Drug Discovery and Evaluation: Pharmacological Assays PDF
Preview Drug Discovery and Evaluation: Pharmacological Assays
H. Gerhard Vogel Wolfgang H. Vogel (Eds.) Drug Discovery and Evaluation Springer-Verlag Berlin Heidelberg GmbH H. Gerhard Vogel • Wolfgang H. Vogel (Eds.) Drug Discovery and Evaluation Pharmacological Assays Including a CD-ROM Springer Prof. Dr. med. Dr.s.c. H. Gerhard Vogel Johann Wolfgang Goethe UniversiUit Frankfurt D-60569 Frankfurt am Main Philipps UnversWit Marburg D-35037 Marburg Germany Professor Dr. Wolfgang H. Vogel Department of Pharmacology Jefferson Medical College Thomas Jefferson University Philadelphia, PA 19107 USA ISBN 978-3-662-03335-7 Library of Congress Cataloging-in-Publication Data Vogel, H. Gerhard, 1927- Drug discovery and evaluation: pharmacological assays I Hans Gerhard Vogel, Wolfgang H. Vogel. p. cm. Includes bibliographical references and index. ISBN 978-3-662-03335-7 ISBN 978-3-662-03333-3 (eBook) DOI 10.1007/978-3-662-03333-3 1. Pharmacological, Experimental. I. Vogel, Wolfgang H., 1930-. II. Title [DNLM: I. Drug Design. 2. Drug Evaluation. 3. Drug Screening. QV 744 V878d 1997] RM301.25.V641997 615'.7-dc20 DNLM/DLC for Library of Congress This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other way, and storage in data banks. Duplication of this publication or parts there of is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. © Springer-Verlag Berlin Heidelberg 1997 Originally published by Springer-Verlag Berlin Heidelberg New York in 1997 Softcover reprint of the hardcover 1st edition 1997 The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publisher cannot guarantee the accuracy of any information about dosage and application thereof contained in this book. In every individual case the user must check such information by consulting the relevant literature. Cover Design: Struve & Partner, Heidelberg Typesetting: Camera ready by Ulrich Kunkel SPIN 10500913 27/3137 5 4 3 2 1 0 -Printed on acid free paper - Preface This book is intended to be an aid for experienced phannacologists as well as for newcomers in the field of experimental pharmacology. The student in pharmacol ogy, the pharmacist and the medicinal chemist will find a survey of pharmacologi cal assays that can be used for a given indication and for which methods have demonstrated their relevance. The researchers working in special fields of pharma cology will find assays in other, unfamiliar areas which might help to expand their own research. Certain therapeutic domains, such as cardiovascular, respiratory and renal dis orders, psychiatry and neurology, peripheral nerve function, pain and rheumatic diseases, metabolic and endocrine diseases including diseases of the gastrointesti nal tract, are discussed in this book. Each chapter is divided into pharmacological classes, e.g., anxiolytics, anti-epi leptics, neuroleptics, antidepressants, or anti-Parkinson drugs. For each class, in vitro methods, tests on isolated organs and in vivo methods are described. For each method the purpose and rationale are given first, followed by a de scription of the procedure, evaluation of the data, modifications of the method de scribed in the literature, and the relevant references. If possible, a critical assess ment of the method based on personal experience is added. The hints for modifi cations of the method and the extended reference list will be of value for the expe rienced pharmacologist. A few words for the justification for a book of this kind: In 1959, AJ. Lehman, Director of the Division of Pharmacology at the Food and Drug Administration, USA, wrote: ... Pharmacologists are individualists. Like most scientists they are seldom willing to copy each other's techniques in detail, and so their methods vary from one to the other. Nevertheless. there are basic principles and techniques which must be applied to establish the safety of a new drug. Visitors could also read a sticker in his office: You too can learn pharmacology, in only three lessons: each of them lasting ten years. Pharmacologists have always used methods from neighboring disciplines; in the past, e.g. from anatomy, pathology, surgery, zoology and predominantly physiol ogy. Useful methods also came from electrophysiology and the behavioral sci ences. Earlier drug discovery was almost exclusively based on animal experiments, clinical observations and serendipity. In recent years, a major input has come from biochemistry. The effect of many drugs in human therapy could be explained biochemicaly as effects on specific en zymes or receptors. With the detection of more and more receptor SUbtypes, the activity spectrum of a single compound became more and more complicated. At present, molecular biology provides pharmacologists with human receptors and ion VI Preface channels expressed in mammalian cells in culture. This avoids the apparently exist ing species differences, but the multitude of natural and perhaps artificial subtypes raises the question of physiological and pathological relevance. The challenge for the pharmacologist always will be to correlate in vitro data with in vivo findings, bearing in mind the old saying: "In vitro simplicitas, in vivo veritas The effects found in tissue cultures are quite often not typical for an in to. tact organism. Pharmacologists, especially in industry, have the task to find new drugs for hu man therapy by using appropriate models. Pharmacological models have to be relevant, that means they should predict the intended therapeutic indications. A pharmacological model can be considered relevant or correlational, if the effects obtained correlate with results observed in human therapy. To be relevant or "correlational", a model has to fulfill some basic criteria: • First, the model must be sensitive in a dose-dependent fashion to standard com pounds that are known to possess the desired therapeutic property. • Second, the relative potency of known active agents in the model should be comparable to their relative potency in clinical use. • Third, the model should be selective, i.e. the effects of known agents in this therapeutic indication should be distinguishable from effects of drugs for other indications. Positive data with a new compound allow the prediction of a thera peutic effect in patients. If new assays are applied to indications for which no effective drug is known, there must be sufficient evidence that this model is relevant for the pathological status in this indication. The methods presented in this book have been selected according to these crite ria. Considerable discussion is going on about the necessity of animal experiments. One has to accept that only the whole animal can reflect the complexity of a hu man being. Even an experiment with human volunteers is only a model, albeit a highly relevant one, to investigate therapeutic effects in patients. The degree of relevance increases from isolated molecules (e.g. receptors or enzymes) to organ elles, to organs up to conscious animals and human volunteers. Without any doubt, animal experiments are necessary for the discovery and evaluation of drugs. However, they should be performed only if they are necessary and well conceived. In chapter N, regulations existing in various countries concerning the care and use of laboratory animals are listed. Furthermore, guidelines for anesthesia, blood collection and euthanasia in laboratory animals are given. In carrying out animal experiments, one must adhere strictly to these guidelines. Following these rules and planing the experiments well, will eliminate or minimize pain and discomfort to the animal. The methods described in this book had the welfare of the animals as well as the benefit of the procedure for the well-being of mankind in mind. Here, we would like to express our sincere thanks to all colleagues who con tributed to this book. Their names and positions are given alphabetically below. Autumn 1996 H. Gerhard Vogel Wolfgang H. Vogel The Editors H. Gerhard Vogel received his license in pharmacy at the University of Erlangen and his license in medicine as well as his doctorate in medicine at the University of Tiibingen, Germany. After working in the Institute for Pharmacology and Toxicol ogy at the University of Tiibingen and as a physician in a city hospital, he joined the pharmaceutical company, Hoechst AG. First, he worked in general pharmacol ogy, then he became head of endocrinology, head of the department of pharmacol ogy, and finally director of preclinical evaluation and development of Hoechst AG. In these positions, he cooperated with many research laboratories worldwide, es pecially in the USA, Great Britain, France, Japan, India and China. In 1967, he completed his habilitation for pharmacology and toxicology at the Philipps Uni versity, Marburg, and was later nominated as honorary professor at the University of Marburg and at the Johann Wolfgang Goethe University, Frankfurt, Germany. At both universities, he lectured to students of medicine, dentistry, and human biology. He published many original papers, more than 100 on biomechanics and the biochemistry of connective tissue. In his manifold positions, he obtained a thorough insight into the broad spectrum of pharmacological assays to detect and to evaluate potential drugs. This provided him with the expertise to write this book together with W. H. Vogel, Jefferson Medical College, Philadelphia, and several colleagues from industry and academia. Wolfgang H. Vogel received his Ph. D. in organic chemistry at the Institute of Technology in Stuttgart, Germany. During his postdoctoral fellowships in the De partments of Pharmacology at the Colleges of Medicine in Syracuse and Chicago, USA, he acquainted himself with pharmacology and toxicology. He worked in biochemical pharmacology at the National Institutes of Health in Bethesda for 2 years. In 1967, he joined the Department of Pharmacology of Jefferson Medical College of Thomas Jefferson University in Philadelphia. He rose from associate professor over full professor to acting chairman of the department. Due to his in terest and research in psychiatry, the title of professor of psychiatry and human be havior was awarded to him. In 1994, he was briefly acting chairman of the Institute of Toxicology at the University of Marburg, Germany. He lectures to medical and graduate students, residents and physicians during postgraduate seminars and has published widely in the field of stress, and the biochemical and genetic basis of behavior and addictions. His academic background, combined with the extensive industrial experience of H. G. Vogel, has greatly benefitted the completion of this book. List of contributors Albus Udo Hoechst-Marion-Roussel, Cardiovascular Research, D-65926 Frankfurt Bartlett Robert R. Hoechst-Marion-Roussel, Immunopharmacology, D-65174 Wiesbaden Bickel Martin Hoechst-Marion-Roussel, Metabolism Research, D-65926 Frankfurt Brioni, Jorge D. Abbott Laboratories, Neuroscience Research, Abbott Park, USA Gogelein Heinz Hoechst-Marion-Roussel, Cardiovascular Research, D-65926 Frankfurt Greger R. Institute of Physiology, University of Freiburg, D-79104 Freiburg Herling Andreas W. Hoechst-Marion-Roussel, Metabolism Research, D-65926 Frankfurt Hock Franz Jakob Hoechst-Marion-Roussel, General Pharmacology, D-65926 Frankfurt Hropot Max Hoechst-Marion-Roussel, Cardiovascular Research, D-65926 Frankfurt Huger Francis P. Hoechst-Marion-Roussel, Neuroscience Research, Sommerville, NJ, USA Jablonka Bernd Hoechst-Marion-Roussel, Cardiovascular Research, D-65926 Frankfurt Just Melitta Hoechst-Marion-Roussel, Cardiovascular Research, D-65926 Frankfurt Linz Wolfgang Hoechst-Marion-Roussel, Cardiovascular Research, D-65926 Frankfurt Maas Jochen Hoechst-Marion-Roussel, Pharma Research, D-65926 Frankfurt McGaugh James L. Center for Neurobiology of Learning and Memory, University of California at Irvine, Irvine, Ca 92717, USA Muller Gunter Hoechst-Marion-Roussel, Metabolism Research, D-65926 Frankfurt Raiss Ruth X. Hoechst-Marion-Roussel, Osteoarthritis Research, Wiesbaden D-65174 v. Rechenberg W. Hoechst-Marion-Roussel, General Pharmacology, D-65926 Frankfurt Rudolphi K.A. Hoechst-Marion-Roussel, Cardiovascular Research. D-65926 Frankfurt Scholkens Bernward Hoechst-Marion-Roussel, Cardiovascular Research, D-65926 Frankfurt Seeger Karl Hoechst-Marion-Roussel, Pharma Research, D-65926 Frankfurt Wirth Klaus Hoechst-Marion-Roussel, Cardiovascular Research, D-65926 Frankfurt Contents Chapter A Cardiovascular activity ...................................................................................... . A.I Cardiovascular analysis ................................................................. . A.l.1 In vitro methods ................................................................................ . A.l.l.1 aj-Adrenoreceptor binding................................................................ I A.1.l.2 <l:z-Adrenoreceptor binding. ......... .... ........ ........... ..... .......... ..... ........... 3 A.l.l.3 Imidazoline receptor binding............................................................. 5 A.1.1.4 ~-Adrenoreceptor binding .............................................. ................... 7 A.l.l.5 ~I-Adrenoreceptor binding ................................................................ 9 A.1.1.6 ~2-Adrenoreceptor binding ................................................................ 10 A.1.1.7 Adenosine Al receptor binding .......................................................... 11 A.1.1.8 Adenosine A2 receptor binding .......................................................... 13 A.I.l.9 Inhibition of adenosine uptake in human erythrocytes ...................... 14 A.1.l.IO Inhibition of the angiotensin-converting enzyme in vitro ....... .......... 15 A.1.l.I1 Angiotensin II receptor binding.............. ............................. .............. 17 A.1.1.12 Renin-inhibitory activity using human kidney renin and a synthetic substrate .................................................................... 18 A.1.l.13 PAF binding assay ............................................................................. 20 A.1.l.I4 Endothelin receptor antagonism ............... ................... ............. ......... 20 A. l.l.I 5 Nitric oxide formation by cultured endothelial cells ......................... 23 A. 1.1.16 Inhibition of Na+/H+ exchange ........................................................... 25 A.l.l.I6.1 Inhibition of Na+/H+ exchange in thrombocytes ................................ 26 A.1.1.16.2 Inhibition of Na+/H+ exchange in cholesterol activated rabbit erythrocytes .......................... ............................................................. 26 A.l.l.I6.3 Sodium influx into cultured cardiac myocytes .................................. 27 A.1.1.16.4 Inhibition of Na+/H+ exchange into cultured aortic endothelial cells ................................................................................. 27 A. 1. 1.17 Inhibition of phosphodiesterase ......................................................... 28 A.1.1.18 Stimulation of heart membrane adenyl ate cyclase ............................ 29 A.1.1.l9 Patch clamp technique in isolated cardiac myocytes......................... 31 A.l.2 Studies in isolated organs .................................................................. 32 A.1.2.1 a-Sympatholytic activity in isolated vascular smooth muscle .......... 32 A.l.2.2 a-Sympatholytic activity in the isolated guinea pig seminal vesicle ................................................................................... 33 A.I.2.3 a-Sympatholytic activity in the isolated vas deferens of the rat... ..... 34 A.I.2.4 a-Sympatholytic activity in the isolated rat spleen ........................... 35 A.I.2.5 ~I-Sympatholytic activity in isolated guinea pig atria ....................... 35 A.I.2.6 ~2-Sympatholytic activity in the isolated tracheal chain .................... 36 A.1.2.7 Angiotensin converting enzyme inhibition in the isolated guinea pig ileum ................................................................................ 37 A.I.2.8 Vasorelaxing activity of various agents including potassium channel openers ................................................................................. 38
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