomimetics, along with the known structure of NGF will allow improved modeling. These improved models will be used in an iterative, quantitative, structure-activity approach to find more active, more specific compounds. Compounds demonstrating close matches to NGF-based queries in terms of structure and chemical features are obtained for biological testing. Testing includes profiles of neurotrophic activity, dependence on p75 and Trk receptor activation, and patterns of ERK, AKT, and JNK activation.

Alzheimer's Therapeutics

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

151

FIGURE 18.2 A. NGF loop 4 crystal structure (stick) with chemical features. B. One compound from the NCI database that fulfills chemical-feature requirements, but likely represents a poor steric fit. C. A compound from the NCI database with better shape and chemical complementarity.

152

Drug Discovery

RESULTS

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Our prototype loop 1 peptidomimetic, "P7," functions as an NGF partial agonist (i.e., dose-response curves reach less than 100% efficacy of the NGF maximum) (Longo et al., 1997). Evidence indicates that P7 acts via p75 receptors: (a) P7 activity requires NGF amino-acid sequence; (b) P7 biological activity requires dimeric conformation; (c) activity is lost with small changes in structure; (d) activity of NGF and P7 are blocked to a similar extent by the p75 antibody 9651; (e) activity of P7 is lost using DRG neurons derived from p75 -/- mice; and (f) consistent with its partial agonist profile, P7 in the presence of added NGF partially blocks NGF. Our prototype loop 4 peptidomimetic, "P92," also functions as a NGF partial agonist. Evidence indicates that P92 acts via TrkA receptors: (a) P92 activity requires NGF sequence; (b) P92 biological activity requires dimeric conformation; (c) activity of NGF and P92 is blocked to a similar extent by the TrkA inhibitor K252a; (d) P92 stimulates TrkA tyrosine phosphorylation; (e) consistent with its partial agonist profile, P92 partially blocks activity of added NGF; (f) P92 activates ERK and this activity is blocked by K252a; (g) the ERK inhibitor UO126 blocks the neurite-promoting activity of both NGF and P92; and (h) P92 activates AKT and this activity is blocked by K252a. For the purpose of small-molecule library screening, a Silicon Graphics Inc. Octane workstation has been set up. Insight II software (Molecular Simulations Inc.) is being used for protein and peptide-structure analysis. Cerius 2 (MSI) and Catalyst (MSI) programs are being used for query building and small-molecule library screening, in conjunction with an Oracle 3.0 database server. The following databases containing compounds already present in multiconformer format have been purchased (number of compounds indicated in parentheses): National Cancer Institute Library (123,000) and Maybridge Chemical Corp. (53,000). One of the largest available databases, Chapman and Hall Combined Chemical Dictionary (CCD; 350,000), is available only in single conformer format. A 3-month processing run in which this database was converted to the multiconformer format on our workstation has been completed. Additional single conformer format databases have been obtained, and are currently in the process of being converted to multiconformer format. Sets of queries corresponding to NGF loop domains 1 and 4 have been created. These queries have been used to probe the NCI, Maybridge, and CCD libraries. Approximately 40 compounds of interest have been identified. Procurement and biological testing are underway.

Alzheimer's Therapeutics

153

DISCUSSION

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

"Agonist lead compounds" will be defined as those hits having the following properties: (a) eliciting neurite outgrowth and/or survival of NGF-responsive neurons at doses of < 10 uM; (b) activating NGF-related signal transduction pathways; and (c) demonstration that activity and signaling are dependent upon p75 or TrkA. "Antagonist lead compounds" will be those that inhibit NGF binding and activity. Both types of compounds will contribute, in subsequent studies, to development of potent NGF agonists, partial agonists or antagonists. Comparisons of the structures of either agonists, or antagonists to NGF loop structures will suggest chemical modifications designed to create more potent agonists or to use features of an antagonist to create an agonist. For example, if activity requires a dimeric structure, one approach would be to use the structure of a potent NGF receptor-binding antagonist to create a bivalent compound with the same loop-mimicking features. While these kinds of pharmaceutical chemistry studies are beyond the scope of the present proposal, the "lead compounds" generated here will trigger these efforts. The extent to which NGF itself and NGF mimetics function as dimeric versus monomeric ligands remains an important question. NGF exists in solution as a dimer. Receptor activation consists of ligand-induced receptor dimerization (TrkA-TrkA or TrkA-p75) and ligand-induced alteration in receptor conformation leading to TrkA autophosphorylation. Recent peptidomimetic studies suggest that TrkA activation can be triggered by monomeric compounds, suggesting the possibility that some degree of TrkA activation may be achieved by ligand-induced alteration in receptor conformation without necessarily inducing receptor dimerization (Maliartchouk et al., 2000a). Less is known about mechanisms of p75 "activation." Since the role of altered receptor conformation is not well established for TrkA or p75, it remains possible that a ligand can affect activation acting as either a dimer or monomer. Thus, while P7 and P92 activity is dependent on the dimer structure, it remains possible that this dimeric structure merely more closely mimics the NGF loop structure, compared with the monomer, and that the dimer is, in effect, functioning as a "monomer." Given that dimeric- versus monomeric-based mechanisms of p75 and TrkA activation are not fully established, both monovalent and bivalent library compounds will be assessed. One "risk" in applying neurotrophin mimetics to AD therapeutics is that it is unknown which profile of mimetic activity (i.e., full agonist, partial agonist, or antagonist) will be optimal for decreasing neuronal death and network loss in vivo. Studies in aging and neurodegenerative disease animal models, made possible by the present studies, will determine if NGF full or partial agonists prevent neuronal death and promote the

154

Drug Discovery

s/ in de

x. ph

p

neurotrophic phenotype. The observations that NGF interaction with p75 promotes neuronal death in certain contexts and that cholinergic function (Yeo et al., 1997; Greferath et al., 2000) and central nervous system (CNS)neurite outgrowth (Walsh, Krol, Crutcher, & Kawaja, 1999) are increased in p75 -/- mice suggest that partial, rather than full, p75 agonists may be particularly advantageous. Regardless of activity profiles, the availability of small molecule compounds acting at NGF receptors will provide powerful tools for answering these critical questions. Moreover, availability of NGF antagonists will have major impacts on approaches to chronic pain, neuromas, and other neuropathological and NGF-regulated immune system processes.

REFERENCES

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

Cummings, J. L., Vinters, H. V., Cole, G. M, & Khachaturian, Z. S. (1998). Alzheimer's disease: Etiologies, pathophysiology, cognitive reserve, and treatment opportunities. Neurology, 51, S2-17. Greferath, U., Bennie, A., Kourakis, A., Bartlett, P. R, Murphy, M., & Barrett, G. L. (2000). Enlarged cholinergic forebrain neurons and improved spatial learning in p75 knockout mice. European Journal ofNeuroscience, 12, 885-893. Guner, O. F. (2000). Pharmacophore: Perception, development, and use in drug design. La Jolla, CA: Internet University Line. Hughes, R. A., & O'Leary, P. D. (1999). Exploiting neurotrophic factors for the treatment of neurodegenerative conditions: An Australian perspective. Drug Development Research, 46, 268-276. Kieber-Emmons, T., Murali, R., & Greene, M. I. (1997). Therapeutic peptides and peptidomimetics. Current Opinions in Biotechnology, 8, 435-441. Longo, E M., Vu, K., & Mobley, W. C. (1990). The in vitro biological effect of nerve growth factor is inhibited by synthetic peptides. Cell Regulation, 1, 189-195. Longo, F. M., Manthorpe, M., Xie, Y. M., & Varon, S. (1997). Synthetic NGF peptide derivatives prevent neuronal death via a p75 receptor-dependent mechanism. Journal ofNeuroscience Research, 48,1-17. Maliartchouk, S., Debeir, T, Beglova, N., Cuello, A. C., Gehring, K., & Saragovi, H. U. (2000a). Genuine monovalent ligands of TrkA nerve growth factor receptors reveal a novel pharmacological mechanism of action. Journal of Biological Chemistry, 275, 9946-9956. Maliartchouk, S., Feng, Y., Ivanisevic, L., Debeir, T, Cuello, A. C., Burgess, K., & Saragovi, H. U. (2000b). A designed peptidomimetic agonistic ligand of TrkA nerve growth factor receptors. Molecular Pharmacology, 57, 385-391. McDonald, N. Q., & Chao, M. V. (1995). Structural determinants of neurotrophin action. Journal of Biological Chemistry, 270, 19669-19672. Smith, D. E., Roberts, J., Gage, F. H., & Tuszynski, M. H. (1999). Age-associated neuronal atrophy occurs in the primate brain and is reversible by growth factor gene therapy. Proceedings of the National Academy of Sciences (USA), 96, 10893-10898.

Alzheimer's Therapeutics 155

155 155

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Walsh, F. S., Krol, K. M., Crutcher, K. A., & Kawaja, M. D. (1999). Enhanced neurotrophin-induced axon growth in myelinated portions of the CNS in mice lacking the p75 neurotrophin receptor. Journal ofNeuroscience, 19, 4155-4168. Wrighton, N. C., Balasubramanian, P., Barbone, F. P., Kashyap, A. K., Farrell, F. X., Jolliffe, L. K., Barrett, R. W., & Dower, W. J. (1997). Increased potency of an erythropoietin peptide mimetics through covalent dimerization. Nature Biotechnology, 15, 2161-1265. Xie, Y. M., & Longo, F. M. (1997). Neurotrophic activity of synthetic peptide derivatives corresponding to NGF loop region 92-98. Society for Neurosciences Abstracts, 23,1701. Xie, Y. M., & Longo, F. M. (2000). Neurotrophin small-molecule mimetics. In F. J. Seil (Ed.), Neural plasticity and regeneration (pp. 333-347). The Netherlands: Elsevier Science B.V. Xie, Y, Tisi, M. A., Yeo, T. T., & Longo, F. M. (2000). NGF loop 4 dimeric mimetics activate ERK and AKT and prevent neuronal death. Journal of Biological Chemistry, 275, 29868-29874. Yeo, T. T., Chua-Couzens, J., Valletta, J., Butcher, L. L., Bredesen, D. E., Mobley, W. C., & Longo, F. M. (1997). Absence of p75NTR causes increased basal forebrain cholinergic neuron size, ChAT activity and target innervation. Journal of Neuroscience, 17, 7594-7605. Zhang B, Salituro G, Szalkowski, Li, Z., Zhang, Y, Royo, I., Vilella, D., Diez, M. T., Pelaez, F, Ruby, C., Kendall, R. L., Mao, X., Griffin, P., Calaycay, J., Zierath, J. R., Hech, J. V., Smith, R. G., & Moller, D. E. (1999). Discovery of a small molecule insulin mimetic with antidiabetic activity in mice. Science, 284, 974-977.

ACKNOWLEDGMENTS

ht

tp

://

w

w

w

.c

NGF mimetic studies in our laboratory are supported by the Institute for the Study of Aging, the Alzheimer's Association, the John Douglas French Foundation, and the Veteran's Administration.

p

x. ph

s/ in de

m

ru

/fo

vn

4a ll.

m

.c he

w

w

w

://

tp

ht This page intentionally left blank

he m

.c

w

w

w

://

tp

ht

m

ru

/fo

vn

4a ll.

p

Drug Development— Preclinical

x. ph

s/ in de

Ill

p

x. ph

s/ in de

m

ru

/fo

vn

4a ll.

m

.c he

w

w

w

://

tp

ht This page intentionally left blank

19

s/ in de

Disease: Breakthrough

x. ph

Treatment of Alzheimer's

p

Antioxidants in the

Potential of Indole-3-

ru

m

Propionic Acid

w

.c

he m

4a ll.

vn

/fo

Daniel G. Chain, Douglas Galasko, Eyal Neria, Miguel Pappolla, Paul E. Bendheim, and Burkhard Poeggeler

w

w

ABSTRACT

ht

tp

://

Much evidence points to the central role of amyloid-p protein (A(3induced toxicity in the pathogenesis of Alzheimer's disease (AD). Treatment strategies that target A(3 include: (a) decreasing its production; (b) increasing its clearance; (c) inhibiting its toxic fibrillar aggregation; (d) preventing oxidative damage and cytotoxicity. A promising new drug candidate to target A(3 is indole-3-propionic acid, a naturally occurring potent scavenger of damaging hydroxyl radicals and an effective inhibitor of A(3 fibrillogenesis.1 Numerous in vitro and animal studies indicate that indole-3-propionic acid inhibits aggregation of A(3, protects human cortical neurons from the toxicity of A(3, and importantly, protects the brain

1

Mindset Pharmaceuticals is developing indole-3-propionic acid for the treatment of AD. 159

160

Drug Development—Preclinical

ru

m

s/ in de

x. ph

p

from oxidative damage produced by a variety of powerful, oxidative, stress-inducing neurotoxins. Moreover, indole-3-propionic acid belongs to a unique class of molecules that have no pro-oxidant activity. This is in contrast to other well-known antioxidants, including vitamin C, vitamin E, glutathione, and other indole compounds, all of whose metabolites have pro-oxidant activity. Indole-3-propionic acid, a deamidation product of tryptophan, is a naturally occurring molecule produced by bacteria in the gastrointestinal tract. It is normally present in blood and cerebrospinal fluid and, importantly, has been tested at pharmacological concentrations in laboratory animals without demonstrating evidence of toxicity. In this chapter, we review the importance of oxidative stress in AD, and compare known properties of indole-3-propionic with other antioxidants that have been tested or are under development for the treatment of AD. We also summarize the status of indole-3-propionic acid's development as a pharmaceutical in the treatment of AD.

vn

/fo

INTRODUCTION

ht

tp

://

w

w

w

.c

he m

4a ll.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder that has a major impact on public health. Its prevalence is rising with the increasing longevity of the population. Clinically, AD is characterized by a relentless impairment of memory and reasoning that generally begins insidiously and can progress for a decade or longer. In most cases, AD starts after the age of 60, and the incidence increases with each subsequent decade. In at least 15% of cases of AD, the medical history reveals a relative affected by the disease. In some families, the disease starts earlier (in the fifth or sixth decade) and affects individuals in an autosomal dominant pattern. Current research indicates a linkage either directly to the gene encoding the [3-amyloid precursor protein (APP) or to the presenilin genes, whose protein products are critical in the processing of APP and/or its toxic fragments, the (3-amyloid peptides (Selkoe, 1997,1999). Underlying its clinical presentation, AD is characterized by a series of dramatic structural abnormalities in the brain, including gross atrophy discernible both on the surface and in cortical and subcortical structures. In addition, AD brain is characterized by prominent morphological abnormalities at the cellular level, including a prominent loss of neuron populations in the entorhinal cortex, the hippocampus, the high-order association cortices of the temporal, frontal, and parietal regions, and some subcortical nuclei in the basal forebrain, thalamus, and brain stem. The neuronal damage and the attending loss of synaptic density disable several neural systems essential to learning and retrieval of memories.

Antioxidants in the Treatment of AD

161

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Histologically, the diseased neurons are seen to contain cytoplasmic neurofibrillary tangles composed of paired helical filaments. The most prominent anatomic change, however, consists of amyloid plaques containing degenerated neuronal fragments surrounding a small, dense core of (3-amyloid peptide fibrillar aggregates (Dickson, 1997). Markers of oxidative stress and reactive glial cells are prominent proximal to these amyloid deposits (Pappolla et al., 1992). A large body of genetic, biochemical, cellular, and molecular biological data supports the hypothesis that the self-aggregating [i-amyloid (A[3) peptide is the toxic molecule responsible for a cascade of events leading eventually to neural degeneration in AD. Misprocessing of Afi, such as could occur with increased production of a longer, more aggregating form of the peptide, or impaired clearance of A(3 from the brain, results in a pathological increase in the brain concentration of this toxic molecule and initiation of the cascade of events leading to neural degeneration. Although further research is required to clarify the molecular mechanism(s) of A(3 toxicity, there is much evidence that toxicity depends on some degree of oligomer or aggregate formation by A(3 and the generation of damaging free radicals. The production of reactive oxygen species (ROS) and free radicals is increasingly implicated in several major neurodegenerative diseases including Parkinson's disease, Friedreich's ataxia, amyotrophic lateral sclerosis, and AD. ROS and, in particular, hydroxyl radicals, damage lipids, proteins, nucleic acids, and other cellular constituents. Mammalian cells contain physiological defense mechanisms that detoxify these free radicals, but oxidative stress results when the amounts generated exceed the capacity of the physiological defenses. Oxidative damage may impair neuronal function in many ways, for example, inducing membrane disturbances, loss of membrane homeostasis, and in promoting aggregation of proteins such as amyloid beta (Ap) and tau (Markesbery & Carney, 1999; Mattson, Pedersen, Duan, Culmsee, & Camandola, 1999). Several lines of evidence point to the importance of ROS as mediators of cell damage in AD, with Af} seen to play a major role in producing an oxidative microenvironment around senile plaques in affected brain areas. A(3 toxicity is likely to involve overproduction of ROS by neurons and, possibly, microglia activation (Johnstone, Gearing, & Miller, 1999). Thus, antioxidant therapy has the potential to reduce or inhibit the damage to neurons and slow the progression of AD.

DISCUSSION Antioxidant Therapeutic Approaches in Alzheimer's Disease Several observational and epidemiological studies suggest beneficial effects of antioxidants on cognition and AD. Vitamin E, ginkgo biloba,

162

Drug Development—-Preclinical

selegiline, and estrogen are putative antioxidant therapies that have been studied in clinical trials for the treatment of AD.

vn

/fo

ru

m

s/ in de

x. ph

p

Vitamin E Decreased risk ratios for developing the disease have been found in population-based studies in people who took vitamin E (Morris et al., 1998), vitamin C (Paleogolos et al., 1998), or both. Although the dose and duration of exposure to antioxidants varied markedly among subjects, the overall effect appears to be toward lowered risk of dementia. A multicenter, controlled, clinical trial of patients with moderate-to-severe AD showed that treatment with 2000 units/day of vitamin E (alpha-tocopherol) delayed the time to reach clinical milestones, such as institutionalization and loss of activities of daily living (ADD (Sano et al., 1997). The results of this trial were encouraging in that they raise the possibility that the antioxidant properties of vitamin E may lead to neuroprotection and slow the rate of clinical progression of AD. Nevertheless, there are several potential disadvantages to treatment with vitamin E. For example, vitamin E forms pro-oxidant intermediates in the presence of transition metals such as F2++ (Neuzil, Thomas, & Stocker, 1997). Moreover, its blood-brain barrier penetrability is poor (Pappert et al., 1996).

ht

tp

://

w

w

w

.c

he m

4a ll.

Ginko biloba Ginkgo biloba extract (Egb 761) is a well-defined plant extract containing two major groups of constituents (i.e., flavonoids and terpenoids). Egb 761 has been shown to protect cultured cells from A(3-induced neurotoxicity via antioxidant actions (Bastianetto et al., 2000). Recent studies suggest that the neuroprotective properties of Egb 761 are not only attributable to the antioxidant NO-scavenging properties of its flavonoid constituents, but also via their ability to inhibit NO-stimulated protein kinase C (PKC) activity. Egb 761 has been tested in various controlled, clinical trials of AD, with modest but positive results reported in a number of cases (Maurer, Ihl, Dierks, & Frolich, 1997; Le Bars et al., 1997; Haase, Halama, & Horr, 1996). Further studies are required to test the long-term benefit of this putative therapeutic for AD. Selegiline

A beneficial effect of selegiline (L-deprenyl) in AD has been reported in several clinical studies. In a trial carried out by the Alzheimer's Disease Cooperative Study (ADCS), selegiline was found to delay important medical milestones for people with moderately severe AD (Sano et al., 1997). Notably, however, the drug had no effect on cognition, suggesting that its positive effects may result from other, more general health improvements, such as cardiovascular effects from its antioxidant activity. Supporting

Antioxidants in the Treatment of AD

163

this idea, a recent report showed that none of the lesions critical for AD diagnosis, such as counts of neuritic plaques, neurofibrillary tangles, or beta-A(3 load, were influenced by selegiline treatment when the drug was tested in a double-blind trial (Alafuzoff et al., 2000). Estrogen

he m

Indole-3-propionic Acid

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Estrogen-mediated neuroprotection has been described in many neuronal culture systems with protection against A(3-induced toxicity and oxidative stress. In animal models, estrogens have been shown to attenuate neuronal death in rodent models of cerebral ischaemia, traumatic injury, and Parkinson's disease. Increasingly, a direct antioxidant activity is implicated in the neuroprotective properties of estrogen, although other mechanisms are probably involved, including activation of the nuclear estrogen receptor, altered expression of bcl-2 and related proteins, and activation of mitogen-activated and other signal transduction pathways (Green & Simpkins, 2000). Several reports from small clinical trials have suggested that estrogen-replacement therapy may be useful for the treatment of AD in women. The results of a recently published, randomized, double-blind, placebo-controlled, clinical trial conducted by the ADCS, however, found that estrogen-replacement therapy did not slow disease progression nor did it improve global, cognitive, or functional outcomes in women with mild-to-moderate AD (Mulnard et al., 2000).

ht

tp

://

w

w

w

.c

Recent studies led to the identification of indole-3-propionic acid as an extremely potent antioxidant that acts as a scavenger of hydroxyl radicals (Poeggeler et al., 1999; Chyan et al., 1999). The most potent, naturally occurring antioxidant known, indole-3-propionic acid, is several orders of magnitude more powerful than vitamin E in scavenging hydroxyl radicals. Importantly, indole-3-propionic acid does not generate pro-oxidative intermediates in the process. In the presence of transition metals, most common, free radical scavengers, such as ascorbate, trolox, and glutathione, are subject to auto-oxidation, which promotes the formation of free radicals rather than inhibits their formation. This results in overproduction of free radicals in the presence of these antioxidants. Indole-3-propionic acid, in contrast, does not promote the formation of free radicals in the presence of transition metals. Uniquely, indole-3-propionic acid is not susceptible to the same decarboxylation step that occurs following oxidation of indole-3-acetic acid or other indole acids, and that results in the formation of carbon-centered radicals capable of reducing iron and promoting hydroxyl radical formation (see Figure 19.1). Instead, indole-3-propionic acid acts as a safe antioxidant through electron transfer from indole-3-propionic acid to the hydroxyl radical,

264 Drue Development—Preclinical

164

s/ in de

x. ph

p

FIGURE 19.1 Pro-oxidant activity of indole-3-acetic acid: The acetic acid, cation radical undergoes decarboxylation to form a carbon-centered skatole radical. Skatole radicals can then undergo subsequent reactions to promote formation of other free radicals, such as the hydroxyl radical.

/fo

ru

m

detoxifying the reactive hydroxyl radical to form a nontoxic hydroxyl anion and an indolyl cation radical. The latter can react with superoxide anion to form kynnuric acid (see Figure 19.2).

tp

://

w

w

w

.c

he m

4a ll.

vn

Dual Action Indole-3-propionic acid can protect cultured neurons from the toxicity of AP (Chyan et al., 1999). The molecule penetrates the brain and has been shown in vivo to protect the brain against a host of potent neurotoxins (Poeggeler et al, unpublished). At least part of an explanation for its neuroprotective mechanism is the discovery that indole-3-propionic acid is, additionally, a potent inhibitor of amyloid-fibril formation in vitro. Thus, the observed neuroprotective effect of indole-3-propionic acid in vivo most likely results from a dual mechanism of action. This unique, dualaction phenomenon targets two steps in the amyloid cascade that are central to the pathogenesis of AD—p-amyloid fibril formation and oxidative stress. As such, indole-3-propionic acid is particularly appealing as a drug candidate for AD.

ht

Bioavailability

The natural concentration of indole-3-propionic acid in human plasma and cerebrospinal fluid (CSF) has been determined in several independent studies, using HPLC methods (Morita et al., 1990; Morita, Kawamoto, & Yoshida, 1992). No report, however, has determined concentration in plasma and CSF in the same subject, so that the CSF/plasma ratio has not been directly determined. In one study (Morita et al., 1990), concentrations of indole-3-propionic acid were determined in normal subjects (n = 31), diabetic patients (n = 140), and pregnant women (n = 20). Total concentrations (free + protein-bound) were 183 ng/ml in controls, 149 ng/ml in

165

s/ in de

x. ph

p

Antioxidants in the Treatment of AD

/fo

ru

m

FIGURE 19.2 Mechanism of hydroxyl radical scavenging by indole-3-propionic acid.

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

diabetics, and 140 ng/ml in pregnant women. The results indicated substantial interindividual variability, however. Some of this variability is due to differences in intestinal flora and dietary tryptophan intake. Accordingly, there appears to be less variability in serial plasma concentrations of indole-3-propionic acid in the same individual measured repeatedly over 4 weeks. Indole-3-propionic acid was also measured in human CSF of epileptic patients who underwent diagnostic pneumoencephalography (Young, Anderson, Gauthier, & Purdy, 1980). Mean concentrations were 0.63 ng/ml in lumbar CSF and 0.48 ng/ml in cisternal CSF. Lumbar and cisternal concentrations were highly correlated in each subject, although the study showed significant interindividual variability as was also the case in plasma measurements. Extrapolating from the plasma and CSF studies, the ratio between the mean concentration of indole-3-propionic acid in plasma and in CSF is in the range of 300:1. Importantly, the concentration of indole-3-propionic acid in the CSF, which, as noted above, rarely exceeds nanomolar concentrations, can be increased significantly by systemic administration of the compound (Poeggeler, unpublished). A dose-dependent rise in the concentration of indole-3-propionic acid in the brain was observed after the administration of the sodium salt to rats via gavage feeding. The concentration of IPA increased from an endogenous level of 7.1 ± 4.6 nM to 35 ± 9 nM 1 hour after a dose of 1 mg/kg, and to 2953 ± 382 nM 1 hour after a dose of 100 mg/kg, indicating that indole-3-propionic acid penetrates through the blood-brain barrier adequately after oral administration.

166

Drug Development—-Preclinical

Safety

p

Preliminary safety data are available for indole-3-propionic acid from in-vivo studies in mice, rats, and dogs, indicating that the compound is safe at pharmacological doses. Poeggeler (unpublished data) has observed no signs of acute toxicity in rodents administered the compound, either orally or systemically, at pharmacological doses. The safety profile of indole-3-propionic acid is being further tested as part of the current preclinical program.

x. ph

Preclinical Development

/fo

ru

m

s/ in de

Mindset Biopharmaceuticals is supporting the accelerated development of indole-3-propionic acid as a possible therapeutic agent for AD. The near-term goals are to: (a) Examine the effects of the compound in a PS1/APP double-transgenic mouse model of AD (Holcomb et al., 1998); (b) complete the regulatory required components of the preclinical development program; (c) file an Investigational New Drug (IND) application; and (d) upon acceptance of the IND, initiate Phase la human safety trials. Human Clinical Studies

w

.c

he m

4a ll.

vn

The initial Phase I clinical trials will evaluate the safety, tolerability, and pharmacokinetics of single and multiple doses of indole-3-propionic acid in healthy volunteers. A Phase Ib study, designed and executed by the ADCS, will evaluate various doses in mild-to-moderate AD patients and correlate a possible effect of indole-3-propionic acid on a number of biomarkers that may eventually prove useful to monitor disease progression and drug effect.

w

Gateway to Identifying New Biomarkers

ht

tp

://

w

The development of indole-3-propionic acid, which because of its extreme potency, lack of pro-oxidant activity, and blood-brain barrier penetrability, may represent a breakthrough in antioxidant therapy for this disease, may also set new directions toward identification and validation of novel and improved biomarkers of AD. The proposed ADCS Phase Ib trial for indole-3-propionic acid aims to use several biomarker measures to evaluate the extent of antioxidant and, possibly, neuroprotective properties of this compound. Oxidative damage usually involves transient, unstable, free radicals and their reactive intermediates, which are difficult to measure because of their lability. Isoprostanes are free-radical derived isomers of prostaglandins. F-2 isoprostanes are chemically stable, and have been shown to be sensitive and specific markers of in vivo [lipid] peroxidation. Patients with probable and possible AD have been shown to have significantly higher

Antioxidants in the Treatment of AD

167

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

cerebrospinal fluid, plasma, and urine concentrations of a major isoprostane, 8/12-iso-iPF2-VI, than matched controls (Pratico et al., 2000). Moreover, the concentrations of 8,12-iso-iPF2-VI in cerebrospinal fluid, plasma, and urine were intercorrelated, suggesting that oxidative damage in the brain could be conveniently evaluated by a blood test. Cerebrospinal fluid, 8,12-iso-iPF2-VI, directly correlated with CSF concentrations of the microtubule-associated protein tau, a marker of neuronal or axonal damage. Finally, AD patients homozygous for the apo E-e4 allele had significantly higher concentrations of 8,12-iso-iPF2-VI than patients with no or one allele. These findings in living patients with mild-to-moderate dementia indicate that patients with clinical diagnosis of AD have increased lipid peroxidation. The concentrations of certain proteins from lesions in AD are altered in cerebrospinal fluid, even at early stages of dementia. For example, the amount of tau in cerebrospinal fluid was found to be increased in AD relative to controls, most likely reflecting release from damaged axons or soma, tangle formation, or both (Galasko, 1998). A(3(1 is decreased in cerebrospinal fluid, perhaps because brain deposits act as a sink and bind secreted Af3 (| 42) (Andreasen et al., 1999). The concentrations of tau and AF3 42)can remain stable over several weeks or months once altered (Andreasen et al., 1999; Sunderland et al., 1999). Treatments that achieve neuroprotection in AD could result in decreases in the amount of tau and increases in the amount of A(3 (14?) . Collectively, these data support the premise that biomarkers, especially in cerebrospinal fluid, and possibly also in blood, can be used to provide evidence for a biological action of an antioxidant and antiamyloid fibrillogenesis in AD. Importantly, biomarker data may help to support further development of Phase II studies for indole-3-propionic acid and other therapeutic candidates in AD. Indole-3-propionic acid in AD patients provides a special opportunity to explore the relationship between doses of medication and central antioxidant effects. It also promises to contribute to our knowledge regarding the effects of an antioxidant/antifibrillogenic compound on indices of oxidative stress, such as F2-isoprostanes, and on indices of neuronal damage, such as cerebrospinal fluid tau or A(3(| 42).

REFERENCES Alafuzoff, I., Helisalmi, S., Heinonen, E. H., Reinikainen, K., Hallikainen, M, Soininen, H., & Koivisto, K. (2000). Selegiline treatment and the extent of degenerative changes in brain tissue of patients with Alzheimer's disease. European journal of Clinical Pharmacology, 55, 815-819.

168

Drug Development—Preclinical

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Andreasen, N., Hesse, C, Davidsson, P., Minthon, L., Wallin, A., Winblad, B., Vanderstichele, H., Vanmechelen, E., & Blennow, K. (1999). Cerebrospinal fluid beta amyloid (1-42) in Alzheimer's disease: Differences between earlyand late-onset Alzheimer's disease and stability during course of the disease. Archives of Neurology, 56, 673-680. Andreasen, N., Minthon, L., Clarberg, A., Davidsson, T., Gottfries, J., Vanmechelen, E., Vanderstichele, H., Winblad, B., & Blennow, K. (1999). Sensitivity, specificity and stability of CSF-tau in a community-based patient sample. Neurology, 53,1488-1494. Bastianetto, S., Ramassamy, C, Dore, S., Christen, Y, Poirier, J., & Quirion, R. (2000). The ginko biloba extract (Egb 761) protects hippocampal neurons against cell death induced by (3-amyloid. Journal ofNeuroscience, 2,1882-1890. Chyan, Y. J., Poeggeler, B., Omar, R. A., Chain, D. G., Frangione, B., Ghiso, J., & Pappolla, M. A. (1999). Potent neuroprotective properties against the Alzheimer beta-amyloid by an endogenous melatonin-related iridole structure, indole-3-propionic acid. Journal of Biological Chemistry, 274,21937-21942. Dickson, D. W. (1997). The pathogenesis of senile pathogenesis. Journal of Neuropathology and Experimental Neurology, 56, 321-339. Galasko, D. (1998). Cerebrospinal fluid levels of A beta 42 and tau: Potential markers of Alzheimer's disease. Journal of Neural Transmission Supplement, 53, 209-221. Green, P. S., & Simpkins, J. W. (2000). Neuroprotective effects of estrogens: Potential mechanisms of action. International Journal of Developmental Neuroscience, 18, 347-358. Haase, J., Halama, P., & Horr, R. (1996). Effectiveness of brief infusions with Gingko biloba Special Extract RGb 761 in dementia of the vascular and Alzheimer type. Gerontology and Geriatrics, 29, 302-309. Holcomb, L., Gordon, M. N., McGowan, E., Yu, X., Benkovic, S., Jantzen, P., Wright, K., Saad, I., Mueller, R., Morgan, D., Sanders, S., Zehr, C., O'Campo, K., Hardy, J., Prada, C. M., Eckman, C., Younkin, S., Hsiao, K., & Duff, K. (1998). Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes. Nature Medicine, 4, 97-100. Johnstone, M., Gearing, A. J., & Miller, K. M. (1999). A central role for astrocytes in the inflammatory response to beta-amyloid; chemokines, cytokines and reactive oxygen species are produced. Journal of Neuroimmunology, 93,182-193. Le Bars, P. L., Katz, M. M., Berman, N., Itil, T. M., Freedman, A. M., & Schatzberg, A. F. (1997). A placebo-controlled, double blind, randomized trial of an extract of Gingko biloba for dementia. Journal of the American Medical Association, 278, 1327-1332. Markesbery, W. R., & Carney, J. M. (1999). Oxidative alterations in Alzheimer's disease. Brain Pathology, 9,133-146. Mattson, M. P., Pedersen, W. A., Duan, W., Culmsee, C., & Camandola, S. (1999). Cellular and molecular mechanisms underlying perturbed energy metabolism and neuronal degeneration in Alzheimer's disease and Parkinson's disease. Annals of the New York Academy of Sciences, 893,154-175. Maurer, K., Ihl, R., Dierks, T., & Frolich, L. (1997). Clinical efficacy of Ginko biloba

Antioxidants in the Treatment of AD 169

169

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

special extract Egb 761 in dementia of the Alzheimer's disease type. Journal of Psychiatric Research, 31, 645-655. Morris, M. C., Beckett, L. A., Scherr, P. A., Herbert, L. E., Bennett, D. A., Field, T. S., & Evans, D. A. (1998). Vitamin E and vitamin C supplement use and risk of incident in Alzheimer's disease. Alzheimer's Disease and Associated Disorders, 12,121-126. Morita, I., Kawamoto, M., Hattori, M., Eguchi, K., Sekiba, K., & Yoshida, H. (1990). Determination of tryptophan and its metabolites in human plasma and serum by high performance liquid chromatography with automated sample cleanup system. Journal of Chromatography, 526, 367-374. Morita, I. M., Kawamoto, M., & Yoshida, H. (1992). Difference in the concentration of tryptophan metabolites between maternal and umbilical foetal blood. Journal of Chromatography, 576, 334-339. Mulnard, R. A., Cotman, C. W., Kawas, C., van Dyck, C. H., Sano, M., Doody, R., Koss, E., Pfeiffer, E., Jin, S., Gamst, A., Grundman, M., Thomas, R., & Thai, L. J. (2000). Estrogen replacement therapy for treatment of mild to moderate Alzheimer disease: A randomized controlled trial. Journal of the American Medical Association, 283,1007-1015. Neuzil, J., Thomas, S. R., & Stocker, R. (1997). Requirement for, promotion, or inhibition by alpha-tocopherol of radical-induced initiation of plasma lipoprotein lipid peroxidation. Free Radicals in Biology and Medicine, 22, 57-71. Paleogolos, M., Gumming, R. G., & Lazarus, R. (1998). Cohort study of vitamin C intake and cognitive impairment. American Journal of Epidemiology, 148,45-50. Pappert, E. J., Tangney, C. C., Goetz, C. G., Ling, Z. D., Lipton, J. W., Stebbins, G. T., & Carvey, P. M. (1996). Alpha-tocopherol in the ventricular cerebrospinal fluid of Parkinson's disease patients: Dose-response study and correlations with plasma levels. Neurology, 47,1037-1042. Pappolla, M. A., Omar, R. A., Kim, K. S., & Robakis, N. K. (1992). Immunohistochemical evidence of oxidative stress in Alzheimer's disease. American Journal of Pathology, 140, 621-628. Poeggeler, B., Pappolla, M. A., Hardeland, R., Rassoulpour, A., Hodgkins, P. S., Guidetti, P., & Schwarcz, R. (1999). Indole-3-propionate: A potent hydroxyl radical scavenger in rat brain. Brain Research, 815, 382-388. Pratico, D., Clark, C. M., Lee, V. M.-Y, Trojanowski, J. Q., Rokach, J., & Fitzgerald, G. A. (2000). Increased 8,12 iso-PF2alpha-VI in Alzheimer's disease. Annals of Neurology, 48, 809-812. Sano, M., Ernesto, C., Thomas, R. G., Klauber, M. R., Schafer, K., Grundman, M., Woodbury, P., Growdon, J., Cotman, C. W., Pfeiffer, E., Schneider, L. S., & Thai, L. J. (1997). A controlled trial of selegiline, alpha-tocopherol, or both as treatment for Alzheimer's disease. New England Journal of Medicine, 336,1216-1222. Selkoe, D. J. (1997). Alzheimer's disease: Genotypes, phenotypes, and treatments. Science, 275(5300), 630-631. Selkoe, D. J. (1999). Translating cell biology into therapeutic advances in Alzheimer's disease. Nature, 399(Suppl), A23-A31. Sunderland, T., Wolozin, B., Galasko, D., Levy, J., Dukoff, R., Bahro, M., Lasser, R., Motter, R., Lehtimaki, T., & Seubert, P. (1999). Longitudinal stability of CSF tau levels in Alzheimer's patients. Biological Psychiatry, 46, 750-755.

170

Drug Development—Preclinical

Young, S. N., Anderson, G. M., Gauthier, S., & Purdy, W. C. (1980). The origin of indoleacetic acid and indolepropionic acid in rat and human cerebrospinal fluid. Journal ofNeurochemistry, 34,1087-1092.

ACKNOWLEDGMENTS

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Mindset Biopharmaceuticals is supported through a program-related investment by the Institute for the Study of Aging and by grants from the Israel-US Binational Research and Development Foundation and the U.S. National Institute of Aging.

20

s/ in de

for Treatment and as

x. ph

Ml Muscarinic Agonists

p

AF150(S) and AF267B:

ru

m

Disease-Modifying Agents in Alzheimer's

vn

/fo

and Related Diseases

w

w

ABSTRACT

.c

he m

4a ll.

A. fisher, Z. Pittel, R. Brandeis, N. Bar-Ner, H. Sonego, I. Marcovitch, N. Natan, and R. Raring

ht

tp

://

w

Ml muscarinic agonists may provide a rational therapeutic strategy in Alzheimer's disease (AD). Such potential may be detected in our Ml agonists: AF102B (Cevimeline: FDA-approved for treatment of Sjogren's syndrome), AF150(S), and AF267B. These compounds promote the neurotrophic and nonamyloidogenic amyloid precursor protein (APPs)processing pathways; prevent (3-amyloid (Ap)- and oxidative stress-induced apoptosis; show neurotrophic-like effects; decrease tau protein hyperphosphorylation; and restore cognitive impairments with an excellent safety margin in several animal models for AD. Remarkably, a 19-month chronic treatment with AF15CKS) in aged microcebes (probably the best AD model) restored cognitive impairments. In apolipoprotein E-deficient mice, prolonged treatment with AF150(S) restored memory impairments, cholinergic hypofunction, and tau hyperphosphorylation. In rabbits, with A(3 sequence identical to the human Af}, both AF267B > AF150(S) reduced 171

272

Drug Development—Preclinical

s/ in de

x. ph

p

significantly cerebrospinal fluid (CSF) A(3(142) and A|3a.40), while AF102B reduced only A(3(1.40). Finally, AF102B reduced significantly CSF Aj3(total) in AD patients. Thus, such Ml agonists have beneficial effects on three major hallmarks of AD (cognition, A(3, and tau). Our study aims to expand the preclinical database and to enhance the potential of AF150(S) and AF267B in treatment (learning and memory) and as disease-modifying agents (modulation of A|3, tau phosphorylation, and neuroprotection, in vitro and in vivo). Therapeutic strategies of AD, based on Ml agonists alone or in drug combinations, will revolutionize our current concepts. These may also be expanded for other neurological, neuropsychiatric diseases and autoimmune diseases with a documented cholinergic deficiency.

INTRODUCTION

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

Alzheimer's disease (AD) is characterized, inter alia, by synaptic loss, neuritic plaques consisting of p-amyloid peptide (A(3), neurofibrillary tangles containing hyperphosphorylated tau proteins, and loss of cholinergic neurons in the basal forebrain (Perry et al., 1998; Ladner & Lee, 1998). One of the neurochemical hallmarks of AD is a presynaptic cholinergic hypofunction, while postsynaptic Ml muscarinic receptors (Ml mAChR) are relatively unchanged (Perry et al., 1998; Ladner & Lee, 1998). Since Ml mAChR have an important role in cognition and, in particular, in shortterm memory, which is impaired in AD, Ml muscarinic agonists may be useful in treatment of the disease state (Fisher, 1997, 1999, 2000a, 2000b; Emmerling, Schwartz, Speigel, & Callahan, 1997). Such strategy should be less affected by the extent of degeneration of presynaptic cholinergic terminals, and, thus, may represent a more rational approach than the FDAapproved acetylcholinesterase inhibitors (AChE-Is) for treatment for AD. However, some agonists showed disappointing clinical results in AD. This may be due, perhaps, to: lack of selectivity to Ml mAChR (e.g., milameline); low intrinsic activity (e.g., alvameline); very low bioa variability and extensive metabolism (e.g,. xanomeline; also M4>M1 agonist); and narrow safety margin (e.g., all of the above and sabcomeline) (Fisher, 1997, 1999,2000). These major limitations may have precluded proper testing of the clinical concept underlying the use of Ml agonists in AD treatment. Recent studies showed a relation between loss of cholinergic hypofunction in AD brains, formation of A(3, and hyperphosphorylation of tau proteins (Ladner & Lee, 1998; Fisher, 1999, 2000; Emmerling et al., 1997; Sabbagh, Galsako, & Thai, 1997; Pa via, de Ceballos, & de la Cuesta, 1998; Hellstrom-Lindahl, 2000; Nitsch, Deng, Tennis, Schoenfield, & Growdon, 2000). These indicate that Ml muscarinic agonists may have a new clinical merit by providing an unexplored therapy in AD.

AF150(S) and AF267B

173

s/ in de

x. ph

p

We have designed and developed selective Ml agonists, the AF series, which are rigid analogs of acetylcholine (ACh), including: AF102B (the first reported Ml agonist; Cevimeline); FDA-approved for treatment of Sjogren's syndrome (Fox, 2000), and the project drugs, AF150(S) and AF267B (Figure 20.1) (Fisher, 1999, 2000). These compounds via activation of Ml mAChR mediate, inter alia, learning and memory with an excellent safety margin, modify A(3 processing and decrease in tau protein hyperphosphorylation (Fisher 1997,1999,2000). Therefore, the working hypothesis of our study focuses on the idea that our new Ml agonists may be useful both in treatment of AD and as disease-modifying agents.

RESULTS AND DISCUSSION

Neurotrophic Properties of the Tested Compounds

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

We have shown that carbachol (CCh) and AF102B induce differentiation in PC12 cells that were stably transfected with Ml mAChR (PC12M1) cells, and these neurotrophic-like effects are synergistic with neurotrophins (Gurwitz et al., 1995; Fisher, Brandeis, Chapman Pittel, & Michaelson, 1998). We have now expanded these findings to AF150(S) and AF267B. The results demonstrate that growth factors and muscarinic agonists induce neurite outgrowth via distinct intracellular pathways, which crosstalk with each other (Fisher et al., 1999). There is a significant difference between the cellular response to nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) (these induce differentiation) and epidermal growth factor (EGF; induces proliferation) of PC12M1 cells. Interestingly, the proliferating profile of EGF was changed in the presence of these muscarinic agonists as together they induced an accelerated differentiation. This may indicate that activation of Ml mAChR leads to activation of intracellular signaling pathway that results, finally, in differentiation. One such possible pathway is a change in tyrosine phosphorylation of extracellular signal-regulated kinases (ERKs), in response to EGF, from a transient to a sustained activation after combined activation of both receptors (Fisher, 1999).

Ml Agonists, Modulation of Amyloid Precursor Protein Processing At least two major pathways control the processing of amyloid precursor proteins (APPs): (a) cleavage by a-secretase of APP in the middle of its (3-amyloid region to produce the secreted, neurotrophic, and neuroprotective APP fragment (a-APPs); and (b) cleavage to form A(3 via activation of (3- and y-secretases (Sabbagh et al., 1997)

Drug Development—Preclinical

x. ph

p

174

FIGURE 20.1 The AF series vs. Acetylcholine.

s/ in de

Effects in vitro

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

Stimulation of Ml mAChRs can increase formation of a-APPs, preventing the formation of A(3 (Nitsch, Slack, Wurtman, & Growdon, 1992; Buxbaum et al., 1992; Haring et al., 1994, 1995, 1998; Eckols et al., 1995; Muller, Mendla, Farber, & Nitsch, 1997). An increased secretion of a-APPs in various in vitro systems resulted in decreased synthesis of A|3, following treatment with muscarinic agonists (Hung et al., 1993; Wolf et al., 1995). Additionally, Ml-selective agonists may alter APP processing in the cortex and hippocampus, where Ml mAChRs are abundant (Farber, Nitsch, Schulz, & Wurtman, 1995; Pittel, Heldman, Barg, Haring, & Fisher, 1996). A 1000-fold lower concentration of AF102B versus CCh enhance APP secretion in brain slices prepared from cerebral cortex (Pittel et al., 1996). In rat cortical and hippocampal primary cell cultures, AF150(S) and AF267B were more potent in elevating APPs than CCh and oxotremorine (both M2>M1 agonists) and physostigmine (AChE-I). Moreover, APP secretion was not induced following exposure of neurons prepared from spinal cord to muscarinic agonists, as these neurons do not contain Ml mAChR. Taken together, the results emphasize the unique importance of Ml mAChR activation in APP secretion. Ml muscarinic stimulation activates at least two transduction pathways that lead to a-APP secretion, as well as protein kinase C (PKQdependent and mitogen-activated protein kinase-dependent pathways. These pathways operate in parallel and converge with transduction pathways of neurotrophins, resulting in enhancement of APP secretion, when both muscarinic agonist and neurotrophins stimulate their respective receptors (Haring et al., 1995,1998). The Cholinergic System and A(3 Metabolism: Some Preclinical and Clinical Studies

Studies in vivo also support a linkage between the cholinergic system and AB metabolism:

AF150(S) and AF267B

175

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

1. Activation of PKC-decreased AP levels in transgenic mice that produce elevated human A(}(1_42) (Savage et alv 1998). Several laboratories are evaluating whether a decrease in AP should occur in such animals, following stimulation of Ml mAChR, since PKC activation is mediated also by this receptor. 2. Chronic cholinergic hypofunction in rats, induced by either the cholinotoxin AF64A (studies from our lab) or the immunotoxin 192 IgGsaporin, produced a decrease in ot-APPs that may indicate a reduction in a-secretase activity (Rossner et al., 1997; Lin, Georgievska, Mattson, & Isacson, 1999). 3. RS-86 (a muscarinic agonist with limited Ml selectivity) attenuated the effects of 192 IgG-saporin on APPs (Lin et al., 1999). 4. In rabbits, where the sequence of A(3(142) is identical to human A[3, a chronic cholinergic hypofunction induced by 192 IgG-saporin elevated PA in cortex and hippocampus (Beach et al., 2000). 5. In rabbits, both AF267B > AF150(S) significantly decreased CSF AP (142) and Ap (140) levels, while AF102B reduced only Ap(140) (Beach, Walker, Potter, & Fisher, unpublished observations). To the best of our knowledge, this is the first study that showed such an effect in vivo. 6. Chronic treatments with Ml agonists (AF102B and talsaclidine) significantly reduced CSF Ap in AD patients (Nitsch et al., 2000; Hock, Maddalena, Deng, Growdon, & Nitsh, 2000). These pioneering studies may indicate that Ml agonists have an important role in affecting AP processing, probably by reducing AP burden in AD patients. No other compounds were reported with such a unique profile in AD patients. Moreover, physostigmine (AChE-I) and hydroxychloroquine (anti-inflammatory drug) did not affect CSF AP levels when tested in AD patients in the same study of AF102B (Nitsch et al., 2000). Additionally, Aricept and galanthamine did not show significant effects on CSF AP levels in AD patients (Blennow et al., 2000).

ht

tp

The Effects of Ap and Oxidative Stress on Ml mAChR-Mediated Effects Subtoxic levels of AP disrupted mAChR coupling to G-proteins linked to phospholipid hydrolysis (IPs) without producing neuronal cell injury or death (Kelly et al., 1996). This may lead to decreased signal transduction, to a reduction in levels of trophic a-APPs, and a generation of more AP (Jope, 1996; Kelly et al., 1996; Fisher, 1997; Ladner & Lee, 1998). Some of these "vicious cycles" may be blocked by Mi-selective agonists (Fisher, 1997,1999, 2000b). Effects on Ml mAChR-Mediated Signaling The role of the mAChR subtype(s) involved in Ap-induced disruption of mAChR coupling to G-proteins was not elucidated, since studies used

176

Drug Development—Preclinical

m

s/ in de

x. ph

p

primary cell culture that may contain a mixture of mAChR subtypes. Therefore, we studied the effects of A(3(25 35) and A|3(]_42) on Ml mAChRmediated IPs and intracellular calcium [Ca2"^ elevations, in two types of cell cultures stably transfected with Ml mAChR, CHO and PC12 (Pittel, Haring, Natan, Marcovitch, & Fisher, 2000). A(3(2535) and A(3 (142) inhibited IPs and [Ca2+]j elevations induced by CCh, being less effective in inhibiting AF150(S)-induced effects. While ineffective on basal IPs and [Ca2+]., both peptides (50-500 nM) significantly inhibited Ml mAChR-induced IPs release by 20% to 30%, yet higher concentrations were ineffective. AP(M2) (1-10 uM), in a concentration-dependent manner, inhibited [Ca2+]. elevation up to 50%. A(3(142)-induced inhibition of Ml mAChR-mediated IPs release was blocked by antioxidants (e.g., vitamin E, propyl gallate, and PBN). These data confirm the findings by Kelly et al. (1996), yet unlike that study, the apparent disruptive effect of AP on Ml mAChR-induced IPs release is confined to only low Ap concentrations.

/fo

ru

Prevention of Cell Death and Apoptosis Induced by Starvation Alone, and in Presence of Aft or Oxidative Stress

://

w

w

w

.c

he m

4a ll.

vn

Apoptosis can be induced, inter alia, by oxidative insults, trophic-factor withdrawal, and aggregated AP (Mattson, 1997). Apoptosis may mediate the death of neurons in AD, and AP that are neurotoxic can be one of the causes for cell death (Mattson, 1997). Activation of Ml mAChR in PC12M1 cells inhibited apoptosis induced by growth-factor deprivation (Lindenboim, Pinkas-Kramarski, Sokolovsky, & Stein, 1995). This Ml mAChR-mediated effect blocks apoptosis-induced caspase activation by phosphatidylinositol 3-kinase and mitogen-activated protein kinase/extracellular signaling-regulated kinase (LeLoup et al., 2000). We hypothesized that apoptosis induced by insults, such as growth factor deprivation in combination with AP or oxidative stress induced by H2O2, can also be attenuated by Ml agonists.

ht

tp

Cell Viability. Starvation alone of PC12M1 cells induced about 20% to 30% reduction of cell viability, and this cell death was further exacerbated by Ap(25 35) to about 50% of control cells. CCh, AF150(S), and AF267B significantly protect the cells from death. Addition of NGF (50 ng/ml), as well as addition of antioxidants such as vitamin E, protected the cells from AP(25 35)-induced toxicity, indicating that the toxicity by AP may involve reactive-oxidative species (ROS) (Haring, Pittel, Eizenberg, & Fisher, 2000).

Apoptosis. We have confirmed apoptotic cells using methods such as DAPI (4,6-diamidino- 2-phenylindole), TUNEL, and flow cytometry (Haring et al., 2000). Ap-treated cells showed the morphology of apoptotic cells that

AF150(S) and AF267B

177

x. ph

p

shrinked and lost their processes. CCh, AF150(S), and AF267B protect these cells from apoptosis and the neuritic-like processes are well observed. A 200% increase in apoptotic cells was detected after starvation of PC 12 and PC12M1 cells, with a further increase of 50% after treatment with A(3(25 35) or A(3(142). In PC12M1 but not PC12 cells, these muscarinic agonists completely reversed apoptosis induced by starvation alone or combined with AP, indicating activation of Ml mAChR. In addition, after starvation alone or combined with AP, the fraction of apoptotic cell population was decreased by muscarinic agonists and this effect was blocked by atropine, indicating the involvement of mAChR. Taken together, our results show that:

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

1. Ml mAChR-mediated IPs and [Ca2^ elevations are impaired by A(3 (low concentrations), probably via disruption of Ml mAChR-Gq/11 coupling. 2. The impairment of IP elevation by AP (low concentrations) is less pronounced for AF150(S) than for carbachol. 3. This impairment in coupling and Ap-induced neurotoxicity (at slightly higher concentration) may imply formation of ROS, since it can be attenuated by antioxidants. 4. AP and H2O2 may cause apoptosis via a similar mechanism that probably involves ROS, as both insults were attenuated by antioxidants or the H2O2-scavenging enzyme catalase. 5. Activation of Ml mAChR in PC12M1 cells, but not endogenous mAChRs present in PCI2 cells (probably m4), is responsible for the protecting effects against starvation-, AP-, and oxidative stressinduced apoptosis. 6. The attenuation of Ap- and oxidative stress-induced apoptosis via Ml mAChR is a novel finding. This may add a significant new value for our compounds in AD therapy.

ht

tp

://

The mechanism whereby activation of Ml mAChR inhibits apoptosis induced by ROS or AP is poorly understood (see also Van et al., 2000; LeLoup et al., 2000). Furthermore, it is not yet clear why Ml agonists can more efficiently prevent Ap-induced toxicity (cell death and apoptosis) than Ap-induced uncoupling of Ml mAChR-G-protein(s). At this stage, it cannot be ruled out that: (a) the disruption induced by AP of Ml mAChRG protein coupling is less important for its toxic effect than originally envisaged; (b) other early signal-transduction pathways are more relevant to the role of Ml mAChR in prevention of Ap-induced neurotoxicity; (c) there are different signaling pathways responsible for these two effects; and (d) we have unveiled an unknown signaling pathway. The answers for these await further investigation.

178

Drug Development—Preclinical

Ml mAChR-Dephosphorylation of Tau Proteins

m

s/ in de

x. ph

p

Activation of Ml mAChRs decreases tau protein phosphorylation. This was first shown by us in PC12M1 cells (Sadot et al., 1996) and confirmed in vitro (cell cultures) (Forlenza, Spink, Oleson, Anderton, & Lovestone, 1998) and in vivo (in apolipoprotein E (apoE)-deficient mice) (Genis, Fisher, & Michaelson, 1999). Thus, activation of Ml mAChRs may provide a novel treatment strategy for AD by modifying tau processing in the brain. The effects on tau proteins suggest a link between Ml mAChR-mediated signal transduction system(s) and the neuronal cytoskeleton via regulation of phosphorylation of tau microtubule-associated protein. Moreover, this may indicate a dual role for Ml agonists: as inhibitors of vicious cycles induced by A(3 and overactivation of certain kinases (e.g., GSK3-(3) and/or down-regulation of phosphatases, respectively (Fisher, 1999; Johnson & Hartigan, 1998; Forlenza et al., 1998; Lovestone & Reynolds, 1997).

ru

Ml Muscarinic Agonists in Animal Models for AD

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

AF102B, AF150(S), and AF267B restored memory and learning deficits in several animal models that mimic cholinergic and/or other deficits reported in AD. These agonists have a wider safety margin (>200-500 fold) when compared with other compounds of the same class (Fisher, 1997, 1999, and our unpublished results). From two chronic studies with AF150(S) in apoE-deficient mice and the aged primate, Microcebus murinus, it can be predicted that: (a) AF150(S) may have disease-modifying properties (Fisher et al., 1998; Genis et al., 1999; Fisher, Kealler, & Bons, 2000); and (b) no tolerance is expected to occur in AD patients when treated with partial Ml agonists, such as AF150(S) and AF267B. Remarkably, the aged Microcebus murinus develop parenchymal A(3 plaques and abnormally phosphorylated tau proteins within the cortex. Neuronal loss, a basal cholinergic degeneration (e.g., cholinergic hypofunction) and behavioral and cognitive impairments accompany these pathological alterations (Bons et al., 1995; Silhol et al., 1996; Giannakopoulos et al., 1997). In this unique model (perhaps the best natural model for AD), AF150(S) has positive effects on cognition and behavior, and its effects do not diminish with time. On the contrary, an increase in performance and the number of responders is observed (Fisher et al., 2000). Finally, the pharmacokinetic profile of AF150(S) and AF267B indicate a relatively long half-life, high bioavailability, and fast brain penetration. In summary, these agonists are suitable for treatment of AD. Conclusions, Positions, and Future Perspectives A treatment of AD patients with Ml muscarinic agonists is probably not wrong, yet it requires a critical reassessment. Failure of some muscarinic

AF150(S) and AF267B 179

179

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

agonists that, a priori, had major clinical limitations cannot be used against this therapeutic strategy, as these cannot be considered Ml selective. The pharmaceutical history teaches us that sometimes the first generation, even when it failed, paved the way to more effective second-generation therapeutics with similar mechanism, providing there is a sensible hypothesis that can support such a strategy. Notably, recent findings indicate that the basic approach may still be valid, yet the original cholinergic hypothesis regarding Ml agonists was perhaps oversimplistic. Cholinergic hypofunction in AD is critical for the pathogenesis of nonfamilial AD. In this context, activation of Ml mAChR could beneficially modulate dysfunctions that are associated with AD, including A(3 and tau proteins, ApoE, as well as some processes involving certain G-proteins and neurotrophins (Fisher, 1999, 2000a, 2000b). Several therapeutic strategies in AD are focused on modulation of APP metabolism and secretion, A(3 formation, and/or aggregation. In this context, the ability of Ml agonists to stimulate APPs secretion and AP reduction might be a viable strategy to lower Ap. This can be concluded from the study of AF102B (Nitsch et al, 2000) and talsaclidine (Hock et al, 2000) in AD patients. Ml-selective agonists may allow testing effects on AP and tau in AD patients, perhaps more directly than with other tools. This may modify the cholinergic hypothesis in AD that was limited to a symptomatic treatment, and ignored the added value of Ml agonists as potential disease-modifying agents. Studies with highly selective Ml agonists in animal models mimicking various aspects of the AD pathology (e.g., transgenic animals that express one or several pathogenic proteins) may test this unifying hypothesis. A safe and selective Ml agonist should be tested in a therapy aimed to slow down the progression of AD or the onset of dementia in various target populations at risk. AF102B, AF150(S), and AF267B and some other highly selective Ml agonists may fulfill such acceptance criteria. A muscarinic treatment of AD will modify current opinions about such treatments. It may also expand their use in polypharmacy and in other neurological, neuropsychiatric diseases and autoimmune diseases with a documented cholinergic deficiency. In this context, it is relevant to mention the recent FDA approval of Cevimeline (AF102B) for treatment of dry mouth in Sjogren's syndrome. This drug might prove to be effective in the future in other diseases, such as AD.

REFERENCES Beach, T. G., Potter, P. E., Kuo, Y, Emmerling, M. R., Durham, R. A., Webster, S. D., Walker, D. G., Sue, L. I., Scott, S., Layne, K. J., & Roher, A. E. (2000).

180

Drug Development—Preclinical

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Cholinergic deafferentation of the rabbit cortex: A new animal model of Abeta deposition. Neuroscience Letters, 31, 9-12. Blennow, K. (2000). CSF biochemical markers for the early detection of Alzheimer's disease. 6th International Stockholm/Springfield Symposium on Advances in Alzheimer Therapy, April 5-8, 2000. Bons, N., Jallageas, V., Maestre-Frances, N., Silhol, S., Fetter, A. & Delacourte, A. (1995). Microcebus murinus, a convenient laboratory animal for the study of Alzheimer's disease. Alzheimer's Research, 1, 83-87. Buxbaum, J. D., Oishi, M, Chen, H. I., Pinkas-Kramarski, R., Jaffe, E. A., Gandy, S. E., & Greengard, P. (1992). Cholinergic agonists and interleukin 1 regulate processing and secretion of the Alzheimer beta-A4 amyloid protein precursor. Proceedings of the National Academy of Sciences (USA), 89,10075-10078. Eckols, K., Bymaster, F. P., Mitch, C. H., Shannon, H. E., Ward, J. S., & DeLapp, N. W. (1995). The muscarinic Ml agonist xanomeline increases soluble amyloid precursor protein release from CHO Ml cells. Life Sciences, 57,1183-1190. Emmerling, M. R., Schwartz, R. D., Speigel, K., & Callahan, M. J. (1997). New perspectives on developing muscarinic agonists for treating Alzheimer's disease (database). Alzheimer's Disease, 2(4). Farber, S. A., Nitsch, R. M., Schulz, J. G., & Wurtman, R. J. (1995). Regulated secretion of (3-amyloid precursor protein in rat brain. Journal of Neuroscience, 15, 7442-7451. Fisher, A. (1997). Muscarinic agonists for the treatment of Alzheimer's disease: Progress and perspectives. Expert Opinion on Investigational Drugs, 6,1395-1411. Fisher, A. (1999). Muscarinic receptor agonists in Alzheimer's disease. More than just symptomatic treatment. Central Nervous System Drugs, 12,197-214. Fisher, A., Brandeis, R., Chapman Pittel, Z., & Michaelson, D. M. (1998). Ml muscarinic agonist treatment reverses cognitive and Cholinergic impairments of apolipoprotein E-deficient mice. Journal of Neurochemistry, 70,1991-1997. Fisher, A., Kealler, E., & Bons, N. (2000, July 9-13). Cognitive and behavioral improvements in the aged primate Microcebus murinus following one year treatment with the Ml muscarinic agonist, AF150(S). World Conference on AD: Washington, DC. Fisher, A. (2000a). Ml Muscarinic agonists—their potential in treatment and as disease-modifying agents in Alzheimer's disease. Drug Development Research, 50, 291-297. Fisher, A. (2000b). Therapeutic strategies in Alzheimer's disease: Ml muscarinic agonists. Japanese Journal of Pharmacology, 84,101-112. Forlenza, O., Spink, J., Oleson, O., Anderton, B. H., & Lovestone, S. (1998). Muscarinic agonists reduce tau phosphorylation in transfected cells and in neurons. Neurobiology of Aging, 19, S218. Fox, R. I. (2000). Sjogren syndrome: New approaches to treatment [on-line]. http://www.medscape.com/medscape/Rheumatology/ Available: TreatmentUpdate/2000/tuOl/toc-tuOl.html; http://www.centerwatch.com/ drugs/dru600.htm; http://www.nacds.org/news/memo.html Genis, I., Fisher, A., & Michaelson, D. M. (1999). Site-specific dephosphorylation of tau in apolipoprotein E-deficient and control mice by Ml muscarinic agonist treatment. Journal of Neurochemistry, 12, 206-213.

AF150(S) and AF267B

181

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Giannakopoulos, P., Silhol, S., Jallageas, V., Mallet, ]., Bons, N., Bouras, C., & Delaere, P. (1997). Quantitative analysis of tau protein-immunoreactive accumulations and beta-amyloid protein deposits in the cerebral cortex of the mouse lemur, Microcebus murinus. Ada Neuropathologica, 94,131-139. Gurwitz, D., Haring, R., Pinkas-Kramarski, R., Stein, R., Heldman, E., Karton, Y., & Fisher, A. (1995). NGF-dependent neurotrophic-like effects of AF102B, an Ml muscarinic agonist, in PC12M1 cells. Neuroreport 6, 485^188. Haring, R., Gurwitz, D., Barg, J., Pinkas-Kramarski, R., Heldman, E., Pittel, Z., Wengier, A., Meshulam, H., Karton, Y, & Fisher, A. (1994). Amyloid precursor protein secretion via muscarinic receptors: Reduced desensitization using the Ml-selective agonist AF102B. Biochemical Biophysical Research Communications, 203, 652-658. Haring, R., Gurwitz, D., Barg, J., Pinkas-Kramarsky, R., Heldman, E., Pittel, Z., Danenberg, H. D., Wengier, A., Meshulam, H., Marciano, D., Karton, Y, & Fisher, A. (1995). NGF promotes amyloid protein secretion via muscarinic receptor activation. Biochemical Biophysical Research Communications, 213, 15-23. Haring, R., Pittel, Z., Eizenberg, O., & Fisher, A. M. (2000, July 9-13). M? muscarinic agonists protect PC12M1 cells from growth factor deprivation and beta-amyloidinduced apoptosis. World Conference on AD, Washington DC. Haring, R., Fisher, A., Marciano, D., Pittel, Z., Kloog, Y, Zuckerman, A., Eshhar, N., & Heldman, E. (1998). Mitogen-activated protein kinase-dependent and protein kinase C-dependent pathways link the ml muscarinic receptor to amyloid precursor protein secretion. Journal of Neurochemistry, 71, 2094-2103. Hellstrom-Lindahl, E. (2000). Modulation of beta-amyloid precursor protein processing and tau phosphorylation by acetylcholine receptors. European Journal of Pharmacology, 393, 255-263. Hock, C., Maddalena, A., Deng, M., Growdon, J. M., & Nitsch, R. M. (2000, February 18-20). Treatment with the selective muscarinic agonist talsaclidine decreases cerebrospinal fluid levels of total amyloid beta-peptide in patients with Alzheimer's disease. Proceedings of the 9th Meeting of the International Study Group on the Pharmacology of Memory Disorders Associated with Aging. Zurich, Switzerland. Hung, A. Y, Haass, C., Nitsch, R., Qiu, W. Q., Citron, M., Wurtman, R. J., Growdon. J. H., & Selkoe, D. J. (1993). Activation of protein kinase C inhibits cellular production of the amyloid beta-protein. Journal of Biological Chemistry, 268, 22959-22962. Johnson, G. V. W., & Hartigan, J. A. (1998). Tau protein in normal and Alzheimer's disease brain: An update. Alzheimer's Disease Review, 3,125-141. Jope, R. S. (1996). Cholinergic muscarinic receptor signaling by phosphoinositides signal transduction system in Alzheimer's disease. Alzheimer's Disease Review, 1,2-14. Kelly, J. F, Furukawa, K., Barger, S. W., Rengen, M. R., Mark, R. J., Blanc, E. M., Roth, G. S., & Matson, M. P., (1996). Amyloid beta-peptide disrupts carbacholinduced muscarinic cholinergic signal transduction in cortical neurons. Proceedings of the National Academy of Sciences (USA), 96, 6753-6758. Ladner, C. J., & Lee, J. M. (1998). Pharmacological drug treatment of Alzheimer

182

Drug Development—Preclinical

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

disease: The cholinergic system revisited. Journal of Neuropathology and Experimental Neurology, 57, 719-731. LeLoup, C., Michaelson, D. M, Fisher, A., Hartman, T., Beyreuther, K., & Stein, R. (2000). Ml muscarinic receptors blocks caspase activation by phosphatidylinositide 3-kinase and MAPK/ERK-independent pathways. Cell Death & Differentiation, 7, 825-833 Lin, L., Georgievska, B., Mattsson, A., & Isacson, O. (1999). Cognitive changes and modified processing of amyloid precursor protein in the cortical and hippocampal system after cholinergic synapse loss and muscarinic receptor activation. Proceedings of the National Academy of Sciences (USA), 96,12108-12113. Lindenboim, L., Pinkas-Kramarski, R., Sokolovsky, M., & Stein, R. (1995). Activation of muscarinic receptors inhibits apoptosis in PC12M1 cells. Journal of Neurochemistry, 64, 2491-2499. Lovestone, S., & Reynolds, C. H. (1997). The phosphorylation of tau: A critical stage in neurodevelopment and neurodegenerative processes. Neuroscience, 78, 309-324. Mattson, M. P. (1997). Central role of oxyradicals in the mechanism of amyloid beta-peptide cytotoxicity. Alzheimer's Disease Review, 2,1-14. Muller, D. M., Mendla, K., Farber, S. A., & Nitsch, R. M. (1997). Muscarinic Ml receptor agonists increase the secretion of the amyloid precursor protein ectodomain. Life Sciences, 60, 985-991. Nitsch, R. N., Slack, B. E., Wurtman, R. J., & Growdon, J. H. (1992). Release of Alzheimer amyloid precursor derivatives stimulated by activation of muscarinic acetylcholine receptors. Science, 58, 304-307. Nitsch, R. M., Deng, M., Tennis, M., Schoenfield, D., & Growdon, J. H. (2000). The selective muscarinic Ml agonist AF102B decreases levels of total A (beta) in cerebrospinal fluid of patients with Alzheimer's disease. Annals of Neurology, 48, 913-918. Pavia, J., de Ceballos, M., & de la Cuesta, F. S. (1998). Alzheimer's disease: Relationship between muscarinic cholinergic receptors, beta-amyloid and tau proteins. Fundamentals of Clinical Pharmacology, 12, 473-481. Perry, E., Court, J., Goodchild, R., Griffiths, M., Jaros, E., Johnson, M., Lloyd, S., Piggott, M., Spurden, D., Ballard, C., McKeith, L, & Perry, R. (1998). Clinical neurochemistry: Developments in dementia research based on brain bank material. Journal of Neural Transmission, 105, 915-933. Pittel, Z., Heldman, E., Barg, J., Haring, R., & Fisher, A. (1996). Muscarinic control of amyloid precursor protein secretion in rat cerebral cortex and cerebellum. Brain Research, 742, 299-304. Pittel, Z., Haring, R., Natan, N., Marcovitch, L, & Fisher, A. (2000). Beta-amyloids impair Ml muscarinic receptor-mediated signaling yet beta-amyloidsinduced cell death is attenuated by Ml agonists. Society for Neurosciences Abstracts. Rossner, S., Ueberham, U., Yu, J., Kirazov, L., Schliebs, R., Perez-Polo, R., & Bigl, V. (1997). In vivo regulation of amyloid precursor protein secretion in rat neocortex by cholinergic activity. European Journal of Neurosciences, 9, 2125-2134. Sabbagh, M. N., Galsako, D., & Thai, L. J. (1997). Beta-amyloid and treatment opportunities for Alzheimer's disease. Alzheimer Disease Review, 3, 1-19.

AF150(S) and AF267B

183

vn

ACKNOWLEDGMENT

/fo

ru

m

s/ in de

x. ph

p

Sadot, E., Gurwitz, D., Barg, J., Behar, L., Ginzburg, L, & Fisher, A. (1996). Activation of ml-muscarinic acetylcholine receptor regulates tau phosphorylation in transfected PC12 cells. Journal of Neurochemistry, 66, 877-880. Savage, M. J., Trusko, S. P., Howland, D. S., Pinsker, L. R., Mistretta, S., Reaume, A. G., Breenberg, B. D., Siman, D., & Scott, R. W. (1998). Turnover of amyloid beta-protein in mouse brain and acute reduction of its level by phorbol ester. Journal ofNeuroscience, 185,1743-1752. Silhol, S., Calenda, A., Jallageas, V., Mestre-Frances, N., Bellis, M., & Bons, N. (1996). Beta-amyloid protein precursor in Microcebus murinus: Genotyping and brain localization. Neurobiology of Disease, 3,169-182. Wolf, B. A., Wertkin, A. M., Jolly, Y. C., Yasuda, R. O., Wolfe, B. B., Konrad, R. J., Manning, D., Ravi, S., Williamson, J. R., & Lee, V. M.-Y. (1995). Muscarinic regulation of Alzheimer's disease amyloid precursor protein secretion and amyloid beta-protein production in human neuronal NT2N cells. Journal of Biological Chemistry, 270, 4916-4922. Yan, X.-Z., Xiao, R., Dou, Y, Wang, S.-D., Qiao, Z.-D., & Qiao. J.-T. (2000). Carbachol blocks beta-amyloid fragment 31-35-induced apoptosis in cultured cortical neurons. Brain Research Bulletin, 51, 465-470.

ht

tp

://

w

w

w

.c he

m

4a ll.

Supported in part by the Institute for the Study of Aging, New York, NY, USA.

21

x. ph

s/ in de

Cognition in Mature and Aged Rats

p

ABS-2057lEffectson

vn

/fo

ru

m

Ciaran M. Regan

4a ll.

INTRODUCTION

ht

tp

://

w

w

w

.c

he m

Valproic acid (VPA), a branched-chain carboxylic acid, is an effective antiepileptic agent that is especially useful in the treatment of childhood petit mal seizures (Loscher, 1999). Despite the extensive use of VPA, no conclusive mechanism has been described for its antiseizure activity. Studies with cultured neural cells have revealed VPA to elicit a profound neuritogenic action, coupled with proliferative arrest at a restriction point in the Gl-phase of the cell cycle (Regan, 1985; Martin & Regan, 1991). This neuritogenic effect could be enhanced by the introduction of a triple bond into one chain, producing a 4-pentynoic acid derivative, and n-alkyl chains of increasing length (Bojic et al., 1998). When the n-alkyl chain length was extended to 5 carbon units (2-n-pentyl-4-pentynoic acid; ABS-205), a dose-dependent increase in neural cell adhesion molecule (NCAM) prevalence was observed at points of cell-cell contact. These simple modifications to the basic structure of VPA have provided an analogue, ABS-205, with in vitro actions not unlike those of the neurotrophins, as such growth factors augment neurite formation and increase NCAM prevalence (Doherty, Mann, & Walsh, 1988; Park, Lucka, Reutter, & Horstkorte, 1997). The neurotrophin-like actions of ABS-205 suggested this VPA analogue to be a putative cognition-enhancing drug. Synapse growth, aided by cell-adhesion molecule function, is required for the induction and 184

ABS-205: Effects on Cognition

185

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

maintenance of long-term potentiation (LTP) and during memory consolidation in all paradigms and species investigated to date (Geinisman, deToledo-Morrell, & Morrell, 1991; O'Malley, O'Connell, & Regan, 1998; O'Malley, O'Connell, Murphy, & Regan, 2000; Schuster, Krug, Hassan, & Schachner, 1998; Skibo, Da vies, Rusakov, Stewart, & Schachner, 1998; Toni, Buchs, Nikonenko, Bron, & Muller, 1999). NCAM, for example, has been implicated in the consolidation of passive-avoidance learning in the chick and rat, as intracerebroventricular administration of anti-NCAM in the 6 to 8 hour posttraining period eliminates recall (Doyle, Nolan, Bell, & Regan, 1992; Scholey, Rose, Zamani, Bock, & Schachner, 1993). This period of consolidation coincides with the transient, two-fold increase in spine formation observed in the molecular layer of the rat dentate gyrus and a doubling of NCAM-immunopositive synapses of the chick lobus parolfactorius (Skibo et al, 1998; O'Malley et al, 1998, 2000). Although the in vitro actions of neurotrophins and ABS-205 suggest they may have cognition-enhancing potential, there is little in vivo behavioral evidence for induced neuritogenesis and increased NCAM prevalence to be predictive endpoints for such an effect. We now provide in vivo evidence that demonstrates ABS-205 to be an effective cognitionenhancing agent. Moreover, we demonstrate that chronic administration of ABS-205 improves learning-associated neuroplasticity through enhanced glycosylation of NCAM with a2,8-linked homopolymers of polysialic acid (PSA). This posttranslational modification of NCAM is required for synapse growth, induction and maintenance of LTP, and consolidation of avoidance and spatial learning paradigms (Fox, O'Connell, Murphy, & Regan, 1995a; Becker et al., 1996; Murphy, O'Connell, & Regan, 1996; Theodosis, Bonhomme, Vitiello, Rougon, & Poulain, 1999). Moreover, age-related loss of NCAM polysialylation has been implicated in the decline of neuroplastic potential (Fox, Kennedy, & Regan, 1995b; Ni Dhuill et al., 1999).

ht

INFLUENCE OF ABS-205 ON SCOPOLAMINECOMPROMISED PASSIVE-AVOIDANCE LEARNING

The effect of ABS-205 in periods of synapse growth and NCAM-mediated memory consolidation was investigated by determining its ability to ameliorate scopolamine-induced amnesia of the one-trial, step-through, lightdark model of the passive-avoidance paradigm (Fox et al., 1995a). As the amnesic action of scopolamine is specific to a 6-hour posttraining period (Doyle & Regan, 1993), the ability of ABS-205 to attenuate this amnesia was determined by administering the drug at the 3-hour posttraining time and assessing its effect on recall at 24 hours following task acquisition. In this

186

Drug Development—Preclinical

x. ph

p

task, acute administration of ABS-205 reversed the scopolamine-induced amnesia in a dose-dependent manner. This antiamnesic effect could not be attributed to a direct influence on cholinergic mechanisms, since serial, whole-blood samples obtained from a jugular vein cannula indicated ABS-205 plasma Cmax to be attained within 15 to 30 minutes following a single intraperitoneal injection, and the drug was virtually eliminated within 2 hours. Moreover, a SpectrumScreen® radioligand-binding study indicated ABS-205 to lack any significant receptor affinity and suggested its action to be mediated through a specific but unknown receptor, as has been proposed previously (Bojic et al., 1998).

s/ in de

INFLUENCE OF ABS-205 ON WATER MAZE LEARNING

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

This spatial learning task has been described in detail previously (Morris, Garrud, Rawlins, & O'Keefe, 1982; Murphy et al., 1996). Each training session started with the rat facing the wall of the water maze at one of three locations, and the time taken to find the hidden platform, within a 60-second period, was defined as the escape-latency time. Escape latencies were measured over 5 trials in each training session with intertrial intervals of 300 seconds. The animals were observed over a total of four training sessions spaced 24 hours apart. This was followed by a transfer test to examine recall of the platform position 3 days after the final training session. Here, the platform was removed and the time spent in each quadrant was measured by a computerized tracking system. Visuomotor controls were required to locate a white platform raised above the water surface (2 cm) in the same quadrant used for the training trials. To investigate the influence of ABS-205 on water maze learning, the drug was administered immediately prior to training on each of 4 consecutive days. At a dose of 84 mg/kg, the time required to locate the platform was significantly reduced by the fifth trial on each training day, as compared with the saline controls (Figure 21.1). Spatial learning was similarly improved at a dose of 50.4 mg/kg, but only on the first day of training. When recall of the task was determined 3 days after the final training session, animals that previously received 84 mg/kg spent 40.9% of the test time in the target quadrant, as compared with 29.7% for the control animals (Figure 21.1). To determine if the cognition-enhancing effects of ABS-205 on spatial learning were enduring, animals were administered a daily intraperitoneal injection of either 16.8 mg/kg or 50.4 mg/kg ABS-205 over the period from postnatal day 40 to postnatal day 80, and training commenced 24 hours following the last drug administration. Chronic administration of ABS-205 showed no effect on animals' weight gain over the period of administration,

on Cognition

187

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

ABS-205: Effects

ht

tp

://

w

w

w

.c

FIGURE 21.1 Influence of subacute administration of ABS-205 on water maze performance. Panel A illustrates the escape latencies observed in the first training session. The escape latencies for the various treatment groups on the fifth trial of each of the four training sessions are shown in Panel B. All values are expressed as the mean ± SEM (n = 8). Panel C shows the percentage time spent in the target and opposite quadrant during the 60-second period of the recall test (6 < n < 8). Values significantly different (P < 0.05) from the saline-treated control are indicated with an asterisk.

and examination of peripheral tissue samples, taken at time of sacrifice, revealed no overt signs of structural damage. In the lung, normal respiratory bronchioles, alveolar sacs, and alveoli were visible. Structurally intact proximal and distal tubules were obvious in the kidney samples and intact hepatocytes with varying amounts of glycogen were numerous in the liver. As with the subacute spatial-learning study, these animals

188

Drug Development—Preclinical

x. ph

INFLUENCE OF ABS-205 ON NEURAL CELL ADHESION MOLECULE POLYSIALIC ACID EXPRESSION

p

exhibited a significantly better performance on the final trial in each of the first three sessions at both of the doses employed, the effect persisting into the final session at the higher dose. This latter group also exhibited superior recall of the platform location during the transfer test (data not shown, but see Murphy et al., 2000).

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

To determine if the improved spatial learning was related to enhanced neuroplasticity, the frequency of polysialylated neurons in the infragranular zone of the dentate gyrus and in layer II of the entorhinal and perirhinal cortex was investigated. The frequency of hippocampal and cortical polysialylated neurons was determined by immunohistochemistry, using an anti-PSA monoclonal antibody (generous gift of Prof. Rougon; Rougon, Dubois, Buckley, Magnani, & Zollinger, 1986), as described previously (Fox et al., 1995a; O'Connell et al., 1997). Cell counts were expressed per unit area of the dentate granule cell layer (0.15 mm2) and per unit length of layer II of the cortex (10 mm). In the dentate gyrus, significantly more polysialylated neurons were observed in the infragranular zone at both doses employed. Chronic treatment with 16.8 mg/kg ABS-205 resulted in an approximate frequency increase of 11% (71.2 ± 2.4 vs. 64.0 ± 2.9 cells/unit area), and this increased to approximately 16% (74.3 ± 2.6 vs. 64.0 ± 2.9 cells/unit area) at a dose of 50.4 mg/kg. This effect was specific to ABS-205, as the methylpropyl-4yn-VPA analogue, which lacks a neurotrophic action (Bojic et al., 1998), was without effect on polysialylated cell frequency. The effect was not specific to the dentate gyrus. In layer II of the entorhinal and perirhinal cortex, at -8.1 and -8.6 mm below bregma, polysialylated cell frequency was dramatically increased. At level -8.1 mm, animals treated chronically with ABS-205 (50.4 mg/kg) were found to express an approximate 70% increase in polysialylated neurons, as compared with rats receiving methylpropyl-4-yn-VPA (302.5 ± 30.3 vs. 178.2 ± 20.2 cells/unit length). At level -8.6 mm, the increase was smaller, at approximately 11% (296.4 ± 32.3 vs. 242.2 ± 16.8 positive cells/unit length), but, nonetheless, significantly differed from controls. When compared with the P40 rat (Fox et al., 1995b), these findings indicated an approximate 50% reduction in the normal loss of polysialylated cells in the rat from P40 to P80 (loss of 117.5 vs. 241.8 cells/unit length in drug-treated vs. control) (data not shown, but see Murphy et al., 2000).

ABS-205: Effects on Cognition

189

INFLUENCE OF ABS-205 ON SPATIAL LEARNING IN THE AGED RAT

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Using the same spatial maze-training procedure outlined above, the cognition-enhancing effect of ABS-205 on aged, male Wistar rats (aged 20 months) was determined. Separate groups of animals were dosed daily with 16.8 mg/kg, 50.4 mg/kg, or 84 mg/kg, by the intraperitoneal route, for 40 days prior to training. The final injection was administered 24 hours prior to the first training session. Chronic administration of ABS-205 showed no effect on the weight gain or general condition of the animals. At a dose of 84 mg/kg ABS-205, the time required to locate the platform was significantly reduced by the fifth trial of the second, third, and fourth training sessions, as compared with the control animals who received saline (Figure 21.2). Spatial learning was similarly improved at a dose of 50.4 mg/kg, but only on the fifth trial of the third and fourth training sessions. Chronic ABS-205 treatment, therefore, significantly enhanced the performance of aged animals in a spatial task in a manner similar to that observed with mature animals and in the same dose range. The ABS-205induced improvements of spatial learning in aged animals could not be attributed to improved visuomotor ability, as the swim speeds recorded for drug- and saline-treated animals were indistinguishable in untrained animals. Moreover, analysis of swim angle to platform position, a measure of search-strategy efficiency, indicated ABS-205-treated animals to orient more correctly to the platform position, which was significant at the highest dosage employed. When recall of the task was determined 7 days after the final training session, the ABS-205-treated animals significantly increased their searching strategy in the target quadrant that formerly contained the platform. Moreover, task recall was improved in a dose-dependent manner, as animals treated with 16.8 mg/kg, 50.4 mg/kg, and 84 mg/kg spent 26%, 35.8%, and 46.2%, respectively, of the search time, respectively, in the correct target quadrant, as compared with 18.3% for the saline-treated group.

CONCLUSION The introduction of a triple bond into the 2-position of one of the n-alkyl chains of VPA, and the extension of the second n-alkyl chain to 5 carbon units appears to convert this effective anticonvulsant into a unique cognition-enhancing agent. The memory-improving effects of ABS-205 are observed in a dose range of 50-84 mg/kg, which is an order of magnitude higher than those of other cognition-enhancing agents, such as tacrine or

Drug Development—Preclinical

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

290

ht

tp

://

w

w

w

FIGURE 21.2 Influence of chronic administration of ABS-205 on water maze performance in the aged Wistar rat. Panel A illustrates the escape latencies achieved on the fifth trial of each of the four training sessions. The times spent in the target and opposite quadrants during the 60-second period of the recall test is shown in Panel B. All values are expressed as the mean ± SEM (n = 8), and those significantly different (P < 0.05) from the saline-treated control are indicated with an asterisk.

nefiracetam (Eaggar, Levy, & Sahakain, 1992; Yamada & Nabeshima, 1996). Yet this dosage range is well within that required to control seizures by VPA and at which no cognition-enhancing effects have been observed (Calandre, Dominguez-Granados, Gomez-Rubio, & Molina-Font, 1990; Craig & Tallis, 1994). By contrast, experimental studies with rodents suggested that anticonvulsant doses of VPA cause amnesia for the passiveavoidance task (Balakrishnan & Pandhi, 1997). ABS-205 would seem to have

ABS-205: Effects on Cognition

191

p

all the neuroplastic qualities of the neurotrophins, but to be without their drawback of impermeability to the blood-brain barrier (Pardridge, 1994). The ability of ABS-205 to attenuate the age-related decline in NCAMpolysialylation state and to improve spatial learning in mature and aged rats suggests it to have a significant advantage in slowing onset of agerelated degenerative deficits by enhancing neuroplasticity, with the concomitant of improved cognition

x. ph

REFERENCES

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

Balakrishnan, S., & Pandhi, P. (1997). Effects of nimodipine on the cognitive dysfunction induced by phenytoin and valproate in rats. Methods and Findings in Experimental Clinical Pharmacology, 19, 693-697. Becker, C. G., Artola, A., Gerardy-Schahn, R., Becker, T., Welzl, H., & Schachner, M. (1996). The polysialic acid modification of the neural cell adhesion molecule is involved in spatial learning and hippocampal long-term potentiation. Journal of Neuroscience Research, 45,143-152. Bojic, U., Ehlers, K., Ellerbeck, U., Bacon, C. L., O'Driscoll, E., O'Connell, C., Berezin, V, Kawa, A., Lepekhin, E., Bock, E., Regan, C. M., & Nau, H. (1998). Studies on the teratogen pharmacophore of valproic acid analogues: Evidence of interactions at a hydrophobic centre. European Journal of Pharmacology, 354,2889-2900. Calandre, E. P., Dominguez-Granados, R., Gomez-Rubio, M., & Molina-Font, J. A. (1990). Cognitive effects of long-term treatment with phenobarbital and valproic acid in school children. Acta Neurologica Scandanavia, 81, 504-506. Craig, I., & Tallis, R. (1994). Impact of valproate and phenytoin on cognitive function in elderly patients: Results of a single-blind randomized comparative study. Epilepsia, 35, 381-390. Doherty, P., Mann, D. A., & Walsh, F. S. (1988). Comparison of the effects of NGF, activators of protein kinase C, and a calcium ionophore on the expression of Thy-1 and N-CAM in PC12 cultures. Journal of Cell Biology, 107, 333-340. Doyle, E., Nolan, P. M., Bell, R., & Regan, C. M. (1992). Intraventricular infusions of anti-neural cell adhesion molecules in a discrete posttraining period impair consolidation of a passive avoidance response in the rat. Journal of Neurochemistry, 59, 1570-1573. Doyle, E., & Regan, C. M. (1993). Cholinergic and dopaminergic agents which inhibit a passive avoidance response attenuate the paradigm-specific increases in NCAM sialylation state. Journal of Neural Transmission, 92, 33-49. Eaggar, S. A., Levy, R., & Sahakain, B. J. (1992). Tacrine in Alzheimer's disease. Acta Neurologica Scandanavia, 139, 75-80. Fox, G. B., O'Connell, A. W., Murphy, K. J., & Regan, C. M. (1995a). Memory consolidation induces a transient and time-dependent increase in the frequency of NCAM polysialylated cells in the adult rat hippocampus. Journal of Neuwchennstry, 65, 2796-2799. Fox, G. B., Kennedy, N., & Regan, C. M. (1995b). Polysialylated neural cell adhesion molecule expression by neurons and astroglial processes in the rat

192

Drug Development—Preclinical

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

dentate gyrus declines dramatically with increasing age. International Journal of Developmental Neuroscience, 13, 663-672. Geinisman, Yv deToledo-Morrell, L., & Morrell, F. (1991). Induction of long-term potentiation is associated with an increase in the number of axospinous synapses with segmented postsynaptic densities. Brain Research, 566, 77-88. Loscher, W. (1999). Valproate: A reappraisal of its pharmacodynamic properties and mechanisms of action. Progress in Neurobiology, 58, 31-59. Martin/ M. L., & Regan, C. M. (1991). The anticonvulsant valproate teratogen restricts the glial cell cycle at a defined point in the mid-Gl phase. Brain Research, 554, 223-228. Morris, R. G. M., Garrud, J., Rawlins, N. P., & O'Keefe, J. (1982). Place navigation impaired in rats with hippocampal lesions. Nature, 297, 681-683. Murphy, K. J., O' Connell, A. W., & Regan, C. M. (1996). Repetitive arid transient increases in hippocampal neural cell adhesion molecule polysialylation state following multi-trial spatial training. Journal of Neurochemistry, 67,1268-1274. Murphy, K. J., Fox, G. B., Foley, A. G., O'Connell, A. W., Gallagher, H. C., Griffin, A.-M., Nau, H., & Regan, C. M. (2000). Pentyl-4-yn-valproic acid enhances both spatial and avoidance learning and attenuates age-related NCAMmediated neuroplastic decline within the medial temporal lobe. Submitted. Ni Dhuill, C. M., Fox, G. B., Pittock, S. J., O'Connell, A. W., Murphy, K. J., & Regan, C. M. (1999). Polysialylated neural cell adhesion molecule expression in the dentate gyrus of the human hippocampal formation from infancy to old age. Journal of Neuroscience Research, 55, 99-106. O'Connell, A. W., Fox, G. B., Murphy, K. J., Fichera, G., Kelly, J., & Regan, C. M. (1997). Spatial learning activates neural cell adhesion molecule polysialylation in a cortico-hippocampal pathway within the medial temporal lobe. Journal of Neurochemistry, 68, 2538-2546. O'Malley, A., O'Connell, C., & Regan, C. M. (1998). Ultrastructural analysis reveals avoidance conditioning to induce a transient increase in hippocampal dentate spine density in the 6h post-training period of consolidation. Neuroscience, 87, 607-613. O'Malley, A., O'Connell, C., Murphy, K. J., & Regan, C. M. (2000). Transient spine density increases in the mid-molecular layer of hippocampal dentate gyrus accompany consolidation of a spatial learning task in the rodent. Neuroscience, 99, 229-232. Pardridge, W. M. (1994). New approaches to drug delivery through the bloodbrain barrier. Trends in Biotechnology, 12, 239-245. Park, T.-U., Lucka, L., Reutter, W., & Horstkorte, R. (1997). Turnover studies of the neural cell adhesion molecule NCAM: Degradation of NCAM in PCI2 cells depends on the presence of NCR Biochemistry Biophysics Research Communication, 234, 686-689. Regan, C. M. (1985). Therapeutic levels of sodium valproate inhibit mitotic indices in cells of neural origin. Brain Research, 347, 394-398. Rougon, G., Dubois, C., Buckley, N., Magnani, J. L., & Zollinger, W. (1986). A monoclonal antibody against meningococcus group B polysaccharides distinguishes embryonic from adult NCAM. Journal of Cell Biology, 103, 2429-2437. Scholey, A. B., Rose, S. P., Zamani, M. R., Bock, E., & Schachner, M. (1993). A role

ABS-205: Effects on Cognition

193

4a ll.

ACKNOWLEDGMENTS

vn

/fo

ru

m

s/ in de

x. ph

p

for the neural cell adhesion molecule in a late, consolidating phase of glycoprotein synthesis six hours following passive avoidance training of the young chick. Neuroscience, 55, 499-509. Schuster, T., Krug, M., Hassan, H., & Schachner, M. (1998). Increase in proportion of hippocampal spine synapses expressing neural cell adhesion molecule NCAM180 following long-term potentiation. Journal of Neurobiology, 37, 359-372. Skibo, G. G., Davies, H. A., Rusakov, D. A., Stewart, M. G., & Schachner, M. (1998). Increased immunogold labelling of neural cell adhesion molecule isoforms in synaptic active zones of the chick striatum 5-6 hours after one-trial passive avoidance training. Neuroscience, 82,1-5. Theodosis, D. T., Bonhomme, R., Vitiello, S., Rougon, G., & Poulain, D. A. (1999). Cell surface expression of polysialic acid on NCAM is a prerequisite for activity-dependent morphological neuronal and glial plasticity. Journal of Neuroscience, 19,10228-10236. Toni, N., Buchs, P.-A., Nikonenko, I., Bron, C. R., & Muller, D. (1999). LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite. Nature, 402, 421-424. Yamada, K., & Nabeshima, T. (1996). Nefiracetam (DM-9384): Anovel antiamnesic drug. Central Nervous System Drug Reviews, 2, 322-342.

ht

tp

://

w

w

w

.c

he m

This work was supported by Enterprise Ireland, American Biogenetic Sciences, Inc., Institute for the Study of Aging Inc., and by a Phase I Small Business Innovation Grant (SBIR) from the National Institute on Aging (NIA), a member Institute of the National Institutes of Health (NIH). We thank E. Drews for her technical assistance and U. Ellerbeck for synthesis of the two compounds studied.

22

A Treatment of

s/ in de

Alzheimer's Disease

x. ph

p

Calpain Inhibitors:

/fo

ru

m

Ottavio Arancio

4a ll.

vn

ABSTRACT

ht

tp

://

w

w

w

.c

he m

Evidence suggests that the impairment of synaptic communication between neurons may significantly contribute to the symptoms and progressive deterioration associated with Alzheimer's disease (Liu, Passant, Risberg, Warkentin, & Brun, 1999; Rapoport, 1999; Selkoe, 1994). Good candidates as possible causes of the disease are a particular type of protein, called calpains. They have the ability to destroy most of the cellular-protein pool at abnormally high calcium levels and to regulate the production of |3-amyloid precursor protein, one of the neuropathologic hallmarks of the disease (Nixon et al., 1994; Wang, 2000). In so doing, they would be responsible for mediating early molecular events of the illness. Using hippocampal neurons in culture derived from the APP(K670M:N671L)/PS1(M146L) mouse model of Alzheimer's disease, I have found that the overexpression of the two mutated proteins causes a reduction in the frequency of spontaneous release of neurotransmitter. Thus, I propose to inhibit calpain activity to test whether it is possible to restore normal communication between cells.

INTRODUCTION Neuropathologic hallmarks of Alzheimer's disease (AD) are loss of neurons, decrease of synapse number, deposition of amyloid protein in senile 194

Calpain Inhibitors: A Treatment of AD

195

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

plaques and within the walls of the cerebral microvasculature, and presence of neurofibrillary tangles (NFT) (Liu et al., 1999; Rapoport, 1999; Selkoe, 1994). Interestingly, synaptic alterations are highly correlated with the severity of clinical dementia, whereas other important variables, such as senile plaques, and NFT are involved to a lesser extent (Masliah & Terry, 1993). These observations support the idea that a block of synaptic disruption might stop or slow-down AD progression. Good candidates as possible causes of the synaptic impairment in AD are cytosolic calcium-activated cystein proteases, called calpains (Nixon et al., 1994). At physiologic calcium levels, they eliminate abnormal and potentially toxic proteins at the membrane-cytoskeleton interface. At abnormally high calcium levels, however, they would destroy most of the cellular-protein pool, causing synaptic impairment in various neurological diseases, including AD (Nixon et al., 1994; Wang, 2000). Calpains cleave amyloid precursor protein (APP) (Nixon et al., 1994) and two enzymes that regulate its phosphorylation, CAM-Kinase Hoc (CAMK-IIcc and c-AMP-dependent protein kinases (PKA) (Gandy, Czernik, & Greengard, 1998; Wang & Yuen, 1997). Calpains, in addition, fragment second messengers involved in signal transduction, like protein kinase C (PKC) (Pontremoli et al., 1987), phospholipase C -1, - 2, Beta-3 (Banno, Nakashima, Hachiya, & Nozawa, 1995), and transcription factors, such as c-Jun, c-Fos, and IKappaB (Carillo et al., 1994; Lin, Brown, & Siebenlist, 1995). Finally, calpains degrade spectrin (Nixon, 1986) and MAP 2 (Johnson, Litersky, & Jope, 1991), two proteins that have been associated with neuronal architecture (Aoki & Siekevitz, 1985). Further evidence in favor of calpain involvement in AD is provided by observations obtained from brains of AD patients, where the activated form of one type of calpains, called calpain I, is significantly increased, and antibodies against another type of calpains, called calpain II, bind extensively to NFT (Nixon et al., 1994). These observations suggest the hypothesis that a block of calpain activity should stop the evolution of AD. During the last 5 years, excellent opportunities to develop a therapy of AD have been provided by animal models of the disease. The transgenie mouse, which expresses the familial Swedish mutation in APP(K670M:N671L), has received much attention (Hsiao et al., 1996). It shows both defects in spatial reference and alternation tasks, together with senile plaques in cortical and limbic structures, and an increase of the levels of amyloid beta at 9 to 10 months (Hsiao et al., 1996). It also presents long-term potentiation (LTP) impairment and synapse loss (Chapman et al., 1999). Analysis of a different transgenic model, the presenilin 1 (PS1) (M146L) mouse, indicates that the mutated but not the wild-type (WT) PS1 expression increases brain amounts of amyloid beta 42, and amyloid beta 43 after 5 months (Duff et al., 1996). Synaptosomes prepared from

Drug Development—Predinical

296

s/ in de

x. ph

p

these transgenic animals exhibit enhanced elevations of cytoplasmic calcium levels following exposure to depolarizing agents, amyloid beta-peptide, and a mitochondrial toxin, 3-nitro-propionic acid (Begley, Duan, Chan, Duff, & Mattson, 1999). Interestingly, large plaques develop in the cortex and hippocampus of younger transgenic mice when the PS1(M146L) mutation is associated with the APP(K670M:N671L) mutation (Holcomb et al., 1998). This finding indicates that double mutants have a faster phenotype than single mutants, APP or PS1. Because of this feature, double APP(K670M:N671L)/PS1(M146L) mutants provide an important platform for testing of various therapeutic approaches to AD.

MATERIAL AND METHODS Cell Culture Preparation

he m

4a ll.

vn

/fo

ru

m

Cell cultures were prepared from 1-day-old mouse pups. Cells were dissociated through enzymatic treatment (0.25% trypsin) and subsequent trituration. They were plated on glass coverslips previously coated with poly(D-lysine) and laminin. Hippocampal cells were grown in medium containing 84% Eagle's minimum essential medium (MEM), supplemented with 10% heat-inactivated fetal calf serum, 45 mM glucose, 1% MEM vitamin solution, and 2 mM glutamine. After 24 hours, this medium was replaced by a medium containing 0.1% 5-fluorouridine, 1% glutamine, and 2% B27-nutrient in DMEM. 0.5 mM kynurenic acid was included in the culture medium.

.c

Electrophysiological Analysis

ht

tp

://

w

w

w

Electrophysiological studies were done 10 to 15 days after plating, as previously described (Arancio, Kandel, & Hawkins, 1995). Miniature, excitatory, postsynaptic currents (mEPSCs) and evoked postsynaptic currents (EPSCs) were recorded with a Warner amplifier (model PC-501A) (CT). mEPSCs were measured automatically by computer using a program from Synaptosoft. Genotyping

Animals utilized to obtain cell cultures were transgenic mice expressing the APP mutation (K670M:N671L) (Hsiao et al., 1996), as well as the human PS1 mutation (M146L) (Duff et al., 1996) and the double mutation APP(K670M:N671L)/PS1(M146L) (mAPP/mPSl). APP-transgenic mice were paired with PS1-transgenic mice. Because of mechanisms of gene segregation, pups showed four different genotypes: APP (K670M:N671L) mutants; PS1 (M146L) mutants; and APP(K670M:N671L)/PS1(M146L) double-mutant, nontransgenic mice (WT). The pups were genotyped

Calpain Inhibitors: A Treatment of AD 197

197

vn

/fo

ru

m

s/ in de

x. ph

p

through the polymerase chain reaction (PCR) technique to identify transgene-positive mice (Duff et al, 1996). DNA samples were prepared from the tail tips (>0.25 cm) of individual mice. Each tail tip was placed in 500 ul of tail lysis buffer (0.1 M Tris-HCl pH 8.0, 5 mM EDTA pH 8.0, 0.1% SDS, and 0.2 M Nad), with proteinase K at 1 mg/ml. The samples were digested for 2 to 4 hours. DNA was extracted by 500 ul of a phenol:chlorophorme:isoamyl alcohol 25:24:1 mix, then precipitated and washed with ethanol. The final DNA pellet was resuspended in 200 ul of water. Each PCR reaction contained 1.5 (1 of DNA and 24 ul of primers mix (total reaction volume = 25 ul). APP transgenes were detected with a forward primer 5'-GAGGACTGACCACTC and a reverse primer 5'-GAACCCACATCTTCTGC. Detection of PS1 transgenes used a forward primer 5'-CATGTCGACTAGGCGGCCGCGGGGATC and a reverse primer 5'CATCCGCGGTCGACTCTAGAGA. In all cases, amplification involved 35 cycles at 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 45 seconds. Following amplification, electrophoresis was performed through a 2% agarose gel stained with ethidium bromide, and bands corresponding to different genotypes were photographed under ultraviolet light.

4a ll.

RESULTS

ht

tp

://

w

w

w

.c

he m

To test whether synaptic transmission properties in mouse hippocampal neurons in culture resemble those of other well-known preparations, such as slices and in vivo systems, I have studied both spontaneous and evoked synaptic currents in the culture system. I have found that cultured neurons formed synapses as detected by spontaneous (Figure 22.1 A) and evoked synaptic currents (Figure 22.IB). Similar to slices and in vivo preparations, EPSCs were blocked by APV and CNQX, two drugs that block NMD A and AMPA type of glutamate receptors, respectively (Figure 22.IB). High-frequency stimulation (three 50Hz pulses, 2-second trains of depolarization at 20-second intervals in 0 Mg2+) of the presynaptic neuron induced LTP (Figure 22.1C), a form of long-lasting enhancement of synaptic transmission that is widely studied as a cellular model of learning (Bliss & Collingridge, 1993). The average potentiation at 30 minutes was 160% of baseline transmission (n = 5). Similar to slice or in vivo preparation, finally, LTP in culture was blocked by the NMDA-receptor antagonist, D-APV (n = 4), and by postsynaptic injection of the calcium chelator, BAPTA (n = 4). Thus, I have concluded that cultured hippocampal neurons in culture can be used as a model to study mechanisms of synaptic transmission. It is generally accepted that mEPSCs directly result from the postsynaptic response to a single quantum of transmitter (Katz, 1966). A variation in

Drug Development—Preclinical

x. ph

p

298

ru

m

s/ in de

FIGURE 22.1 Synaptic transmission in murine hippocampal neuron in culture. A) Examples of mEPSCs. B) Examples of EPSCs after a 10-msec step depolarization of the presynaptic cell eliciting an inward current. C) Time course of EPSC amplitude before and after potentiation by tetanic stimulation (filled circles). Potentiation was blocked by extracellular application of APV (open circles).

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

their frequency is associated with a change in probability of quantal transmission from the presynaptic terminal, whereas a modification in their amplitude is related to postsynaptic changes (i.e., modification of the postsynaptic receptor number and/or change in the opening properties of the associated channel). Therefore, I have studied whether mEPSC frequency and/or amplitude are modified in cultured neurons from double mAPP/mPSl mice. Data obtained in the laboratory during the last year have shown that the average basal frequency of mEPSCs is increased in 10- to 15-day-old cultures derived from newborn transgenic mAPP/mPSl mice (F = 11.2 mEPSC/min; n = 6), compared with cultures from single mAPP (F = 5.3 mEPSC/min; n = 4) or mPSl (F = 5.0 mEPSC/min; n = 6) mice, as well as cultures from WT mice (F = 5.1 mEPSC/min; n = 8) (Figure 22.2). These results suggest that the probability of release per release site is increased in the double-transgenic model. The increase occurs at early developmental stages because cultured cells were taken from newborn animals and they were recorded after 10 to 15 days in vitro. To further investigate spontaneous release of neurotransmitter, I have also analyzed mEPSC frequency over time. Cultures from double-transgenic animals showed a progressive increase of the mEPSC frequency over time (326% of the initial value at 45 minutes), compared with cultures from single mutants (88% in mAPP mice, and 85% in PS1 animals at 45 minutes), and cultures from WT mice (86% at 45 minutes) (Figure 22.3). These results are consistent with the increase in basal mEPSC frequency shown in Figure 22.2, and suggest that changes in mEPSCs are ongoing at this stage in culture. Interestingly, the mEPSC increase precedes any morphological and behavioral change occurring only as the animal becomes an adult.

Calpain Inhibitors: A Treatment of AD

m

s/ in de

x. ph

p

199

vn

/fo

ru

FIGURE 22.2 Basal mEPSC frequency is increased in double-transgenic mAPP/mPSl mice (filled circles), compared with mAPP (open circles), mPSl (open diamonds), and WT (filled diamonds) mice.

ht

tp

://

w

w

w

.c

he m

4a ll.

Since a variation in mEPSc amplitude might indicate changes in the postsynaptic membrane, I have also investigated whether mEPSC amplitude was modified in the double-transgenic mAPP/mPSl mice. Different than mEPSc frequency, mEPSC amplitude was not modified in the dou-

FIGURE 22.3 mEPSC frequency increases progressively in double mAPP/mPSl mice (filled circles), compared with mAPP (open circles), mPSl (open diamonds), and WT (filled diamonds) mice.

Drug Development—Preclinical

200

ble mutants (Figure 22.4). Thus, these results suggest that postsynaptic receptor properties are not changed in transgenic mAPP/mPSl mice.

DISCUSSION

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Until recently, the strategies for developing interventions in AD focused primarily on symptomatic treatment for the middle and late stages of the disease. AD patients suffering from depression, anxiety, delusion, hallucination, aggression, or sleep disturbance benefit most from drugs indicated specifically for those conditions. The next drug generation will directly interfere with the causes of the disease and will, eventually, delay the onset or slow the progression of the disease by intervening in the causes of the disease. One of the important targets for developing a causal therapy for AD is the loss of synapses. Research in my laboratory has shown that frequency of spontaneous release of neurotransmitter is reduced in hippocampal neurons in culture, suggesting that presynaptic release mechanisms are modified in cultured neurons from mAPP/mPSl animals. These results are particularly interesting in view of using changes in mEPSC frequency as a tool to test whether a specific treatment can rescue their modification. Calpains may play a major role in the pathogenesis of AD (Nixon et al., 1994; Wang, 2000). A model for their involvement in AD is illustrated in Figure 22.5. Calpain inhibitors have been successfully used in models of

FIGURE 22.4 Basal mEPSC amplitude is not changed in mAPP/mPSl mice (filled circles), compared with mAPP (open circles), mPSl (open diamonds), and WT (filled diamonds) mice.

Calpain Inhibitors: A Treatment of AD

p

201

s/ in de

x. ph

FIGURE 22.5 Model for a role of abnormal calpain activation in AD. Ca2+ elevation activates calpains that would be responsible for the degradation of most of the cellular-protein pool, including cytoplasmic substrates, transcription factors, and cytoskeletal substrates.

w

.c

REFERENCES

he m

4a ll.

vn

/fo

ru

m

vascular disorders (i.e., cerebral and myocardial ischemia, hypertension, platelet aggregation), degenerative disorders (i.e., muscular dystrophy, cataract), acute renal failure, infections, and cancer (Wang & Yuen, 1997). Based on the findings described in this chapter, I plan to test whether a treatment with calpain inhibitors is able to prevent or revert changes of spontaneous release of neurotransmitters occurring in synapses from double mAPP/mPSl animals. To further examine the possibility of utilizing calpain inhibitors as a treatment of AD, I will also examine their effects on amyloid-beta peptides, 42 and 43, as well as on [Ca2+]. levels.

ht

tp

://

w

w

Aoki, C, & Siekevitz, P. (1985). Ontogenetic changes in the cyclic adenosine 3',5'monophosphate-stimulatable phosphorylation of cat visual cortex proteins, particularly of micro tubule-associated protein 2 (MAP 2): Effects of normal and dark rearing and of the exposure to light. Journal of Neuroscience, 5, 2465-2483. Arancio, O., Kandel, E. R., & Hawkins, R. D. (1995). Activity-dependent long-term enhancement of transmitter release by presynaptic 3', 5' -cyclic GMP in cultured hippocampal neurons. Nature, 376, 74-80. Banno, Y, Nakashima, S., Hachiya, T., & Nozawa, Y (1995). Endogenous cleavage of phospholipase C-beta 3 by agonist-induced activation of calpain in human platelets. Journal of Biological Chemistry, 270, 4318-4324. Begley, J. G., Duan, W., Chan, S., Duff, K., & Mattson, M. P. (1999). Altered calcium homeostasis and mitochondrial dysfunction in cortical synaptic compartments of presenilin-1 mutant mice. Journal ofNeurochemistry,72,1030-1039. Bliss, T. V. P., & Collingridge, G. L. (1993). A synaptic model of memory: Longterm potentiation in the hippocampus. Nature, 361, 31-39.

202

Drug Development—Preclinical

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Carillo, S., Pariat, M., Steff, A. M., Roux, P., Etienne-Julan, M., Lorca, T., & Piechaczyk, M. (1994). Differential sensitivity of FOS and JUN family members to calpains. Oncogene, 9,1679-1689. Chapman, P. F, White, G. L., Jones, M. W., Cooper-Blacketer, D., Marshall, V. J., Irizarry, M., Younkin, L., Good, M. A., Bliss, T. V., Hyman, B. T., Younkin, S. G., & Hsiao, K. K. (1999). Impaired synaptic plasticity and learning in aged amyloid precursor protein transgenic mice. Nature Neuroscience, 2, 271-276. Duff, K., Eckman, C, Zehr, C, Yu, X., Prada, C. M., Perez-tur, J., Hutton, M., Buee, L., Harigaya, Y, Yager, D., Morgan, D., Gordon, M. N., Holcomb, L., Refolo, L., Zenk, B., Hardy, J., & Younkin, S. (1996). Increased amyloid-beta42(43) in brains of mice expressing mutant presenilin 1. Nature, 383, 710-713. Gandy, S., Czernik, A. J., & Greengard, P. (1998). Phosphorylation of Alzheimer disease amyloid precursor peptide by protein kinase C and Ca2+/calmodulindependent protein kinase II. Proceedings of the National Academy of Sciences (USA), 85,6218-6221. Holcomb, L., Gordon, M. N., McGowan, E., Yu, X., Benkovic, S., Jantzen, P., Wright, K., Saad, L, Mueller, R., Morgan, D., Sanders, S., Zehr, C., O'Campo, K., Hardy, J., Prada, C. M., Eckman, C., Younkin, S., Hsiao, K., & Duff, K. (1998). Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes. Nature Medicine, 4, 97-100. Hsiao, K., Chapman, P., Nilsen, S., Eckman, C., Harigaya, Y, Younkin, S., Yang, F., & Cole, G. (1996). Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science, 274, 99-102. Johnson, G. V., Litersky, J. M., & Jope, R. S. (1991). Degradation of microtubuleassociated protein 2 and brain spectrin by calpain: A comparative study. Journal of Neurochemistry, 56,1630-1638. Katz, B. (1966). Quantal nature of chemical transmission. In G. Wald (Ed.), Nerve, muscle and synapse (pp. 129-134). New York: McGraw-Hill. Lin, Y. C., Brown, K., & Siebenlist, U. (1995). Activation of NF-kappa B requires proteolysis of the inhibitor I kappa B-alpha: Signal-induced phosphorylation of I kappa B-alpha alone does not release active NF-kappa B. Proceedings of the National Academy of Sciences (USA), 92, 552-556. Liu, X., Passant, U., Risberg, J., Warkentin, S., & Brun, A. (1999). Synapse density related to cerebral blood flow and symptomatology in frontal lobe degeneration and Alzheimer's disease. Dementia and Geriatric Cognitive Disorders, 1, 64-70. Masliah, E., & Terry, R. D. (1993). Role of synaptic pathology in the mechanisms of dementia in Alzheimer's disease. Clinical Neuroscience, 1,192-198. Nixon, R. A. (1986). Fodrin degradation by calcium-activated neutral proteinase (CANP) in retinal ganglion cell neurons and optic glia: Preferential localization of CANP activities in neurons. Journal of Neuroscience, 6,1264-1271. Nixon, R. A., Saito, K. I., Grynspan, F, Griffin, W. R., Katayama, S., Honda, T., Mohan, P. S., Shea, T. B., & Beermann, M. (1994). Calcium-activated neutral proteinase (calpain) system in aging and Alzheimer's disease. Annals of the New York Academy of Sciences, 747, 77-91. Pontremoli, S., Melloni, E., Michetti, M., Sparatore, B., Salamino, F, Sacco, O., &

Calpain Inhibitors: A Treatment of AD 203

203

s/ in de

x. ph

p

Horecker, B. L. (1987). Phosphorylation and proteolytic modification of specific cytoskeletal proteins in human neutrophils stimulated by phorbol 12myristate 13-acetate. Proceedings of the National Academy of Sciences (USA), 84, 3604-3608. Rapoport, S. I. (1999). In vivo PET imaging and postmortem studies suggest potentially reversible and irreversible stages of brain metabolic failure in Alzheimer's disease. European Archives of Psychiatry and Clinical Neurosciences, 249, 46-55. Selkoe, D. J. (1994). Normal and abnormal biology of the beta-amyloid precursor protein. Annual Review of Neurosciences, 17, 489-517. Wang, K. K. (2000). Calpain and caspase: Can you tell the difference? Trends in Neuroscience, 23, 20-26. Wang, K. K., & Yuen, P. W. (1997). Development and therapeutic potential of calpain inhibitors. Advances in Pharmacology, 37,117-152.

ru

m

ACKNOWLEDGMENT

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

This work has been supported by the Institute for the Study of Aging, New York, NY.

s/ in de

x. ph

Femtomolar-Acting Neuroprotective Peptides: Application for Inhibition

p

23

m

of Alzheimer's Disease

w

.c

he m

4a ll.

vn

/fo

ru

Illana Gozes, Sharon Furman, Ruth A. Steingart, Albert Pinhasov, Inna Vulih, Jacob Romano, Roy Zaltzman, Rachel Zamostiano, Eliezer Giladi, Sara Rubinraut, Matt Fridkin, Janet Hauser, and Douglas E. Brenneman

w

w

ABSTRACT

ht

tp

://

Alzheimer's disease (AD) is characterized by death of selected brain cells that normally supply messenger molecules, forming short-term memory. The focus of the current work is the discovery and characterization of very short, readily available proteins (termed peptides) that provide protection to nerve cells at very low concentrations (among the lowest described to date). A major brain natural-protective short protein is vasoactive intestinal peptide (VIP) that affords neuronal defense through activation of proteins derived from glial cells (the brain support cells), such as activity-dependent neurotrophic factor (ADNF) and activity-dependent neuroprotective protein (ADNP). Drug design identified small and modified peptide fragments derived from VIP, ADNF, and ADNP, which can protect against damage in cell and animal models. The specific aim of the study is to 204

Femtomolar-Acting Neuwprotective Peptides

205

m

s/ in de

x. ph

p

optimize and choose the lead compounds. Tests include: simplified tissueculture systems, brain-penetration assessments, and effects on short-term memory, in models relevant to AD. Selected lead compounds are further tested for mechanisms of protection, as well as potential side effects, and put forth for future clinical evaluations. Key findings include: (1) Lead compounds have been chosen: (a) the ADNP-derived peptide, NAP, (b) the fat-modified VIP, SNV, (c) the shortened VIP-derivative, stearyl-KKYL-NH2, and (d) the ADNF-related peptide, ADNF-9; (2) formulations for nasal spray administration have been optimized; (3) studies on mechanisms of protection identified selective gene activation and defense against inflammation. Advanced geneticengineering techniques are now utilized for the identification of novel interacting molecules, with NAP, the first lead planned for clinical trials, as the target.

/fo

ru

INTRODUCTION

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

Vasoactive intestinal peptide (VIP, 28 amino acid), originally discovered in the intestine, was later found to be a major brain peptide having neuroprotective activities (Gozes et al., 1999a). Animals that have been exposed to a VIP antagonist or transgenic animals that underexpress VIP exhibit cognitive impairments. VIP gene-expression patterns are developmentally determined, associated with synaptogenesis (Blondel et al., 2000), dependent on synaptic activity and animal physical activity (Eilam, Davidson, Gozes, & Segal, 1999) and are reduced with aging. Lipophilic "fatty" derivatives of VIP cross the blood-brain barrier, and provide nerve-cell protection and accelerate learning and memory in animal models (Gozes et al., 1996a, 1997a; Gozes, Fridkin, Hill, & Brenneman, 1999a; Gozes et al., 1999b). Drug application can be intranasal (Gozes et al., 1996a, 1999b). Recently, a peptide containing the active core of VIP was disclosed (stearyl-KKYL-NH2) that is about seven times smaller than VIP and may offer an exciting candidate for future drug development. To exert neuroprotective activity in the brain, VIP requires glial cells. Glial cells secrete protective proteins following exposure to VIP. Activitydependent neurotrophic factor (ADNF) is a recently isolated factor secreted by glial cells under the action of VIP. ADNF is the most potent neuroprotective protein complex described to date (Brenneman & Gozes, 1996; Gozes & Brenneman, 1996b; Gozes et al., 1997b). This protein, isolated by sequential chromatographic methods, was named ADNF, since it protected neurons from death associated with blockade of electrical activity. A novel ADNF peptide (ADNF-9, SALLRSIPA) was identified, a 9-aminoacid-long peptide with activity that surpasses that of the ADNF protein

206

Drug Development—Preclinical

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

with regard to potency and a broader range of effective concentration (Brenneman et al., 1998). Furthermore, ADNF-9 exhibits protective activity in AD-related systems ((3-amyloid toxicity, Brenneman et al., 1998; presenilin 1 mutation, Guo et al., 1999; apolipoprotein E (ApoE) deficiencies, Bassan et al., 1999, genes that have been associated with AD onset and progression). Activity-dependent neuroprotective protein (ADNP) is another glial mediator of VIP-associated neuroprotection (Bassan et al., 1999). The human ADNP gene was characterized (Zamostiano et al., 2001) and an 8-amino-acid peptide derived from ADNP, NAP (NAPVSIPQ, sharing structural and functional similarities with ADNF-9), was identified. NAP is suggested to be the most potent neuroprotectant described to date in an animal model of ApoE deficiency (knock-out mice, Bassan et al., 1999). Since the lipid carrier, ApoE, has been implicated as a risk factor in AD, and since the knock-out mice exhibit short-term memory deficits that are ameliorated by chronic peptide treatment, NAP holds promise for future treatment against AD-associated short-term memory deficits. The initial effort of the current research was the optimization of the lead peptide compounds in relevant cell-culture systems and animal models. Lead compounds included: stearyl-Nle17-VIP (SNV), stearylKKYL-NH2, ADNF-9, and NAP.

he m

MATERIALS AND METHODS

ht

tp

://

w

w

w

.c

Peptide Syntheses. VIP (HSDAVFTDNYTRLRKQMAVKKYLNSILN-NH2), Stearyl-Nle17-VIP [SNV, Stearyl-HSDAVFTDNYTRLRKQ-NleAVKKYLNSILN-NH2)1, Stearyl-KKYL-NH2, ADNF-9 (SALLRSIPA), and NAP (NAPVSIPQ) were synthesized by automatic procedure as before (Gozes et al., 1999b). Animal models. A cholinotoxicity model included a single injection (i.c.v.) of ethylcholine aziridium (AF64A), a week's recovery, and then daily intranasal administration of NAP (0.5-1 pg/animal/day) or other peptides. After an additional week of peptide treatment, the animals were subjected to two daily tests in the water maze and the time required to reach a hidden platform was recorded over a period of 4 to 5 days using the HVS video tracking system (HVS Image Ltd., Hampton, UK). During the test period, animals were given an intranasal administration of the peptide an hour before the daily tests (Gozes et al., 1996a, 1999b; Gozes, Giladi, Pinhasov, Bardea, & Brenneman, 2000). A second model included the ApoE-deficient mice (e.g., Gozes et al., 1997a, 1999b; Bassan et al., 1999). A third model included head trauma, presenting a risk factor for the development of AD (Beni-Adani et al., 2000). Biodistribution studies were performed as described (Gozes et al., 1996a, 1999b, 2000).

Femtomolar-Acting Neuroprotective Peptides

207

RESULTS

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Table 23.1 compares SNV and NAP in cell cultures, both compounds exhibiting a broad range of neuroprotection. Original results indicated that ADNF-9 is unstable (Brenneman et al., 1998); therefore, it is subject to further modifications (see below). Similarly, stearyl-KKYL-NH2 is subject to further drug-stabilization studies. Lead compound no. 1: NAP. Figure 23.1 demonstrates the video tracking of 15 individual rats (divided into three experimental groups), finding the hidden platform on the second daily trial (indicative of short-term memory) in a round water maze. Results demonstrated that the path the animals travel is significantly decreased following NAP treatment in the cholintoxicity model (Gozes et al., 2000). Similarly, daily NAP injections to newborn ApoE-deficient mice for the first 2 weeks of life enhanced memory when tested at 3 to 4 weeks of age (Bassan et al., 1999); and in the closed head-injury model in mice, a single NAP injection after injury dramatically reduced mortality and facilitated clinical recovery (Beni-Adani et al., 2000). Water accumulation (edema) was reduced by about 70% in the NAP-treated mice. Magnetic resonance imaging (2 weeks following the head trauma) demonstrated significant brain-tissue recovery in the NAP-treated animals (about two-fold better improvement, as compared with sham-treated controls). Taken together, NAP showed brain-protective effects. Interestingly, a significant effect was observed on memory in control mice (not suffering from any cognitive impairment, Gozes et al., 2000). Pharmacokinetics, formulation, and toxicology. NAP penetrated into the brain and exhibited a half-life of at least 15 to 25 minutes (Gozes et al., 2000). Further studies identified a vehicle of choice for intranasal administration. Vehicles compared were: (a) 20% isopropranol/5% sefsol (e.g., Gozes et al., 2000) and (b) a mixture containing 7.5 mg/ml sodium chloride, 1.7 mg/ml citric acid monohydrate, 3.0 mg/ml disodium phosphate dehydrate, and 0.2 mg/ml of a 50% benzalkonium chloride solution. The second formulation is used for intranasal peptide administration as in the case of vasopressin (Physician's Desk Reference). Results showed no significant differences between the two vehicles. We have also identified a metered-dose nasal dispenser (using the Children NasalCrom "nasal spray," Pharmacia & Upjohn, Kalamazoo, MI). A 14-day intranasal toxicity study of NAP in rats performed by MPI Research (Mattawan, MI) has been completed (NIA support). Under the conditions of this study, intranasal dosing of NAP up to 40 ug/rat/day was not toxic. Additional experiments with increasing times (30 days), doses (1-4 mg/kg), and measurements, including various hematology, clinical chemistry, ophthalmology, organ weight, macroscopic and microscopic pathology evaluations, and pharmacokinetics in rats and dogs, are now carried out (repeated dose-toxicity for 30 days, MPI, NIA support).

208

Drug Development—Preclinical

TABLE 23.1 Lead Compounds: Comparison of Protective Concentrations in Nerve Cell Cultures 1 NAPVSIPQ: Name: NAP

2 Stearyl-Nle17-VIP: Name: SNV

x. ph

s/ in de

Model: rat pheochromocytoma (PCI 2 cells)—nerve cell-like Insult: iodoacetate (ischemia) lO-'MO-8 M (Sigalov et al., 2000)

p

Model: rat pheochromocytoma (PC12 cells)—nerve cell-like Insult: hydrogen peroxide (oxidative stress) 10-17-10-14 M (Steingart et al., 2000) lO^-lO-8 M (Steingart et al., 2000)

ru

m

Model: rat pheochromocytoma (PC12 cells)—nerve cell-like Insult: TNF (inflammation) 10-15 M (Beni-Adani et al., 2000)

he m

4a ll.

vn

/fo

Model: human neuroblastoma—nerve cell-like Insult: glutathione (GSH) depletion 10-14-10-13M;10-10-10-7M (Offen et al., 2000) 10-14-10-" M; lO'MO-8 M (Offen et al., 2000)

://

w

w

w

.c

Model: rat brain nerve cells (cerebral cortex) Insult: p-amyloid peptide (the Alzheimer's disease neurotoxin) 10-16 and 10'15M (Bassan et al., 1999) >1Q-15-10-14M (Gozes et al., 1999). 9 10-iM O- M (Zemlyak, Furman, Brenneman, & Gozes, 2000) 100-1000-fold more potent than VIP

ht

tp

Model: rat brain nerve cells (cerebral cortex) Insult: tetrodotoxin (electrical blockade) 10-i«-io-i4 M (Bassan et al., 1999) and lower. Model: rat brain nerve cells (cerebral cortex) Insult: N-methyl-D-aspartate (excitotoxicity) 10-ift-iO-8 M (Bassan et a\f 1999) Model: Mouse cerebellar granule cells. Insult: 6-hydroxydopamine 10-13-10-" M (Offen et al., 2000)

Femtomolar-Acting Neuroprotective Peptides

m

s/ in de

x. ph

p

209

he m

4a ll.

vn

/fo

ru

FIGURE 23.1 Video tracking of 5 individual rats for each treatment group, finding the hidden platform in a round water maze. Five animals are shown for each treatment: controls treated with saline intranasal administration, AF64A treated with saline intranasal administration, and AF64A treated once daily with NAP (0.5 (g/rat/day). Results shown are of the second daily trial, on the third testing day (Gozes et al, 2000).

ht

tp

://

w

w

w

.c

Lead Compound no. 2: Stearl-Nle17-VIP, SNV. Comprised of the VIP molecule with two modifications, an N-terminal attachment of stearic acid and an exchange of the methionine in position 17 with norleucine, SNV exhibits increased stability and bioavailability, as well as a high degree of neuroprotection both in vitro (Table 23.1) and in vivo (e.g., Gozes et al., 1999a, 1999b; Ashur-Fabian, Perl, Lilling, Fridkin, & Gozes, 1999; Hill et al., 1999; Gressens et al., 1999; Offen et al., 2000; Sigalov, Steingart, Fridkin, Brenneman, & Gozes, 2000). Like NAP, SNV enhanced learning and memory in the cholinotoxicity model, the ApoE-deficiency model, and in naive animals (Gozes et al., 1996a; Gozes et al., 1997a). Furthermore, a single injection (i.p.) of SNV to developing mice exposed to an excitotoxic lesion (excess glutamate analog) resulted in neuroprotection (Gressens et al., 1999). Pharmacokinetics and toxicology. Previous studies have shown intact SNV 15 minutes after intranasal administration (Gozes et al., 1996a), and safety studies indicated no side effects at the biologically active dose and higher doses (Gozes et al., 1994). Pipeline compounds. Preliminary results indicate that an all D-amino acids ADNF-9 may be an orally available neuroprotective peptide (Brenneman,

210

Drug Development—Preclinical

Hauser, & Gozes, 2000; Gozes et al., 2000). In addition, stearyl-KKYLNH2, the very short pep tide chain, also presents an attractive lead for further drug design.

DISCUSSION

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

From a point of view of drug development, one important aspect is the mechanism of VIP-related, peptide-based neuroprotection. SNV. VIP binding to brain slices was displaced, in part, by SNV, implicating specific VIP-like, SNV-binding sites (Hill et al., 1999). Cyclic GMP was suggested to mediate, in part, the VIP-induced neuroprotective activity. SNV is 100 to 1000-fold more potent than VIP in increasing cGMP production and is 2 to 3-fold more efficacious (Ashur-Fabian et al., 1999). Thus, cGMP may be associated with the increased neuroprotective potency of SNV (Gozes et al., 1996a). Furthermore, SNV protected against buthionine sulfoximine (BSO), implicating glutathione (GSH) involvement in SNV-mediated neuroprotection. Indeed, BSO is a selective inhibitor of GSH synthesis and causes a marked reduction in GSH in neuroblastoma cells and declines cellular viability by 70% to 90% (Offen et al., 2000). SNV may also be associated, in part, with immunornodulation (Wang et al., 1999). Finally, we have now shown that SNV increases the content of ADNP mRNA (the NAP-containing protein mRNA) in PCI2 cells that are subjected to an ischemic insult (Sigalov et al., 2000). ADNF-9. ADNF-9 increased chaperonin expression (heat shock protein 60). Chaperonins may provide cellular protection against the p-amyloid peptide toxicity (Zamostiano et al., 1999). This effect of ADNF-9 may present a more general modulation of cellular gene expression through the activation of transcription factors (Glazner, Camandola, & Mattson, 2000). ADNF-9 protected against oxidative stress by maintaining mitochondrial function (Guo et al., 1999; Guo & Mattson, 2000) and through a reduction in the accumulation of intracellular-reactive oxygen species (Glazner et al., 1999). Furthermore, ADNF-like molecules increased axonal elongation through cAMP-mediated mechanisms in sensory neurons (White, Walker, Brenneman, & Gozes, 2000), triggered the release of NT-3 by hippocampal neurons, and regulated the levels of NMDA-receptor subunits, NR2A and NR2B, thereby controlling synapse formation (Blondel et al., 2000). It was thus hypothesized that ADNF secreted by glia is required during early development, in order for hippocampal neurons to achieve full morphological and synaptic maturation. Antibody studies suggested that ADNF-like molecules mediate VIP neuroprotective and neurotrophic activities (e.g., White et al., 2000; Blondel et al., 2000; Steingart, Solomon, Brenneman, Fridkin, & Gozes, 2000). However, in vivo studies

Femtomolar-Acting Neuroprotective Peptides

211

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

indicated that ADNF-9 is not as efficacious as NAP (Bassan et al., 1999; Gozes et al., 2000), suggesting that ADNF-9 should be further modified for drug development. NAP. As indicated above, the mechanism of neuroprotection by VIP may involve cGMP production. It is now shown that femtomolar concentrations of NAP increase cGMP production in neuroglial cells (AshurFabian, Sigalov, Brenneman, & Gozes, 1999). NAP protected against oxidative stress induced by dopamine (Offen et al., 2000) or by oxygen peroxide (Steingart et al., 2000), and provided significant neuroprotection against BSO toxicity. These results indicate that neuroprotection by VIP/NAP may involve raising cellular resistance against oxidative stress (Offen et al., 2000). NAP, as well as ADNF-9, protected human neurons against oxidative stress caused by H2O2 or increased expression of amyloid precursor protein in Down Syndrome neurons (Pelsman, Fernandez, Gozes, Brenneman, & Busciglio, 1998). In the closed head-injury model, NAP protected against increases in TNFa, increases that are inflammatory in nature. Furthermore, NAP protected against TNFoc-induced PC12 cell death (Beni-Adani et al., 2000). In ApoE-deficient mice and in rats suffering from cholinotoxicity, NAP (as well as SNV, Gozes et al., 1997a) provided protection against a reduction in choline acetyl transferase activity (Bassan et al., 1999; Gozes et al., 2000). Finally, femtomolar concentrations of NAP promoted neurite outgrowth in rat hippocampal cultures (SmithSwintosky, Gozes, Brenneman, & Plata-Salaman, 2000). In conclusion, neurotrophic peptides with increased stability and bioavailability are suggested as future therapeutics. Preclinical studies are now aimed at strengthening the data with additional models and development of assay systems for the pharmacokinetic evaluations. Further experiments are aimed at identifying the target-binding proteins, as well as developing pipeline drugs and clinical protocols.

ht

tp

REFERENCES Ashur-Fabian, O., Perl, O., Lilling, G., Fridkin, M., & Gozes, I. (1999). SNV, a lipophilic superactive VIP analog, acts through cGMP to promote neuronal survival. Peptides, 20, 629-633. Ashur-Fabian, O., Sigalov, E., Brenneman, D. E., & Gozes, I. (1999). Neuroscience Letters, 54, S3. Bassan, M., Zamostiano, R., Davidson, A., Pinhasov, A., Giladi, E., Perl, O., Bassan, H., Blat, C, Gibney, G., Glazner, G., Brenneman, D. E., & Gozes, I. (1999). Complete sequence of a novel protein containing a femtomolar-activitydependent neuroprotective peptide. Journal ofNeurochemistry, 72,1283-1293. Beni-Adani, L., Gozes, I., Cohen, Y, Assaf, Y, Steingart, R. A., Brenneman, D. E., Eizenberg, O., Trembolver, V, & Shohaml, E. (in press). A peptide derived

222

Drug Development—Preclinical

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

from activity-dependent neuroprotective protein (ADNP) ameliorates injury response in closed head injured mice. Journal of Pharmacology and Experimental Therapeutics, Blondel, O., Collin, C, McCarran, B., Zhu, S., Zamostiano, R., Gozes, I., Brenneman, D. E., & McKay R. D. G. (2000). A glia-derived signal regulating neuronal differentiation. Journal of Neuroscience, 20, 8012-8020. Brenneman, D. E., & Gozes, I. (1996). A femtomolar-acting neuroprotective peptide. Journal of Clinical Investigation, 97, 2299-2307. Brenneman, D. E., Hauser,}., Neale, E., Rubinraut, S., Fridkin, M, Davidson, A., & Gozes, I. (1998). Activity-dependent neurotrophic factor: Structure-activity relationships of femtomolar-acting peptides. Journal of Experimental Therapeutics, 285, 619-627. Eilam, R., Davidson, A., Gozes, I., & Segal, M. (1999). Locomotor activity causes a rapid increase in the expression of vasoactive intestinal peptide in the rat brain. Hippocampus, 9, 534-541. Glazner, G. W., Boland, A., Dresse, A. E., Brenneman, D. E., Gozes, I., & Mattson, M. P. (1999). Activity-dependent neurotrophic factor peptide (A.DNF9) protects neurons against oxidative stress-induced death. Journal of Neurochemistry, 6, 2341-2347. Glazner, G. W., Camandola, S., & Mattson, M. P. (2000). Nuclear factor-kappaB mediates the cell survival-promoting action of activity-dependent neurotrophic factor peptide-9. Journal of Neurochemistry, 75,101-108. Gozes, I., Bachar, M., Bardea, A., Davidson, A., Rubinraut, S., Fridkin, M., & Giladi, E. (1997a). Protection against developmental retardation in apolipoprotein E-deficient mice by a fatty neuropeptide: Implication for early treatment of Alzheimer's disease. Journal of Neurobiology, 33, 329-342. Gozes, I., Bardea, A., Reshef, A., Zamostiano, R., Zhukovsky, S., Rubinraut, S., Fridkin, M., & Brenneman, D. E. (1996a). Novel neuroprotective strategy for Alzheimer's disease: Inhalation of a fatty neuropeptide. Proceedings of the National Academy of Sciences (USA), 93, 427-432. Gozes, I., & Brenneman, D. E. (1996b). Activity-dependent neurotrophic factor (ADNF): An extracellular neuroprotective chaperonin? Journal of Molecular Neuroscience, 7, 235-244. Gozes, I., Davidson, A., Gozes, Y., Mascolo, R., Barth, R., Warren, D., Hauser, J., & Brenneman, D. E. (1997b). Antiserum to activity-dependent neurotrophic factor produces neuronal cell death in CNS cultures: Immunological and biological specificity. Developmental Brain Research, 99,167-175. Gozes, I., Fridkin, M., Hill, J. M., & Brenneman, D. E. (1999a). Pharmaceutical VIP: Prospects and problems. Current Medicinal Chemistry, 6,1019-1034. Gozes, I., Fridkin, M., Westphal, H., Glowa, J., Reshef, A., Zhukovsky, S., Waner, T., Niska, A., Rubinrout, S., Lilling, G., Davidson, A., Glazer-Steiner, R., Moody, T. W., Rostene, W., & Brenneman, D. E. (1994). Neuronal VIP: From gene to sexual behavior, memory and clinical applications. The Proceedings of the International Symposium on VIP, PACAP and related regulatory peptides, pp. 314-324. Gozes, I., Giladi, E., Pinhasov, A., Bardea, A., & Brenneman, D. E. (2000). Activitydependent neurotrophic factor: Intranasal administration of femtomolar-

Femtomolar-Acting Neuroprotective Peptides 213

213

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

acting peptides improve performance in a water maze. Journal of Pharmacology and Experimental Therapeutics, 293,1091-1098. Gozes, I., Perl, O., Giladi, E., Davidson, A., Ashur-Fabian, O., Rubinraut, S., & Fridkin, M. (1999b). Mapping the active site in vasoactive intestinal peptide to a core of four amino acids: Neuroprotective drug design. Proceedings of the National Academy of Sciences (USA), 96, 4143-4148. Gressens, P., Besse, L., Robberecht, P., Gozes, I., Fridkin, M., & Evrard, P. (1999). Neuroprotection of the developing brain by systemic administration of vasoactive intestinal peptide derivatives. Journal of Pharmacology and Experimental Therapeutics, 288,1207-1213. Guo, Q., Sebastian, L., Sopher, B. L., Miller, M. W., Glazner, G. W., Ware, C. B., Martin, G. M., & Mattson, M. P. (1999). Neurotrophic factors [activity-dependent neurotrophic factor (ADNF) and basic fibroblast growth factor (bFGF)] interrupt excitotoxic neurodegenerative cascades promoted by a PS1 mutation. Proceedings of the National Academy of Sciences (USA), 96, 4125-4130. Guo, Z. H., & Mattson, M. P. (2000). Neurotrophic factors protect cortical synaptic terminals against amyloid and oxidative stress-induced impairment of glucose transport, glutamate transport and mitochondrial function. Cerebral Cortex, 10, 50-57. Hill, J. M., Lee, S. J., Dibbern, D. A., Fridkin, M., Gozes, L, & Brenneman, D. E. (1999). Pharmacologically distinct vasoactive intestinal peptide binding sites: CNS localization and role in embryonic growth. Neuroscience, 93, 783-791. Offen, D., Sherki, Y., Melamed, E., Fridkin, M., Brenneman, D. E., & Gozes, I. (2000). Vasoactive intestinal peptide (VIP) prevents neurotoxicity in neuronal cultures: Relevance to neuroprotection in Parkinson's disease. Brain Research, 854, 257-262. Pelsman, A., Fernandez, G., Gozes, I., Brenneman, D. E., & Busciglio, J. (1998). Journal of Neurochemistry, 72, S54. Sigalov, E., Steingart, R. A., Fridkin, M., Brenneman, D. E., & Gozes, I. (2000). VIPrelated protection against iodoacetate toxicity in pheochromocytoma (PCI2) cells: A model for ischemic/hypoxic injury. Journal of Molecular Neuroscience, 15,147-154. Smith-Swintosky, V. L., Gozes, I., Brenneman, D. E., & Plata-Salaman, C. R. (2000). Society for Neuroscience, 317, 6. Steingart, R. A., Solomon, B., Brenneman, D. E., Fridkin, M., & Gozes, I. (2000). VIP and peptides related to activity-dependent neurotrophic factor protect PC 12 cells against oxidative stress. Journal of Molecular Neuroscience, 15,137-145. Wang, H. Y, Jiang, X., Gozes, I., Fridkin, M., Brenneman, D. E., & Ganea, D. (1999). Vasoactive intestinal peptide inhibits cytokine production in T lymphocytes through cAMP-dependent and cAMP-independent mechanisms. Regulatory Peptide, 84, 55-67. White, D. M., Walker, S., Brenneman, D. E., & Gozes, I. (2000). CREB contributes to the increased neurite outgrowth of sensory neurons induced by vasoactive intestinal polypeptide and activity-dependent neurotrophic factor. Brain Research, 868, 31-38. Zamostiano, R., Pinhasov, A., Bassan, M., Perl, O., Steingart, R. A., Atlas, R., Brenneman, D. E., & Gozes, I. (1999). A femtomolar-acting neuroprotective

224

Drug Development—Preclinical

x. ph

p

peptide induces intracellular increases in heat shock protein 60: A potential neuroprotective mechanism. Neuroscience Letters, 264, 9-12. Zamostiano, R., Pinhasov, A., Gelber, E., Steingart, R. A., Seroussi, E., Giladi, E., Bassan, M., Wollman, Y, Eyre. H.}., Mulley, J. C, Brenneman, D. E., & Gozes, I. (2001). Cloning and characterization of the human activity-dependent neuroprotective protein (ADNP). Journal of Biological Chemistry, 276, 708-714. Zemlyak, I., Furman, S., Brenneman, D. E., & Gozes, I. (2000). A novel peptide (NAP) prevents death in enriched neuronal cultures. Regulatory Peptide, 96, 39-43.

s/ in de

ACKNOWLEDGMENTS

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

Supported in part by the Institute for the Study of Aging, New York, NY; BSF, ISF, Ramot of Tel Aviv University; and NIA and NICHD, Bethesda, MD. Professor Illana Gozes is the incumbent of the Lily and Avraham Gildor Chair for the Investigation of Growth Factors. Original studies on VIP derivatives were supported by Fujimoto Pharmaceutical Corp., Osaka, Japan. Patents and patent applications have been placed for all the compounds described.

he m

.c

w

w

w

://

tp

ht

m

ru

/fo

vn

4a ll.

p

Drug Development—

Clinical

x. ph

s/ in de

IV

p

x. ph

s/ in de

m

ru

/fo

vn

4a ll.

m

.c he

w

w

w

://

tp

ht This page intentionally left blank

24

x. ph

of Ex Vivo Nerve Growth

p

A Phase I ClinicalStudy

s/ in de

Factor Gene Therapy for

m

Alzheimer's Disease

vn

/fo

ru

Mark H. Tuszynski

4a ll.

ABSTRACT

://

w

w

w

.c

he m

Cholinergic neuronal degeneration is a prominent component of cell loss in Alzheimer's disease. Nerve growth factor (NGF) can prevent the death of basal forebrain cholinergic neurons after injury or excitotoxic damage, and can reverse age-related spontaneous atrophy of neurons in the system. Ex vivo gene therapy may be an effective means of delivering NGF to the brain in a therapeutic effort to prevent or reduce the extent of cholinergic neuronal decline in Alzheimer's disease.

ht

tp

INTRODUCTION Alzheimer's disease (AD) is the most common neurodegenerative disorder, currently afflicting approximately 4 million people in the United States. Whereas the molecular mechanisms leading to the development of AD remain the subject of active investigation, certain pathological hallmarks of the disease are clearly recognizable. These pathological features include the abnormal accumulation of extracellular, insoluble [3-amyloid, the formation of intraneuronal, neurofibrillary tangles, synapse loss, and cellular degeneration. Cellular degeneration occurs in several neuronal populations in the central nervous system. Among the neuronal populations that degenerate in AD, loss of basal forebrain cholinergic neurons is particularly 217

218

Drug Development—Clinical

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

severe (Bartus, Dean, Beer, & Lippa, 1982; Coyle, Price, & Delong, 1983). Loss of cholinergic neurons in AD correlates with severity of clinical dementia, the density of amyloid plaques in the brain, and the extent of synapse loss (Bartus et al., 1982; Coyle et al., 1983; Mufson & Kordower, 1989; Perry et al., 1978; Terry, Katzman, & Bick, 1994). To date, the only FDA-approved therapies for AD focus on augmenting the function of degenerating cholinergic neurons. Studies outlined in this chapter aim to move beyond compensating for cholinergic neuronal degeneration by attempting to (a) protect cholinergic neurons from degeneration, and (b) augment the function of remaining cholinergic neurons through direct elevation of choline acetyltransferase (ChAT) function in neurons. These two therapeutic interventions may be achievable by the delivery of human nerve growth factor (NGF) to the brain. NGF is a naturally occurring pentameric protein that is normally synthesized by targets of basal forebrain cholinergic innervation. The active component of NGF, (3-NGF (MW 12,250 daltons), mediates the active in vivo biological effects of the molecule. During development of the nervous system, NGF establishes normal patterns of cholinergic projections to the hippocampus and the neocortex. In 1986, it was reported for the first time that the delivery of soluble NGF protein to the brain could completely prevent lesion-induced degeneration of basal forebrain cholinergic neurons in the adult rodent brain. Three different groups of investigators reported that following fimbria-fornix lesions, infusion of mouse NGF intracerebroventricularly prevented the loss of 100% of injured cholinergic neurons (Hefti, 1986; Kromer, 1987; Williams et al., 1986). These findings were closely followed by the report that the infusion of mouse NGF into the aged rodent brain reversed spontaneous, age-related atrophy of basal forebrain cholinergic neurons, and reversed age-associated loss of memory function on a spatial memory task (Fischer et al., 1987). These findings have subsequently been replicated by several independent research groups (Chen & Gage, 1995; Lindner, Kearns, Winn, Frydel, & Emerich, 1996; Markowska, Koliatsos, Breckler, Price, & Olton, 1994; Martinez-Serrano, Fischer, & Bjorklund, 1995; Mervis, Pope, Lewis, Dvorak, Williams, 1991; Rylett, Goddard, Schmidt, & Williams, 1993; Williams, Rylett, Moises, & Tang, 1991). Similar protection of basal forebrain cholinergic neurons was subsequently demonstrated by infusions of recombinant human NGF into the brains of rodents (Barnett et al., 1990). In 1990, two groups of investigators reported that the infusion of murine NGF intracerebroventricularly to the adult primate brain (Macaca fascicularis) also prevented cholinergic degeneration following lesions of basal forebrain neurons (Koliatsos et al., 1990; Tuszynski, U., Amaral, & Gage, 1990). In 1991, it was demonstrated that infusions of recombinant human NGF into the primate ventricular system also prevented lesion-induced degeneration of basal forebrain cholinergic neurons (Tuszynski, U., & Gage, 1991).

Ex Vivo Nerve Growth Factor Gene Therapy for AD 219

219

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Thus, NGF prevented both lesion-induced and spontaneous, age-related degeneration of basal forebrain cholinergic neurons. Further, NGF infusions reversed both lesion-induced memory loss and spontaneous, age-related memory loss in rodents. Based on these findings, NGF administration offers significant potential as a neuroprotective strategy in AD (Appel, 1981; Hefti & Weiner, 1986; Holtzman & Mobley, 1994; Tuszynski, 1998; Tuszynski, 1998; Yuen & Mobley, 1996) In addition, further studies demonstrated that NGF augmented the function of cholinergic neurons. Infusions of NGF into the rodent brain substantially augmented ChAT activity (Dekker, Gage, & Thai, 1992; Dekker, Langdon, Gage, & Thai, 1991; Rylett et al., 1993; Williams, Jodelis, & Donald, 1989; Williams et al., 1991). In vivo microdialysis also demonstrated directly that intracerebroventricular NGF infusions elevated ACh levels in the brain. Thus, the delivery of NGF to the brain offered the potential to both rescue degenerating cholinergic neurons and to augment the function of remaining neurons. Given the potential of NGF to treat AD, preclinical toxicity studies were performed. Several groups independently discovered that infusions of NGF were associated with significant toxicity problems. It should be noted that NGF is a relatively large, polar protein molecule that cannot cross the blood-brain barrier. In order to access responsive neurons in the brain, NGF must be delivered across the blood-brain barrier; this had been achieved by infusing NGF intracerebroventricularly. However, preclinical toxicity studies showed that intracerebroventricular infusions of NGF were associated with four distinct toxicities: (a) Weight loss caused by hypophagia (Williams, 1991); (b) sprouting of sympathetic axons around the cerebral vasculature (Isaacson, Saffran, & Crutcher, 1990; Saffran & Crutcher, 1990); (c) migration of Schwann cells from the peripheral nervous system into the central nervous system (Schwann cells migrated into the subpial space surrounding the spinal cord and medulla, forming an expanding cell layer that was approximately 20 to 40 cells thick along the extent of the medulla and spinal cord (Winkler et al., 1996); these changes spontaneously regressed with time following the discontinuation of NGF infusions); and (d) sprouting of sensory, primary nociceptive axons into the expanding Schwann cell layer in the subpial space (Winkler et al., 1996). These adverse effects, although reversible by discontinuation of NGF intracerebroventricular infusions, were substantial. Three AD patients in Sweden, in fact, received intracerebroventricular infusions of murine NGF (Jonhagen et al., 1998; Olsen et al., 1992). These patients developed a pain syndrome that was likely attributable to the sprouting of sensory nociceptive axons into the subpial space, and also exhibited weight loss. As a result, intracerebroventricular NGF infusions had to be discontinued in these patients. In addition, another patient with Parkinson's disease

220

Drug Development—Clinical

ht

tp

://

w

w

w

.c he

m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

received a 23-day infusion of NGF intraputaminally to support an adrenal autograft (Olsen et al., 1991). Reportedly the patient suffered no adverse events as a result of this NGF infusion. Given these significant adverse effects, intracerebroventricular NGF infusions do not appear to be a practical or clinically useful means of NGF delivery to the central nervous system for human clinical trials. Over the last decade, ex vivo gene therapy has been developed as an alternative method for delivering NGF to the brain (Chen & Gage, 1995; Kawaja, Rosenberg, Yoshida, & Gage, 1992; Rosenberg et al., 1988; Tuszynski, Smith, Roberts, McKay, & Mufson, 1998; Tuszynski & Gage, 1995; Roberts, Senut, U., & Gage, 1996; Tuszynski, Senut, Ray, U., & Gage, 1994). In first rodent and then primate studies, primary fibroblasts were genetically modified using retroviral vectors to produce and secrete significant quantities of human P-NGF growth factor. When genetically modified cells are grafted into the brain, they have been shown to prevent lesioninduced cholinergic degeneration in the adult rodent brain, to reverse spontaneous age-related atrophy of basal forebrain cholinergic neurons in aged rats, and to ameliorate age-related mnemonic deficits in rats (Chen & Gage, 1995; Kawaja et al., 1992; Rosenberg et al., 1988). Several groups have reported similar findings (Emerich, Hammang, Baetge, & Winn, 1994; Emerich et al., 1994; Hoffman, Breakefield, Short, & Aebischer, 1993; Kordower et al., 1994; Lindner et al., 1996; Martinez-Serrano et al., 1995). More recently, we have extended these studies to primate systems (Smith, Roberts, Gage, & Tuszynski, 1999; Tuszynski et al., 1998; Tuszynski & Gage, 1995; Tuszynski et al., 1996,1994). Injections of autologous primate fibroblasts, genetically modified to produce (3-NGF, substantially reduce basal forebrain cholinergic neuronal degeneration. This effect is significant at a 1-month postlesion time point in adult Macaca fascicularis monkeys. The effect has been observed to persist in one monkey studied 8 months postlesion. In addition, recent studies have shown that spontaneous atrophy of basal forebrain cholinergic neurons occurs in the rhesus monkey, and that ex vivo NGF gene therapy (grafting autologous fibroblasts producing human NGF) will reverse spontaneous age-related atrophy of basal forebrain cholinergic neurons in monkeys (Smith et al., 1999). In these recent studies in aged monkeys, ex vivo NGF gene therapy has not caused detectable adverse effects: (a) monkeys do not suffer significant weight loss; and (b) histological analysis of the brain reveals no abnormalities: specifically, migration of Schwann cells does not occur into the subpial space surrounding the brain stem and spinal cord, and sensory and sympathetic axons are not observed to sprout abnormally in the central nervous system. Thus, ex vivo NGF gene therapy is an effective method of preventing or reversing cholinergic neuronal degeneration in the adult primate brain and is not associated with adverse effects in rodent and primate studies that have been conducted to date.

221

Ex Vivo Nerve Growth Factor Gene Therapy for AD 221

IN VIVO GENE EXPRESSION

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Grafts of primary fibroblasts transduced to express human NGF have been shown to sustain NGF in vivo gene expression for at least 18 months in the rodent central nervous system (Tuszynski & Conner, unpublished obervations). These findings are based upon measurement of NGF protein by two-site ELISA. In addition, these grafts sustain NGF messengerRNA production for at least 6 months in vivo, as demonstrated by in situ hybridization. The grafts have also been demonstrated to sustain NGF mRNA expression by RT-PCR for at least 14 months in vivo. In primate systems, ex vivo NGF gene therapy has been demonstrated to sustain NGF protein production in the brain in the rhesus money for at least 1 year, by two-site ELISA. In no case have grafts of primary fibroblasts transduced to express human NGF been shown to form tumors after in vivo grafting. In an experience base of 100 grafts of autologous, NGF-secreting fibroblasts to the primate brain, tumor formation has not been observed. An experience base of more than 100 grafts of primary fibroblasts transduced to express NGF to the rodent brain has similarly never resulted in tumor formation. When NGF-transduced primary fibroblasts are grafted to the rodent spinal cord, these grafts have shown a slow increase in size over time points of 1 year; this expansion in graft size is caused by continued growth of responsive axons into the grafts, and is not caused by abnormal growth or tumor formation in primary, nonimmortalized, genetically modified fibroblasts. Thus, the available data suggests that ex vivo NGF gene therapy is an effective means of preventing loss of basal forebrain cholinergic neurons and of augmenting cholinergic function in the primate brain. In animals, this procedure is safe and well tolerated. Based on these data, clinical trials of ex vivo NGF gene therapy in AD at the University of California, San Diego, have been proposed and approved by the Food and Drug Administration for initiation in 2001. This research program aims to determine whether gene therapy delivering human NGF to the AD brain will prevent or reduce the rate of decline of cholinergic neuronal degeneration, and promote the cholinergic phenotype of remaining neurons.

REFERENCES Appel, S. H. (1981). A unifying hypothesis for the cause of amyotrophic lateral sclerosis, parkinsonism, and Alzheimer disease. Annals of Neurology, 10,499-505. Barnett, J., Baecker, P., Routledge-Ward, C, Bursztyn-Pettegrew, H., Chow, J., Nguyen, B., Bach, C., Chan, H., Tuszynski, M. H., Yoshida, K., Rubalcava, R., & Gage, F. H. (1990). Human beta nerve growth factor obtained from a

222

Drug Development—Clinical

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

baculovirus expression system has potent in vitro and in vivo neurotrophic activity. Experimental Neurology, 110,11-24. Bartus, R., Dean, R. L., Beer, C, & Lippa, A. S. (1982). The cholinergic hypothesis of geriatric memory dysfunction. Science, 217, 408-^17. Chen, K. S., & Gage, F. H. (1995). Somatic gene transfer of NGF to the aged brain: Behavioral and morphological amelioration. Journal of Neuroscience, 15, 2819-2825. Coyle, J. T., Price, P. H., & Delong, M. R. (1983). Alzheimer's disease: A disorder of cortical cholinergic innervation. Science, 219,1184-1189. Dekker, A. J., Gage, F. H., & Thai, L. J. (1992). Delayed treatment with nerve growth factor improves acquisition of a spatial task in rats with lesions of the nucleus basalis magnocellularis: Evaluation of the involvement of different neurotransmitter systems. Neuroscience, 48,111-119. Dekker, A. J., Langdon, D. J., Gage, F. H., & Thai, L. J. (1991). NGF increases cortical acetylcholine release in rats with lesions of the nucleus basalis. Neuroreport, 2, 577-580. Emerich, D. F, Hammang, J. P., Baetge, E. E., & Winn, S. R. (1994). Implantation of polymer-encapsulated human nerve growth factor-secreting fibroblasts attenuates the behavioral and neuropathological consequences of quinolinic acid injections into rodent striatum. Experimental Neurology, 130,141-150. Emerich, D. W., Winn, S., Harper, J., Hammang, J. P., Baetge, E. E., & Kordower, J. H. (1994). Implants of polymer-encapsulated human NGF-secreting cells in the nonhuman primate: Rescue and sprouting of degenerating cholinergic basal forebrain neurons. Journal of Comparative Neurology, 349,148-164. Fischer, W., Wictorin, K., Bjorklund, A., Williams, L. R., Varon, S., & Gage, F. H. (1987). Amelioration of cholinergic neuron atrophy and spatial memory impairment in aged rats by nerve growth factor. Nature, 329, 65-68. Hefti, F. (1986). Nerve growth factor (NGF) promotes survival of septal cholinergic neurons after fimbrial transection. Journal of Neuroscience, 6, 2155-2162. Hefti, F, & Weiner, W. J. (1986). Nerve growth factor and Alzheimer's disease. Annals of Neurology, 20, 275-281. Hoffman, D., Breakefield, X. O., Short, M. P., & Aebischer, P. (1993). Transplantation of a polymer-encapsulated cell line genetically engineered to release NGF. Experimental Neurology, 122,100-106. Holtzman, D. M., & Mobley, C. M. (1994). Neurotrophic factors and neurologic disease. Western Journal of Medicine, 161, 246-254. Isaacson, L. G., Saffran, B. N., & Crutcher, K. A. (1990). Intracerebral NGF infusion induces hyperinnervation of cerebral blood vessels. Neurobiology of Aging, 11, 51-55. Jonhagen., M. E. N., Amberla, K., Backman, L., Ebendal, T., Meyerson, B., Olson, L., Seiger, A., Shigeta, M., Theodorsson, E., Viitanen, M., Winblad, B., & Wahlund, L. (1998). Intracerebroventricular infusion of nerve growth factor in three patients with Alzheimer's disease. Dementia and Geriatric Cognitive Disorders, 9,246-257. Kawaja, M., Rosenberg, M., Yoshida, K., & Gage, F. H. (1992). Somatic gene transfer of nerve growth factor promotes the survival of axotomized septal neurons and the regeneration of their axons in adult rats. Journal of Neuroscience, 12,2849-2864.

Ex Vivo Nerve Growth Factor Gene Therapy for AD 223

223

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Koliatsos, V. E., Nauta, H. J., Clatterbuck, R. E., Holtzman, D. M, Mobley, W. C, & Price, D. L. (1990). Mouse nerve growth factor prevents degeneration of axotomized basal forebrain cholinergic neurons in the monkey. Journal of Neuroscience, 10, 3801-3813. Kordower, J. H., Winn, S. R., Liu, Y.-T., Mufson, E. J., Sladek, J. R., Hammang, J. P., Baetge, E. E., & Emerich, D. F. (1994). The aged monkey basal forebrain: Rescue and sprouting of axotomized basal forebrain neurons after grafts of encapsulated cells secreting human nerve growth factor. Proceedings of the National Academy of Sciences (USA), 91,10898-10902. Kromer, L. F. (1987). Nerve growth factor treatment after brain injury prevents neuronal death. Science, 235, 214-216. Lindner, M. D., Kearns, C. E., Winn, S. R., Frydel, B., & Emerich, D. F. (1996). Effects of intraventricular encapsulated hNGF-secreting fibroblasts in aged rats. Cell Transplantation, 5, 205-23. Markowska, A. L., Koliatsos, V. E., Breckler, S. J., Price, D. L., & Olton, D. S. (1994). Human nerve growth factor improves spatial memory in aged but not in young rats. Journal of Neuroscience, 14, 4815-4824. Martinez-Serrano, A., Fischer, W., & Bjorklund, A. (1995). Reversal of age-dependent cognitive impairments and cholinergic neuron atrophy by NGF-secreting neural progenitors grafted to the basal forebrain. Neuron, 15, 473-484. Mervis, R. F, Pope, D., Lewis, R., Dvorak, R. M., & Williams, L. R. (1991). Exogenous nerve growth factor reverses age-related structural changes in neocortical neurons in the aging rat. A quantitative Golgi study. Annals of the New York Academy of Sciences (USA), 640, 95-101. Mufson, E. J., & Kordower, J. H. (1989). Nerve growth factor expressing human basal forebrain neurons: Pathologic alterations in Alzheimer's and Parkinson's diseases. In Alzheimer's disease and related disorders (pp. 401-414). New York: Alan R. Liss, Inc. Olson, L., Backlund, E.-O., Ebendal, T., Freedman, R., Hamberger, B., Hansson, P., Hoffer, B., Lindblom, U., Meyerson, B., Stromberg, I., Sydow, O., & Sieger, A. (1991). Intraputaminal infusion of nerve growth factor to support medullary autografts in Parkinson's disease. Archives of Neurology, 48, 373-381. Olson, L., Nordberg, A., von Hoist, H., Backman, L., Ebendal, T., Alafuzoff, I., Amberla, K., Hartvig, P., Herlitz, A., & Lilja, A. (1992). Nerve growth factor affects llC-nicotine binding, blood flow, EEG, and verbal episodic memory in an Alzheimer's patient. Journal of Neural Transmission, 4, 79-95. Perry, E. K., Tomlinson, B. E., Blessed, G., Bergmann, K., Gibson, P. H., & Perry, R. H. (1978). Correlation of cholinergic abnormalities with senile plaques and mental test scores in senile dementia. British Medical Journal, 2,1457-1459. Rosenberg, M. B., Friedmann, T., Robertson, R. C., Tuszynski, M., Wolff, J. A., Breakefield, X. O., & Gage, F. H. (1988). Grafting genetically modified cells to the damaged brain: Restorative effects of NGF expression. Science, 242,1575-1578. Rylett, R. J., Goddard, S., Schmidt, B. M., & Williams, L. R. (1993). Acetylcholine synthesis and release following continuous intracerebral administration of NGF in adult and aged Fischer-344 rats. Journal of Neuroscience, 13, 3956-3963. Saffran, B. N., & Crutcher, K. A. (1990). NGF-induced remodeling of mature uninjured axon collaterals. Brain Research, 525,11-20.

224

Drug Development—Clinical

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Smith, D. E., Roberts, J., Gage, F. H., Tuszynski, M. H. (1999). Age-associated neuronal atrophy occurs in the primate brain and is reversible by growth factor gene therapy. Proceedings of the National Academy of Sciences (USA), 96,10893-10898. Terry, R. D., Katzman, R., & Bick, K. L. (1994). Alzheimer disease. New York: Raven Press. Tuszynski, M. H. (1998). Gene therapy for neurological disease. Annals of the New York Academy of Sciences (USA), 835,1-11. Tuszynski, M. H. (1998). Gene therapy: Applications to the neurosciences and to neurological disease. The Neuroscientist, 4, 398-407. Tuszynski, M. H., Smith, D. E., Roberts, J., McKay, H., & Mufson,. E. (1998). Targeted intraparenchymal delivery of human NGF by gene transfer to the primate basal forebrain for 3 months does not accelerate (3-amyloid plaque deposition. Experimental Neurology, 154, 573-582. Tuszynski, M. H., & Gage, F. H. (1995). Maintaining the neuronal phenotype after injury in the adult CNS. Molecular Neuwbiology, 10,151-167. Tuszynski, M. H., Roberts, J., Senut, M. C, U., H.-S., & Gage, F. H. (1996). Gene therapy in the adult primate brain: Intraparenchymal grafts of cells genetically modified to produce nerve growth factor prevent cholinergic neuronal degeneration. Gene Therapy, 3, 305-314. Tuszynski, M. H., Senut, M. C., Ray, J., U., H.-S., & Gage, F. H. (1994). Somatic gene transfer to the adult primate CNS: In vitro and in vivo characterization of cells genetically modified to secrete nerve growth factor. Neurobiology of Disease, 1, 67-78. Tuszynski, M. H., U., H.-S., & Gage, F. H. (1991). Recombinant human nerve growth factor infusions prevent cholinergic neuronal degeneration in the adult primate brain. Annals of Neurology, 30, 625-636. Tuszynski, M. H., U., H. S., Amaral, D. G., & Gage, F. H. (1990). Nerve growth factor infusion in primate brain reduces lesion-induced cholinergic neuronal degeneration. Journal ofNeuroscience, 10, 3604-3614. Williams, L. R. (1991). Hypophagia is induced by intracerebroventricular administration of nerve growth factor. Experimental Neurology, 113, 31-37. Williams, L. R., Jodelis, K. S., & Donald, M. R. (1989). Axotomy-dependent stimulation of choline acetyltransferase activity by exogenous nerve growth factor in adult rat basal forebrain. Brain Research, 498, 243-255. Williams, L. R., Rylett, R. J., Moises, H. C., & Tang, A. H. (1991). Exogenous NGF affects cholinergic transmitter function and Y-maze behavior in aged Fischer 344 male rats. Canadian Journal of Neurological Sciences, 18, 403-407. Williams, L. R., Varon, S., Peterson, G. M., Wictorin, K., Fisher, W, Bjorklund, A., & Gage, F. H. (1986). Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria-fornix transection. Proceedings of the National Academy of Sciences (USA), 83, 9231-9235. Winkler, J., Ramirez, G. A., Kuhn, H. G., Peterson, D. A., Day-Lollini, P. A., Stewart, G. R., Tuszynski, M. H., Gage, F. H., & Thai, L. J. (1996). Reversible induction of Schwann cell hyperplasia and sprouting of sensory and sympathetic neurites in vivo after continuous intracerebroventricular administration of nerve growth factor. Annals of Neurology, 40,128-139. Yuen, E. C., & Mobley, W. C. (1996). Therapeutic potential of neurotrophic factors for neurological disorders. Annals of Neurology, 40, 346-354.

25

s/ in de

Alzheimer's Disease

x. ph

Levels in Patients With

p

Lowering Homocysteine

he m

INTRODUCTION

4a ll.

vn

/fo

ru

m

Paul Aisen, Mary Sana, Ramon Diaz-Arrastia, and William Jagust

ht

tp

://

w

w

w

.c

Alzheimer's disease (AD) is the most common form of dementia and the greatest source of disability in aging. The incidence increases with age, ranging from less than 1% at age 65 to 8% after age 85. Given this increasing rate of disease with age, as well as the fact that the aging population (over age 65) is the fastest growing segment in our society, it is apparent that this disease will reach epidemic proportions. In addition, there is a 3- to 5-year period of mild but significant cognitive impairment that precedes the diagnosis; the societal trend toward a growing technological complexity suggests that even minimal cognitive loss may lead to significant disability. The need to find treatment to minimize the impact of dementia and cognitive loss associated with aging and neurodegeneration is imperative, and there is growing evidence that lowering homocysteine may be one mechanism for providing benefit. This chapter will: 1. Outline the role of homocysteine in cardiovascular, neurologic, and cerebrovascular disease; 2. Summarize the data about homocysteine in AD and in cognitive status; 225

226

Drug Development—Clinical

3. Describe the primary intervention for hyperhomocysteinemia and propose it as an empirical treatment for AD, including the technical difficulties in studying such an intervention; and 4. Propose future directions for evaluating this mechanism in cognitive deterioration in aging and dementia.

x. ph

p

HOMOCYSTEINE IN CARDIOVASCULAR AND NEUROLOGIC DISEASE

o

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

[Z

s/ in de

Homocysteine is a sulfur amino acid; it is involved in methionine and cysteine metabolism, and plays a role in methylation reactions. Folate, as well as vitamins B,. and B,, are cofactors in metabolic reactions involving homocysteine; deficiencies of these vitamins may result in elevation of homocysteine in blood. Elevated homocysteine is a sensitive marker of B12 and folate deficiency. Mild hyperhomocysteinemia is an important risk factor for peripheral vascular disease (Bots, Launer, Lindemons, Hofman, & Grobbee, 1997; Clarke, Daly, Robinson, et al., 1991; Moghadasian, McManus, & Frohlich, 1997), cardiovascular disease (Bostom, Silbershatz, & Rosenberg, et al., 1999b; Bots et al., 1997; Clarke et al., 1991; Eikelboom, Lonn, Genest, Hankey, & Yusuf, 1999; Hoogeveen, Kostense, Beks, et al., 1998), and stroke (Bostom, Rosenberg, Silbershatz, et al., 1999a; Clarke et al., 1991; Selhub, Jacques, Bostom, et al., 1996), similar in effect to hypercholesterolemia and smoking (Boers, 1997). Mechanisms may include a direct toxic effect of mild hyperhomocysteinemia on endothelial cells, as well as increased thrombin formation, free radical generation, and adhesiveness of inflammatory cells (Bellamy & McDowell, 1997). A number of factors are known to be associated with hyperhomocysteinemia, including methylenetetrahydrofolate reductase (MTHFR) genotype (Jacques, Bostom, Williams, et al., 1996), nutritional status (Selhub, Jacques, Wilson, Rush, & Rosenberg, 1993), renal function (Norlund, Grubb, Fex, et al., 1998), age (Selhub et al., 1993; Jacques, Rosenberg, Rogers, et al., 1999a; Joosten, Lesaffre, & Riezler, 1996), gender (Jacques et al., 1999a), and smoking (Kato, Dnistrian, Schwartz, et al., 1999). Regardless of the contributing factors, treatment with high doses of folate and vitamins B]2 and B6 reduces blood levels of homocysteine. A recent meta-analysis indicated that supplementation lowers homocysteine levels by a mean of 30%, varying from 16% to 40% depending on initial levels (Anonymous, 1998). In view of the well-documented association between hyperhomocysteinemia and vascular disease, it is not surprising that elevated levels of homocysteine are found in individuals with vascular dementia, compared with age-matched controls (Clarke et al., 1998; Lehmann, Gottfries, & Regland, 1999; Nilsson, Gustafson, Faldt, et al., 1996). C->

Lowering Homocysteine Levels in Patients With AD 227

227

HOMOCYSTEINE IN ALZHEIMER'S DISEASE

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

At least five studies have demonstrated that hyperhomocysteinemia is associated with AD in the absence of folate or B12 deficiency (Clarke et al., 1998; Joosten, Lesaffre, Riezler, et alv 1997; Lehmann et alv 1999; McCaddon, Da vies, Hudson, Tandy, & Cattell, 1998; Nilsson et al., 1996). When agebased norms for plasma homocysteine are divided into tertiles, the relative risk of AD in subjects with homocysteine levels in the highest fertile, compared with the lowest, is 4.5 (Clarke et al., 1998). Elevated plasma homocysteine occurs in neuropathologically confirmed cases of AD, in the absence of any vascular neuropathology (Clarke et al., 1998). Among AD cases, higher levels of homocysteine are associated with more rapid radiologic disease progression (Clarke et al., 1998). In nondemented elderly subjects, there is a negative correlation between blood levels of homocysteine and cognitive function (Lehmann et al., 1999; Budge et al., 2000). The epidemiological studies cited above cannot indicate whether the relationship between homocysteine elevations and AD is causal. Indeed, it is possible that inadequate nutrition related to dementia contributes to elevated homocysteine in AD; if so, homocysteine may still contribute to disease progression, as evidence indicates that mild hyperhomocysteinemia is directly neurotoxic (Lipton, Kim, Choi, et al., 1997). However, longitudinal assessment of homocysteine levels in AD subjects indicate that levels remain stable (Clarke et al., 1998), suggesting that elevations are not caused by cognitive dysfunction.

w

.c

LOWERING HOMOCYSTEINE

ht

tp

://

w

w

Supplementation with folate and vitamins B12 and B6 will lower homocysteine levels, and elevated levels occurring in elderly subjects with normal vitamin levels will fall in response to that supplement regimen (Naurath et al., 1995). There is some evidence from retrospective analyses to suggest that vitamin supplementation may provide cognitive benefit in patients with AD. Dr. Shelia Jin of the Alzheimer's Disease Cooperative Study (ADCS) has conducted meta-analyses across ADCS studies to examine use of folate and B complex vitamins. Among 1,145 participants of ADCS trials, 383 (33%) reported regular use of such supplements. Longitudinal Alzheimer's Disease Assessment Scale-Cognitive subscale (ADAS-Cog) data was available on 639 participants, of whom 248 (38%) took supplements. Those using supplements show a trend toward slower annual rate of decline on the ADAS-Cog (users, 4.4 points per year; nonusers, 5.4 points per year, P = 0.13). Such supplements generally contain between 100 and 400-ug folate, less than 10-ug B12, and 1- to 2-mg B6.

228

Drug Development—Clinical

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

A pilot study has been initiated in the United Kingdom to determine whether folate and B]2 supplementation reduces blood homocysteine levels in AD. However, the U.K. data may not be applicable to the U.S. population. The Food and Drug Administration has required folate fortification of grain products (140-ug folic acid per 100 gm) since January 1998. This fortification is estimated to provide an additional 70- to 120-ug folic acid in the typical diet. Data collected from participants in the Framingham Health Study suggests that fortification has resulted in a significant (-7%) reduction in homocysteine levels in the United States; use of standard multivitamins causes further reduction (Jacques, Selhud, Bostom, Wilson, & Rosenberg, 1999b). The ADCS has completed an application to NIA proposing a large, multicenter trial using supplementation with folate, B]2, and B>6 to lower homocysteine levels in patients with AD. The primary outcome in this study will be the longitudinal decline in the cognitive portion of the ADAS-Cog. However, the work from the ADCS and the results of the Jacques et al. study (1999b) illustrates potential logistic problems in conducting this trial, which must be assessed before the trial is finalized. Specifically, as many as one third of patients with AD may be taking supplements that may minimize the ability to reduce homocysteine levels. Additional supplementation with grain fortification may further reduce our ability and may not be fully realized even in the meta-analysis, which includes participants from as early as 1993. While these sources of supplementation are substantially less than the supplements proposed in the ADCS trial, it is imperative that the trial design address these nutritional trends. The Institute for the Study of Aging is currently supporting a pilot study of high-dose supplements in AD subjects at four ADCS sites. This is an unblinded study to determine the effect of high-dose supplements on homocysteine metabolism in subjects with probable AD. A total of 80 participants will be selected (40 multivitamin users and 40 nonvitamin users) at the four participating sites (Columbia University, Georgetown University, University of Texas Southwestern Medical Center, and the University of California, Davis). The treatment regimen is identical to that proposed here; the duration of treatment is 8 weeks. The primary measures, determined before and after treatment, are biochemical: fasting and postmethionine-loading homocysteine levels, as well as vitamin levels, S-adenosylhomocysteine, and S-adenosylmethionine. In addition to confirming the impact of supplementation on fasting homocysteine and on postmethionine homocysteine in this population, this study will determine whether supplementation lowers homocysteine levels in subjects already taking standard supplements. Final decisions for the ADCS study regarding study design (e.g., inclusion of subjects taking standard multivitamins) will be made after review of the results of this pilot study.

Lowering Homocysteine Levels in Patients With AD

229

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

The three supplements are inexpensive and well tolerated. The goal in selecting doses for these studies was to maximize reduction in blood homocysteine without significant likelihood of toxicity. After reviewing the data, we chose to use a daily regimen of folate (5 mg), vitamin B6 (50 mg) and, vitamin B12 (1 mg). Recently, a meta-analysis examined the relationship between dose of vitamin supplements and homocysteine reduction, reviewing 12 published reports (Anonymous, 1998). The authors concluded that supplementation with 0.5- 5-mg folate and about 0.5-mg B12 reduces blood homocysteine by a quarter to a third. None of the studies examined in the meta-analysis examined the impact of vitamin doses on postmethionine homocysteine, and the authors did not suggest an appropriate dose of vitamin B6. The recommended daily allowance of folate is 400 ug. As discussed above, since 1998 grains in the U.S. have been fortified with folate, providing the typical adult with approximately 100 ug daily. Typical multivitamin and B-complex vitamin preparations contain 100- 400-ug folate. In preparation for the VISP (Vitamin Stroke Prevention) trial, a pilot study examined the issue of folate dose. In subjects with acute stroke, a folate dose of 5 mg was associated with a reduction in homocysteine of 3.68 nmol/mL, compared with 2.91 for folate 2.5 mg, and 1.96 for folate 125 ug. Preliminary data from the Women's Antioxidant Cardiovascular Study (WACS) indicate that high-dose supplementation significantly lowers homocysteine levels in U.S. adults, despite grain fortification. WACS is a large, randomized, prospective study of vitamin supplements in the prevention of cardiovascular disease among high- risk subjects. In the last 2 years, an arm has been added to this trial to investigate the effect of high-dose supplementation with folate (2.5 mg), vitamin B6 (50 mg), and vitamin B12 (1 mg). Of note, there has been no apparent toxicity associated with this regimen. Both the VISP and WACS studies utilize a folate dose of 2.5 mg. In view of the safety and low cost of folate, this study opted for a dose of 5 mg daily. In selecting a dose of B6, we aimed to assure maximal reduction of postmethionine homocysteine, without significant risk of toxicity. We could find no reports of B6 toxicity at chronic doses below 100 mg daily. The VISP study administers 25 mg daily, while the WACS study uses 50 mg. An oral daily dose of 1-mg B12 is sufficient to replenish B12 stores even in individuals with impaired absorption. Thus, this dose will assure adequate B12 levels and will protect against the precipitation of B12 deficiency that can occur with folate therapy. For comparison, the WHS investigators selected a daily dose of 1 mg, while the VISP study uses 400 ug. There is minimal toxicity associated with the use of folate and B vitamins, even in high doses. Chronic, high-dose vitamin B6 therapy may cause peripheral neuropathy, but only at doses greater than 100 mg daily

Drug Development—Clinical

230

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

(Bernstein, 1990). There is no significant risk of precipitating B12 deficiency with the use of a regimen combining 5-mg folate with 1-mg B12. As noted above, the regimen proposed is quite similar to that used in the WACS study, and there has been no observed toxicity to date in that trial. A single epidemiological study has suggested an association between high blood levels of B12 and mortality (Zeitlin, Frishman, & Chang, 1997), but the significance of this finding with regard to use of oral vitamin supplements is unclear. Another important aspect of designing this supplementation study is to accommodate typical medications used in AD. Three drugs have been approved by the FDA for the treatment of AD: tacrine, donepezil, and rivastigmine. These drugs are cholinesterase inhibitors. By boosting brain levels of the neurotransmitter acetylcholine, they improve cognitive function. They are considered to be symptomatic treatments; they have not been demonstrated to slow the progression of the disease. The supplementation trial is based on the hypothesis that homocysteine reduction will have a disease-modifying effect in AD. However, the proposed design of this study will not distinguish between a symptomatic effect and a disease-modifying effect. It is likely that the optimal treatment for AD will involve combination therapy, incorporating symptomatic treatment with one or more neuroprotective agents. Indeed, it is now common practice to treat AD patients with donepezil plus vitamin E. If a regimen of high-dose folate and B vitamin supplements is shown to have a neuroprotective effect in AD, it is likely that the treatment would be used in combination with donepezil and/or vitamin E. Therefore, the pilot study and the ADCS proposal allows continuation of standard treatment for AD, including donepezil and vitamin E. Whether it would be appropriate to have a true placebo group, not allowing donepezil, is a matter of debate. Disallowing such treatment would vastly complicate recruitment and might be unacceptable to some sites. In any event, we do not believe that allowing such treatment will interfere with the study objectives. Vitamin E treatment does not have a significant impact on cognitive decline as measured by the ADAS-Cog (Sano, Ernesto, Thomas, et al., 1997), the primary outcome measure in this trial. We also expect that stable cholinesterase therapy (started at least 3 months before enrollment, remaining stable throughout the trial) will not have a major impact on cognitive decline.

FUTURE DIRECTIONS The data described above includes some evidence that higher homocysteine levels may be associated with better cognition in the elderly. However,

Lowering Homocysteine Levels in Patients With AD 231

231

m

s/ in de

x. ph

p

the full extent of the cognitive benefit to be provided by lowering levels with supplementation is unknown. Lower levels are associated with reduced risk of cardiovascular disease and cerebrovascular disease. Several ongoing studies will assess the degree to which supplementation can lower these risks by further reducing homocysteine levels. These findings may provide hints as to mechanisms by which cognition could also be maximized. If ongoing trials of high doses of supplementation continue to prove to be safe-particularly in aging populations, one could envision that this intervention may be a viable treatment strategy for primary prevention of cognitive loss and dementia in aging. Primary prevention trials for cognitive loss or dementia in the elderly require large sample sizes and long observation periods. Relatively safe agents, such as the supplementation regimen proposed here, would have great advantage in such trials.

ru

REFERENCES

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

(Anonymous, 1998). Lowering blood homocysteine with folk acid based supplements: Metaanalysis of randomised trials. Homocysteine Lowering Trialists' Collaboration. British Medical Journal, 316, 894-898. Bellamy, M. R, & McDowell, I. F. (1997). Putative mechanisms for vascular damage by homocysteine. Journal of Inherited Metabolic Disorders, 20, 307-315. Bernstein, A. L. (1990). Vitamin B6 in clinical neurology. Annals of the New York Academy of Sciences, 585, 250-260. Boers, G. H. (1997). The case for mild hyperhomocysteinaemia as a risk factor. Journal of Inherited Metabolic Disorders, 20, 301-306. Bostom, A. G., Rosenberg, I. H., Silbershatz, H., Jacques, P. R, Selhub, J., D'Agostino, J. B., Wilson, P. W., & Wolf, P. A. (1999a). Nonfasting plasma total homocysteine levels and stroke incidence in elderly persons: The Framingham Study. Annals of Internal Medicine, 131, 352-355. Bostom, A. G., Silbershatz, H., Rosenberg, I. H., Selhub, J., D'Agostino, R. B., Wolf, P. A., Jacques, P. R, & Wilson, P. R (1999b). Nonfasting plasma total homocysteine levels and all-cause and cardiovascular disease mortality in elderly Framingham men and women. Archives of Internal Medicine, 159,1077-1080. Bots, M. L., Launer, L. J., Lindemans, J., Hofman, A., & Grobbee, D. E. (1997). Homocysteine, atherosclerosis and prevalent cardiovascular disease in the elderly: The Rotterdam Study. Journal of Internal Medicine, 242, 339-347. Budge, M., Johnson, C, Hogervorst, E., de Jager, C., Milwain, E., Iversen, S. D., Barnetson, L., King, E., & Smith, A. D. (2000). Plasma total homocysteine and cognitive performance in a volunteer elderly population. Annals of the New York Academy of Sciences, 903, 407-410. Clarke, R., Daly, L., Robinson, K., Naughten, E., Cahalane, S., Fowler, B., & Graham, 1. (1991). Hyperhomocysteinemia: An independent risk factor for vascular disease. New England Journal of Medicine, 324(17), 1149-1155.

232

Drug Development—Clinical

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Clarke, R., Smith, A. D., Jobst, K. A., Refsum, H., Sutton, L., & Ueland, P. M. (1998). Folate, vitamin B12, and serum total homocysteine levels in confirmed Alzheimer disease. Archives of Neurology, 55,1449-1455. Eikelboom, J. W., Lonn, E., Genest, }., Jr., Hankey, G., & Yusuf, S. (1999). Homocyst(e)ine and cardiovascular disease: A critical review of the epidemiologic evidence. Annals of Internal Medicine, 131, 363-375. Hoogeveen, E. K., Kostense, P. J.,Beks, P. J., Mackaay, A. J., Jakobs, C, Bouter, L. M., Heine, R. J., & Stehouwor, C. D. (1998). Hyperhomocysteinemia is associated with an increased risk of cardiovascular disease, especially in non-insulindependent diabetes mellitus: A population-based study. Arteriosclerosis, Thrombosis and Vascular Biology, 18,133-138. Jacques, P. E, Bostom, A. G., Williams, R. R., et al. (1996). Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation, 93, 7-9. Jacques, P. E, Rosenberg, I. H., Rogers, G., Selhub, J., Bowman, B. A., Gunter, E W., Wright, J. D., & Johnson, C. L. (1999a). Serum total homocysteine concentrations in adolescent and adult Americans: Results from the third National Health and Nutrition Examination Survey. American Journal of Clinical Nutrition, 69, 482^189. Jacques, P. E, Selhub, J., Bostom, A. G., Wilson, P. W., & Rosenberg I. H. (1999b). The effect of folic acid fortification on plasma folate and total homocysteine concentrations. New England Journal of Medicine, 340,1449-1454. Joosten, E., Lesaffre, E., & Riezler, R. (1996). Are different reference intervals for methylmalonic acid and total homocysteine necessary in elderly people? European Journal of Haematology, 57, 222-226. Joosten, E., Lesaffre, E., Riezler, R., et al. (1997). Is metabolic evidence for vitamin B-12 and folate deficiency more frequent in elderly patients with Alzheimer's disease? Journal of Gerontology and Biological Medicine Sciences, 52, M76-M79. Kato, I., Dnistrian, A. M., Schwartz, M., et al. (1999). Epidemiologic correlates of serum folate and homocysteine levels among users and non-users of vitamin supplement. International Journal of Vitamins and Nutritional Research, 69, 322-329. Lehmann, M., Gottfries, C. G., & Regland, B. (1999). Identification of cognitive impairment in the elderly: Homocysteine is an early marker. Dementia and Geriatric Cognitive Disorders, 10,12-20. Lipton, S. A., Kim, W. K., Choi, Y. B., et al. (1997). Neurotoxicity associated with dual actions of homocysteine at the N- methyl-D-aspartate receptor. Proceedings of the National Academy of Sciences (USA), 94, 5923-5928. McCaddon, A., Davies, G., Hudson, P., Tandy, S., & Cattell, H. (1998). Total serum homocysteine in senile dementia of Alzheimer type. International Journal of Geriatric Psychiatry, 13, 235-239. Moghadasian, M. H., McManus, B. M., & Frohlich, J. J. (1997). Homocysteine and coronary artery disease. Clinical evidence and genetic and metabolic background. Archives of Internal Medicine, 157, 2299-2308. Naurath, H. J., Joosten, E., Riezler, R., Stabler, S. P., Allen, R. H., & Lindenbaum, J. (1995). Effects of vitamin B]2, folate, and vitamin B6 supplements in elderly people with normal serum vitamin concentrations. Lancet, 346, 85-89.

Loivering Homocysteine Levels in Patients With AD 233

233

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Nilsson, K., Gustafson, L., Faldt, R., Andersson, A., Brattstrom, L., Lindgren, A., Israelsson, B., & Hultberg, B. (1996). Hyperhomocysteinaemia: A common finding in a psychogeriatric population. European Journal of Clinical Investigation, 26, 853-859. Norlund, L., Grubb, A., Fex, G., Leksell, H., Nilsson, J. E., Schenck, H., & Hultgerg, B. (1998). The increase of plasma homocysteine concentrations with age is partly due to the deterioration of renal function as determined by plasma cystatin C. Clinical Chemistry and Laboratory Medicine, 36,175-178. Sano, M., Ernesto, C., Thomas, R. G., Glauber, M. R., Schafer, K., Grundman, M., Woodburg, P., Growden, J., Cotman, C. N., & Thak J. (1997a). A controlled trial of selegiline, alpha-tocopherol, or both as treatment for Alzheimer's disease. New England Journal of Medicine, 336,1216-1222. Selhub, J., Jacques, P. R, Bostom, A. G., et al. (1996). Relationship between plasma homocysteine, vitamin status and extracranial carotid-artery stenosis in the Framingham Study population. Journal of Nutrition, 126,1258S-1265S. Selhub, J., Jacques, P. E, Wilson, P. W., Rush, D., & Rosenberg, I. H. (1993). Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. Journal of the American Medical Association, 270, 2693-2698. Zeitlin, A., Frishman, W. H., & Chang, C. J. (1997). The association of vitamin B]2 and folate blood levels with mortality and cardiovascular morbidity incidence in the old old: The Bronx aging study. American Journal of Therapeutics, 4,275-281.

s/ in de

Modulators in Mild

x. ph

Therapeutic Potential of Positive AMPA Receptor

p

26

Cognitive Impairment

ru

m

and Alzheimer's Disease

.c

w

ABSTRACT

he m

4a ll.

vn

/fo

Steven A. Johnson, Gary Lynch, Gary A. Rogers, and Ursula V. Staubli

ht

tp

://

w

w

Currently approved anticholinesterase drugs for Alzheimer's disease (AD) are minimally effective for only a portion of the target patient population. They can produce side effects that may limit dose and discourage compliance. New pharmacological approaches are needed to address the cognitive deterioration and the underlying disease process. The glutamatergic system is fundamentally involved in learning, memory, and cognition, and is also adversely affected by the underlying disease process. Members of a family of AMPA-type glutamate receptor-modulating compounds, termed AMPAKINE® compounds", have been shown to facilitate formation and prolong retention of several kinds of memory in young rodents, and to improve the impaired cognitive performance of aged rodents to the level of the young animals. AMPAKINE treatment can enhance the production of neurotrophic factors in the rodent central nervous system. In three single-dose, Phase I, clinical safety studies of Ampalex® (CX516), the drug was safe and significantly enhanced recall of nonsense syllables in 234

Therapeutic Potential of Positive AMPA Receptor Modulators 235

235

x. ph

p

healthy young and elderly volunteers. In addition, CX516 enhanced the performance of young volunteers in tests of visual-spatial, odor- and visual-recognition memory. CX516 also improved attention, verbal memory, and cognition after a 4-week treatment of medicated schizophrenia patients who typically have underlying cognitive impairment that is not treated by antipsychotic medication. Together, these studies suggest AMPAKINE compounds may have therapeutic utility in patients with mild cognitive impairment (MCI), the earliest, clinically defined group with memory impairment beyond that expected for normal individuals of the same age and education, but who do not yet meet clinical criteria for AD.

s/ in de

OVERVIEW

he m

4a ll.

vn

/fo

ru

m

This chapter briefly reviews preclinical and clinical results that suggest positive modulators of the AMPA-type glutamate receptor (AMPAKINE® compounds) may have therapeutic utility in patients with MCI or AD. After a brief discussion of the electrophysiological attributes that broadly define AMPAKINE compounds as a pharmacological class, various in vivo experiments that support the concept of AMPAKINE compounds as memoryenhancing drugs are presented. Finally, the results of recent clinical safety and pilot efficacy studies with the AMPAKINE CX516 are briefly reviewed.

INTRODUCTION

ht

tp

://

w

w

w

.c

In the aging population, there is a continuum of cognitive decline, ranging from normal (for the general age group) to mild cognitive impairment (MCI) to dementia or probable Alzheimer's disease (AD). Patients with MCI represent the earliest clinically defined group with memory impairment beyond that expected for normal individuals of the same age and education, but yet do not meet clinical criteria for AD. The memory deficits in the MCI population are clinically discernable (Petersen et al., 1999) and can interfere with daily functioning (Sherwin, 2000). MCI patients appear to have a greatly increased risk of developing AD. Whereas approximately 1% to 2% of the normal elderly population will be diagnosed with AD every year, 10% to 15% or more of MCI patients will progress to AD each year (Petersen, 2000). One recent study suggests the risk of progressing to AD in the MCI population may be as high as 25% (Jelic et al., 2000). Since AD is a progressive disorder, it is clearly important to identify and treat affected individuals as early as possible. Currently approved drugs (anticholinesterases) for AD, which target the cholinergic deficit in the AD brain, are effective for only a portion of

Drug Development—Clinical

236

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

the target patient population. In addition, anticholinesterase drugs produce side effects that may limit dose and/or discourage patient compliance. Furthermore, it appears this class of drugs is effective only during administration. In one study with Donepezil, scores returned to baseline within 3 weeks of washout after 6 weeks of therapy (Greenberg et al., 2000). In a second, longer study of Donepezil, patients improved for 6 to 9 months, but then gradually deteriorated (Rogers, Doody, Pratt, & leni, 2000). These studies and others suggest that treatment with anticholinesterase drugs produces little or no modification of the underlying disease process, and that the drugs will be less useful as the disease progresses. Thus, new pharmacological approaches are needed to address the limitations of current therapeutics. In addition to the cholinergic system, it is now clear that other neurotransmitter systems, including the glutamatergic system, are affected in the AD brain and, perhaps, the pre-AD brain. Studies have documented significant reductions of AMPA and NMDA-receptor (NMDA-R) mRNA or protein in various AD brain regions (e.g., Yasuda et al., 1995; Ulas & Cotman, 1997; Thorns, Mallory, Hansen, & Masliah, 1997). Furthermore, several studies show that the loss of glutamate receptors precedes neuronal loss (Pelligrini-Giampietro, Bennett, & Zukin, 1994; Ikonomovic et al., 1997), even in subclinical patients that had sufficient neuropathology to meet AD criteria (Armstrong, Ikonomovic, Sheffield, & Wenthold, 1994). There is good evidence for synaptic loss during normal aging (Masliah, Mallory, Hansen, DeTeresa, & Terry, 1993) and there is a strong correlation between synapse loss and cognitive impairment (Terry et al., 1991). Glutamate is recognized as the major excitatory neurotransmitter in the brain, where it is fundamentally involved in learning and memory, as well as synaptic plasticity. Preclinical evidence shows that enhancing glutamate neurotransmission can promote learning and memory. Thus, the glutamate system is an obvious target for therapy to restore memory and cognition that is compromised in patients with MCI or AD.

AMPAKINES Experimental work has shown that positive modulation of AMPA-receptors (AMPA-R) makes it easier for afferent activity to unblock the voltagedependent NMDA-Rs often colocalized with AMPA-Rs (Arai & Lynch, 1992). Thus, AMPA-R facilitation indirectly enhances, in a use-dependent fashion, NMDA-R function. This has the effect of reducing the amount of afferent activity needed to induce long-term potentiation (LTP), a variant of synaptic plasticity widely regarded as a substrate of many forms of memory. Centrally active AMPA-R modulators (AMPAKINE compounds)

Therapeutic Potential of Positive AMPA Receptor Modulators

237

ht

tp

://

w

w

w

.c he

m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

would be predicted to promote the encoding of memory. This reasoning prompted efforts to synthesize compounds that could cross the blood brain-barrier and enhance AMPA-R functioning in vivo. Many compounds have now been synthesized that show positive modulatory effects on AMPA-R function in various in vitro and in vivo tests. AMPAKINE compounds reach the brain by all routes of administration in rodents, including oral. Positron emission tomography studies with rats show that some are capable of reaching the brain within a few minutes of intraperitoneal or oral administration (Staubli et al., 1994b; Rogers et al., 1997). At the receptor level, AMPAKINE compounds bind to one or more modulatory sites distinct from the agonist/antagonist site or the benzothiadiazide (e.g., cyclothiazide) site of the AMPA-R complex (Arai et al., 1996). Several AMPAKINE compounds, including CX516, have been extensively tested at concentrations as high as 100 uM for competition with specific ligands to other neurotransmitter receptors. No significant interactions with other receptors have been seen, suggesting those compounds tested have good specificity for the AMPA receptor. Electrophysiological studies using hippocampal pyramidal membrane patches show that AMPAKINE compounds increase the peak and prolong the duration of glutamate-induced inward currents (Arai et al., 1994). Glutamate is absolutely required; AMPAKINE compounds alone have no agonist activity, and thus are use-dependent enhancers of AMPA-R activity. Similarly, in in vitro hippocampal slice experiments, AMPAKINE compounds increase the amplitude and prolong the duration of field excitatory postsynaptic responses (Arai et al., 1994), indicating the receptor effects are mirrored at the synapse. Moreover, the net facilitory effects are amplified across multisynaptic circuits. Using the acute, in vitro, hippocampal slice model, responses evoked in field CA1 of the hippocampal slice by stimulation of the Schaffer-commissural inputs (monosynaptic responses) were compared with responses to perforant path stimulation relayed through the dentate gyrus and field CA3 to CA1 (trisynaptic responses) in the same slice. The effect of CX516 on the polysynaptic response was more than three-fold greater, suggesting that AMPAKINE compounds probably have much greater effects on complex brain operations than on simple reflex-like functions (Sirvio, Larson, Quach, Rogers, & Lynch, 1996).

AMPAKINES ENHANCE LTP AND MEMORY IN RODENTS Pharmacologically increasing the size and duration of AMPA-R currents should promote NMDA-R activity, facilitate LTP, and enhance memory encoding. This was the fundamental hypothesis behind the development

Drug Development—Clinical

238

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

of AMPA-R-modulating compounds as memory-enhancing drugs. A number of different AMPAKINE compounds, including CX516, have been shown to facilitate the induction of LTP in hippocampal slices. Importantly, several AMPAKINE compounds have been shown to facilitate the induction of LTP in vivo in awake, freely moving rats with permanently implanted electrodes (Staubli et al., 1994b). Furthermore, doses that facilitated LTP in vivo were also sufficient to enhance performance of rats in a spatial memory task (eight-arm radial maze) and an odor-matching task (Staubli et al., 1994b). Additional studies using other AMPAKINE compounds have strengthened the concept that AMPA-R facilitation will enhance the encoding of several types of memory. In addition to odor-matching (long-term) and eight-arm maze (short-term) tasks, various AMPAKINE compounds have been shown to enhance performance in the Morris water maze (Staubli, Rogers, & Lynch, 1994a), a conditioned eyeblink paradigm (Shors, Servatius, Thompson, Rogers, & Lynch, 1995), and a conditioned fear-learning paradigm (Rogan, Staubli, & LeDoux, 1997), the latter two being classical examples of long-term memory. As discussed briefly above, there is good evidence for a decline in the number of synapses with aging (Masliah et al., 1993). Similarly, it is well known that various types of memory show age-related declines in performance. An important question for the development of memory-enhancing Pharmaceuticals is whether age-related cognitive decline can be reversed by drug therapy. This question was addressed by testing the effects of the AMPAKINE CX516 on the performance of middle-aged rats in an eight-arm radial maze task (Granger et al., 1996); a group of young vehicle-treated rats served as a further control. When administered vehicle, there was a clear deficit in the ability of the middle-aged rats to recall the spatial location of rewards in the maze 5 or 8 hours after acquisition, compared with the young rats; whereas the young rats scored well, the middle-aged rats scored poorly at 'chance' level. However, when the middle-aged rats were administered CX516 before acquisition, they scored significantly better during recall, and, indeed, were not significantly different from the young control rats. Thus, the performance deficit of the middle-aged rats in a spatial-memory task was significantly improved, essentially to the level of young controls, by treatment with the drug. Two additional sets of preclinical studies support a profile for therapeutic utility of CX516 in age-related memory disorders. In the first (Hampson, Rogers, Lynch, & Deadwyler, 1998a, 1998b), rats that were extensively trained in a delayed nonmatch-to-sample task were administered CX516 or vehicle on alternate days over a period of 4 weeks. The rats were scored on their ability to remember the position of a lever in a shuttle box after a random, variable delay. Although the rats had reached a performance plateau during initial training, after CX516 administration they began to

Therapeutic Potential of Positive AMPA Receptor Modulators

239

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

consistently improve day after day. Furthermore, the performance remained at the higher level even on the alternate vehicle-administration days. This was clearly not due to residual CX516 because the drug has a 20minute half-life in rats. Finally, after cessation of alternate-day CX516 treatment, the rats were given only vehicle for another 7 days, during which time their performance remained at the higher performance level induced by the drug. In addition, the rats had surgically implanted electrodes in the hippocampus, which enabled electrophysiological recording during the task. Interestingly, the average neuronal firing rate increased during CX516 treatment, did not decrease on alternate vehicle days, and remained higher during the 7-day vehicle-treatment period at the end of the study (Hampson et al., 1998b). Together, the results suggest the presence of a long-term, pharmacodynamic effect that is unrelated to the relatively short pharmacokinetic half-life of CX516 in rats. This long-term "carry-over" effect has also been seen in the clinic (see below). Whereas the mechanism for the carry-over effect of CX516 is unknown, it is possibly related to a recently discovered consequence of AMPA-R upmodulation, namely enhancement of neurotrophin synthesis in the brain. Previous studies have shown that NGF and BDNF are upregulated as a consequence of intense neuronal activity, such as during a seizure (Gall & Isackson, 1989), but also by less intense activation due to afferent stimulation (Patterson, Grover, Schwartz Kroin, & Bothwell, 1992). This raised the question of whether AMPA-R modulating compounds could also enhance neurotrophin expression. Several different AMPAKINE compounds have now been shown to increase the prevalence of BDNF mRNA in vitro in cultured hippocampal slices, as well as in vivo in aged rats and middle-aged mice after peripheral administration (Lauterborn, Lynch, Vanderklish, Arai, & Gall, 2000). The effect was mediated via AMPA-Rs, because specific AMPA-R antagonists, but not NMDA-R antagonists, blocked AMPAKINE induction of neurotrophins. Other studies have demonstrated similar effects in vivo with CX516, the AMPAKINE compound currently under evaluation in the clinic (Johnson, Luu, & Herbst, 1998). AMPA-R modulation may well promote the production of other molecules involved in neuronal health and synaptic plasticity. In a recent study, Hoist et al. (1998) demonstrated that an AMPAKINE compound stimulated transcription of NCAM, which is known to have roles in maintenance of LTP and synaptic plasticity. Together, these results show that peripheral administration of AMPAKINE compounds can upregulate neurotrophins in the CNS of both young and aged rodents. It remains to be determined whether such modulation of neurotrophins by CX516 and other AMPAKINE compounds will slow age- or disease-related neurodegenerative processes.

Drug Development—Clinical

240

CLINICAL STUDIES OF THE AMPAKINE CX516

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

After rigorous safety testing in rodents and monkeys, CX516 was administered to young (25 to 35 years) and elderly (65 to 76 years) male volunteers in separate Phase I safety trials (Lynch et al., 1996; Lynch et al., 1997). The drug was well tolerated at all doses. Volunteers were tested for their ability to recall lists of nonsense syllables 5 minutes after hearing the list, both before and after drug or placebo administration. CX516 dose dependently enhanced both young and elderly participants' recall of syllables. In addition, whereas elderly placebo participants had much poorer recall than young placebo participants, the majority of the high-dose elderly volunteers had delayed-recall scores that were similar to the young controls. As stated above, CX516 also raised the poor performance of middleaged rats in the eight-arm radial maze task to that of young control rats, suggesting the drug was able to offset age-related impairments in spatial memory (Granger et al., 1996). In a third Phase I trial, two daily doses of CX516 or vehicle were administered to young volunteers whose performance in several tests of memory, motor skills, and general intellect was assayed in a within-subjects design (Ingvar et al., 1997). CX516 enhanced performance in tests of visual association, odor recognition, and visuospatial maze acquisition. Motor skills, cued recall of visual recognition, and general intellect were not significantly affected. In all three humansafety trials, subjects scored at chance level when asked to guess whether they had received drug or placebo. CX516 has been recently tested in 19 long-term schizophrenia patients who were concurrently treated with optimal doses of clozapine (Goff et al., in press). Clozapine, the current 'gold-standard' antipsychotic, manages positive symptoms (e.g., hallucinations) and partially manages negative symptoms (e.g., anhedonia, social withdrawal). However, schizophrenics have widespread, multifactorial, neurocognitive impairments that are only partially treated in a subset of patients by clozapine or other modern atypical antipsychotics. Thus, one goal of the pilot study was to improve the cognitive deficit symptomology with CX516. First, there were no treatment-related side effects or abnormal laboratory test results from 28-day administration of CX516 (900 mg TID) in conjunction with clozapine (mean dose, -450 mg/day). This is noteworthy because clozapine alone can produce significant side effects. Second, CX516, compared with placebo, appears to have produced improvements in a number of clinical measures, including the Positive and Negative Symptom Scale (PANSS tot), Scale for Assessment of Negative Symptoms (SANS tot), General Assessment Scale (GAS), and Abnormal Involuntary Movement Scale (AIMS). This is very encouraging because clozapine manages positive and negative symptoms, as well as any antipsychotic medication.

Therapeutic Potential of Positive AMPA Receptor Modulators

241

ru

m

CLINICAL STUDY OF CX516 IN MCI

s/ in de

x. ph

p

Third, the neurocognitive results show moderate-to-large therapeutic effects in measures of attention, distractibility, verbal memory, and executive function. Furthermore, many of these effects were maintained, or even improved, when patients were tested 2 to 3 weeks after the last dose of CX516, which has a pharmacokinetic half-life of about an hour in male volunteers. This result is similar to the carry-over effect seen with CX516 in the delayed nonmatch-to-sample rat studies discussed above. Thus, in this limited number of medicated schizophrenia patients, CX516 appears to improve at least some cognitive deficits, and demonstrates extended pharmacodynamic effects that are evident long after drug cessation. This evidence, combined with the effects in healthy young and elderly male volunteers, suggests CX516 or other AMPAKINE compounds, may prove therapeutically useful in individuals with mild cognitive impairment.

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

A Phase II study of CX516 in patients with MCI is currently being planned. The study will likely use a double-blind, placebo-controlled, parallel-group design involving patients who will be administered CX516 daily for 1 month. After screening, patients will be randomized to placebo or CX516; the latter group will receive placebo for 2 weeks and CX516 for 1 month. A test battery will be given at randomization, after 2 weeks placebo, at weeks 2 and 4 of drug treatment, and at 2 and 4 weeks following cessation of CX516 to examine persistence of any therapeutic benefit. Primary outcome measures are yet to be decided, but will likely be selected from a group currently being used to distinguish normal elderly from MCI and AD, including the Clinical Dementia Rating scale (CDR), the Wechsler Logical Memory test, and the Alzheimer's disease assessment scale-cognitive subscale (ADAS-Cog). In addition, subjects will be tested with a neuropsychological test battery that includes immediate and delayed paragraph recall, word list recall, maze performance, Boston naming test, and category recall.

REFERENCES Aral, A., & Lynch, G. (1992). Factors regulating the magnitude of long-term potentiation. Brain Research, 598,173-184. Aral, A., Kessler, M., Ambros-Ingerson, }., Quon, A., Yigiter, E., Rogers, G., & Lynch, G. (1996). Effects of centrally active benzoylpyrrolidine drug on AMPA receptor kinetics. Neuroscience, 75, 573-585. Arai, A., Kessler, M., Xizo, P., Ambros-Ingerson,}., Rogers, G., Lynch, G. (1994). A centrally active drug that modulates AMPA receptorgated currents. Brain Research, 638, 343-346.

Drug Development—Clinical

242

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Armstrong, D. M., Ikonomovic, M. D., Sheffield, R., & Wenthold, R. J. (1994). AMPA-selective glutamate receptor subtype immunoreactivity in the entorhinal cortex of patients with Alzheimer's disease. Brain Research, 639, 207-216. Gall, C, & Isackson, P. J. (1989). Limbic seizures increase neuronal production of MRNA for nerve growth factor. Science, 245, 758-760. Goff, D. C., Leahy, L., Berman, I., Posever, J. A., Herz, L, Leon, A. C., Johnson, S. A., & Lynch, G. (in press). Journal of Clinical Psychopharmacology. Granger, R. S., Deadwyler, S., Davis, M., Moskovitz, B., Kessler, M., Rogers, G., & Lynch, G. (1996). Facilitation of glutamate receptors reverses age associated memory impairment in rats. Synapse, 22, 332-337. Greenberg, S. M., Tennis, M. K., Brown, L. B., Gomez-Isla, T., Hayden, D. L., Schoenfeld, D. A., Walsh, K. L., Corwin, C., Daffner, K. R., Friedman, P., Meadows, M. E., Sperling, R. A., & Growdon, J. H. (2000). Donepezil therapy in clinical practice: A randomized cross-over study. Archives of Neurology, 57, 94-99. Hampson, R. E., Rogers, G., Lynch, G., & Deadwyler, S. A. (1998a). Facilitative effects of ampakine CX516 in rats. Journal ofNeuroscience, 18, 2740-2747. Hoist, B. D., Vanderklish, P. W., Krushel, L. A., Zhou, W., Langdon, R. B., McWhirter, J. R., Edelman, G. M., & Crossin, K. L. (1998). Allosteric modulation of AMPA-type glutamate receptors increases activity of the N-CAM. Proceedings of the National Academy of Sciences (USA), 95, 2597-2602. Ikonomovic, M. D., Mizukami, K., Davies, P., Hamilton, R., Sheffield, R., & Armstrong, D. M. (1997). The loss of GLUR2(3) immunoreactivity precedes neurofibrillary tangle. Journal of Neuropathology and Experimental Neurology, 56,1018-1027. Ingvar, M., Ambros-Ingerson, J., Davis, M., Granger, R., Kessler, M., Rogers, G. A., Schehr, R. S., & Lynch, G. (1997). Enhancement by an ampakine of memory encoding in humans. Experimental Neurology, 146, 553-559. Jelic, V., Johansson, S., Almkvist, O., Shiegeta, M., Julin, P., Nordberg, A., Winblad, B., & Wahlund, L. (2000). Quantitative electroencephalography in mild cognitive impairment: Longitudinal changes and possible prediction of Alzheimer's disease. Neurobiology of Aging, 21, 533-540. Johnson, A. A., Luu, N. T., & Herbst, T. A. (1998). Society for Neuroscience Abstract, #511.3. Lauterborn, J. C., Lynch, G., Vanderklish, P., Arai, A., & Gall, C. M. (2000). Positive modulation of AMPA receptors increases neurotrophin expression. Journal of Neuroscience, 20, 8-21. Lynch, G., Kessler, M., Rogers, G., Ambros-Ingerson, J., Granger, R., & Schehr, R. S. (1996). Psychological effects of a drug that facilitates brain AMPA receptions. International Clinical Psychopharmacology, 11,13-19. Lynch, G.., Granger, R., Ambros-Ingerson, J., Davis, C. M., Kessler, M., & Schehr, R. (1997). Evidence that a positive modulator of AMPA type glutamate receptors improves delayed recall. Experimental Neurology, 145, 89-92. Masliah, E., Mallory, M., Hansen, L., DeTeresa, R., & Terry, R. (1993). Quantitative synaptic alterations in the human cortex during aging. Neurology, 43,192-197. Patterson, S. L., Grover, L. M., Schwartzkroin, P. A., & Bothwell, M. (1992). Neurotrophin expression in rat hippocampal slices: A stimulus paradigm. Neuron, 9,1081-1088.

Therapeutic Potential of Positive AMPA Receptor Modulators

243

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Pelligrini-Giampietro, D. E., Bennett, M. V., & Zukin, R. S. (1994). AMPA/Rainate receptor gene expression in Alzheimer's disease hippocampus. Neuroscience, 61,41-49. Petersen, R. C, Smith, G. E., Waring, S. C, Ivnik, R. J., Tangalos, E. G., & Kokmen, E. (1999). Mild cognitive impairment: Clinical characterization and outcome. Archives of Neurology, 56, 303-308. Petersen, R. C. (2000). Mild cognitive impairment: Transition between aging and Alzheimer's disease. Neurologica, 15, 93-101. Rogan, M. T., Staubli, U. V., & LeDoux, J. E. (1997). AMPA receptor facilitation accelerates fear learning. Journal of Neuroscience, 17, 5928-5935. Rogers, G. A., OThorell, J., Johnstrom, P., Eriksson, L., Ingvar, M., & Stone-Elander, S. (1997). Journal of Labeled Compounds & Radiopharmacology, 40, 645-647. Rogers, S. L., Doody, R. S., Pratt, R. D., & leni, J. R. (2000). Long term efficacy and safety of donepezil in treatment of Alzheimer's disease. European Neuropsychopharmacology, 10,195-203. Shors, T. J., Servatius, R. J., Thompson, R. R, Rogers, G., & Lynch, G. (1995). enhanced glutaminergic neurotransmission facilitates classical conditioning. Neuroscience Letters, 186,153-156. Sirvio, J., Larson, J., Quach, C. N., Rogers, G. A., & Lynch, G. (1996). Effects of pharmacologically facilitating glutaminergic transmission in the hippocampus. Neuroscience, 74,1025-1035. Sherwin, B. B. (2000). Mild cognitive impairment: Potential pharmacological treatment options. Journal of the American Geriatric Society, 48, 431-441. Staubli, U., Rogers, G., & Lynch, G. (1994a). Central modulated glutamate receptors facilitate induction of long-term potentiation. Proceedings of the National Academy of Sciences (USA), 91, 777-781. Staubli, U., Perez, Y., Xu, R, Rogers, G., Ingvar, M., Stone-Elander, S., & Lynch, G. (1994b). Facilitation of glutamate receptors enhances memory. Proceedings of the National Academy of Sciences (USA), 91,11158-11162. Terry, R. D., Masliah, E., Salmon, D. P., Butters, N., DeTeresa, R., Hill, R., Hansen, L., & Katzman, R. (1991). Physical basis of cognitive alterations in Alzheimer's disease. Annals of Neurology, 30, 572-580. Thorns, V., Mallory, M., Hansen, L., & Masliah, E. (1997). Alterations in glutamate receptor 2/3 subunits and amyloid precursor protein. Ada Neuropathologica, 94, 539-548. Ulas, J., & Cotman, C. W. (1997). Decreased expression of NMDA receptor type 1 mRNA in select regions of Alzheimer brain. Neuroscience, 79, 973-982. Yasuda, R. P., Ikonomovic, M. D., Sheffield, R., Rubin, R. T., Wolfe, B. B., & Armstrong, D. M. (1995). Reduction of AMPA-selective glutamate receptor subunits in the entorhinal cortex of patients. Brain Research, 678,161-167.

27 The Alzheimer's Disease

x. ph

p

Atorvastatin Treatment Trial:

s/ in de

Scientific Basis and Position on the Use of HMG-CoA

ru

m

Reductase Inhibitors

/fo

(Statins) That Do or

4a ll.

vn

Do Not Cross the

he m

Blood-Brain Barrier

tp

://

w

w

w

.c

D. Larry Sparks, Donald J. Connor, Patrick ]. Browne, Dawn R. Wasser, Jean E. Lopez, and Marwan N. Sabbagh

ht

ABSTRACT Increased circulating cholesterol has been long linked to an increased risk of coronary artery disease (CAD), and is now linked to an increased risk of developing Alzheimer's disease (AD). We first showed the neuropathologic link between CAD and AD as increased incidence of cerebral senile plaques in both disorders. We then showed that AD-like neuropathology occurred in the brains of cholesterol-fed rabbits, which included increased (3-amyloid (A(3). Currently, essentially all transgenic mouse models of AD exhibit enhanced A(3 pathology if cholesterol diet is administered. Culture studies clearly show that excess cholesterol enhances p-metabolism of 244

The AD Atorvastatin Treatment Trial

245

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

amyloid precursor protein (APP) and production of p-amyloidogenic peptides, and that sufficiently reducing cholesterol levels by inhibition of synthesis completely inhibits all (3-metabolism of APP. Our finding that the elevated levels of Ap in rabbits fed cholesterol diet could be cleared from the brain by resuming a control diet prompted the hypothesis that lowering cholesterol levels in the blood of AD patients may be of some clinical benefit. Accordingly, we have initiated a double-blind treatment trial evaluating atorvastatin Na+ among 120 mild to moderately impaired AD subjects randomized to one of two groups receiving placebo or active drug once daily. Atorvastatin is one of a general class of HMG-CoA reductase inhibitor drugs called statins that lower cholesterol by inhibition of synthesis. We chose to use atorvastatin in this AD treatment trial because it does not cross the blood-brain-barrier, and believe it would be ill advised to use a statin that does. This position stems from the observations that excess cholesterol inhibits cholesterol synthesis and increases A (3 production, that AP kills cells, in part, by inhibiting cholesterol synthesis, and that statins acting at the neuronal level could further exacerbate degeneration in AD by further inhibition of necessary cholesterol synthesis. Identifying risk factors for the development of Alzheimer's disease (AD) is important as they can provide clues for therapeutic approaches that may delay or prevent AD. One important risk factor we have identified is high cholesterol levels (Sparks, 1997). We initially demonstrated neuropathologic lesions in nondemented individuals with heart disease identical to those found in AD (Sparks et al, 1990; Sparks, 1999). Epidemiologic surveys now suggest an association between AD risk and heart disease (Breteler, Claus, Grobbee, & Hofman, 1994; Skoog et al., 1996), as well as a link between a high fat/cholesterol diet and the risk of developing AD (Petot et al., 2000). More globally, a growing body of epidemiologic and clinical evidence suggests that cerebrovascular changes could play a role in AD pathogenesis (Ott et al., 1997). High cholesterol levels may lead to cerebrovascular alterations that increase the likelihood of AD. Apolipoprotein E (Apo E) allotype, a known risk factor for AD, is also known to elevate circulating cholesterol levels (Sing & Davignon, 1985). Although there are numerous reports of lower or no difference in circulating cholesterol levels in AD (Romas, Tang, Berglund, & Mayeux, 1999; Carantoni et al., 2000), many reports also suggest there are increased cholesterol levels in the blood of AD patients (Kuo et al., 1998; Evans et al., 2000). One may make sense of these inconsistent reports based on the observation that it is previously elevated cholesterol concentration in the serum that increases the risk of AD three-fold (Notkola et al., 1998). A more recent study also suggests that elevated cholesterol levels in midlife increases the risk of AD (Kivipelto et al., 2000). Furthermore, increased cholesterol has been observed in the AD brain as a function of Apo E allotype (Sparks,

Drug Development—Clinical

246

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

1997; Kuo et al., 1998), and increased circulating cholesterol in AD patients correlates to the levels of A|3 found in the brain (Kuo et al., 1998) and blood (Arvanitakis, Lucas, & Graff-Radford, 2000). In order to test more carefully the hypothesis that high cholesterol contributes to the pathogenesis of AD, we established an animal model that has a physiology similar to the human. The cholesterol-fed rabbit brain exhibits a number of neuropathologic alterations similar to those reported in the AD brain, including accumulation of A(3 immunoreactivity (Sparks et al., 1994; Sparks, Koou, Roher, Martin, & Lukas, 2000). Cholesterol-fed mouse models have, for the most part, confirmed the early rabbit findings; essentially, every transgenic mouse model of AD exhibits increased production and accumulation of A(3 in the brain (Durham, Parker, Emmerling, Bisgaier, & Walker, 1998; Fishman et al., 1999; Li, Zeigler, Lindsey, & Fukuchi, 1999; Shie, LeBoeuf, Leverenz, & Jin, 1999; Bales et al., 2000; Refolo et al., 2000). Further studies in rabbits suggest that removal of cholesterol from the diet, after prolonged cholesterol administration, leads to the clearance of A0 from the brain (Sparks, 1996). These observations, with the culture studies outlined below, suggested to us that it might be of value to test the hypothesis that cholesterol-lowering drugs (statins) can delay the progression of AD, stabilize or improve cognition in elderly individuals with mild-to-moderate AD-like dementia, and provide the scientific basis for the ongoing AD treatment trial using atorvastatin. The statins are a class of HMG-CoA reductase inhibitor medications that lower plasma cholesterol and lipoprotein levels by inhibiting the rate-limiting step in the synthesis of cholesterol. We have initiated a Phase II trial assessing the efficacy of the statin, atorvastatin Na+, on cognitive function in AD patients. Cholesterol-lowering agents, such as atorvastatin, are especially attractive as therapeutic targets in the treatment of AD because they are commercially available, well tolerated, widely used, relatively inexpensive, and easy to take. Atorvastatin and associated agents could have disease-modifying effects, thus slowing the disease progression or improving symptoms. To investigate this, we are performing a double-blind, placebo-controlled study of 120 individuals with mild-tomoderate cognitive impairment and a clinical diagnosis of AD. Participants meeting eligibility criteria will be randomized into two groups, receiving either placebo or atorvastatin once daily for 12 months, and receive wellestablished, standardized, quarterly neuropsychological, cognitive, and patient safety measures. An important question has arisen as to the wisdom of using a statin that crosses the blood-brain barrier (BBB) or one that does not in the treatment of AD. This is coupled with the question of whether statins crossing the BBB have a direct effect in the central nervous system (CNS). Our position is based on the hypothesis that it is excess cholesterol outside of a

The AD Atorvastatin Treatment Trial

247

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

neuron (emanating from the blood) that produces AD-like pathologic change within the effected neuron. We propose that, if it is cholesterol coming from the blood causing the pathophysiologic alterations in AD brain, then reducing cholesterol in the blood would be the safest way to reduce cholesterol in the brain. Accordingly, we chose to investigate a statin (atorvastatin) that does not cross the BBB (Knopp, 1999), and contend that it would be ill-advised to inhibit cholesterol synthesis within CNS proper (by use of a statin that does cross the BBB). Among the six widely prescribed statins, evidence suggest that lovastatin, simvastatin, and cerivastatin cross the BBB and that atorvastatin, prevastatin, aand fluvastatin do not (Harrison & Ashton, 1994; Saheki, Terasaki, Tamai, & Tsuji, 1994; Knopp, 1999). One clinical study suggested the BBB permeability of a statin correlated to CNS side effects (Saheki et al, 1994). However, another clinical investigation found no such correlation (Kostis, Rosen, & Wilson, 1994). A more recent clinical investigation suggests that treatment with a BBB-permeable statin caused a relative decrease in cognitive ability (Wardle et al., 2000), but another investigation found no significant changes in cognitive function among cognitively normal individuals treated with the same statin (Muldoon et al., 2000). In the latter study, there were small reductions in attention and psychomotor speed in the treatment group noted, but were likely a resultant of the placebo (nontreatment) group performing better on these measures (Muldoon et al., 2000). Nevertheless, recent epidemiologic data suggest there is decreased prevalence of dementia among individuals using the HMG-CoA reductase inhibitors (Jick, Zornberg, Jick, Seshadro, & Drachman, 2000; Wolozin, Kellman, Ruosseau, Celesia, & Siegel, 2000), and irrespective of BBB permeability in AD (Wolozin et al., 2000). Most of the scientific basis for using a statin that does not cross the BBB comes from cell-culture studies, where neurons and other cells are challenged with a variety of agents. For the purpose of this discussion, we focus on cholesterol, p-amyloidogenic peptides, and HMG-CoA reductase inhibitors. Interpretation of this culture data is based on a number of principles. The first is that A(3 is made in the cell that it kills or makes susceptible to degeneration. The second, is that culture studies with cholesterol added would mimic an increase of cholesterol in the extracellular space (as if from the blood). Finally, culture studies with HMG-CoA reductase inhibitors added would mimic intracellular effects of the drug as if it crossed the BBB. Under normal conditions, a neuron in situ or in culture will synthesize all the cholesterol that it requires, and there is a balance between alpha and beta metabolism of APP, where the predominant by-product would be a secreted form of APP (APPs), although minor amounts of amyloidogenic peptides, A^ N, are also produced. Bodivitz and Klein (1996) were

Drug Development—Clinical

248

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

the first to show in culture that cholesterol caused a shift of APP metabolism from alpha to beta products. It has now been generally shown that cholesterol causes an increase in (3-secretase activity, and possibly, y-secretase activity to produce increased intracellular (3-amyloidogenic peptides (Racchi et al., 1997; Simons et al., 1998; Urmoneit, Turner, & Dyrks, 1998; Frears, Stephens, Walters, Da vies, & Austen, 1999; Austen, Frears, & Da vies, 2000; Beyreuther, 2000; Galbete et al., 2000). It has been shown that this cholesterol effect on APP metabolism correlated to the dose of cholesterol applied and that the p-amyloidogenic effect of cholesterol is not mediated via the LDL-receptor (Racchi et al., 1997). The effects of Ap on cultured cells reveal that the peptide leads to cell death, at least in part, via disruption of cholesterol metabolism (Janciauskiene & Wright, 1998) and inhibition of cholesterol synthesis (Koudinova, Berezov, & Koudinov, 1996; Janciauskiene & Wright, 1998). A(32535 applied to PC 12 cells causes cell death, mediated by increased influx of Ca++, and this flux of Ca++ is inhibited in a dose-dependent manner by cholesterol (Zhou & Richardson, 1996). It has recently been confirmed that Ca++ influx caused by A|325 35 is blocked by cholesterol (Kawahara & Kuroda, 2000). Statins as HMG-CoA reductase inhibitors block A(3 production in cultured cells (Simons et al., 1998; Frears et al., 1999; Austen et al., 2000; Bergmann, Runz, Jakala, & Hartmann, 2000), suggesting that cholesterol is required for A(3 production and implying a link between cholesterol, A(3, and AD (Keller, Simons, Michel, & Simons, 2000), but kill such cells by inhibition of cholesterol synthesis (Michikawa & Yanagisawa, 1999a). Statins have been shown to cause dose-dependent cell death in culture (Michikawa & Yanagisawa, 1999b), and this toxic effect of statins is inhibited by addition of mevalonate. Furthermore, at sublethal doses of statin, if E3 is the isoform of Apo present, cells survive, but if the E4 form is present, cells degenerate (Michikawa & Yanagisawa, 1999a). It is suggested that the presence of the Apo-E4 isoform causes death of the cell by further attenuation of de novo cholesterol synthesis (Michikawa & Yanagisawa, 1999a). Based on the foregoing cell culture and animal studies, we suggest that direct inhibition of cholesterol synthesis within CNS proper would be ill-advised. Too much statin in a neuron would kill it by inhibition of normal necessary cholesterol synthesis. In AD, abnormal production of A(3 would synergistically inhibit cholesterol synthesis with the concurrent use of centrally acting statins, and, therefore, augment cell death. The main hypothesis serving as the foundation for this trial is that it is circulating levels of cholesterol that induce the cascade of physiochemical derangements, leading to Ap production and, thereafter, the symptoms of AD. Therefore, reducing cholesterol at its source (the blood), and not at the neuronal level, would be the appropriate approach. By lowering

The AD Atorvastatin Treatment Trial

249

cholesterol levels in the circulation of AD patients, we would be using the blood as a sponge to reduce levels in the brain, thereby passively and safely attenuating AP production in the brain.

REFERENCES

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Arvanitakis, Z., Lucas, J. A., & Graff-Radford, N. R. (2000). Correlation between serum cholesterol parameters and plasma amyloid beta protein. Neurobiology of Aging, 21,596. Austen, B. M., Frears, E. R., & Davies, H. (2000). Cholesterol upregulates production of Abeta 1-40 and 1-42 in transfected cells. Neurobiology of Aging, 21, S254. Bales, K. R., Fishman, C, DeLong, C, Du, Y., Jordan, W., & Paul, S. M. (2000). Dietinduced hyperlipidemia accelerates amyloid deposition in the APPv717f transgenie mouse model of Alzheimer's disease. Neurobiology of Aging, 21, S139. Bergmann, C., Runz, H., Jakala, P., & Hartmann, T. (2000). Diversification of gamma-secretase versus beta-secretase inhibition by cholesterol depletion. Neurobiology of Aging, 21, S278. Beyreuther, K. (2000). Physiological function of APP processing. Neurobiology of Aging, 21,569. Bodovitz, 5., & Klein, W. L. (1996). Cholesterol modulates alpha-secretase cleavage of amyloid precursor protein. Journal of Biological Chemistry, 271, 4436-4440. Breteler, M. M., Claus, J. J., Grobbee, D. E., & Hofman, A. (1994). Cardiovascular disease and distribution of cognitive function in elderly people: The Rotterdam study. British Medical Journal, 308,1604-1608. Carantoni, M., Zuliani, G., Maunari, M. R., D'Elia, K., Palmieri, E., & Fellin, R. (2000). Alzheimer disease and vascular dementia: Relationships with fasting glucose and insulin levels. Dementia and Geriatric Cognitive Disorders, 11, 176-180. Durham, R. A., Parker, C. A., Emmerling, M. R., Bisgaier, C. L., & Walker, L. C. (1998). Effect of age and diet on the expression of beta-amyloid 1-40 and 1-42 in the brains of apolipoprotein-E-deficient mice. Neurobiology of Aging, 19, 5281. Evans, R. M., Emsley, C. L., Gao, 5., Sohata, A., Hall, K. S., Farlow, M. R., & Hendrie, H. (2000). Serum cholesterol, APOE genotype, and the risk of Alzheimer's disease: A population-based study of African Americans. Neurology, 54, 240-242. Fishman, C. E., White, S. L., DeLong, C. A., Cummins, D. J., Jordan, W. H., Bales, K. R., & Paul, S. M. (1999). High fat diet potentiates fi-amyloid deposition in the APP V717F transgenic mouse model of Alzheimer's disease. Society for Neurosciences Abstract, 25,1859. Frears, E. R., Stephens, D. J., Walters, C. E., Davies, H., & Austen, B. M. (1999). The role of cholesterol in the biosynthesis of p-amyloid. Neuroreport, 10,1699-1705. Galbete, J. L., Martin, T. R., Peressini, E., Modena, P., Bianchi, R., & Forloni , G. (2000). Cholesterol decreases secretion of the secreted form of amyloid precursor protein by interfering with glycosylation in the protein secretory pathway. Biochemical Journal, 348, 307-313.

Drug Development—Clinical

250

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Harrison, R. Wv & Ashton, C. H. (1994). Do cholesterol-lowering agents affect brain activity? A comparison of simvastatin, pravastatin and placebo in healthy volunteers. British Journal of Clinical Pharmacology, 37, 231-236. Janciauskiene, S., & Wright, H. T. (1998). Inflammation, antichymotrypsin, and lipid metabolism: Autogenic etiology of Alzheimer's disease. Bioessays, 20, 1039-1046. Jick, H., Zornberg, G. L., Jick, S. S., Seshadri, S., & Drachman, D. A. (2000). Statins and the risk of dementia. Lancet, 356,1627-1631. Kawahara, M, & Kuroda, Y. (2000). Characterization of channel formation by Alzheimer's beta-amyloid protein: Inhibition by estradiol. Neurobiology of Aging, 21, 552. Keller, P., Simons, M., Michel,B., & Simons, K. (2000). The role of sphingolipidcholesterol microdomains (RAFTS) in processing and transport of the amyloid precursor protein APR Neurobiology of Aging, 21, SI 79. Kivipelto, M., Helkala, E.-L., Hallikainen, M., Alhainen, K., Tuomilehto, }., Nissinen, A., & Soininen, H. (2000). Elevated systolic blood pressure and high cholesterol levels at midlive are risk factors for late-life dementia. Neurobiology of Aging, 21,5174. Knopp, R. H. (1999). Drug treatment of lipid disorders. New England Journal of Medicine, 341, 489-511. Kostis, J. B., Rosen, R. C., & Wilson, A. C. (1994). Central nervous system effects of HMG CoA reductase inhibitors: Lovastatin and pravastatin on sleep and cognitive performance in patients with hypercholesterolemia. Journal of Clinical Pharmacology, 34, 989-996. Koudinova, N. V., Berezov, T. T., & Koudinov, A. R. (1996). Multiple inhibitory effects of Alzheimer's peptide Abetal-40 on lipid biosynthesis in cultured human HepG2 cells. FEBS Letters, 395, 204-206. Kuo, Y.-M., Emmerling, M. R., Bisgaier, C. L., Essenburg, A. D., Lampert, H. C., & Roher, A. E. (1998). Elevated low-density lipoprotein in Alzheimer's disease correlates with brain A-beta 1-42 levels. Biochemistry Biophysical Research Communication, 252, 711-715. Li, L., Zeigler, S., Lindsey, R. J., & Fukuchi, K. (1999). Effects of an atherogenic diet on amyloidosis in transgenic mice overexpressing the C-terminal portion of p-amyloid precursor protein. Society for Neurosciences Abstract, 25,1859. Michikawa, M., & Yanagisawa, K. (1999a). Apolipoprotein E4 isoform-specific actions on neuronal cells in culture. Mechanisms of Aging and Development, 107, 233-243. Michikawa, M., & Yanagisawa, K. (1999b). Inhibition of cholesterol production but not of nonsteroid isoprenoid products induces neuronal cell death. Journal of Neurochemistry, 72, 2278-2285. Muldoon, M. R, Barger, S. D., Ryan, C. M., Flory, J. D., Lehoczky, J. P., Matthews, K. A., & Manuck, S. B. (2000). Effects of lovastatin on cognitive function and psychological well-being. American Journal of Medicine, 108, 538-546. Notkola, I.-L., Sulkava, R., Pekkanen, J., Erkinjuntti, T., Ehnholm, C., Kivnen, P., Tuomilehto, J., & Nissinen, A. (1998). Serum total cholesterol, apolipoprotein Eepsilon4 allele, and Alzheimer's disease. Neuroepidemiology, 17,14-20. Ott, A., Breteler, M. M. B., de Bruyne, M. C., van Harskamp, R, Grobbee, D. E., &

251

The AD Atorvastatin Treatment Trial

ht

tp

://

w

w

w

.c he

m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Hofman, A. (1997). Atrial fibrilation and dementia in a population-based study. The Rotterdam study. Stroke, 28, 316-321. Petot, G. J., Cook, T. B., Chen, C. H., Riedel, T. M., Debanne, S. M., Koss, E., Lerner, A. J., & Friedland, R. P. (2000). A high fat diet during adulthood increases risk for Alzheimer's disease for those with the ApoeE4 allaele. Neurobiology of Aging, 21,5246. Racchi, M., Baetta, R., Salvietti, N., lanna, P., Franceschini, G., Paoletti, R., Fumagalli, R., Govoni, S., Trabucchi, M., & Soma, M. (1997). Secretory processing of amyloid precursor protein is inhibited by increase in cellular cholesterol content. Biochemical Journal, 322, 893-898. Refolo, L. M., Pappolla, M. A., Malester, B., LaFrancois, J., Bryant-Thomas Wang, R., Sambamurti, K., & Duff, K. (2000). Hypercholesterolemia accelerates Alzheimer's amyloid pathology in a transgenic mouse model. Neurobiology of Disease, 7, 321-331. Romas, S. N., Tang, M. X., Berglund, L., & Mayeux, R. (1999). APOE genotype, plasma lipids, lipoproteins and AD in community elderly. Neurology, 53, 517-521. Saheki, A., Terasaki, T., Tamai, I., & Tsuji, A. (1994). In vivo and in vitro bloodbrain barrier transport of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase inhibitors. Pharmacological Research, 11, 305-311. Shie, F. S., LeBoeuf, R. C., Leverenz, J. B., & Jin, L. W. (1999). Effects of cholesterol feeding on histopathologic hallmarks of Alzheimer's disease in (3-amyloid precursor protein (APP) transgenic mice. Society for Neurosciences Abstracts, 25,1859. Simons, M., Keller, P., De Strooper, B., Beyreuther, K., Dotti, C. G., & Simons, K. (1998). Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons. Proceedings of the National Academy of Sciences (USA) 95, 6460-6464. Sing, C. F, & Davignon, J. (1985). Role of apolipoprotein E polymorphism in determining normal plasma lipid and lipoprotein variation. American Journal of Human Genetics, 37, 268-285. Skoog, I., Lernfelt, B., Landahl, S., Palmertz, B., Andreasson, L.-A., Nilsson, L., Persson, G., Oden, A., & Svanborg, A. (1996). 15-year longitudinal study of blood pressure and dementia. Lancet, 347,1141-1145. Sparks, D. L. (1996). Intraneuronal p-amyloid immunoreactivity in the CNS. Neurobiology of Aging, 17, 291-299. Sparks, D. L. (1997). Coronary artery disease, hypertension, ApoE, and cholesterol: A link to Alzheimer's disease? Annals of the New York Academy of Sciences, 826, 128-146. Sparks, D. L. (1999). Vascular related and mediated alterations in Alzheimer's disease. Cerebral cortex (pp. 733-772). New York: Raven Press. Sparks, D. L., Hunsaker, J. C., Scheff, S. W., Kryscio, R. J., Henson, J. L., & Markesbery, W. R. (1990). Cortical senile plaques in coronary artery disease, aging and Alzheimer's disease. Neurobiology of Aging, 11, 601-607. Sparks, D. L., Kou, Y.-M., Roher, A., Martin, T. A., & Lukas, R. J. (2000). Alterations of Alzheimer's disease in the cholesterol-fed rabbit, including vascular inflammation. Preliminary observations. Annals of the Neiu York Academy of Sciences, 903, 335-344.

Drug Development—Clinical

252

ht

tp

://

w

w

w

.c

he m

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

Sparks, D. L, Scheff, S. W., Hunsaker III, J. C, Liu, H., Landers, T., & Gross, D. R. (1994). Induction of Alzheimer-like (3-amyloid immunoreactivity in the brains of rabbits with dietary cholesterol. Experimental Neurology, 126, 88-94. Urmoneit, B., Turner, J., & Dyrks, T. (1998). Cationic lipids (lipofectamine) and disturbance of cellular cholesterol and sphinglmyelin distribution modulates gamma-secretase activity within amyloid precursor protein in vitro. Prostaglandins & Other Lipid Mediators, 55, 331-343. Wardle, J., Rogers, P., Judd, P., Taylor, M. A., Rapoport, L., Green, M., & Nicholson Perry, K. (2000). Randomized trial of the effects of cholesterol-lowering dietary treatment on psychological function. American Journal of Medicine, 108, 547-553. Wolozin, B., Kellman, W., Ruosseau, P., Celesia, G. G., & Siegel, G. (2000). Decreased prevalence of Alzheimer disease associated with 3-hydroxy-3methyglutaryl coenzyme A reductase inhibitors. Archives of Neurology, 57, 1439-1443. Zhou, Y, & Richardson, J. S. (1996). Cholesterol protects PCI2 cells from betaamyloid induced calcium disordering and cytotoxicity. Neuroreport, 7, 2487-2490.

28

p

A Double-Blind,

x. ph

Randomized Investigation

s/ in de

of the Effects of Testosterone or Placebo in Male Patients

ru

m

With Mild-to-Moderate

vn

/fo

Alzheimer's Disease

he m

w

.c

STUDY AIM

4a ll.

Bruce Miller

ht

tp

://

w

w

Alzheimer's disease (AD) is an urgent national health priority. There are currently 4 million affected victims in the United States, and with the increasing size of the elderly population, it is anticipated that there will be 14 million AD patients by the year 2050 if effective therapy is not found. The current annual cost of AD in the United States is estimated at $1 billion. There is a compelling need for therapies that prevent, defer the onset, slow the progression, or improve the symptoms of AD. Hormonal therapies have been the focus of recent research attention. Evidence suggests that estrogen replacement therapy (ERT) in postmenopausal women reduces the risk of AD, and small-scale neuropsychological investigations suggest that ERT in women with AD enhance cognition. Androgens may also have important effects on cognition, behavior, and mood in men. Testosterone administration to hypogonadal men significantly decreases negative mood scores. Testosterone is the primary androgenic secretory product of the testes. It is converted to estrogen by aromatases 253

Drug Development—Clinical

254

/fo

ru

m

s/ in de

x. ph

p

in the brains of human males; thus, men have lifelong estrogen sources. Testosterone levels decline throughout life, however, and central nervous system estrogen levels decrease concomitantly. Based on the emerging evidence that ERT enhances cognition in women with AD, we propose to investigate the potential cognition-enhancing effects of testosterone in men with AD. We hypothesize that administration of testosterone will raise central nervous system estrogen and androgen levels and will have beneficial cognitive and mood effects in men with AD.a The effects of testosterone on cognition in normal elderly men are unknown and a normal control group will be included in the clinical trial for comparison purposes. We propose a double-blind, randomized, placebo-controlled trial of testosterone in elderly men with AD. The treatment trial will be 6 months in duration, and will include cognitive and neuropsychiatric measures, as well as neuroimaging at baseline and at study end. The study objective is to determine the cognitive and neuropsychiatric effects of testosterone therapy in elderly men with mild and moderate AD. It is part of a multisite study being conducted at both UCLA and UCI Medical Centers.

vn

BACKGROUND

ht

tp

://

w

w

w

.c he

m

4a ll.

Several epidemiological studies provide evidence that ERT decreases the likelihood that postmenopausal women will develop AD, dela)/s the onset of AD in these women, as well as reduces its severity (Paganini-Hill & Henderson, 1994; Tang et al., 1996; Yaffe, Sawaya, Lieberburg, & Grady, 1998). Estrogen replacement therapy also has been shown to have cognitive benefit in elderly women without AD (Henderson, Watt, & Buckwalter, 1996; Sherwin, 1988; Kampen & Sherwin, 1994). Estrogens have many effects of potential therapeutic relevance in AD. They have rieurotropic effects, stimulating neurite growth and synapse formation. Estrogen also increases choline acetyltransferase activity in the basal forebrain, an area prominently affected in AD (Luine, 1985). The effects of testosterone on cognition have not been extensively studied. Men in general perform better in visuospatial/mathematical tests, while women are better in language performance (verbal fluency), measures of perceptual speed, (Halpern, 1992; Linn & Petersen, 1985) and spatial memory, defined as memory for object locations. Gouchie and Kimura (1991) found that men with lower salivary testosterone levels performed better than men with higher levels on measures of spatial and mathematical ability, whereas women with high testosterone levels scored higher on these same measures. Improvement in spatial cognition has been found upon testosterone administration (Janowsky, Oviatt, & Orwall, 1994). Hypogonadal men show improved verbal fluency and improved measures of

Investigation of the Effects of Testosterone or Placebo

255

he m

SIGNIFICANCE

4a ll.

vn

/fo

ru

m

s/ in de

x. ph

p

energy, positive feelings, and friendliness, and decreased negative mood aspects, including anger, nervousness, and irritability upon testosterone administration (Alexander et al., 1998; Wang et al., 1996). These data suggest that testosterone may influence mood and some aspects of cognition. There has been no research on testosterone and AD. In general, the frequency of sexual activity declines in AD (Ballard et al. 1997), suggesting lowered libido possibly reflecting decreased testosterone levels. There is a consistent decrease in plasma testosterone levels in men with age (Vermeulen, 1991). However, serum concentrations of estrogen do not change significantly with age in men (Gray, Feldman, McKinlay, & Longcope, 1991). Since patients with AD are elderly, they are likely to have diminished serum testosterone levels. Testosterone replacement therapy will lead to an increase of serum and brain testosterone, as well as increased estradiol levels. The conversion of androgen to estrogen in the male brain is catalyzed by aromatase enzymes that are concentrated in the bed nucleus of the stria terminalis, the medial and cortical amygdala, and in the periventricular, preoptic, and medial preoptic nuclei of the hypothalamus. Assuming a related distribution of aromatase activity in the human brain, direct effects of elevated serum testosterone are most likely to affect mood, motivation, and emotion, with secondary effects on cognition.

://

w

w

w

.c

The potential cognitive and neuropsychiatric benefits of testosterone replacement therapy will be determined in men with mild and moderate AD in this study. If testosterone is found to be helpful, it could be used to possibly prevent or treat AD.

ht

tp

METHODS General Study Design

We used a double-blind, randomized, placebo-controlled, 6-month study of male patients with mild and moderate AD. Methods of Data Analysis The primary analysis will be an intent-to-treat analysis. All tests will be two-tailed and a Type I error rate of 0.05 will be used. Multiway analysis of variance will be used to compare primary outcome measures between

Drug Development—Clinical

256

the treated and nontreated groups. In the event of early termination, the analysis will be performed using last-analysis-carried-forward (LOCF). A complete analysis then will be performed.

Subject Selection

x. ph

p

The UCSF Alzheimer's Center, the UCSF/Mt. Zion Memory Disorders Clinic, and the San Francisco VAMC will select a total of 24 AD patients per year for 3 years, with a total of 72 AD patients.

s/ in de

Inclusion/Exclusion Criteria Inclusion criteria includes:

Male gender; Age 50 years old or greater; NINCDS-ADRDA criteria for probable AD; MMSE score equal to or above 15; Proficient English to be able to perform cognitive testing; A caregiver must be available to monitor and administer medication and accompany the subject to every clinical visit; 7. All subjects must be stable on concomitant medications for 1 month prior to starting the study medication; and 8. No history of psychiatric or non-AD neurological illness.

.c he

m

4a ll.

vn

/fo

ru

m

1. 2. 3. 4. 5. 6.

Exclusion criteria includes:

ht

tp

://

w

w

w

1. Subjects with known prostate cancer; 2. Subjects with an abnormal prostate as evidenced by prostatic symptoms, prostatic masses, or induration on rectal examination (performed within 60 days of baseline), or elevated levels of prostate specific antigen (PSA >4 mg/ml) or a urine flow rate of less than 8 ml/sec., or an International Prostate Symptom Score (I-PSS) >25. If the urine flow rate is 50%; 5. Subjects with current or recent major psychiatric illness (i.e., manic depressive states, schizophrenia); 6. Subjects with significant, uncontrolled systemic illness (i.e., chronic renal failure, chronic liver disease, poorly controlled diabetes, or poorly controlled congestive heart failure);

Investigation of the Effects of Testosterone or Placebo

257

x. ph

p

7. Subjects with a history of alcoholism or substance abuse within the past year; 8. Subjects who are taking other drugs that might interfere with the results of the study (i.e., antiandrogen, estrogens, or p450 enzyme inducers, barbiturates); 9. Subjects who are greater than 140% or less than 80% of their ideal body weight based on Metropolitan Life tables; and 10. Subjects with generalized skin disease that may affect absorption of T-gel (i.e., psoriasis) or a known skin intolerance to alcohol. 11. Subjects with a morning prolactin level >40 ng/ml.

s/ in de

Subject Recruitment

ru

m

Source. Potential AD subjects will be recruited from patients seen at the UCSF Alzheimer's Center, the UCSF-Mt. Zion Memory Disorders Clinic, and the San Francisco VAMC.

4a ll.

vn

/fo

Initial contact method. Potential subjects will be informed of the study by an investigator during their visit at one of the source sites listed above, and will be offered the opportunity to return for an initial screening visit.

Consent Process and Documentation

://

w

w

w

.c

he m

At the initial screening visit, the consent form will be reviewed with prospective subjects by an investigator or other trained research staff, and will be signed prior to participation in the study. The signed consent form will be kept in a confidential coded manner. In the case of conserved patients, the conservator, as well as the patient, will be asked to sign the consent form.

tp

Procedures

ht

Study Procedures

All subjects are anticipated to have a range of serum testosterone with -50% having levels