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Welch • Eckelman

Medical Imaging/Radiology

Targeted Molecular Imaging Michael J. Welch • William C. Eckelman Targeted Molecular Imaging covers the development of novel diagnostic approaches that use an imaging probe and agent to noninvasively visualize cellular processes in normal and disease states. It discusses the concept, development, preclinical studies, and, in many cases, translation to the clinic of targeted imaging agents.

The many case studies that form the core of this book deal with the development and translation of non-nuclear probes and radiotracers; other sections address critical topics such as In vitro studies, small animal research, and the application of targeted probes for nuclear, optical and MRI imaging. The chapters use a common format to demonstrate how various investigators approach the comprehensive task of validating a new targeted probe.

Targeted Molecular Imaging is a timely resource for a rapidly advancing field, and addresses: •

arious methods of validating a new targeted probe through examples from human studies V with imaging of breast cancer, cardiovascular disease, and neurodegenerative diseases

•

asic principles, disease models, imaging studies in animals, imaging in initial human B studies, and the application of molecular imaging in pharmacy and drug discovery

•

In vitro studies, small animal studies, and targeted radiopharmaceuticals

Using these case studies, investigators can generalize and apply the information to their own specific targeted probe. The insights provided by the contributors, experts who have developed these approaches in their own groups, help guide scientists planning to translate imaging agents from the concept stage to clinical application.

Targeted Molecular Imaging

Edited by

K11953 ISBN 978-1-4398-4195-2

90000

9 781439 841952

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Targeted Molecular Imaging

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ImagIng In medIcal dIagnosIs and Therapy William R. Hendee, Series Editor Quality and safety in radiotherapy

Informatics in medical Imaging

Todd Pawlicki, Peter B. Dunscombe, Arno J. Mundt, and Pierre Scalliet, Editors ISBN: 978-1-4398-0436-0

George C. Kagadis and Steve G. Langer, Editors ISBN: 978-1-4398-3124-3

adaptive radiation Therapy X. Allen Li, Editor ISBN: 978-1-4398-1634-9

Quantitative mrI in cancer Thomas E. Yankeelov, David R. Pickens, and Ronald R. Price, Editors ISBN: 978-1-4398-2057-5

Informatics in medical Imaging George C. Kagadis and Steve G. Langer, Editors ISBN: 978-1-4398-3124-3

adaptive motion compensation in radiotherapy Martin J. Murphy, Editor ISBN: 978-1-4398-2193-0

Image-guided radiation Therapy Daniel J. Bourland, Editor ISBN: 978-1-4398-0273-1

Targeted molecular Imaging Michael J. Welch and William C. Eckelman, Editors ISBN: 978-1-4398-4195-2

Forthcoming titles in the series Informatics in radiation oncology

stereotactic radiosurgery and radiotherapy

Bruce H. Curran and George Starkschall, Editors ISBN: 978-1-4398-2582-2

Stanley H. Benedict, Brian D. Kavanagh, and David J. Schlesinger, Editors ISBN: 978-1-4398-4197-6

Image processing in radiation Therapy Kristy Kay Brock, Editor ISBN: 978-1-4398-3017-8

proton and carbon Ion Therapy Charlie C.-M. Ma and Tony Lomax, Editors ISBN: 978-1-4398-1607-3

monte carlo Techniques in radiation Therapy

cone Beam computed Tomography Chris C. Shaw, Editor ISBN: 978-1-4398-4626-1

handbook of Brachytherapy Jack Venselaar, Dimos Baltas, Peter J. Hoskin, and Ali Soleimani-Meigooni, Editors ISBN: 978-1-4398-4498-4

Joao Seco and Frank Verhaegen, Editors ISBN: 978-1-4398-1875-6

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IMagIng In MedIcal dIagnosIs and Therapy William R. Hendee, Series Editor

Targeted Molecular Imaging

Edited by

Michael J. Welch William C. Eckelman

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

A TA Y L O R & F R A N C I S B O O K

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Cover Image: From “Targeting Norepinephrine Transporters in Cardiac Sympathetic Nerve Terminals” by David M. Raffel.

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2012 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Version Date: 20111222 International Standard Book Number-13: 978-1-4398-4196-9 (eBook - PDF) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint.

Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers.

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Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

v

Contents Series Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

part i 1

In Vitro Studies

In Vitro Approaches to Site-Specific Imaging Agents. . . . . . . . . . . . . . . . . . . . . 3 Raymond E. Gibson

part ii Small Animal Studies 2

Small Animal Tumor Models for Use in Imaging . . . . . . . . . . . . . . . . . . . . . . . 19 Cheryl L. Marks

3

Animal Models in Cardiology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Wengen Chen, Henry Gewirtz, Mark Smith, and Vasken Dilsizian

4

Imaging Diabetes in Small Animals: Models, Methods, and Targets. . . . . . . 71 Kooresh I. Shoghi

part iii

Radiopharmacology and Radiopharmacy

5 Targeted Radiopharmaceuticals: Advances in Radiopharmacology and Radiopharmacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Cathy S. Cutler and Sally W. Schwarz

part iV C ase Studies on Developing Targeted Nonnuclear Probes 6 Targeted Optical Molecular Imaging with Cytate: From Bench toward Bedside. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Yang Liu and Samuel Achilefu

7

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Molecular Imaging of P-Selectin with Contrast Ultrasound for the Detection of Recent Tissue Ischemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Brian Davidson and Jonathan R. Lindner

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vi

8

Evolution of Magnetic Nanoparticles: From Imaging to Therapy . . . . . . . . . 139 Yali Li and Jason R. McCarthy

part V C ase Studies on Developing Targeted Radiotracers 9 Targeting Prostate Cancer with Small Molecule Inhibitors of Prostate-Specific Membrane Antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 John W. Babich, John L. Joyal, and R. Edward Coleman

10

Imaging Infection Based on Expression of Thymidine Kinase . . . . . . . . . . . 175 Chetan Bettegowda, Youngjoo Byun, Catherine A. Foss, George Sgouros, Richard F. Ambinder, and Martin G. Pomper

11 Design Criteria for Targeted Molecules: Muscarinic Cholinergic Systems Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 William C. Eckelman

12

Radioligands for the Vesicular Monoamine Transporter Type 2. . . . . . . . . . 217 Michael R. Kilbourn

13

Radiopharmaceuticals for Imaging Proliferation . . . . . . . . . . . . . . . . . . . . . . 231 Kenneth A. Krohn, John R. Grierson, Mark Muzi, and Jeffrey L. Schwartz

14

[99mTc]TRODAT: Dopamine Transporter Imaging Agent for SPECT. . . . . . . . 253 Hank F. Kung and Karl Ploessl

15

Development of Radiotracers and Fluorescent Probes for Imaging Sigma-2 Receptors In Vitro and In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 Robert H. Mach and Kenneth T. Wheeler

16

Steroid Hormone Ligands for the Estrogen Receptor . . . . . . . . . . . . . . . . . . 279 David A. Mankoff, Jeanne M. Link, and Hannah M. Linden

17 Targeting Norepinephrine Transporters in Cardiac Sympathetic Nerve Terminals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 David M. Raffel

18 PET Quantification of Myocardial Metabolism: Challenges and Opportunities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 Arun K. Thukkani and Robert J. Gropler

19 Development of Receptor-Avid Peptide Radiotracers for Targeting Human Cancers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 Wynn A. Volkert, Thomas P. Quinn, C. Jeffrey Smith, and Timothy J. Hoffman

Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

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vii

Series Preface The Taylor & Francis Group series of books in medical physics is an effort by the publisher and the series editor to describe and explain scientific and technological developments in medical imaging and radiation therapy. It is the goal of this series to identify and illustrate the improvements that these developments provide and promise for patient care. Several books have been published in the medical physics series, and many more are in progress.

The book Targeted Molecular Imaging is an important addition to the medical physics series. It is widely understood that molecular imaging is an emerging frontier of biomedical research with important potential ramifications for medical diagnosis and therapy.

Techniques of molecular imaging are revealing new knowledge about normal and abnormal structure and function at the cellular and molecular levels, and are providing promising mechanisms for guiding the delivery of new therapies to aberrant cells and tissues.

This book clarifies these techniques and their potential for expanding scientific knowledge and improving the care of patients suffering from a variety of diseases

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and disabilities. We are very pleased to add Targeted Molecular Imaging to the Taylor & Francis Group series in medical physics.

Selecting the editors for this book was relatively easy.

We wanted two individuals who are well versed in molecular imaging and have a broad perspective on the discipline and its potential contributions to biomedical science and clinical care. We also wanted editors who are well known and respected as medical researchers, and who have experience in translating research into products and procedures that are useful in patient care. Our first choice as editors was Michael Welch and William Eckelman. We are delighted that they agreed to undertake the challenge of designing and editing this book.

We intend to provide a book that will clarify the principles and techniques of molecular imaging and its applications to biomedical research and, potentially, clinical medicine. We hope that readers will agree that we have achieved our intent.

William Hendee, PhD Series Editor

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ix

Preface When Bill Hendee, the series editor, discussed with us a book on molecular imaging, we felt it essential to select a format that distinguished this text from the several other publications that have appeared recently on the subject. The majority of contributions to this book discuss the concept, development, preclinical studies, and, in many cases, translation to the clinic of targeted imaging agents. The first four chapters relate to specific in vitro approaches needed to develop agents; three chapters discuss animal models available for preclinical development while the fourth relates to the translation of nuclear probes to human use applications. The remaining chapters consist of case studies on the development and translation of targeted agents. The majority of these chapters relate to nuclear probes; one deals with optical approaches, another with ultrasound approaches, and yet another with the application of nanoparticles. If one examines the molecular imaging probes that have been studied in humans, the majority are nuclear probes.

There are several reasons for this, one of the major ones being that the amount of mass administered is much less than for the other techniques and so obtaining approval from the U.S. Food and Drug Administration

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for human studies is less expensive and time consuming than for agents using optical, ultrasound, and magnetic resonance approaches.

The case studies in this publication involve targets for a variety of diseases, from oncology to cardiac disease to neurological disease as well as inflammation and infection. As the number of targets increase in the postgenomic era and proteins that are either mutated or constitutively increased in disease are identified, it is important to validate targeting to those proteins and demonstrate that external imaging will monitor the key process with high sensitivity. These chapters using a common format demonstrate how various investigators approach the comprehensive task of validating a new targeted probe. The value of these case studies should be measured by the degree to which the new investigator can generalize from these studies to their own specific targeted probe. We believe that this will be substantial.

It is our hope that this book will provide valuable guidance to scientists planning to translate imaging agents from concept to the clinic and to graduate students, postdoctoral fellows, and junior faculty members who are becoming involved in this rapidly expanding field.

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xi

Acknowledgments We appreciate the efforts of all the contributors to this book; they all have invested a significant amount of time in the preparation of these informative chapters. We also thank Mrs Kari Alca of the Division of

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Radiological Sciences, Department of Radiology at Washington University St. Louis, for the many hours that she has spent editing and converting these chapters into a uniform format.

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xiii

Editors Dr. Michael J. Welch is the codirector of the Division of ­ Radiological Sciences and a professor of radiology at the ­ Washington University School of Medicine’s Mallinckrodt Institute of Radiology in St. Louis, Missouri. He is the recipient of several honors, including the Georg Charles de Hevesy Nuclear Medicine Pioneer Award, Paul C. Aebersold Award, American Chemical Society’s St. Louis, Midwest and National Awards for Nuclear Chemistry, and the Cassen Award. He has served for many years on the editorial board of the Journal of Nuclear Medicine. Dr. Welch received his bachelor’s and graduate degrees from Cambridge University, and his doctorate in radiochemistry from the University of London. His primary research involves the development of high-resolution PET imaging techniques, with a focus on the developments of novel PET detectors and systems suitable for imaging of small laboratory animals.

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Dr. William C. Eckelman is a professor of radiology at the University of California, San Diego and the president of Molecular Tracer LLC. A pioneer in Tc-99m radiopharmaceutical development, he and colleagues developed the Instant Tc-99m Kit, which became the basis for all subsequent 99mTc radiopharmaceutical kits, and targeted receptor-binding radiotracers for human use for both single-photon emission computed tomography and positron emission tomography, including the first neuroreceptor image in humans. Dr. Eckelman has been the editor-in-chief of Nuclear Medicine and biology since 1985. He received numerous awards including the Paul C. Aebersold Award, the Institute of Clinical PET Distinguished Scientist Award, the American College of Nuclear Physicians Corporate Achievement Award, the Georg deHevesy Nuclear Pioneer Award, the Great Golden Seal of Padua University in recognition of contributions to Radiopharmaceutical Development, and the Society of Radiopharmaceutical Sciences President’s Award.

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xv

Contributors Samuel Achilefu Departments of Radiology and Biomedical Engineering Washington University School of Medicine St. Louis, Missouri Richard F. Ambinder Johns Hopkins University School of Medicine Baltimore, Maryland John W. Babich Molecular Insight Pharmaceuticals Cambridge, Massachusetts Chetan Bettegowda Johns Hopkins University School of Medicine Baltimore, Maryland Youngjoo Byun College of Pharmacy Korea University Chungnam, South Korea Wengen Chen Diagnostic Radiology and Nuclear Medicine University of Maryland Medical Center Baltimore, Maryland R. Edward Coleman Department of Radiology Duke University Durham, North Carolina Cathy S. Cutler Research Reactor Center University of Missouri Columbia, Missouri

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Brian Davidson Division of Cardiovascular Medicine Oregon Health and Science University Portland, Oregon Vasken Dilsizian Diagnostic Radiology and Nuclear Medicine University of Maryland Medical Center Baltimore, Maryland William C. Eckelman Molecular Tracer, LLC Bethesda, Maryland Catherine A. Foss Johns Hopkins University School of Medicine Baltimore, Maryland Henry Gewirtz Diagnostic Radiology and Nuclear Medicine University of Maryland Medical Center Baltimore, Maryland Raymond E. Gibson Gibson Imaging Apps, LLC Holland, Pennsylvania John R. Grierson Department of Radiology University of Washington Seattle, Washington Robert J. Gropler Mallinckrodt Institute of Radiology Washington University School of Medicine St. Louis, Missouri

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xvi

Timothy J. Hoffman Department of Internal Medicine University of Missouri and H.S. Truman Memorial VA Hospital Columbia, Missouri John L. Joyal Molecular Insight Pharmaceuticals Cambridge, Massachusetts Michael R. Kilbourn Department of Radiology University of Michigan Medical School Ann Arbor, Michigan Kenneth A. Krohn Departments of Radiology and Radiation Oncology University of Washington Seattle, Washington Hank F. Kung Departments of Radiology and Pharmacology University of Pennsylvania Philadelphia, Pennsylvania Yali Li Center for Systems Biology Harvard Medical School Massachusetts General Hospital Charlestown, Massachusetts Hannah M. Linden Division of Medical Oncology University of Washington Medical Center and Seattle Cancer Care Alliance Seattle, Washington

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Jonathan R. Lindner Division of Cardiovascular Medicine and Department of Biomedical Engineering Oregon Health and Science University Portland, Oregon Jeanne M. Link Division of Nuclear Medicine University of Washington Medical Center Seattle, Washington Yang Liu Departments of Radiology and Biomedical Engineering Washington University School of Medicine St. Louis, Missouri Robert H. Mach Department of Radiology Washington University School of Medicine St. Louis, Missouri David A. Mankoff Division of Nuclear Medicine University of Washington Medical Center and Seattle Cancer Care Alliance Seattle, Washington Cheryl L. Marks Division of Cancer Biology National Cancer Institute Bethesda, Maryland Jason R. McCarthy Center for Systems Biology Harvard Medical School Massachusetts General Hospital Charlestown, Massachusetts Mark Muzi Department of Radiology University of Washington Seattle, Washington

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xvii

Karl Ploessl Department of Radiology University of Pennsylvania Philadelphia, Pennsylvania Martin G. Pomper Johns Hopkins University School of Medicine Baltimore, Maryland Thomas P. Quinn Department of Biochemistry University of Missouri and H.S. Truman Memorial VA Hospital Columbia, Missouri David M. Raffel Department of Radiology University of Michigan Medical School Ann Arbor, Michigan Jeffrey L. Schwartz Department of Radiation Oncology University of Washington Seattle, Washington Sally W. Schwarz Department of Radiology Washington University School of Medicine St. Louis, Missouri George Sgouros Johns Hopkins University School of Medicine Baltimore, Maryland

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Kooresh I. Shoghi Mallinckrodt Institute of Radiology Washington University School of Medicine St. Louis, Missouri Mark Smith Diagnostic Radiology and Nuclear Medicine University of Maryland Medical Center Baltimore, Maryland C. Jeffrey Smith Department of Radiology University of Missouri and H.S. Truman Memorial VA Hospital Columbia, Missouri Arun K. Thukkani Mallinckrodt Institute of Radiology Department of Internal Medicine Washington University School of Medicine St. Louis, Missouri Wynn A. Volkert Department of Radiology University of Missouri and H.S. Truman Memorial VA Hospital Columbia, Missouri Kenneth T. Wheeler Wheeler Scientific Consultants, Inc. Winston-Salem, North Carolina

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I

In Vitro Studies

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3

In Vitro Approaches t 1. In Vitro Approaches to Site-Specific Imaging Agents

Raymond E. Gibson

1.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.2 Determining the Site Concentration/Affinity of Radiotracer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.2.1 Physiological Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.2.2 Tissue Homogenate Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1.2.3 Soluble Target Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1.2.4 Cell Culture Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1.2.5 Autoradiography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1.3 Blood–Brain Barrier and Cellular Permeability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1.3.1 Lipophilicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1.3.2 Efflux Pumps. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1.4 Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 1.5 Nonwash Autoradiography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 1.6 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 References ���������������������������������������������������������������������������������������������������������������������������������������������������������������������� 12

By now, the information needed to develop site-directed (receptor, transporter, enzyme) radiotracers is well known (Eckelman and Gibson 1993, Eckelman 1981, 2003, Fowler et al. 2003, Patel and Gibson 2008).

Although I will focus on developing site-directed radiotracers, the principles apply to developing any sitedirected molecular probe (nuclear, optical, and the more speculative site-specific magnetic resonance and ultrasound contrast agents). In general, site-directed radiotracers are based upon receptor antagonists, uptake blockers, and enzyme inhibitors (functionally equivalent to the binding of an antagonist to a soluble receptor).

Targeted Molecular Imaging. Edited by Michael J. Welch and William C. Eckelman © 2012 Taylor & Francis Group, LLC. ISBN: 978-1-4398-4195-2

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Receptor agonists can be used (e.g., opiates), but, since many agonists undergo internalization, this will limit the nature of the information that can be obtained (e.g., blockade of receptor binding by unlabeled compounds may be possible, but quantification of receptor in vivo may not be). In a similar fashion, while enzyme substrates, for example, [11C]palmitate (Hoffman et al. 1977, Ter-Pogossian et al. 1980), [11C]acetate (Pike et al. 1982), and [11C]glucose (Herrero et al. 2002) can be useful, the metabolism of the radiotracer complicates analysis and certainly these radiotracers cannot be used to quantitate the binding-site concentration. Although these issues are primarily of concern for in vivo imaging and thus not treated herein, one should be aware of the caveats before embarking on the in vitro methods useful for developing new site-specific probes.

Chapter 1

1.1 Introduction

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4 Targeted Molecular Imaging The main information needed for the design of sitedirected probes are the concentration of the site of interest, the affinity of the putative molecular probe for the site of interest, the lipophilicity of the putative probe, whether or not the molecular probe is a substrate for an efflux pump (e.g., the P-glycoprotein pump), and the metabolic fate of the putative probe (stability of the

1.2

Determining the Site Concentration/Affinity of Radiotracer

When a new target is identified as being of interest for the development of a site-specific radiotracer, the most significantly needed information is the concentration of the target. It is axiomatic that if the target-binding site is not present in a tissue, then it is not possible to image it, regardless of how “wonderful” the properties of a radiotracer are (e.g., very high affinity, suitable lipophilicity, etc.). It should also be obvious that very low concentrations of target must require very high affinities, and this relationship has been well established over the years, both theoretically (Eckelman et al. 1979a,b) and experimentally (Francis et al. 1982).

For example, the dopamine D2 receptor in the caudate nucleus is of sufficiently high concentration that it is well imaged in vivo using radiotracers of modest affinity such as raclopride (Dean et al. 1997), while the concentration of receptor present in extrastriatal sites, for example, cortex, is sufficiently low that much higher affinity radiotracers are needed to provide in vivo visualization of those receptor populations in humans (Halldin et al. 1995, Olsson et al. 1999). Therefore, not only must one determine the concentration of the target protein in a tissue, but also the affinity of putative ligands that can subsequently be derivatized to ­produce the imaging probes.

Occasionally, the target is sufficiently mature that there is information concerning receptor concentrations and, perhaps, lead compounds suitable for initial exploration. This is the case for the dopamine D2 receptor ([3H]spiperone: Lyon et al. 1986, Chugani et al. 1988) in brain, muscarinic acetylcholine receptor ([3H]3-quinuclidinyl benzilate) in heart (Gibson et al.

1979) and brain (Pelham and Munsat 1979, Gibson et al. 1984), the β-adrenoceptors ([3H]alprenolol: U’Prichard et al. 1978, [125I]Iodohydroxypindolol: Ezrailson et al. 1981) in heart, and the estradiol receptor ([3H]estradiol: Eckelman et al. 1979b) for estradiol receptor-containing tumors However, when the target is new, the characterization of the target becomes an interesting case of boot-strap-lifting. The concentration of target protein cannot be determined

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probe and presence or absence of interfering metabolites). For radiotracers including isotopes of short halflife, access to rapid synthetic methods and purifications are also needed. We will discuss in this chapter the various in vitro methods that are available to generate the information needed prior to investing time and resources to a new development project.

without an appropriate tool, usually a tritiated radioligand, and the lack of an in vitro radiotracer-binding assay makes it difficult to develop a high-affinity ligand suitable for determining the concentration of the target ­protein. Initial efforts are focused therefore on ­finding high-affinity lead compounds that may lead to radioligands.

1.2.1

Physiological Methods

Most initial efforts that lead to radioligands are not oriented toward developing radioligands, but are the result of pharmaceutical exploration of new targets with the intent of finding potent drug candidates, or for the pharmacological characterization of a new receptor. ­ Nonetheless, pharmaceutical companies recognize the utility of radioligands which are often used in moderatethroughput assays to generate compounds with improved properties over those identified in initial screens.

The most common approach to obtaining initial leads for the development of potential high-affinity drugs is to screen compounds for their effect on a physiological response. Historically these have been effects of a response such as muscle contraction, effects on a second messenger for which there is an assay, for example, inhibition of c-AMP production or phosphotidyl inositol turnover, or on subsequent downstream responses, for example, Ca-efflux. The latter is particularly useful since Ca-influx can be monitored using fluorescence technologies that are amenable to highthroughput assays (e.g., FLIPR, Bednar et al. 2004).

Initial leads, most often identified through robotic assays in which entire compound libraries are screened, may exhibit affinities that are in the micromolar (μM) range (i.e., measurable inhibition of response occurs in concentrations of several μM).

Using well-established methods in medicinal chemistry (Martin 1981, Evans et al. 1988, Lin et al. 2002, Neamati and Barchi 2002), such modest leads are modified to increase affinity. These increases in ­affinity are demonstrated using the physiological or

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In Vitro Approaches to Site-Specific Imaging Agents 5

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An interesting aside to the NR2B story is that no in vivo imaging radiotracer for this target has been demonstrated. Several potential positron-emitting radiotracers have been reported, but specific in vivo binding have not been demonstrated (Haradahira et al.

2002, Årstad et al. 2006, Layton et al. 2006). I would suggest from the very slow kinetics of association demonstrated in the in vitro binding of NR2B radiotracers result in in vivo radiotracers with binding kinetics too slow to provide specific binding, that is, if significant binding to the receptor-binding sites requires hours of exposure and if either the radiotracer plasma clearance is too rapid or the radiotracer is labeled with carbon-11, site-specific binding in vivo will not be observed before clearance or decay obviates the ability to see the specific signal.

1.2.2

Tissue Homogenate Assay

There are excellent publications on radiotracer assays (Braestrup and Squires 1978, Pasternak 1980, Luthin and Wolfe 1984) and thus we will not provide an extensive exposition of these here. The most frequently used in vitro target-specific assay is often referred to as the “grind and bind” assay. When the target protein is part of the cell membrane, radioligand binding to the target protein can be assayed using homogenates of the target tissue (e.g., corpus striatum for the dopamine D2 receptor) or cells expressing the receptor (e.g., the NR2B receptor cited earlier). Radioligand is incubated with the membrane homogenates in a suitable physiological medium, in the absence and presence of a ­competitive unlabeled ligand. Once equilibrated, the radioligand bound to the receptor in the membranes is separated from the unbound usually by filtration over glass fiber filters. The specific signal is enhanced by a washing procedure using several volumes of incubation buffer (with no radioligand or competitive ligand present). The primary limiting factor in this form of in vitro radioligand assay is that the dissociation rate of the radioligand must be sufficiently slow so that the washing procedure used to reduce nonspecific binding does not wash away the specific binding. To minimize this, washing buffer usually used is ice cold, the lower temperature reducing the dissociation of specifically bound radioligand.

The most significant information that is obtained from a tissue homogenate assay is the affinity of the radioligand (usually expressed as Kd) and the concentration of binding site (Bmax). When cell lines are used as the source of receptor, this concentration may not be

Chapter 1

­ iochemical-based screening assays. And herein lies b the first caution: apparent affinities determined using nonradiotracer assays do not represent true affinity.

One interesting example of this is the development of antagonist of the glutamate receptor subtype, the NR2B receptor (Laube et al. 1997).

The NR2B receptor is a potential therapeutic target for pain (Parsons 2001), and may have positive influence in the treatment of Parkinson’s disease (SteeceCollier et al. 2000). Screening and medicinal chemistry refinements, using the fluorescence methodology FLIPR, provided antagonists with affinities <10 nM. Interestingly, despite considerable synthetic effort, an apparent floor to the affinity was found in the nM range. When a radioligand was developed, equilibrium conditions were established and it was found that many of the antagonists in which FLIPR estimates of affinity were near 1 nM exhibited much higher affinity. The kinetics of association of the antagonists is slow, and the fluorescent assay with whole cells did not permit incubation times sufficient to provide accurate estimations of affinity (Kiss et al. 2005).

Estimation of affinity using physiological measures when in error generally errs on the side of underestimating the affinity of compounds. One could reasonably assume that this should not be a problem. After all, if one selects a potential imaging radiotracer on the basis of an assay that underestimates the affinity, then the resulting compound should be better than expected. However, in the case of the NR2B antagonists, many different structural categories varying greatly in physicochemical properties appeared to have the same affinity as determined by the physiological methodology. There was no basis upon which to correctly select reasonable candidates for radiolabeling.

Once the radiotracer assay was established, compounds that appeared to have the same affinity sorted themselves out to cover affinities ranging over 100fold. Thus, the physiological assay did not provide sufficient discrimination to allow rational selection.

While other factors may lead to the disagreement between affinities determined via physiological versus radiotracer assays, for example, inability to estimate nonspecific binding in the physiological assay or ­complexities in the coupling of second effectors as compared to binding at the receptor site, the slow association kinetics and inability to equilibrate binding in the physiological assay are adequate to explain the inability of this assay to provide knowledge of the affinities to allow selection of the best candidate for preparing a site-specific radiotracer.

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6 Targeted Molecular Imaging particularly useful since it may not relate to the concentration expressed in target tissue in vivo. Therefore, the receptor concentration needs to be determined in the tissue is of interest. Using tissue from the target of interest, for example, dopamine D2 receptor in the caudate nucleus (of either an appropriate model animal, e.g., rodent, dog, and/or nonhuman primate, or from humans) and the homogenate assay, the effective concentration in the target in vivo can be calculated.

The Bmax obtained from the tissue homogenate assay is the number of femtomoles of radioligand that specifically bind to the small amount of tissue that is present in the incubation tube (usually between 1 and 10 mg of tissue, wet weight). The initial Bmax can therefore be expressed as femtomoles bound per milligram of tissue, or picomoles bound per gram of tissue. If we now assume that the receptor is uniformly distributed throughout the tissue, and that the specific gravity is 1, the result immediately is expressed as nanomolar.

While the assumption of uniform distribution of receptor may not be strictly true, for example, membrane receptors do not occupy intracellular space, imaging techniques are resolution-limited, and volume averaging effectively averages the binding protein concentration through the voxel volume. The best example of concordance between in vitro measured concentration and in vivo determined concentration is the dopamine D2 receptor that has been determined by both methods to exhibit concentrations of 15–20 nM (Wong et al. 1986, Farde et al. 1987).

1.2.3

Soluble Target Assay

For soluble targets, the difficulty is in how one separates the bound radioligand from the free pools.

Among the techniques used are the centrifugal assay particularly used in the work on the steroid receptors, and typified by corticosteroid binding (Gardner and Wittliff 1973, Anderson et al. 1980) and equilibrium ­dialysis (Katzenellenbogen et al. 1982).

Centrifugal assays comprise two varieties: (1) sucrose gradient and (2) activated charcoal. In the former, separation of bound from free radioligand is achieved by ultracentrifugation: Following equilibration of the radioligand with receptor, the incubation mixture is layered onto a density gradient (usually sucrose which is benign to proteins). Separation of radioligand bound to receptor from free radioligand is effected by high g-forces which cause the heavy molecular weight species (receptor) to settle through the gradient. Free radiotracer remains on the top of the gradient. The

K11953_Book.indb 6

l­ imitation of the assay is that density-gradient centrifugation usually takes many hours during which time lower affinity radioligands will dissociate.

The second variation on the centrifugal assay involves treating the incubation mixture with dextrancoated activated charcoal (DCC) which absorbs lipophilic compounds, a characteristic of most radioligands.

Radioligand bound to receptor is not absorbed onto the DCC (unless too much is used). Centrifugation on a bench-top unit is sufficient to pellet the DCC. Radioligand bound to the receptor is then determined by assaying the supernatant. Similar to the sucrose-­ gradient assay, low-affinity radioligands are likely to dissociate during incubation with charcoal, thus giving erroneous data. Despite being prone to error with inconsistencies in assay conditions (Thorpe 1987), this assay is the standard one for slowly dissociating radioligands bound to soluble receptors (Johnson et al. 1975, Carola and McGuire 1977).

A viable alternative, particularly for lower affinity radioligands, is the dialysis assay. Dialysis membranes are commercially available which allow small molecular weight species to diffuse through, but retain higher molecular weight species. Dialysis can be accomplished using low-volume cells in which dialysis membrane separates two compartments, one containing the binding site of interest, and the second compartment containing the radioligand solution.

An alternate approach is to use dialysis tubing. In this case, the enzyme or soluble receptor is placed inside a tube made by tying each end. The binding-site containing tube is then placed in a large container containing the radioligand in physiological medium. The system is allowed to equilibrate, during which time radioligand migrates across the membrane to bind to the soluble receptor. Unbound radioligand is at the same concentration on either side of the membrane.

Bound ligand (representing both specific and nonspecific) is obtained from the difference in radioligand concentration between the binding-site compartment and that not containing the protein. The specifically bound radioligand is determined as in the filtration assay by including saturation concentrations of an unlabeled drug to block the specific binding of the radioligand. Much like the filtration assay, Bmax and Kd are obtained by plotting the relationship between free radioligand concentration and the concentration of radioligand bound to the specific site in the dialysis tube or chamber, and fitting this binding curve with an appropriate model (e.g., single-site binding isotherm).

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In Vitro Approaches to Site-Specific Imaging Agents 7

1.2.4

Cell Culture Assay

When binding sites are present in cell membranes, the usual method for determining binding parameters is via the tissue homogenate assay. However, when the binding site is internal to the cell, for example, a cytosolic enzyme, maintaining cell integrity offers a method to determine the binding of radioligand to enzyme which would be otherwise quite difficult to accomplish. The centrifugal assay and dialysis assay described earlier may be useful, but obviously have limitations. By contrast, using whole cells provides an assay with similar characteristics to the dialysis assay, but the advantages are that (1) one is assaying the binding site of interest in its natural mileu and (2) times for equilibration of most reasonably lipophilic radioligands are considerably shorter, often being no more than several hours.

An excellent example of this type of binding assay is provided by studies on radiolabeled inhibitors of the enzyme farnesyl transferase (FPTase). This particular enzyme was considered an interesting potential cancer target since the protein which is farnesylated, Ras, has been shown to exhibit mutations in tumors (Bos 1989).

Reversible inhibitors for the enzyme were developed which exhibited high affinity, modest lipophilicity, and structures amenable to the synthesis of radiotracers

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(Williams et al. 1999). As part of the drug development program, an in vitro cell-based assay was developed using a radio-iodinated derivative of a reversible FPTase inhibitor (Lobell et al. 2003). Equilibration of cells with the radioligand and inhibitors at various concentrations provided the relative affinity of the unlabeled inhibitors. The IC50 values (concentration of unlabeled inhibitor that blocked the binding of radioligand by 50%) correlated well with the efficacy of these compounds in inhibiting cell proliferation. We conducted saturation assays using the radioligand but the methodology we used did not permit assessing the Bmax in cell culture, though it clearly can be done. While the apparent affinity of radioligand can thus be determined, the other parameter of interest, Bmax, has little value since the concentration of enzyme in cells grown in tissue culture may not reflect the concentration enzyme naturally present in targets. Nonetheless, we were able to prepare and characterize radioligands for the enzyme FPTase that provided enzyme-mediated localization in tissues in vivo. It should be obvious that, when the concentration of binding site is as high or higher than occurs in target tissues in vivo, a radioligand that does not provide a specific signal in a tissue culture assay will have little chance of providing specific binding in vivo. However, the relationship between what will provide an in vitro signal in cell culture versus localization in a t­arget tissue has not been ­ established.

1.2.5

Autoradiography

Autoradiography is used to examine the binding of radioligands to binding sites in tissues or cells in which an image of the binding is generated by exposing the radioligand–tissue combination on x-ray film or specially design phosphor-imaging plates. Procedures for autoradiography are much like those used in filtration tissue homogenate assays: tissue on slides is incubated with a radioligand of interest to equilibrium, and, subsequently, nonspecific binding is reduced by washing in buffers without radiotracer. In the same manner as other in vitro assays, the washing procedures which greatly reduce nonspecific binding may be confounding if the off-rate of the radioligand is too fast and specifically bound radiotracer is lost. After drying, the tissues on slides are juxtaposed to films or phosphorimaging plates which can be digitally scanned to provide the desired quantifiable images of radioligand binding. Nonspecific binding is determined either by including a high concentration of competitive ligand

Chapter 1

Interestingly, equilibrium dialysis is the only technique that provides a measure of unbound radioligand. In other techniques, one either creates conditions in which the initial and final concentrations of radioligand are essentially the same or one must calculate the free radioligand concentration (often with some erroneous assumptions); but, in dialysis, one measures the free radioligand concentration. A second advantage is that there is no separation procedure, that is, no washing, and thus this assay may provide a measure of affinity for lower affinity radioligands which would otherwise be washed away in the tissue homogenate assay or dissociate in the centrifugal assay. For example, the binding of [3H]acetylcholine to acetylcholine receptors from the electric organ of the fish Torpedo marmorata with micromolar affinities is easily determined (O’Brien and Gibson 1974).

The primary limitation of this dialysis assay is the time it takes for equilibration. If equilibration times are long, and may be as long as 1–2 days, labile proteins will not survive, denaturing over the time required for equilibration.

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8 Targeted Molecular Imaging or by incubation of tissue sections in which the target of interest has been knocked-out (Kitchen et al. 1997, Ramboz et al. 1998, Lopez-Gimenez et al. 2002, Wong et al. 2003).

The advantage of autoradiography is that it can provide information on the binding of a radiotracer to sites that may be too small to be easily dissected for use in tissue homogenate assays. Of course, the obvious caveat is that if the site is too small to be easily isolated, perhaps it will be too small to be imaged, that is, partial volume averaging of very small structures may obviate in vivo visualization.

In the development of an in vivo imaging radiotracer, a striking feature of autoradiography is that it provides a direct correlate to predict what an image should look like. For example, the in vitro autoradio­ graphy of the histamine H3 receptor with a new radiotracer provided an exact correlate for the in vivo imaging of that receptor population in primates (Figure 1.1; Hamill et al. 2009). We also demonstrated via in vitro autoradiographic studies that mGluR5 radiotracer binds to the cerebellum in species other than rat at sufficiently high concentrations (Patel et al.

2007) that one should expect an image of the distribution of these receptors in the cerebellum of primates, both human and nonhuman. The autoradiographic studies prompted us to determine the Bmax for the mGluR5 in the cerebellum of rat, rhesus monkey, and human brain: 1.4, 11, and 5.1 nM, respectively (Patel et al. 2007). In the rhesus monkey brain, the radiotracer exhibits Kd of 0.1 nM for mGluR5; Bmax/Kd is 120

1.3

(b)

CP

CP

Figure 1.1 Comparison of in vitro and in vivo results of a histamine H3 radiotracer. Left: total binding. Right: nonspecific binding. (a) In vitro autoradiography of radioligand binding to rhesus monkey brain sections. Highest specific signal is associated with the basal ganglia—the caudate, putamen, and globus pallidus, with moderate binding in cortex. (b) In vivo PET image of the the histamine H3 radiotracer showing highest uptake in the basal ganglia and moderate uptake in cortex, recapitulating the autoradiographic results. (Data from Hamill, T.G. et al. 2009. Synapse 63:1122–1132.)

for cerebellum. For comparison, the Bmax/Kd is 210 for rhesus caudate. Imaging studies in rhesus monkey proved specific binding to the mGluR5 in caudate and cerebellum as predicted by the Bmax/Kd values (Hamill et al. 2005). By contrast, the Bmax/Kd for rat cerebellum is 9. The reduction in ratio by more than 10-fold suggests that rat cerebellum mGluR5 would not be imaged in vivo.

Blood–Brain Barrier and Cellular Permeability

The primary characteristics which influence the ability of radiotracers to cross membranes, and thus also to enter the brain, are the radiotracer lipophilicity and the presence of efflux pumps. While there are a number of in vitro methods to examine membrane permeability involving the use of cells (Di et al.

2003), easier methods are available based simply on determining the lipophilicity of the new radiotracer and whether or not the radioligand is a substrate of an efflux pump.

1.3.1 Lipophilicity It is axiomatic the hydrophilic compounds will not cross cell membranes since membranes are composed of bilayer lipids which provide a barrier to the diffusion of hydrophilic compounds. Barring the presence of

K11953_Book.indb 8

(a)

active transport mechanisms (amino acid transport, glucose transport, or the internalization mechanisms associated with peptide-receptor binding and antibody–antigen binding), radiotracers need to have some lipophilic character which will allow them to enter into and pass through cellular membranes. The primary measure of lipophilicity is log P (Waterhouse 2003), the logarithm of the ratio of compound concentration in a lipophilic phase (e.g., 1-butanol or ethylacetate) to that in an aqueous phase (water or buffered physiological medium). The log P can be calculated (see below), or determined experimentally, the two most common methods being equilibration between an organic and aqueous phase or by high-performance liquid chromatography (HPLC).

Equilibration between two phases is quite easy to do: compound or radioligand is mixed into the ­lipophilic

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