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  • 1. Drug Discovery and Evaluation: Pharmacological Assays
  • 2. H. Gerhard Vogel (Ed.)Drug Discovery and Evaluation:Pharmacological AssaysThird Completely Revised, Updated and Enlarged EditionWith 5 Figures and 66 Tables123
  • 3. Hans Gerhard VogelBohl Str. 2873430 AalenGermanyISBN: 978-3-540-70995-4This publication is available also as:Print publication under ISBN 978-3-540-71420-0 andPrint and electronic bundle under ISBN 978-3-540-71419-4Library of Congress Control Number: 2007936031This work is subject to copyright. All rights are reserved, whether the whole or part of the mate-rial is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,broadcasting, reproduction on microfilms or in other ways, and storage in data banks. Duplicationof this publication or parts thereof is only permitted under the provisions of the German CopyrightLaw of September 9, 1965, in its current version, and permission for use must always be obtainedfrom Springer-Verlag. Violations are liable for prosecution under the German Copyright Law.Springer is part of Springer Science+Business Mediaspringer.com© Springer-Verlag Berlin Heidelberg New York 2008The use of registered names, trademarks, etc. in this publication does not imply, even in the ab-sence of a specific statement, that such names are exempt from the relevant protective laws andregulations and therefore free for general use.Product liability: The publishers cannot guarantee the accuracy of any information about the ap-plication of operative techniques and medications contained in this book. In every individual casethe user must check such information by consulting the relevant literature.Printed on acid-free paper SPIN: 11733775 2109 - 5 4 3 2 1 0
  • 4. Preface to the Third EditionThe third edition of the book Drug Discovery and Evaluation. PharmacologicalAssays is presented here. The volume of data has risen considerably compared to the second edition. Inparticular, a large number of assays have been added. Meanwhile, data on SafetyPharmacology have been removed from this edition. An extended discussion of thatimportant aspect of pharmacology was published in the book Drug Discovery andEvaluation. Safety and Pharmacokinetic Assays, edited by H. G. Vogel, F. J. Hock,J. Maas, and D. Mayer, by Springer in 2006. Several of my colleagues provided essential contributions to this edition. In par-ticular I am indebted to S. A. Mousa for rewriting the chapter PharmacologicalAssays in Thrombosis and Hemostasis, S. G. E. Hart, for rewriting the chapter Ac-tivity on Urinary Tract, G. Müller for rewriting and amending the chapter Antidi-abetic Activity, B. Schultz for amending the chapter Ophthalmologic Activity, andJ. Sandow for rewriting the chapter Endocrinology. All chapters have been revisedand thoroughly updated as well. The approach to drug discovery is changing continuously. Decades ago, manydrugs were found by serendipity in clinical trials. Most new drugs, however, werefound by the classical approach in animal experiments. This approach has the ad-vantage of relatively high predictability, but it also has the disadvantage that littleinformation is provided about the molecular mechanisms involved in the observedeffects, and the detection of drugs with new mechanisms always required new mod-els. It is generally believed that the costs of developing new pharmaceutical drugsare exploding, while the output of new drugs is actually decreasing. A changein paradigm, the target-based or mechanism-based drug discovery approach, wastherefore welcomed with great enthusiasm. The techniques of combinatorial chem-istry could generate thousands of compounds to be tested against thousands of tar-gets using high-throughput and ultra-high-throughput technology with tremendouscapacity. This made it highly effective for the identification of target-selective com-pounds. However, despite the fact that this approach is very advantageous from ascientific and practical viewpoint, it did not translate into a high success rate in thediscovery of new drugs. This has naturally led to questions regarding the success oftarget-based drug discovery and, more importantly, a search for alternatives. The target-based approach has therefore been replaced again by the physiology-based approach, the classical drug discovery paradigm, or the function-based ap-proach, which seeks to induce a therapeutic effect by normalizing a disease-specificabnormality. On top of this, important changes in the management of some of the larger drugcompanies have taken place in recent years. Modern managers were installed aschief executive officers and other high-level executives, quite often with little orno biological and technical experience. They were more interested in blockbusters
  • 5. VI Preface to the Third Editionand in shareholders’ value than finding new drugs, especially for rare diseases.Research workers were confronted with cumbersome and inflexible organizationalstructures characterized by regimentation, control, conformity, and excessive bu-reaucracy. Consequently, creativity and productivity decreased in this environment.Recently, however, there are signs that the situation is changing. In this book we ask the question: “Quo vadis, pharmacology?” We suggest giv-ing pharmacologists the freedom, time, and money to carry out their own ideas.We also describe animal models of rare diseases that enable the medically trainedpharmacologist to find appropriate drugs. We are well aware that the rapid progress in biology will once again change themethodological approach in the coming years, and that electronic media will contin-uously help the researcher to access and share information. However, it is becomingmore and more evident that many young pharmacologists have only limited train-ing in classical pharmacological methodologies. When searching for these methods,researchers will only find insufficient information on the methodological details inthe electronic databases currently available. To this end, we hope the current bookmay bridge this gap by comprehensively covering those pharmacological methodsutilized for over more than a hundred years. At this point I would like to express my sincere thanks to all colleagues whocontributed to the new and to the earlier editions of this book. Their names andaffiliations are given in alphabetical order.Spring 2007 H. Gerhard Vogel
  • 6. Preface to the Second EditionThe first edition of “Drug Discovery and Evaluation – Pharmacological Assays” hasbeen well accepted by a broad readership ranging from experienced pharmacologiststo students of pharmacology. Therefore, already after a short period of time thequestion for a second edition arose. The first edition was mainly centered around the personal laboratory experienceof the editors and the contributors. The second edition tries to close evident gaps.The input of biochemistry to pharmacology has grown. Molecular pharmacologyputs more emphasis on the mode of action of drugs, albeit it becomes clear that theactivities of most drugs are not confined to one single mode of action. Studies insingle cells become more and more popular, however, they do not cover the com-plexity of a whole organism. Possible side effects of drugs can be better detectedin whole animals than in single cells. Therefore, the new requirements of the healthauthorities on safety pharmacology put emphasis on experiments not only in wholeanesthetized animals but in conscious ones. The second edition of this book takesnote of these requirements and devotes special chapters for each indication to safetypharmacology. Molecular biology also introduced new methods to pharmacology, such as thepolymerase chain reaction (PCR), reverse PCR, Northern, Western and Southernblotting. Very recently, microarray technology, proteomics, and mass spectroscopywere added as novel in vitro methods. Furthermore, genetically modified animalshave been created which resemble human diseases. Pharmacogenomics has alreadybegun to influence pharmacology and even will have greater input in the future.Special attention is given to these new achievements in various chapters of the book. The editor and the co-editors are well aware that the rapid progress in biologyduring the next decades will change the methodological approach. Electronic mediawill help the researcher for continuous information. However, it becomes more andmore evident, that young pharmacologists have only insufficient training in the clas-sical pharmacological methodology. Searching for these methods, e.g., for safetypharmacology, the researchers cannot find sufficient information on the method-ological details in the electronic data bases currently available. This hook coveringthe pharmacological methods of more than hundred years may be of help. The guidelines concerning the care and use of laboratory animals have been up-dated. A change in paradigm of pharmacological research has been claimed but superi-orities of these new approaches compared to the old ones have still to be proven. Toaddress this, an introductory chapter on new strategies in drug discovery and eval-uation has been added, including combinatorial chemistry; high throughput screen-ing, ultrahigh throughput screening and high content screening; pharmacogenomics,proteomics and array technology. Some critical thoughts on errors in screening pro-cedures have been added.
  • 7. VIII Preface to the Second Edition At this place, we would like to express our sincere thanks to all colleagues whocontributed to the new and to the first edition of this book. Their names and theirpositions are given alphabetically.March 2002 H. Gerhard Vogel Also in the name of the co-editors
  • 8. Preface to the First EditionThis hook is intended to be an aid for experienced pharmacologists as well as fornewcomers in the field of experimental pharmacology. The student in pharmacol-ogy, the pharmacist and the medicinal chemist will find a survey of pharmacologicalassays that can be used for a given indication and for which methods have demon-strated their relevance. The researchers working in special fields of pharmacologywill find assays in other, unfamiliar areas which might help to expand their ownresearch. Certain therapeutic domains, such as cardiovascular, respiratory and renal disor-ders, psychiatry and neurology, peripheral nerve function, pain and rheumatic dis-eases, metabolic and endocrine diseases including diseases of the gastrointestinaltract, are discussed in this hook. Each chapter is divided into pharmacological classes, e.g., anxiolytics, anti-epileptics, neuroleptics, antidepressants, or anti-Parkinson drugs. For each class, invitro methods, tests on isolated organs and in vivo methods are described. For each method the purpose and rationale are given first, followed by a descrip-tion of the procedure, evaluation of the data, modifications of the method describedin the literature, and the relevant references. If possible, a critical assessment of themethod based on personal experience is added. The hints for modifications of themethod and the extended reference list will be of value for the experienced pharma-cologist. A few words for the justification for a book of this kind: In 1959, A.J. Lehman,Director of the Division of Pharmacology at the Food and Drug Administration,USA, wrote: . . . Pharmacologists are individualists. Like most scientists they are sel- dom willing to copy each other’s techniques in detail, and so their meth- ods 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 last- ing ten years. Pharmacologists have always used methods from neighboring disciplines; in thepast, e.g. from anatomy, pathology, surgery, zoology and predominantly physiology.Useful methods also came from electrophysiology and the behavioral sciences. Ear-lier drug discovery was almost exclusively based on animal experiments, clinicalobservations and serendipity. In recent years, a major input has come from biochemistry. The effect of manydrugs 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
  • 9. X Preface to the First Editionactivity spectrum of a single compound became more and more complicated. Atpresent, molecular biology provides pharmacologists with human receptors and ionchannels expressed in mammalian cells in culture. This avoids the apparently exist-ing species differences, hut the multitude of natural and perhaps artificial subtypesraises the question of physiological and pathological relevance. The challenge for the pharmacologist always will be to correlate in vitro datawith in vivo findings, bearing in mind the old saying: “In vitro simplicitas, in vivoveritas”. The effects found in tissue cultures are quite often not typical for an intactorganism. Pharmacologists, especially in industry, have the task to find new drugs for hu-man therapy by using appropriate models. Pharmacological models have to be rele-vant, that means they should predict the intended therapeutic indications. A pharma-cological model can be considered relevant or correlational, if the effects obtainedcorrelate 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 com- parable to their relative potency in clinical use.• Third, the model should be selective, i.e. the effects of known agents in this ther- apeutic indication should be distinguishable from effects of drugs for other indi- cations. Positive data with a new compound allow the prediction of a therapeutic effect in patients.If new assays are applied to indications for which no effective drug is known, theremust be sufficient evidence that this model is relevant for the pathological status inthis indication. The methods presented in this book have been selected according to these criteria. 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 humanbeing. Even an experiment with human volunteers is only a model, albeit a highlyrelevant one, to investigate therapeutic effects in patients. The degree of relevanceincreases from isolated molecules (e.g. receptors or enzymes) to organelles, to or-gans up to conscious animals and human volunteers. Without any doubt, animal experiments are necessary for the discovery and eval-uation of drugs. However, they should be performed only if they are necessary andwell conceived. In Chapter N, regulations existing in various countries concerning the care anduse of laboratory animals are listed. Furthermore, guidelines for anesthesia, bloodcollection and euthanasia in laboratory animals are given. In carrying out animalexperiments, one must adhere strictly to these guidelines. Following these rules andplaning the experiments well, will eliminate or minimize pain and discomfort to theanimal. The methods described in this hook had the welfare of the animals as wellas 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
  • 10. ContentsVolume 1Chapter 0Introduction Strategies in Drug Discovery and Evaluation . . . . . . . . . . 10.1 Historical Approaches in Drug Discovery . . . . . . . . . . 10.2 Classical Pharmacological Approaches . . . . . . . . . . . 30.3 Quo Vadis, Pharmacology? . . . . . . . . . . . . . . . . . . 50.3.1 Managers, Not Leaders . . . . . . . . . . . . . . . . . . . . . 60.3.2 Pressures From Shareholders . . . . . . . . . . . . . . . . . . 60.3.3 Merger Mania . . . . . . . . . . . . . . . . . . . . . . . . . . 60.3.4 Blockbuster Mania . . . . . . . . . . . . . . . . . . . . . . . 60.3.5 Orphan Drugs and Drug Pricing . . . . . . . . . . . . . . . . 60.4 New Approaches in Drug Discovery . . . . . . . . . . . . . 90.4.1 Combinatorial Chemistry . . . . . . . . . . . . . . . . . . . . 90.4.2 High-Throughput Screening, Ultra-High-Throughput Screening, and High-Content Screening . . . . . . . . . . . . 110.4.2.1 Errors in HTS . . . . . . . . . . . . . . . . . . . . . . . . . . 130.4.2.2 Statistical Evaluation of HTS . . . . . . . . . . . . . . . . . . 130.4.3 Technologies for High-Throughput Screening . . . . . . . . . 150.4.3.1 Dispensing Technologies . . . . . . . . . . . . . . . . . . . . 160.4.3.2 Cell-Based Assays . . . . . . . . . . . . . . . . . . . . . . . 160.4.3.3 High-Throughput Assay Technologies for Ion Channels . . . . 170.4.3.4 Detection Methods . . . . . . . . . . . . . . . . . . . . . . . 180.4.4 Pharmacogenomics . . . . . . . . . . . . . . . . . . . . . . . 290.4.5 Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . 350.4.6 Array Technology . . . . . . . . . . . . . . . . . . . . . . . . 370.4.7 NMR Technology . . . . . . . . . . . . . . . . . . . . . . . . 410.5 Errors in Screening Procedures . . . . . . . . . . . . . . . 44Chapter ACardiovascular Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47A.1 Cardiovascular Analysis . . . . . . . . . . . . . . . . . . . 51A.1.1 In Vitro Methods . . . . . . . . . . . . . . . . . . . . . . . . 51A.1.1.1 α 1 -Adrenoreceptor Binding . . . . . . . . . . . . . . . . . . 51A.1.1.2 α 2 -Adrenoreceptor Binding . . . . . . . . . . . . . . . . . . 54A.1.1.3 Electrically Stimulated Release of [3 H]Norepinephrine from Brain Slices . . . . . . . . . . . . . . . . . . . . . . . . 57
  • 11. XII ContentsA.1.1.4 Imidazoline Receptor Binding . . . . . . . . . . . . . . . . . 59A.1.1.5 β-Adrenoreceptor Binding . . . . . . . . . . . . . . . . . . . 62A.1.1.6 β 1 -Adrenoreceptor Binding . . . . . . . . . . . . . . . . . . 64A.1.1.7 β 2 -Adrenoreceptor Binding . . . . . . . . . . . . . . . . . . 65A.1.1.8 Adenosine A1 Receptor Binding . . . . . . . . . . . . . . . . 69A.1.1.9 Adenosine A2 Receptor Binding . . . . . . . . . . . . . . . . 71A.1.1.10 Adenosine A3 Receptor Binding . . . . . . . . . . . . . . . . 73A.1.1.11 Inhibition of Adenosine Uptake in Human Erythrocytes . . . . 74A.1.1.12 Inhibition of Vasopeptidases . . . . . . . . . . . . . . . . . . 75A.1.1.12.1 Inhibition of the Angiotensin-Converting Enzyme in Vitro Purpose and Rationale . . . . . . . . . . . . . . . . . 76A.1.1.12.2 Inhibition of Neutral Endopeptidase (Neprilysin) . . . . . . . 77A.1.1.13 Quantitative Autoradiographic Localization of Angiotensin- Converting Enzyme . . . . . . . . . . . . . . 79A.1.1.14 Angiotensin Antagonism . . . . . . . . . . . . . . . . . . . . 80A.1.1.14.1 Angiotensin II Receptor Binding . . . . . . . . . . . . . . . . 80A.1.1.14.2 AT2 Receptor Binding . . . . . . . . . . . . . . . . . . . . . 84A.1.1.14.3 Angiotensin II Induced Contraction in Isolated Rabbit Aorta . . . . . . . . . . . . . . . . . . . . 85A.1.1.14.4 Angiotensin II Antagonism in Vivo . . . . . . . . . . . . . . 86A.1.1.15 Renin-Inhibitory Activity Using Human Kidney Renin and a Synthetic Substrate . . . . . . . . . . . . . . . . . . . . 88A.1.1.16 PAF Binding Assay . . . . . . . . . . . . . . . . . . . . . . . 90A.1.1.17 Endothelin . . . . . . . . . . . . . . . . . . . . . . . . . . . 91A.1.1.17.1 General Considerations . . . . . . . . . . . . . . . . . . . . . 91A.1.1.17.2 Evaluation of Endothelin Activity . . . . . . . . . . . . . . . 92A.1.1.17.3 Endothelin Receptor Antagonism in Vitro . . . . . . . . . . . 93A.1.1.17.4 Endothelin Receptor Antagonism in Vivo . . . . . . . . . . . 95A.1.1.17.5 Quantitative Autoradiographic Localization of Endothelin-1 Receptor . . . . . . . . . . . . . . . . . . . . 97A.1.1.17.6 Inhibition of Endothelin Converting Enzyme . . . . . . . . . 98A.1.1.18 Nitric Oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . 101A.1.1.18.1 General Considerations on Nitric Oxide . . . . . . . . . . . . 101A.1.1.18.2 Bioassay of EDRF Release . . . . . . . . . . . . . . . . . . . 102A.1.1.18.3 Isolated Arteries with and Without Endothelium . . . . . . . . 103A.1.1.18.4 Nitric Oxide Formation by Cultured Endothelial Cells . . . . 104A.1.1.18.5 Expression of Nitric Oxide Synthase . . . . . . . . . . . . . . 107A.1.1.19 Inhibition of Rho Kinase . . . . . . . . . . . . . . . . . . . . 109A.1.1.20 Inhibition of Na+ /H+ Exchange . . . . . . . . . . . . . . . . 109A.1.1.20.1 Inhibition of Na+ /H+ Exchange in Thrombocytes . . . . . . . 111A.1.1.20.2 Inhibition of Na+ /H+ Exchange in Cholesterol Activated Rabbit Erythrocytes . . . . . . . . . 111A.1.1.20.3 Sodium Influx into Cultured Cardiac Myocytes . . . . . . . . 112A.1.1.20.4 Inhibition of Na+ /H+ Exchange into Cultured Aortic Endothelial Cells . . . . . . . . . . . . . 113A.1.1.20.5 NHE Activity Measured by Intracellular pH in Isolated Ventricular Myocytes . . . . . . . . . . . . . . . . 113A.1.1.20.6 NHE Subtype Specificity . . . . . . . . . . . . . . . . . . . . 114A.1.1.21 Phosphodiesterases . . . . . . . . . . . . . . . . . . . . . . . 115A.1.1.21.1 Inhibition of Phosphodiesterase . . . . . . . . . . . . . . . . 116A.1.1.22 Stimulation of Heart Membrane Adenylate Cyclase . . . . . . 121A.1.1.23 3 H-Forskolin Binding Assay . . . . . . . . . . . . . . . . . . 123
  • 12. Contents XIIIA.1.1.24 Patch Clamp Technique . . . . . . . . . . . . . . . . . . . . . 124A.1.1.24.1 Patch Clamp Technique in Isolated Cardiac Myocytes . . . . . 126A.1.1.24.2 Voltage Clamp Studies on Sodium Channels . . . . . . . . . . 127A.1.1.24.3 Voltage Clamp Studies on Potassium Channels . . . . . . . . 130A.1.1.24.4 Voltage Clamp Studies on Calcium Channels . . . . . . . . . 136A.1.1.24.5 Patch Clamp Studies on Chloride Channels . . . . . . . . . . 138A.1.1.25 Inhibition of Hyperpolarization-Activated Channels . . . . . . 139A.1.1.26 Measurement of Cytosolic Calcium with Fluorescent Indicators . . . . . . . . . . . . . . . . . . . 141A.1.1.27 Measurement of Contractile Force of Isolated Cardiac Myocytes . . . . . . . . . . . . . . . . . . 142A.1.1.28 Adrenomedullin . . . . . . . . . . . . . . . . . . . . . . . . . 143A.1.1.28.1 General Considerations . . . . . . . . . . . . . . . . . . . . . 143A.1.1.28.2 Receptor Binding of Adrenomedullin . . . . . . . . . . . . . 146A.1.1.29 Atrial Natriuretic Factor (ANF) . . . . . . . . . . . . . . . . 147A.1.1.29.1 General Considerations . . . . . . . . . . . . . . . . . . . . . 147A.1.1.29.2 Bioassay for ANF . . . . . . . . . . . . . . . . . . . . . . . . 148A.1.1.29.3 Receptor Binding of ANF . . . . . . . . . . . . . . . . . . . 149A.1.1.29.4 ANF Gene Expression . . . . . . . . . . . . . . . . . . . . . 150A.1.1.29.5 Radioimmunoassay for ANF . . . . . . . . . . . . . . . . . . 151A.1.1.30 Urotensin II . . . . . . . . . . . . . . . . . . . . . . . . . . . 152A.1.1.30.1 General Considerations . . . . . . . . . . . . . . . . . . . . . 152A.1.1.30.2 Rat Thoracic Aorta Bioassay for Urotensin II . . . . . . . . . 153A.1.1.30.3 Intracellular Calcium Mobilization Assay . . . . . . . . . . . 155A.1.1.30.4 Receptor Binding of Urotensin II . . . . . . . . . . . . . . . . 156A.1.1.30.5 Urotensin II Gene Expression . . . . . . . . . . . . . . . . . 158A.1.1.31 Apelin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160A.1.1.31.1 General Considerations . . . . . . . . . . . . . . . . . . . . . 160A.1.1.31.2 Cardiovascular Actions of Apelin . . . . . . . . . . . . . . . 161A.1.1.32 TRP Channels . . . . . . . . . . . . . . . . . . . . . . . . . . 163A.1.1.32.1 General Considerations . . . . . . . . . . . . . . . . . . . . . 163A.1.1.32.2 TRPC Channels . . . . . . . . . . . . . . . . . . . . . . . . . 164A.1.1.32.3 TRPM Channels . . . . . . . . . . . . . . . . . . . . . . . . 166A.1.1.32.4 TRPV Channels . . . . . . . . . . . . . . . . . . . . . . . . . 167A.1.1.32.5 TRPV1 Receptor Assay . . . . . . . . . . . . . . . . . . . . 169A.1.2 Studies in Isolated Organs . . . . . . . . . . . . . . . . . . . 170A.1.2.1 α-Sympatholytic Activity in Isolated Vascular Smooth Muscle 170A.1.2.2 α-Sympatholytic Activity in the Isolated Guinea Pig Seminal Vesicle . . . . . . . . . . . 171A.1.2.3 α-Sympatholytic Activity in the Isolated Vas Deferens of the Rat . . . . . . . . . . . . . 171A.1.2.4 α-Sympatholytic Activity in the Isolated Rat Spleen . . . . . . 173A.1.2.5 α-Sympatholytic Activity in the Isolated Rat Anococcygeus Muscle . . . . . . . . . . . 174A.1.2.6 β1 -Sympatholytic Activity in Isolated Guinea Pig Atria . . . . 175A.1.2.7 β2 -Sympatholytic Activity in the Isolated Tracheal Chain . . . 177A.1.2.8 Angiotensin Converting Enzyme Inhibition in the Isolated Guinea Pig Ileum . . . . . . . . . . . . . . . . 178A.1.2.9 Contractile and Relaxing Activity on Isolated Blood Vessels Including Effects of Potassium Channel Openers . . . . . . . 179A.1.2.10 Isolated Guinea Pig Ureter . . . . . . . . . . . . . . . . . . . 183A.1.2.11 Isolated Corpus Cavernosum . . . . . . . . . . . . . . . . . . 184
  • 13. XIV ContentsA.1.3 Cardiovascular Analysis in Vivo . . . . . . . . . . . . . . . . 187A.1.3.1 Hemodynamic Screening in Anesthetized Rats . . . . . . . . 187A.1.3.2 Blood Pressure in Pithed Rats . . . . . . . . . . . . . . . . . 189A.1.3.3 Antihypertensive Vasodilator Activity in Ganglion-Blocked, Angiotensin II Supported Rats . . . . . 191A.1.3.4 Blood Pressure in Conscious Hypertensive Rats (Tail Cuff Method) . . . . . . . . . . . . . . . . . . . . . . . 192A.1.3.5 Direct Measurement of Blood Pressure in Conscious Rats with Indwelling Catheter . . . . . . . . . . . . . . . . . . . . 193A.1.3.6 Cannulation Techniques in Rodents . . . . . . . . . . . . . . 195A.1.3.6.1 Permanent Cannulation of the Jugular Vein in Rats . . . . . . 195A.1.3.6.2 Permanent Cannulation of the Renal Vein in Rats . . . . . . . 196A.1.3.6.3 Permanent Cannulation of the Portal Vein in Rats . . . . . . . 197A.1.3.6.4 Permanent Cannulation of the Thoracic Duct in Rats . . . . . 197A.1.3.6.5 Portacaval Anastomosis in Rats . . . . . . . . . . . . . . . . 198A.1.3.7 Cardiovascular Analysis in Anesthetized Mice . . . . . . . . . 199A.1.3.8 Blood Pressure in Anesthetized Cats . . . . . . . . . . . . . . 200A.1.3.9 Cardiovascular Drug Challenging Experiments in Anesthetized Dogs . . . . . . . . . . . . . . . . . . . . . . 201A.1.3.10 Hemodynamic Analysis in Anaesthetized Dogs . . . . . . . . 202A.1.3.11 Hemodynamic Measurements in Conscious Dogs . . . . . . . 204A.1.3.12 Hemodynamic Studies in Monkeys . . . . . . . . . . . . . . . 206A.1.3.13 Measurement of Cardiac Output and Regional Blood Flow with Microspheres . . . . . . . . . . . . . . . . . . . . . . . 206A.1.3.14 Carotid Artery Loop Technique . . . . . . . . . . . . . . . . 207A.1.3.15 Measurement of Heart Dimensions in Anesthetized Dogs . . . 209A.1.3.16 Telemetric Monitoring of Cardiovascular Parameters in Rats . 210A.1.3.17 Cardiovascular Effects After Intracerebroventricular Administration . . . . . . . . . 212A.1.3.18 Influence on Orthostatic Hypotension . . . . . . . . . . . . . 213A.1.3.19 Bezold-Jarisch Reflex . . . . . . . . . . . . . . . . . . . . . . 214A.1.3.20 Endotoxin Induced Shock . . . . . . . . . . . . . . . . . . . 216A.1.3.21 Hemorrhagic Shock . . . . . . . . . . . . . . . . . . . . . . . 218A.1.3.22 Tourniquet Shock . . . . . . . . . . . . . . . . . . . . . . . . 219A.1.3.23 Heat Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . 220A.1.3.24 α- and β-Adrenoreceptors in the Mouse Iris . . . . . . . . . . 221A.1.3.25 α2 -Adrenoreceptor Blockade Measured in Vivo by Clonidine-Induced Sleep in Chicks . . . . . . . . . 223A.1.3.26 Activity at β 1 - and β 2 -Adrenoreceptors in the Rat . . . . . . . 223A.1.3.27 β 1 - and β 2 -Sympatholytic Activity in Dogs . . . . . . . . . . 224A.1.3.28 Intrinsic β-Sympathomimetic Activity in Reserpine-Pretreated Dogs . . . . . . . . . . . . . . . . . . 225A.1.3.29 Cat Nictitating Membrane Preparation (Ganglion Blocking Activity) . . . . . . . . . . . . . . . . . . 226A.1.3.30 Assessment of Ganglion-Blocking Activity in the Isolated Bovine Retractor Penis Muscle . . . . . . . . . 228A.1.3.31 Angiotensin II Antagonism . . . . . . . . . . . . . . . . . . . 229A.1.3.32 ACE Inhibition Measured in Vivo in the Rat . . . . . . . . . . 233A.1.3.33 Evaluation of Renin Inhibitors in Dogs . . . . . . . . . . . . . 234A.1.3.34 Evaluation of Renin Inhibitors in Monkeys . . . . . . . . . . 235A.1.3.35 Penile Erection in Rabbits . . . . . . . . . . . . . . . . . . . 237A.1.4 Cardiovascular Safety Studies . . . . . . . . . . . . . . . . . 238
  • 14. Contents XVA.2 Methods to Induce Experimental Hypertension . . . . . . 239A.2.0.1 Acute Renal Hypertension in Rats . . . . . . . . . . . . . . . 239A.2.0.2 Chronic Renal Hypertension in Rats . . . . . . . . . . . . . . 239A.2.0.3 Chronic Renal Hypertension in Dogs . . . . . . . . . . . . . . 240A.2.0.4 Neurogenic Hypertension in Dogs . . . . . . . . . . . . . . . 241A.2.0.5 DOCA-salt Induced Hypertension in Rats . . . . . . . . . . . 242A.2.0.6 Fructose Induced Hypertension in Rats . . . . . . . . . . . . 243A.2.0.7 Genetic Hypertension in Rats . . . . . . . . . . . . . . . . . . 244A.2.0.8 Hypertension Induced by Chronic NO-Synthase Inhibition . . 246A.2.0.9 Pulmonary Hypertension Induced by Monocrotaline . . . . . 247A.2.0.10 Portal Hypertension in Rats . . . . . . . . . . . . . . . . . . . 249A.3 Coronary (Cardiac) Drugs . . . . . . . . . . . . . . . . . . 251A.3.1 Isolated Organs . . . . . . . . . . . . . . . . . . . . . . . . . 251A.3.1.1 Heart-Lung Preparation . . . . . . . . . . . . . . . . . . . . . 251A.3.1.2 Isolated Heart According to Langendorff . . . . . . . . . . . 253A.3.1.3 Coronary Artery Ligation in Isolated Working Rat Heart . . . 259A.3.1.4 Isolated Working Heart Model in Infarcted Rat Heart . . . . . 261A.3.1.5 Relaxation of Bovine Coronary Artery . . . . . . . . . . . . . 262A.3.2 In Vivo Methods . . . . . . . . . . . . . . . . . . . . . . . . 263A.3.2.1 Isoproterenol Induced Myocardial Necrosis in Rats . . . . . . 263A.3.2.2 Myocardial Infarction After Coronary Ligation in Rodents . . 264A.3.2.3 Occlusion of Coronary Artery in Anesthetized Dogs and Pigs . 269A.3.2.4 Acute Ischemia by Injection of Microspheres in Dogs . . . . . 271A.3.2.5 Influence on Myocardial Preconditioning . . . . . . . . . . . 273A.3.2.6 MRI Studies of Cardiac Function . . . . . . . . . . . . . . . . 276A.3.2.7 MRI Studies After Heart and Lung Transplantation . . . . . . 277A.3.3 Ex Vivo Methods . . . . . . . . . . . . . . . . . . . . . . . . 279A.3.3.1 Plastic Casts from Coronary Vasculature Bed . . . . . . . . . 279A.4 Calcium Uptake Inhibiting Activity . . . . . . . . . . . . . 280A.4.0.1 General Considerations . . . . . . . . . . . . . . . . . . . . . 280A.4.1 In Vitro Methods . . . . . . . . . . . . . . . . . . . . . . . . 282A.4.1.1 3 H-Nitrendipine Binding in Vitro . . . . . . . . . . . . . . . . 282A.4.2 Calcium Antagonism in Isolated Organs . . . . . . . . . . . . 286A.4.2.1 Calcium Antagonism on Action Potential of Isolated Guinea Pig Papillary Muscle . . . . . . . . . . . . 286A.4.2.2 Calcium Antagonism in the Isolated Guinea Pig Atrium . . . . 286A.4.2.3 Calcium Antagonism in the Isolated Rabbit Aorta . . . . . . . 287A.4.2.4 Calcium Antagonism in the Isolated Guinea Pig Pulmonary Artery . . . . . . . . . 288A.4.3 In Vivo Methods . . . . . . . . . . . . . . . . . . . . . . . . 289A.4.3.1 Evaluation of Calcium Blockers in the Pithed Rat . . . . . . . 289A.5 Anti-Arrhythmic Activity . . . . . . . . . . . . . . . . . . . 290A.5.0.1 General Considerations . . . . . . . . . . . . . . . . . . . . . 290A.5.0.2 Electrocardiography in Animals . . . . . . . . . . . . . . . . 292A.5.0.3 Aconitine Antagonism in Rats . . . . . . . . . . . . . . . . . 293A.5.0.4 Digoxin-Induced Ventricular Arrhythmias in Anesthetized Guinea Pigs . . . . . . . . . . . . . . . . . . 295A.5.0.5 Strophanthin or Ouabain Induced Arrhythmia . . . . . . . . . 296A.5.0.6 Ventricular Fibrillation Electrical Threshold . . . . . . . . . . 297A.5.0.7 Coronary Artery Ligation, Reperfusion Arrhythmia and Infarct Size in Rats . . . . . . . . . . . . . . . . . . . . . 298
  • 15. XVI ContentsA.5.0.8 Ventricular Arrhythmia After Coronary Occlusion . . . . . . . 301A.5.0.8.1 Ventricular Fibrillation After Coronary Occlusion and Reperfusion in Anesthetized Dogs . . . . . . . . . . . . . 301A.5.0.8.2 Harris Dog Model of Ventricular Tachycardia . . . . . . . . . 303A.5.0.8.3 Protection against Sudden Coronary Death . . . . . . . . . . 304A.5.0.8.4 Ventricular Fibrillation Induced by Cardial Ischemia During Exercise . . . . . . . . . . . . . . 306A.5.0.9 Experimental Atrial Fibrillation . . . . . . . . . . . . . . . . 308A.5.0.9.1 Atrial Fibrillation by Atrial Pacing in Dogs . . . . . . . . . . 308A.5.0.9.2 Atrial Fibrillation in Chronically Instrumented Goats . . . . . 309A.5.0.9.3 Influence on Ultrarapid Delayed Rectifier Potassium Current in Pigs . . . . . . . . . . . . . . . . . . . 311A.5.0.10 Characterization of Anti-Arrhythmic Activity in the Isolated Right Ventricular Guinea Pig Papillary Muscle 312A.5.0.11 Action Potential and Refractory Period in Isolated Left Ventricular Guinea Pig Papillary Muscle . . . 314A.6 Methods to Induce Cardiac Hypertrophy and Insufficiency 316A.6.0.1 Cardiac Hypertrophy and Insufficiency in Rats . . . . . . . . 316A.6.0.1.1 Aortic Banding in Rats . . . . . . . . . . . . . . . . . . . . . 316A.6.0.1.2 Chronic Heart Failure in Rats . . . . . . . . . . . . . . . . . . 318A.6.0.2 Cardiac Hypertrophy and Insufficiency in Mice . . . . . . . . 321A.6.0.2.1 Cardiac Hypertrophy in Mice . . . . . . . . . . . . . . . . . . 321A.6.0.2.2 Chronic Heart Failure in Mice . . . . . . . . . . . . . . . . . 322A.6.0.2.3 Transgenic Mice and Heart Failure . . . . . . . . . . . . . . . 323A.6.0.3 Cardiac Insufficiency in Guinea Pigs . . . . . . . . . . . . . . 324A.6.0.4 Cardiomyopathic Syrian Hamster . . . . . . . . . . . . . . . 325A.6.0.5 Cardiac Failure in Rabbits . . . . . . . . . . . . . . . . . . . 327A.6.0.6 Congestive Heart Failure in Dogs . . . . . . . . . . . . . . . 329A.6.0.7 Cardiac Failure in Pigs . . . . . . . . . . . . . . . . . . . . . 332A.6.0.8 Cardiac Failure in Sheep . . . . . . . . . . . . . . . . . . . . 335A.6.0.9 Cardiac Failure in Monkeys . . . . . . . . . . . . . . . . . . 337A.6.0.10 Cardiac Failure in Other Species . . . . . . . . . . . . . . . . 338A.6.0.11 Hypertrophy of Cultured Cardiac Cells . . . . . . . . . . . . 339A.7 Positive Inotropic Activity (Cardiac Glycosides) . . . . . . 340A.7.0.1 General Considerations . . . . . . . . . . . . . . . . . . . . . 340A.7.1 In Vitro Tests . . . . . . . . . . . . . . . . . . . . . . . . . . 341A.7.1.1 Ouabain Binding . . . . . . . . . . . . . . . . . . . . . . . . 341A.7.1.2 Influence on Na+ /K+ ATPase . . . . . . . . . . . . . . . . . . 341A.7.2 Tests in Isolated Tissues and Organs . . . . . . . . . . . . . . 343A.7.2.1 Isolated Cat Papillary Muscle . . . . . . . . . . . . . . . . . 343A.7.2.2 Isolated Hamster Cardiomyopathic Heart . . . . . . . . . . . 344A.7.2.3 Potassium Loss from the Isolated Guinea Pig Heart . . . . . . 345A.7.3 In Vivo Tests . . . . . . . . . . . . . . . . . . . . . . . . . . 345A.7.3.1 Cardiac Toxicity in Cats (Hatcher’s Method) . . . . . . . . . . 345A.7.3.2 Decay Rate and Enteral Absorption Rate of Cardiac Glycosides . . . . . . . . . . . . . . . . . . . . . 346A.8 Effects on Blood Supply and on Arterial and Venous Tonus 347A.8.1 Models for Stroke and Multi-Infarct Cerebral Dysfunction . . 347A.8.1.1 Cerebral Ischemia by Carotid Artery Occlusion in Mongolian Gerbils . . . . . . . . . . . . . . . . . . . . . . 347A.8.1.2 Forebrain Ischemia in Rats . . . . . . . . . . . . . . . . . . . 349A.8.1.3 Hypoxia Tolerance Test in Rats . . . . . . . . . . . . . . . . . 350
  • 16. Contents XVIIA.8.1.4 Middle Cerebral Artery Occlusion in Rats . . . . . . . . . . . 351A.8.1.5 Photochemically Induced Focal Cerebral Ischemia in Rats . . 353A.8.1.6 Microdialysis and Neuroprotection Experiments After Global Ischemia in Rats . . . . . . . . . . . . . . . . . 355A.8.1.7 Hypoxia/Hypoglycemia in Hippocampal Slices . . . . . . . . 356A.8.1.8 Measurement of Local Cerebral Blood Flow and Glucose Utilization in Rats . . . . . . . . . . . . . . . . . 356A.8.1.9 Cerebrovascular Resistance in Anesthetized Baboons . . . . . 358A.8.1.10 Effect on Cerebral Blood Flow in Cats (Fluvography) . . . . . 360A.8.1.11 Effect on Cerebral Blood Flow and in Ischemic Skeletal Muscle in Rats (Laser-Doppler-Effect) . . . . . . . . . . . . . 361A.8.1.12 Traumatic Brain Injury . . . . . . . . . . . . . . . . . . . . . 362A.8.1.13 Cerebral Blood Flow Measured by NMRI . . . . . . . . . . . 365A.8.2 Peripheral Blood Supply . . . . . . . . . . . . . . . . . . . . 367A.8.2.1 Perfused Hindquarter Preparation with Sympathetic Nerve Stimulation in Rats . . . . . . . . . . 367A.8.2.2 Effect on Peripheral Blood Flow in Rats . . . . . . . . . . . . 369A.8.2.3 Effect on Peripheral Blood Flow in Anesthetized Dogs . . . . 370A.8.2.4 Effect on Peripheral Blood Supply Measured by Local Oxygen Pressure . . . . . . . . . . . . . . . . . . . 372A.8.2.5 Effect on Mesenteric Blood Flow in Rats . . . . . . . . . . . 372A.8.2.6 Effect on Pulmonary Blood Flow . . . . . . . . . . . . . . . . 374A.8.2.7 Effect on Contractile Force of Ischemic Muscle . . . . . . . . 375A.8.2.8 Effect on Perfusion of Rabbit Ear (Pissemski Method) . . . . 376A.8.2.9 Effect on Venous Tonus In Situ in Dogs . . . . . . . . . . . . 378A.8.3 Arterial Aneurysms . . . . . . . . . . . . . . . . . . . . . . . 379A.8.3.1 General Considerations . . . . . . . . . . . . . . . . . . . . . 379A.8.3.2 Angiotensin II-Induced Aortic Aneurysm in Mice . . . . . . . 380A.8.4 Angiogenesis and Anti-Angiogenesis . . . . . . . . . . . . . 382A.8.4.1 General Considerations . . . . . . . . . . . . . . . . . . . . . 382A.8.4.2 Endothelial Cell Proliferation . . . . . . . . . . . . . . . . . . 383A.8.4.3 Chorioallantoic Membrane Assay . . . . . . . . . . . . . . . 384A.8.4.4 Cornea Neovascularization . . . . . . . . . . . . . . . . . . . 385A.8.4.5 Rat Subcutaneous Air Sac Model . . . . . . . . . . . . . . . . 387A.8.4.6 Mesenteric Window Angiogenesis Model . . . . . . . . . . . 388A.8.4.7 Quantification of Vascular Endothelial Growth Factor-C . . . 388A.8.4.8 Inhibitors of Vascular Endothelial Growth Factor . . . . . . . 389Chapter BPharmacological Assays in Thrombosis and Haemostasis . . . . . . . . . . 393B.1 General Introduction . . . . . . . . . . . . . . . . . . . . . 394B.2 In Vitro Tests . . . . . . . . . . . . . . . . . . . . . . . . . . 394B.2.1 Blood Coagulation Tests . . . . . . . . . . . . . . . . . . . . 394B.2.2 Thrombelastography . . . . . . . . . . . . . . . . . . . . . . 395B.2.3 Chandler Loop . . . . . . . . . . . . . . . . . . . . . . . . . 396B.2.4 Platelet Aggregation and Deaggregation in Platelet-Rich Plasma or Washed Platelets (Born Method) . . 397B.2.5 Platelet Aggregation After Gel Filtration (Gel-Filtered Platelets, GFP) . . . . . . . . . . . . . . . . . . 400B.2.6 Platelet Aggregation in Whole Blood . . . . . . . . . . . . . 401
  • 17. XVIII ContentsB.2.7 Platelet Micro- and Macro- Aggregation Using Laser Scattering . . . . . . . . . . . . . . . . . . . . . 402B.2.8 Fibrinogen Receptor Binding . . . . . . . . . . . . . . . . . . 403B.2.9 Euglobulin Clot Lysis Time . . . . . . . . . . . . . . . . . . . 405B.2.10 Flow Behavior of Erythrocytes . . . . . . . . . . . . . . . . . 405B.2.11 Filterability of Erythrocytes . . . . . . . . . . . . . . . . . . 406B.2.12 Erythrocyte Aggregation . . . . . . . . . . . . . . . . . . . . 407B.2.13 Determination of Plasma Viscosity . . . . . . . . . . . . . . . 408B.3 In Vitro Models of Thrombosis . . . . . . . . . . . . . . . . 408B.3.1 Cone-and-Plate Viscometry Under Shear-Flow Cytometry . . 410B.3.2 Platelet Adhesion and Aggregation Under Dynamic Shear . . 412B.3.3 Cell Adhesion to Immobilized Platelets: Parallel-Plate Flow Chamber . . . . . . . . . . . . . . . . . . 413B.4 In Vivo or Ex Vivo Models . . . . . . . . . . . . . . . . . . 415B.4.1 Stenosis- and Mechanical Injury-Induced Coronary Thrombosis: Folts Model . . . . . . . . . . . . . . 417B.4.2 Stenosis- and Mechanical Injury- Induced Arterial and Venous Thrombosis: Harbauer-Model . . . . . . . . . . . 421B.4.3 Electrical-Induced Thrombosis . . . . . . . . . . . . . . . . . 423B.4.4 FeCl3 -Induced Thrombosis . . . . . . . . . . . . . . . . . . . 424B.4.5 Thrombin-Induced Clot Formation in Canine Coronary Artery 425B.4.6 Laser-Induced Thrombosis . . . . . . . . . . . . . . . . . . . 426B.4.7 Photochemical-Induced Thrombosis . . . . . . . . . . . . . . 427B.4.8 Foreign-Surface-Induced Thrombosis . . . . . . . . . . . . . 428B.4.8.1 Wire Coil-Induced Thrombosis . . . . . . . . . . . . . . . . . 428B.4.8.2 Eversion Graft-Induced Thrombosis . . . . . . . . . . . . . . 429B.4.8.3 Arteriovenous Shunt Thrombosis . . . . . . . . . . . . . . . . 430B.4.8.4 Thread-Induced Venous Thrombosis . . . . . . . . . . . . . . 431B.4.8.5 Thrombus Formation on Superfused Tendon . . . . . . . . . . 432B.4.9 Stasis-Induced Thrombosis (Wessler Model) . . . . . . . . . . 432B.4.10 Disseminated Intravascular Coagulation (DIC) Model . . . . . 434B.4.11 Microvascular Thrombosis in Trauma Models . . . . . . . . . 434B.4.12 Cardiopulmonary Bypass Models . . . . . . . . . . . . . . . 434B.4.13 Extracorporeal Thrombosis Models . . . . . . . . . . . . . . 435B.4.14 Experimental Thrombocytopenia or Leucocytopenia . . . . . 436B.4.15 Collagenase-Induced Thrombocytopenia . . . . . . . . . . . . 437B.4.16 Reversible Intravital Aggregation of Platelets . . . . . . . . . 437B.5 Bleeding Models . . . . . . . . . . . . . . . . . . . . . . . . 438B.5.1 Subaqueous Tail Bleeding Time in Rodents . . . . . . . . . . 438B.5.2 Arterial Bleeding Time in Mesentery . . . . . . . . . . . . . . 439B.5.3 Template Bleeding Time Method . . . . . . . . . . . . . . . . 439B.6 Genetic Models of Hemostasis and Thrombosis . . . . . . . 440B.6.1 Knock-Out Mice . . . . . . . . . . . . . . . . . . . . . . . . 443B.7 Critical Issues in Experimental Models . . . . . . . . . . . 451B.7.1 The Use of Positive Control . . . . . . . . . . . . . . . . . . 451B.7.2 Evaluation of Bleeding Tendency . . . . . . . . . . . . . . . 451B.7.3 Selection of Models Based on Species-Dependent Pharmacology/Physiology . . . . . . . 452B.7.4 Selection of Models Based on Pharmacokinetics . . . . . . . 453B.7.5 Clinical Relevance of Data Derived from Experimental Models . . . . . . . . . . . . . . . . . . . 453B.8 Safety Assays in Thrombosis and Haemostasis . . . . . . . 455
  • 18. Contents XIXChapter CActivity on Urinary Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457C.1 Diuretic and Saluretic Activity . . . . . . . . . . . . . . . . 458C.1.1 In Vitro Methods . . . . . . . . . . . . . . . . . . . . . . . . 458C.1.1.1 Carbonic Anhydrase Inhibition In Vitro . . . . . . . . . . . . 458C.1.2 In Vivo Methods . . . . . . . . . . . . . . . . . . . . . . . . 459C.1.2.1 Diuretic Activity in Rats (LIPSCHITZ Test) . . . . . . . . . . 459C.1.2.2 Saluretic Activity in Rats . . . . . . . . . . . . . . . . . . . . 461C.1.2.3 Diuretic and Saluretic Activity in Dogs . . . . . . . . . . . . 461C.2 Assessment of Renal Function . . . . . . . . . . . . . . . . 462C.2.1 In Vitro Methods . . . . . . . . . . . . . . . . . . . . . . . . 462C.2.1.1 Patch Clamp Technique in Kidney Cells . . . . . . . . . . . . 462C.2.1.2 Perfusion of Isolated Kidney Tubules . . . . . . . . . . . . . 463C.2.1.3 Isolated Perfused Kidney . . . . . . . . . . . . . . . . . . . . 466C.2.2 In Vivo Methods . . . . . . . . . . . . . . . . . . . . . . . . 467C.2.2.1 Clearance Methods . . . . . . . . . . . . . . . . . . . . . . . 467C.2.2.2 Assessment of Glomerular Filtration Rate (GFR) and Renal Blood Flow (RBF) . . . . . . . . . . . . . . . . . . 469C.2.2.2.1 Assessment of GFR by Plasma Chemistry . . . . . . . . . . . 469C.2.2.2.2 Assessment of RBF by Intra- vascular Doppler Flow Probes . 472C.2.2.2.3 Assessment of GFR and RBF by Scintigraphic Imaging . . . . 473C.2.2.3 Assessment of Renal Tubule Functions . . . . . . . . . . . . 474C.2.2.3.1 Urinalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474C.2.2.3.2 Electrolyte Excretion . . . . . . . . . . . . . . . . . . . . . . 477C.2.2.3.3 Fractional Excretion Methods . . . . . . . . . . . . . . . . . 477C.2.2.3.4 Quantitative Electrolyte Excretion . . . . . . . . . . . . . . . 479C.2.2.3.5 Assessment of Tubular Transport Processes using Transport Substrates and Inhibitors . . . . . . . . . . . 480C.2.2.4 Assessment of Renal Concentrating Ability . . . . . . . . . . 481C.2.2.4.1 Solute-Free Water Excretion and Reabsorption . . . . . . . . 481C.2.2.4.2 Assessment of Medullary Osmolarity and Blood Flow . . . . 482C.2.2.5 Micropuncture Techniques in the Rat . . . . . . . . . . . . . 483C.2.2.6 Stop-Flow Technique . . . . . . . . . . . . . . . . . . . . . . 485C.3 Impaired Renal Function . . . . . . . . . . . . . . . . . . . 485C.3.1 Chronic Renal Failure in the Rat . . . . . . . . . . . . . . . . 485C.3.2 Chronic Renal Failure After Subtotal (Five-Sixths) Nephrectomy in Rats . . . . . . . . . . . . . . . 487C.3.3 Experimental Nephritis . . . . . . . . . . . . . . . . . . . . . 490C.3.3.1 General Considerations . . . . . . . . . . . . . . . . . . . . . 490C.3.3.2 Nephrotoxic Serum Nephritis . . . . . . . . . . . . . . . . . . 490C.3.4 Experimental Nephrosis . . . . . . . . . . . . . . . . . . . . 493C.4 Uricosuric and Hypo-Uricemic Activity . . . . . . . . . . . 495C.4.1 In Vitro Methods . . . . . . . . . . . . . . . . . . . . . . . . 495C.4.1.1 Inhibition of Xanthine Oxidase In Vitro Indicating Hypouricemic Activity . . . . . . . . . . . . . . . 495C.4.1.2 Urate Uptake in Brush Border Membrane Vesicles . . . . . . 495C.4.2 In Vivo Methods . . . . . . . . . . . . . . . . . . . . . . . . 496C.4.2.1 Diuretic and Uricosuric Activity in Mice . . . . . . . . . . . . 496C.4.2.2 Hypouricemic Activity After Allantoxanamide Treatment in Rats . . . . . . . . . . . 497
  • 19. XX ContentsC.4.2.3 Hypouricemic and Uricosuric Activity After Potassium Oxonate Treatment in Rats . . . . . . . . . . . . . . . . . . . 497C.4.2.4 Phenol Red Excretion in Rats . . . . . . . . . . . . . . . . . . 498C.4.2.5 Uricosuric Activity in Dalmatian Dogs . . . . . . . . . . . . . 498C.4.2.6 Uricosuric Activity in Cebus Monkeys . . . . . . . . . . . . . 499C.5 Influence on Lower Urinary Tract . . . . . . . . . . . . . . 500C.5.1 In Vivo Studies . . . . . . . . . . . . . . . . . . . . . . . . . 500C.5.1.1 Micturition Studies . . . . . . . . . . . . . . . . . . . . . . . 500C.5.2 Studies in Isolated Organs . . . . . . . . . . . . . . . . . . . 502C.5.2.1 Studies on Renal Pelvis . . . . . . . . . . . . . . . . . . . . . 502C.5.2.2 Propagation of Impulses in the Guinea Pig Ureter . . . . . . . 504C.5.2.3 Studies on Urinary Bladder and Internal Urethral Sphincter . . 505C.5.2.4 Effects on Isolated Urethra . . . . . . . . . . . . . . . . . . . 508C.5.2.5 Effects on External Urethral Sphincter . . . . . . . . . . . . . 510Chapter DRespiratory Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511D.1 In Vitro Tests . . . . . . . . . . . . . . . . . . . . . . . . . . 511D.1.0.1 Histamine (H1 ) Receptor Binding . . . . . . . . . . . . . . . 511D.1.0.2 Muscarinic Receptor Binding . . . . . . . . . . . . . . . . . . 512D.2 Effects on Air Ways . . . . . . . . . . . . . . . . . . . . . . 514D.2.1 Tests in Isolated Organs . . . . . . . . . . . . . . . . . . . . 514D.2.1.1 Spasmolytic Activity in Isolated Guinea Pig Lung Strips . . . 514D.2.1.2 Spasmolytic Activity in Isolated Trachea . . . . . . . . . . . . 515D.2.1.3 Reactivity of the Isolated Perfused Trachea . . . . . . . . . . 518D.2.1.4 Bronchial Perfusion of Isolated Lung . . . . . . . . . . . . . 519D.2.1.5 Vascular and Airway Responses in the Isolated Lung . . . . . 520D.2.2 In Vivo Tests . . . . . . . . . . . . . . . . . . . . . . . . . . 522D.2.2.1 Bronchospasmolytic Activity in Anesthetized Guinea Pigs (Konzett–Rössler method) . . . . . . . . . . . . . . . . . . . 522D.2.2.2 Effect of Arachidonic Acid or PAF on Respiratory Function In Vivo . . . . . . . . . . . . . . . . 524D.2.2.3 Bronchial Hyperreactivity . . . . . . . . . . . . . . . . . . . 525D.2.2.4 Body Plethysmography and Res- piratory Parameters After Histamine- Induced Bronchoconstriction in Anesthetized Guinea Pigs . . . . . . . . . . . . . . . . . . 528D.2.2.5 Pneumotachography in Anesthetized Guinea Pigs . . . . . . . 530D.2.2.6 Airway Microvascular Leakage . . . . . . . . . . . . . . . . 532D.2.2.7 Isolated Larynx In Situ . . . . . . . . . . . . . . . . . . . . . 533D.2.2.8 Animal Models of Asthma . . . . . . . . . . . . . . . . . . . 534D.2.2.8.1 Treatment of Asthma . . . . . . . . . . . . . . . . . . . . . . 534D.2.2.8.2 Prevention of Allergic Asthma Reaction . . . . . . . . . . . . 539D.2.2.9 Bleomycin-Induced Pulmonary Fibrosis . . . . . . . . . . . . 541D.2.2.10 Influence of Cytokines on Lung Fibrosis . . . . . . . . . . . . 544D.2.2.11 Emphysema Models . . . . . . . . . . . . . . . . . . . . . . 547D.2.2.12 Models of Chronic Obstructive Pulmonary Disease (COPD) . 549D.3 Antitussive Activity . . . . . . . . . . . . . . . . . . . . . . 551D.3.0.1 Antitussive Activity After Irritant Inhalation in Guinea Pig . . 551D.3.0.2 Cough Induced by Mechanical Stimulation . . . . . . . . . . 553D.3.0.3 Cough Induced by Stimulation of the Nervus Laryngicus Superior . . . . . . . . . . . . . . . 554
  • 20. Contents XXID.4 Effects on Tracheal Cells and Bronchial Mucus Secretion and Transport . . . . . . . . . . . . . . . . . . . . . . . . . 555D.4.0.1 In Vitro Studies of Mucus Secretion . . . . . . . . . . . . . . 555D.4.0.2 Acute Studies of Mucus Secretion . . . . . . . . . . . . . . . 556D.4.0.3 Studies of Mucus Secretion With Chronic Cannulation . . . . 557D.4.0.4 Bronchoalveolar Lavage . . . . . . . . . . . . . . . . . . . . 558D.4.0.5 Ciliary Activity . . . . . . . . . . . . . . . . . . . . . . . . . 559D.4.0.6 Studies of Mucociliary Transport . . . . . . . . . . . . . . . . 561D.4.0.7 Culture of Tracheal Epithelial Cells . . . . . . . . . . . . . . 562D.4.0.8 Alveolar Macrophages . . . . . . . . . . . . . . . . . . . . . 563D.5 Safety Pharmacology of the Respiratory System . . . . . . 564Chapter EPsychotropic and Neurotropic Activity . . . . . . . . . . . . . . . . . . . . 565E.1 Effects on Behavior and Muscle Coordination . . . . . . . 569E.1.1 Spontaneous Behavior . . . . . . . . . . . . . . . . . . . . . 569E.1.1.1 General Considerations . . . . . . . . . . . . . . . . . . . . . 569E.1.1.2 Observational Assessment . . . . . . . . . . . . . . . . . . . 569E.1.1.3 Safety Pharmacology Core Battery . . . . . . . . . . . . . . . 571E.1.2 Effects on Motility (Sedative or Stimulatory Activity) . . . . . 571E.1.2.1 General Considerations . . . . . . . . . . . . . . . . . . . . . 571E.1.2.2 Method of Intermittent Observations . . . . . . . . . . . . . . 572E.1.2.3 Open Field Test . . . . . . . . . . . . . . . . . . . . . . . . . 573E.1.2.4 Hole-Board Test . . . . . . . . . . . . . . . . . . . . . . . . 576E.1.2.5 Combined Open Field Test . . . . . . . . . . . . . . . . . . . 576E.1.2.6 EEG Analysis From Rat Brain by Telemetry . . . . . . . . . . 577E.1.3 Tests fo