Short protocols in pharmacology and drug discovery: a compendium of methods from current protocols in pharmacology
Gespeichert in:
| Format: | Buch |
|---|---|
| Sprache: | English |
| Veröffentlicht: |
Hoboken, N.J.
Wiley
2007
|
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Beschreibung: | Includes bibliography and index Introduction to drug discovery and development -- Receptor binding -- Enzyme assays -- Isolated tissues -- Animal models of disease -- Pharmacokinetics -- Drug discovery technologies -- Safety pharmacology/toxicology |
| Beschreibung: | Getr. Zählung |
| ISBN: | 0470095261 9780470095263 |
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| 245 | 1 | 0 | |a Short protocols in pharmacology and drug discovery |b a compendium of methods from current protocols in pharmacology |c editorial board, S. J. Enna (editor-in-chief) ; Michael Williams (editor-in-chief) ; John W. Ferkany ; Terry Kenakin ; Roger D. Porsolt |
| 264 | 1 | |a Hoboken, N.J. |b Wiley |c 2007 | |
| 300 | |a Getr. Zählung | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a Includes bibliography and index | ||
| 500 | |a Introduction to drug discovery and development -- Receptor binding -- Enzyme assays -- Isolated tissues -- Animal models of disease -- Pharmacokinetics -- Drug discovery technologies -- Safety pharmacology/toxicology | ||
| 650 | 0 | |a Pharmacology / Laboratory manuals | |
| 650 | 0 | |a Drugs / Design / Laboratory manuals | |
| 650 | 4 | |a Drug Design |v Laboratory Manuals | |
| 650 | 4 | |a Drugs |x Design |v Laboratory manuals | |
| 650 | 4 | |a Pharmacology |v Laboratory Manuals | |
| 650 | 4 | |a Pharmacology |v Laboratory manuals | |
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| 700 | 1 | |a Williams, Michael |e Sonstige |4 oth | |
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Datensatz im Suchindex
| _version_ | 1804136526024540160 |
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| adam_text | CONTENTS
Preface xv
Contributors xxi
1 Introduction to Drug Discovery and Development
1.1 Overview of Drug Discovery and Development 1 1
Pharmacology 1 2
Medicinal Chemistry 1 4
Drug Discovery 1 4
Drug Targets 1 6
Lead Discovery 1 10
Compound Sources 1 10
Biologicals, Antisense, and RNA Interference 1 13
Computational Chemistry 1 14
High Throughput Screening 1 14
Hit to Lead 1 15
Lead Optimization 1 16
Structure Activity Relationships (SARs) 1 16
Drug Metabolism and Pharmacokinetics 1 18
Preclinical Development 1 21
2 Receptor Characterization
2.1 Receptors as Drug Targets 2 1
Receptor Classification and Nomenclature 2 3
Receptor Structure and Motifs 2 3
Receptor Ligands 2 7
Constitutively Active Receptors 2 8
Ligand Receptor Interactions 2 9
Orphan Receptors 2 12
Neurotransmitters, Neurohormones, and Neuromodulators 2 13
Allosteric Ligands 2 14
Human Recombinant Receptors 2 14
Receptor Mutations and Chimeras 2 16
Assessing Receptor Function 2 17
2.2 Receptor Theory and Function 2 20
Origins of Receptor Theory 2 20
Occupancy Theory 2 20
Two State Theory 2 31
Ternary Complex Model of Receptors 2 36
Summary 2 49
23 Practical Aspects of Radioligand Binding 2 49
Fundamentals of Radioligand Binding Assays 2 51
Guidelines for Establishing a Radioligand Binding Assay 2 55
Analysis of Binding Data 2 64
Troubleshooting 2 85
2.4 Measurement of Allosteric Interactions at G Protein Coupled Receptors
Using Radioligand Binding 2 85
Overview of Receptor Allosterism 2 85
Protocols for the Detection of Allosteric Interactions Using Radioligand
Binding 2 94
Basic Protocol 1: Measurement of Allosteric Modulation of Radioligand
Binding Using Saturation Experiments 2 94
Contents Short Protocols in Pharmacology and Drug Discovery
iii
Basic Protocol 2: Measurement of Allosteric Modulation of Radioligand
Binding: Inhibition or Potentiation Experiments 2 97
Basic Protocol 3: Measurement of Allosteric Modulation of Radioligand
Binding to Cloned Receptors in Membranes: Dissociation Kinetic
Studies Using Isotopic Dilution 2 99
Support Protocol 1: Data Analysis of Saturation Binding Experiments 2 101
Support Protocol 2: Data Analysis of Equilibrium Inhibition (or
Potentiation) Experiments 2 103
Support Protocol 3: Data Analysis of Inhibition (or Potentiation)
Experiments Under Nonequilibrium Conditions 2 106
Support Protocol 4: Data Analysis of Dissociation Kinetic Experiments 2 108
Extensions of the Allosteric Model 2 110
2.5 Characterization of Opioid Receptor Binding and Function 2 110
Basic Protocol 1: Measurement of Opiod Receptor Binding to Cloned
Receptors in Membranes 2 110
Alternate Protocol 1: Measurement of Opioid Receptor Binding in Tissue
Membrane Homogenates 2 113
Basic Protocol 2: Measurement of Binding to Cloned Nociceptin/Orphanin FQ
(ORL1) Receptors in Membranes 2 114
Alternate Protocol 2: Flashplate Scintillation Proximity Assay to Measure
ORL1 Receptor Binding 2 115
Basic Protocol 3: Testing Inhibitors of Opioid Receptor Ligand Binding 2 115
Functional Assays 2 116
Basic Protocol 4: Measurement of Opiod Receptor Mediated [ SJGTP^S
Binding Using Basic Flashplates 2 118
Alternate Protocol 3: Measurement of Opioid Receptor Mediated [35S]GTP7S
Binding Using Wheat Germ Agglutinin (WGA) Coated Scintillation
Proximity Assay (SPA) Beads 2 120
Alternate Protocol 4: Measurement of Opioid Receptor Mediated [35S]GTP yS
Binding Using Filtration 2 120
Support Protocol 1: Saponin Enhancement of Opioid Receptor Mediated
[35S]GTP yS Binding 2 121
Basic Protocol 5: Testing Compounds for Functional Activity at Opioid
Receptors Using [35 S]GTP y S Binding 2 122
Support Protocol 2: Data Analysis 2 123
2.6 Characterization of GABAA Receptor Binding 2 133
Strategic Planning 2 133
Basic Protocol: Benzodiazepine Binding in Brain Membranes 2 135
Support Protocol: Preparation of Membranes 2 137
Alternate Protocol 1: Benzodiazepine Binding to All Diazepam Sensitive
GABAA Receptors 2 137
Alternate Protocol 2: Benzodiazepine Binding to Diazepam Sensitive GABAA
Receptor Suptype Containing the al Subunit 2 138
Alternate Protocol 3: Benzodiazepine Binding to Diazepam Sensitive GABAA
Receptor Subtypes Containing the a5 Subunit 2 138
Alternate Protocol 4: Benzodiazepine Binding to All Diazepam Sensitive and
Diazepam Insensitive G AB AA Receptors 2 138
Alternate Protocol 5: Benzodiazepine Binding to All Diazepam Insensitive
GABAA Receptors 2 139
2.7 GABA Transporters Assays 2 144
Basic Protocol: Measurement of GABA Uptake in Transiently Transfected Cell
Lines 2 144
Alternate Protocol: Batch Transfection of Cell Lines for GABA Uptake Assay 2 150
3 Enzyme Assays
3.1 Overview of Signal Transduction 3 2
Protein Functional Modules 3 3
Intracellular Signaling Proteins 3 6
Short Protocols in Pharmacology and Drug Discovery Contents
3.2 Adenylyl and Guanylyl Cyclase Assays 3 22
Basic Protocol 1: Measurement of Adenylyl Cyclase Activity In Vitro 3 22
Support Protocol 1: Preparation and Calibration of Dowex Columns 3 24
Support Protocol 2: Preparation and Calibration of Alumina Columns 3 25
Support Protocol 3: Tissue Preparation 3 26
Support Protocol 4: Cell Preparation 3 26
Basic Protocol 2: Measurement of Guanylyl Cyclase Activity In Vitro 3 27
3.3 Assays for Cyclic Nucleotide Specific Phosphodiesterases (Families 1 to 5) 3 30
Basic Protocol 1: Measurement of PDE1 Activity In Vitro Using Radiolabeled
Substrate and Alumina Acid Columns 3 31
Support Protocol 1: Production of PDE Enzyme in 5/9 Cells 3 34
Support Protocol 2: Production of PDE Enzyme in Transfected Cells 3 34
Support Protocol 3: Titration of PDE 1 Enzyme to Determine Enzyme Working
Concentration 3 34
Support Protocol 4: Determination of KM of PDE1 3 35
Alternate Protocol 1: PDE2 Enzyme Inhibition Assay Using Radiolabeled
Substrate and Alumina Acid Column 3 36
Alternate Protocol 2: PDE3 Enzyme Inhibition Assay Using Radiolabeled
Substrate and Alumina Acid Columns 3 36
Alternate Protocol 3: PDE4 Enzyme Inhibition Assay Using Radiolabeled
Substrate and Alumina Acid Columns 3 36
Alternate Protocol 4: PDE5 Enzyme Inhibition Assay Using Radiolabeled
Substrate And Alumina Acid Columns 3 36
Basic Protocol 2: Measurement of PDE1 Inhibition In Vitro Using Fluorescence
Methods 3 37
3.4 Phosphoinositide Turnover 3 39
Phosphoinositide Signaling Pathways 3 39
Experimental Strategies 3 42
Assays of Receptor Stimulated Phosphoinositide Turnover 3 44
Basic Protocol 1: Incorporation of [3H]Inositol into Phospholipids in
Tissue Slices and Measurement of [3H]Inositol Phosphates 3 44
Alternate Protocol 1: Separation of Individual [3H]Inositol
Polyphosphates 3 45
Basic Protocol 2: [3H]Mvo Inositol Prelabeling Assay in Cultured Cells 3 46
Support Protocol 1: Purification of [3H]Afyo Inositol 3 47
Support Protocol 2: Preparation of Chloride Form Dowex AG1X8
Columns 3 47
Support Protocol 3: Preparation of Formate Form Dowex AG1X8 Columns 3 48
Basic Protocol 3: Estimation of Inositol (1,4,5) Trisphosphate in Tissue
Slices by Mass Assay 3 48
Support Protocol 4: Preparation of Tissue Slices 3 49
Alternate Protocol 2: Inositol (1,5,5) Trisphosphate Mass Assay in
Cultured Cells 3 50
Support Protocol 5: Preparation of Inositol Trisphosphate Binding Protein 3 50
3.5 Nitric Oxide Synthase Assays 3 51
Basic Protocol: Arginine to Citrulline Conversion Assay for Measuring NOS
Activity 3 52
Support Protocol 1: Preparation of NOS Containing Tissue Extracts 3 54
Support Protocol 2: One Step Partial Purification of NOS 3 55
Support Protocol 3: Preparation of NOS From Cultured Cells 3 55
Support Protocol 4: Purification of [3H]Arginine by Thin Layer
Chromatography 3 56
3.6 HIV Protease Assays 3 57
Basic Protocol: Fluorometric Screening to Identify Inhibitors of HIV Protease 3 57
Support Protocol 1: Assay of HIV Protease to Determine Working Enzyme
Concentration 3 60
Support Protocol 2: Data Analysis 3 61
Contents Short Protocols in Pharmacology and Drug Discovery
v
3.7 Topoisomerase Assays 3 62
Basic Protocol 1: Assay of Topoisomerase I Activity 3 63
Basic Protocol 2: Assay of Topoisomerase II Activity 3 64
Support Protocol: Preparation of Mammalian Cell Extracts for Assaying
Topoisomerase Activity 3 64
Basic Protocol 3: In Vivo Determination of Topoisomerase Covalent
Complexes Using the K+/SDS Assay 3 65
Basic Protocol 4: Determination of DNA Cleavage by Purified Topoisomerase I 3 67
Basic Protocol 5: In Vitro Quantitative Determination of Topoisomerase II
Cleavage Using the K+/SDS Assay 3 69
Alternate Protocol: Gel Electrophoresis Determination of Topoisomerase II
Cleavage 3 70
3.8 Cytochrome P450 Assays 3 71
Basic Protocol 1: Evaluation of a Test Compound as an Inhibitor of CYP1A2
Activity Using Phenacetin 3 74
Basic Protocol 2: Evaluation of a Test Compound as an Inhibitor of CYP2A6
Activity Using Coumarin 3 76
Basic Protocol 3: Evaluation of a Test Compound as an Inhibitor of CYP2B6
and CYP2C19 Activities Using S Mephenytoin 3 78
Basic Protocol 4: Evaluation of a Test Compound as an Inhibitor of CYP2C8
Activity Using Paclitaxel 3 79
Basic Protocol 5: Evaluation of a Test Compound as an Inhibitor of CYP2C9
Activity Using Tolbutamide 3 80
Basic Protocol 6: Evaluation of a Test Compound as an Inhibitor of CYP2D6
Activity Using Dextromethorphan 3 82
Basic Protocol 7: Evaluation of a Test Compound as an Inhibitor of CYP2E1
Activity Using Chlorzoxazone 3 83
Basic Protocol 8: Evaluation of a Test Compound as an Inhibitor of CYP3A4
Activity Using Testosterone 3 85
Alternate Protocol 1: High Throughput Screening for CYP Inhibition Using
7 Benzyloxyquinoline 3 86
Alternate Protocol 2: High Throughput Screening for CYP Inhibition Using
Testosterone 3 87
Alternate Protocol 3: Evaluate a Test Compound as a Time Dependent
Inhibitor of CYP Activity 3 90
4 Isolated Tissues
4.1 Overview of Length Tension Relationships in Isolated Tissue 4 1
Isometric Contraction 4 3
Isotonic Recording 4 6
Auxotonic Recording 4 7
Conclusions 4 9
4.2 Isolated Cardiac Muscle Assays 4 9
Basic Protocol 4 9
4.3 Isolated Blood Vessel Assays 4 13
Basic Protocol 1: Ring Preparations of Blood Vessels 4 13
Alternate Protocol: Spiral Strip Preparations of Blood Vessels 4 14
Basic Protocol 2: Perfused Preparations of Blood Vessels 4 16
4.4 a Adrenoceptor Assays 4 18
Basic Protocol 1: Evaluation of a,A Adrenoceptors in Isolated Vas Deferens 4 18
Support Protocol: Setup of Tissue Bath Apparatus for Adrenoceptor Studies 4 20
Alternate Protocol 1: Evaluation of aiA Adrenoceptors in Canine Prostate 4 20
Alternate Protocol 2: Evaluation of x1B Adrenoceptors in Rat Spleen 4 23
Alternate Protocol 3: Evaluation of ot1D Adrenoceptors in Isolated Rat Aorta 4 23
Alternate Protocol 4: Evaluation of a2A Adrenoceptors in Isolated Vas
Deferens by Electrical Stimulation 4 24
Basic Protocol 2: Evaluation of 012 Adrenoceptors by Superfusion of
Radiolabeled Isolated Tissues 4 26
Short Protocols in Pharmacology and Drug Discovery Contents
vi
4.5 B Adrenoceptor Assays 4 29
Basic Protocol 1: (Ji Adrenoceptors: Guinea Pig Left Atria 4 29
Alternate Protocol: Bi Adrenoceptors: Guinea Pig Right Atria 4 31
Basic Protocol 2: B2 Adrenoceptors: Guinea Pig Trachea 4 33
Basic Protocol 3: S2 Adrenoceptors: Rat Uterus 4 35
Basic Protocol 4: Measuring B Adrenoceptor Stimulated Lipolytic Activity 4 36
Support Protocol 1: Isolation and Culture of Primary Preadipocytes and
Adipocytes 4 39
Support Protocol 2: Measuring Adipocyte Differentiation by Nile Red
Staining or Triglyceride Accumulation 4 42
4.6 PI (Adenosine) Purinoceptor Assays 4 43
Basic Protocol: Measurement of Tissue Responsiveness to PI Purinoceptor
Agonists 4 43
Support Protocol 1: Preparation of Guinea Pig Left Atrium 4 46
Support Protocol 2: Preparation of Porcine Left Circumflex Coronary Artery 4 46
Support Protocol 3: Preparation of Guinea Pig Aorta 4 47
Support Protocol 4: Quantitation of Responses 4 47
4.7 Opioid Receptor Assays 4 48
Basic Protocol 1: Measurement of Opioid Activity in the Guinea Pig Ileum 4 48
Basic Protocol 2: Measurement of Opioid Activity in the Mouse Vas Deferens 4 56
4.8 Nicotinic Acetylcholine Receptor Assays 4 62
Basic Protocol 1: The Innervated Guinea Pig Tracheal/Esophageal Isolated
Tissue Preparation to Study Receptors on Autonomic Nerves 4 62
Alternate Protocol 1: Nicotinic Agonist Mediated Effects: Isolated Guinea Pig
Trachealis and Ileum 4 70
Basic Protocol 2: Nicotinic Receptors in Striated Muscle: The Isolated
Diaphragm/Phrenic Nerve Preparation 4 71
Alternate Protocol 2: Nicotinic Receptors in Striated Muscle: The Isolated,
Innervated Guinea Pig Esophagus 4 72
4.9 Muscarinic Receptor Assays 4 72
Basic Protocol 1: Muscarinic Mi Receptors: Canine Saphenous Vein 4 72
Basic Protocol 2: Muscarinic M2 Receptors: Guinea Pig Paced Left Atrium 4 74
Basic Protocol 3: Muscarinic M3 Receptors: Rat Urinary Bladder 4 76
Alternate Protocol 1: Muscarinic M3 Receptors: Guinea Pig Trachea 4 77
Alternate Protocol 2: Muscarinic M3 Receptors: Guinea Pig Ileum 4 78
Basic Protocol 4: Muscarinic M4 Receptors: Rabbit Anococcygeus 4 78
4.10 Histamine Receptor Assays 4 81
Basic Protocol 1: Histamine Hi Receptors: Guinea Pig Ileum 4 81
Basic Protocol 2: Histamine H2 Receptors: Guinea Pig Right Atrium 4 83
Basic Protocol 3: Histamine H? Receptors: Electrically Stimulated Guinea Pig
Ileum 4 85
4.11 Prostanoid Receptor Assays 4 87
Basic Protocol 1: Evaluation of Prostanoid DP Receptor Activity in Human
Washed Platelets 4 87
Basic Protocol 2: Evaluation of Prostanoid EPi Receptors in Guinea Pig
Isolated Trachea (GPT) 4 89
Basic Protocol 3: Evaluation of Prostanoid EP2 Receptor Activity in Isolated
Ring Preparations from the Rabbit Ear Artery 4 92
Basic Protocol 4: Evaluation of Prostanoid EP3 Receptors in Guinea Pig
Isolated Vas Deferens (GPVD) 4 94
Alternate Protocol 1: Evaluation of Prostanoid EP3 Receptors in Guinea Pig
Isolated Aorta (GPA) 4 96
Basic Protocol 5: Evaluation of Prostanoid EP4 Receptor Activity in Isolated
Ring Preparations from the Rabbit Saphenous Vein 4 97
Basic Protocol 6: Evaluation of Prostanoid FP Receptors in Mouse Uterus 4 100
Alternate Protocol 2: Evaluation of Prostanoid FP Receptors in Cat and Dog
Isolated Iris Sphincter (CIS/DIS) 4 102
Contents Short Protocols in Pharmacology and Drug Discovery
vii
Basic Protocol 7: Evaluation of Prostanoid IP Receptors in Human Isolated
Pulmonary Artery (HPA) 4 104
Alternate Protocol 3: Evaluation of Prostanoid IP Receptors in Rat Isolated
Vagus Nerve (RVN) 4 105
Basic Protocol 8: Evaluation of TP Receptor Activity in Human Washed
Platelets 4 107
Alternate Protocol 4: Evaluation of TP Receptor Activity in Isolated Ring
Preparations from the Rabbit Saphenous Vein 4 109
4.12 Cysteinyl Leukotriene Receptor Assays 4 112
Basic Protocol: Measurement of CysLT Induced Contractions in Guinea Pig
Trachea 4 113
Support Protocol: Storage and Spectroscopic Quantification of CysLT Solutions 4 119
4.13 5 Hydroxytryptamine Receptor Assays 4 121
Basic Protocol 1: Inhibition of the 5 HTiA Receptor Mediated Twitch
Response in Guinea Pig Transmurally Stimulated Ileum 4 121
Basic Protocol 2: 5 HTiB Receptor Mediated Contraction of Rat Caudal Artery 4 124
Basic Protocol 3: 5 HTiD Receptor Mediated Relaxation of Precontracted
Guinea Pig Jugular Vein 4 127
Alternate Protocol: 5 HTiD Receptor Mediated Contraction of Rabbit Cerebral
Arteries 4 129
Basic Protocol 4: 5 HT2A Receptor Mediated Contraction of Rabbit Thoracic
Aorta 4 131
Basic Protocol 5: 5 HT2B Receptor Mediated Contraction of the Rat Stomach
Fundus 4 132
Basic Protocol 6: 5 HT2C Receptor Mediated Contraction of the Rat Jugular
Vein 4 134
Basic Protocol 7: 5 HTj Receptor Mediated Contraction of Guinea Pig Heal
Smooth Muscle 4 135
Basic Protocol 8: 5 HT4 Receptor Mediated Relaxation of Rat Esophageal
Tunica Muscularis Mucosa 4 137
Basic Protocol 9: 5 HT7 Receptor Mediated Relaxation of the
Endothelium Denuded, Precontracted Rabbit Jugular Vein 4 139
5 Animal Models of Disease
5.1 Models of Cardiovascular Disease: Measurement of Arrhythmogenic/
Antiarrhythmic Activity in the Guinea Pig and Antihypertensive Activity in
the Conscious Rat 5 2
Basic Protocol 1: Measuring Digoxin Induced Arrhythmias in the Guinea Pig 5 2
Basic Protocol 2: Measuring Antihypertensive Activity in the Instrumented
Conscious Rat 5 3
5.2 Models of Pulmonary Disease: Acute and Chronic Allergic Asthma in the
Monkey and Acute and Chronic Viral Pulmonitis in the Mouse 5 5
Basic Protocol 1: Inhaled Allergen Induced Acute Responses in Primates 5 5
Support Protocol 1: Processing of Bronchoalveolar Lavage (BAL) Samples 5 8
Basic Protocol 2: Repeated Inhaled Allergen Induced Airway
Hyperresponsiveness and Eosinophilia in Primates as a Model of Chronic
Asthma 5 8
Basic Protocol 3: Lung Inflammation and Dysfunction Associated with
Respiratory Syncytial Virus (RSV) Infection in Mice 5 9
Support Protocol 2: Preparation of High Titer Respiratory Syncytial Virus
(RSV) Stocks 5 13
53 Models of Pulmonary Disease: Guinea Pig Models of Asthma 5 14
Strategic Planning 5 15
Basic Protocol 1: Measurements of Pulmonary Insufflation Pressure in
Anesthetized, Paralyzed, Mechanically Ventilated Guinea Pigs 5 16
Alternate Protocol 1: Measurement of Reflex Tracheal Contractions in
Anesthetized, Paralyzed, Mechanically Ventilated Guinea Pigs 5 21
Basic Protocol 2: Induction of Airways Hyperresponsiveness in Guinea Pigs
Through Active Sensitization and Allergen Challenge 5 24
Short Protocols in Pharmacology and Drug Discovery Contents
viii
Basic Protocol 3: Measurement of Cough Reflexes in Conscious Guinea Pigs 5 25
Alternate Protocol 2: Measurement of Cough Reflexes in Anesthetized Guinea
Pigs 5 26
Basic Protocol 4: Measurement of Inflammatory Cell Influx into the Airways by
Bronchoalveolar Lavage (BAL) 5 27
Basic Protocol 5: Quantification of Plasma Extravasation 5 28
5.4 Models of Inflammation: Carrageenan Induced Paw Edema in the Rat 5 29
Basic Protocol 5 29
5.5 Models of Inflammation: Adjuvant Induced Arthritis in the Rat 5 30
Basic Protocol 5 30
5.6 Models of Inflammation: Carrageenan Air Pouch in the Rat 5 32
Basic Protocol 5 32
5.7 Models of Pain: Hot Plate and Formalin Test in Rodents 5 33
Basic Protocol 1: Measurement of Acute Pain Using the Hot Plate Test 5 33
Basic Protocol 2: Measurement of Persistent Pain Using the Formalin Test 5 34
5.8 Models of Pain: Neuropathic Pain in the Rat 5 37
Basic Protocol 1: The Chronic Constriction Injury (CCI) Model of Neuropathic
Pain 5 37
Basic Protocol 2: The Partial Sciatic Ligation (PSL) Model of Neuropathic
Pain 5 38
Basic Protocol 3: The Spinal Nerve Ligation (SNL) Model of Neuropathic
Pain 5 39
Support Protocol 1: Behavioral Assays: Heat Hyperalgesia 5 39
Support Protocol 2: Behavior Assays: Mechano Hyperalgesia 5 40
Support Protocol 3: Behavioral Assays: Mechano Allodynia 5 41
Support Protocol 4: Behavioral Assays: Cold Allodynia 5 42
5.9 Models of Visceral Pain: Colorectal Distension (CRD) 5 42
Basic Protocol 1: EMG Electrode Implantation in the Rat 5 43
Alternate Protocol 1: EMG Electrode Implantation in the Mouse 5 44
Basic Protocol 2: Preparing Rats for CRD 5 45
Alternate Protocol 2: Preparing Mice for CRD 5 46
Basic Protocol 3: CRD Testing Paradigms 5 48
5.10 Models of Pain: Postoperative Pain 5 50
Basic Protocol 5 50
5.11 Models of Affective Illness: Forced Swimming and Tail Suspension Tests in
Rodents 5 53
Basic Protocol: Behavioral Despair Test in the Rat 5 53
Alternate Protocol 1: Forced Swimming Test in the Mouse 5 54
Alternate Protocol 2: Tail Suspension Test in Mice 5 55
5.12 Models of Affective Illness (Depression): Chronic Mild Stress 5 56
Basic Protocol 5 56
5.13 Models of Schizophrenia: Phencyclidine Disruption of Prepulse Inhibition
(PPI) of Startle in Rats 5 58
Strategic Planning: Experimental Materials 5 58
Basic Protocol: Prepulse Inhibition (PPI) of Startle in Rats 5 60
5.14 Models of Schizophrenia: Amphetamine Induced Hyperactivity and
Stereotypies in Mice 5 61
Basic Protocol 1: Antagonism of Amphetamine Induced Hyperactivity in Mice 5 61
Basic Protocol 2: Antagonism of Amphetamine Induced Stereotypies in Mice 5 63
5.15 Models of Anxiety Disorder: Stress Induced Hyperthermia (SIH) in
Group Housed Mice 5 64
Basic Protocol 5 64
5.16 Models of Anxiety Disorder: Ultrasonic Vocalizations of Isolated Rat Pups 5 69
Basic Protocol 5 69
Contents Short Protocols in Pharmacology and Drug Discovery
ix
5.17 Models of Urogenital Dysfunction: Benign Prostatic Hyperplasia (BPH) 5 74
Basic Protocol 1: Measurement of Prostatic Pressure and Blood Pressure in the
Anesthetized Dog 5 74
Alternate Protocol: Hypogastric Nerve Stimulation to Produce Rises in Prostate
Pressure in the Anesthetized Dog 5 76
Basic Protocol 2: Measurement of Prostatic Pressure and Blood Pressure in the
Conscious Dog 5 77
Support Protocol: Measurement of Postural Reflexes in Conscious Dogs 5 79
5.18 Models of Urogenital Dysfunction: Urinary Urge Incontinence (UUI) 5 81
Basic Protocol: Measurement of Pelvic Nerve Stimulated Bladder Contraction
and Cardiovascular Function in the Anesthetized Dog 5 81
Alternate Protocol: Measurement of Pelvic Nerve Stimulated Bladder
Contraction and Cardiovascular Function in the Anesthetized Rat 5 84
Support Protocol: Measurement of Parasympathetic Nerve Induced Salivary
Secretion from the Submandibular Gland of the Dog 5 85
5.19 Models of Urogenital Dysfunction: Volume Evoked Micturition Reflex
(VEMR) in the Conscious Rat 5 87
Basic Protocol 5 87
5.20 Models of Urogenital Dysfunction: Erectile Dysfunction 5 90
Basic Protocol 1: Direct Measurement of Intracavernous and Intra Arterial
Pressures in the Anesthetized Rat 5 90
Alternate Protocol: Direct Measurement of Intracavernous and Intra Arterial
Pressures in the Anesthetized Mouse During Erections 5 100
Basic Protocol 2: Direct Measurement of Intracavernous Pressure in the
Conscious and Freely Moving Rat During Erections 5 100
5.21 Models of Dementia: Delayed Alternation in Aged Rats 5 104
Basic Protocol: Acquisition of an Operant Delayed Alternation Task in Young
and Aged Rats 5 104
Alternate Protocol: Stabilized Performance of an Operant Delayed Alternation
Task in Young and Aged Rats 5 106
5.22 Models of Dementia: Spatial Discrimination Water Maze Test in
Septal Lesioned Rats 5 107
Basic Protocol 5 107
5.23 Models for Eating Disorders: Dietary Hyperphagia and Anorexia Nervosa 5 111
Strategic Planning 5 111
Basic Protocol 1: Induction of Dietary Hyperphagia 5 112
Basic Protocol 2: Induction of Anorexia Nervosa 5 115
5.24 Models of Epilepsy: Electroshock and Chemical Induced Convulsions in the
Mouse 5 118
Basic Protocol 1: Anticonvulsant Determination Using Electroshock Induced
Convulsions in the Mouse 5 118
Alternate Protocol 1: Anticonvulsant Determination by the Method of Limits
Using Electroshock Induced Convulsions in the Mouse 5 119
Basic Protocol 2: Proconvulsant/Anticonvulsant Determination Using
Pentylenetetrazol Induced Clonk and Tonic Convulsions in the Mouse 5 120
Alternate Protocol 2: Anticonvulsant Determination Using
Pentylenetetrazol Induced Clonic Convulsions in the Mouse 5 122
Basic Protocol 3: Anticonvulsant Determination Using a Timed Intravenous
Infusion of Pentylenetetrazol to Induce Convulsions in the Mouse 5 122
Basic Protocol 4: Anticonvulsant Determination Using Bicuculline, Picrotoxin,
or Strychnine Induced Convulsions in the Mouse 5 124
5.25 In Vivo Cancer Models: Three Mouse Models Used in Experimental
Therapeutics 5 126
Strategic Planning 5 126
Basic Protocol 1: Intravenously Implanted Murine P388 Leukemia 5 126
Basic Protocol 2: Subcutaneously Implanted Murine B16 Melanoma 5 128
Basic Protocol 3: Two Examples of Subcutaneously Implanted Human Tumor
Xenografts, LX 1 (Lung) and MX 1 (Breast) Tumors 5 130
Short Protocols in Pharmacology and Drug Discovery Contents
x
6 Pharmacokinetics
6.1 Overview of Pharmacokinetics 6 1
Basic Protocol 1: Compound Administration in Rats 6 2
Support Protocol 1: Methoxyflurane Anesthesia of the Rat 6 4
Basic Protocol 2: Blood Collection in Rats 6 5
Basic Protocol 3: Excreta Collection in Rats 6 7
Basic Protocol 4: Tissue Collection in Rats 6 8
Support Protocol 2: Sample Processing: Radiolabeled Compound 6 12
Support Protocol 3: Sample Processing: Nonradiolabeled Compounds 6 13
Support Protocol 4: Data Calculation 6 14
6.2 Estimating Intestinal Mucosal Permeation of Compounds Using Caco 2 Cell
Monolayers 6 15
Basic Protocol 1: Measurement of the Apparent Permeability Coefficient of a
Compound Across Caco 2 Cell Monolayers 6 16
Support Protocol 1: Testing Caco 2 Monolayer Integrity: Mannitol Flux
Experiment 6 17
Support Protocol 2: Growing Caco 2 Cell Monolayers on Filters 6 21
Support Protocol 3: Starting and Maintaining Caco 2 Cells in Culture 6 22
6.3 Determination of Compound Binding to Plasma Proteins 6 24
Basic Protocol 1: Determination of Plasma Protein Binding by Ultrafiltration 6 25
Basic Protocol 2: Determination of Plasma Protein Binding by Traditional
Equilibrium Dialysis 6 26
Alternate Protocol: Determination of Plasma Protein Binding by 96 Well Plate
Equilibrium Dialysis 6 30
6.4 Determination of the Stability of Drugs in Plasma 6 31
Basic Protocol 6 31
6.5 In Vitro Drug Metabolism Using Liver Microsomes 6 34
Basic Protocol: Measurement of Compound Metabolic Stability Using Liver
Microsomes 6 34
Alternate Protocol: Higher Throughput Screening for Metabolic Stability 6 35
Support Protocol 1: Preparation of Microsomes from Rat Liver 6 37
Support Protocol 2: Determination of Cytochrome P450 (CYP) Content in
Microsomes 6 38
6.6 Hepatic Clearance and Drug Metabolism Using Isolated Perfused Rat Liver 6 39
Basic Protocol: Analysis of Hepatic Clearance Using IPRL 6 39
Support Protocol 1: Viability Evaluation 6 43
Support Protocol 2: Measuring LDH Activity 6 44
7 Drug Discovery Technologies
7.1 Overview of Chemical Diversity 7 2
High Throughput Screening (HTS) 7 3
Natural Products and the Screening of Biodiversity 7 3
Modification of Existing Structures 7 6
Rationally Planned Approaches 7 7
Identification and Exploitation of Basic Pharmacophores 7 8
Combinatorial Chemistry 7 9
Evolutionary Chemistry 7 10
Orphan Ligands and Orphan Receptors 7 10
7.2 Overview on High Throughput Screening 7 22
High Throughput Screening 7 22
Precedents 7 23
Disease Targets 7 23
Compound Libraries 7 24
Contents Short Protocols in Pharmacology and Drug Discovery
xi
Combinatorial Chemistry 7 25
Natural Products 7 26
Compound Storage and Handling 7 27
Mother and Daughter Plates 7 28
Barcodes 7 29
Assay Formats 7 29
Assay Design 7 31
Classical Receptor and Enzyme Assays 7 32
Homogeneous Radioactive Bioassays 7 33
Fluorescence 7 34
Luminescence 7 37
Reporter Gene Assays 7 37
Melanophore Technology 7 38
Cell Based Assays for Ion Channel Targets 7 38
Assay Robustness 7 39
Robotic Versus Automated Workstations 7 39
Hit to Lead Process 7 41
Pharmacological and Pharmaceutical Profiling 7 41
Data Evaluation 7 43
Is It Worth the Effort? 7 43
Outlook and Further Directions 7 44
7.3 Testing for Inverse Agonism with Constitutive Receptor Systems 7 44
GPCR Constitutive Activity 7 44
The Influence of Ligands on Constitutive GPCR Systems 7 49
Optimal Conditions for Constitutively Active Systems 7 51
Advantages and Disadvantages of Constitutively Active GPCR Assay Systems 7 55
Conclusions 7 57
7.4 Overview On the Use of Therapeutic Antibodies in Drug Discovery 7 58
History of Therapeutic Antibodies 7 58
Basic Antibody Structure 7 58
Technologies for Generation of Therapeutic Human Antibodies 7 60
Design Opportunities for an Antibody Therapeutic 7 65
Immunogenicity 7 68
Expression Technologies 7 69
How Do Therapeutic Antibodies Compare with Traditional Drugs? 7 69
Summary 7 73
8 Safety Pharmacology/Toxicology
8.1 Overview of Safety Pharmacology 8 2
Terminology 8 2
Safety Pharmacology Versus Toxicology 8 3
Comparison of Japanese and ICH Guidelines 8 3
CNS Safety Pharmacology 8 4
Cardiovascular Safety Pharmacology 8 5
Respiratory Safety Pharmacology 8 6
Timing of Safety Pharmacology Studies and Good Laboratory Practice 8 6
Ethical and Animal Welfare Issues 8 7
Statistical Evaluation 8 7
Conclusions 8 8
8.2 Gastrointestinal Tract: Gastrointestinal Ulceration in the Rat 8 8
Basic Protocol 8 8
8.3 Gastrointestinal Tract: Intestinal Transit and Ulcerogenic Activity in the Rat 8 10
Basic Protocol 1: Measurement of Intestinal Transit in the Rat 8 10
Basic Protocol 2: Measurement of Ulcerogenic Activity in the Rat 8 11
Short Protocols in Pharmacology and Drug Discovery Contents
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8.4 Toxicology in the Drug Discovery and Development Process 8 12
New Chemical Entities (NCEs; Synthetic Organic Chemicals) 8 13
New Biological Entities (NBEs) 8 17
Models of Drug Effect 8 19
Incorporation of Toxicokinetics into the Toxicity Profile 8 23
Regulatory Guidelines for Toxicology Profiles 8 27
Special Issues for Biotechnology Derived NBEs 8 40
IND Safety Reports 8 45
Summary and Future Approaches 8 45
8.5 Abuse Liability: Place Preference Test in Rodents 8 47
Basic Protocol 1: Place Preference Test in Rats 8 47
Basic Protocol 2: Place Preference Test in Mice 8 49
8.6 Abuse Liability: Self Administration in Monkeys 8 51
Strategic Planning 8 52
Basic Protocol: Self Administration Training and Testing 8 55
Support Protocol 1: Catheter Implantation 8 57
Support Protocol 2: Determination of Catheter Patency Using the Methohexital
Test 8 58
8.7 Vigilance Controlled Quantified EEG in Safety Pharmacology 8 59
Strategic Planning 8 59
Basic Protocol 1: Electrode Implantation 8 63
Basic Protocol 2: Vigilance Controlled Quantified EEG in Implanted Rats 8 67
Support Protocol: Cable Construction 8 70
8.8 The Action Potential of the Purkinje Fiber: An In Vitro Model for Evaluation
of the Proarrhythmic Potential of Cardiac and Noncardiac Drugs 8 70
Basic Protocol: Measurement of Changes in the Action Potential Parameters in
the Purkinje Fiber During Superfusion with a Test Compound 8 71
Alternate Protocol 1: Evaluation of the Proarrhythmic Risk of a Test Compound
on Epinephrine Facilitated Early Afterdepolarizations 8 76
Alternate Protocol 2: Evaluation of the Proarrhythmic Risk of a Test Compound
on the Occurrence of Delayed Afterdepolarizations and/or Triggered
Activities 8 79
8.9 Respiratory Function Assessment in Safety Pharmacology 8 80
Functional Disorders of the Respiratory System 8 84
Techniques for Measuring Changes in Respiratory Function 8 85
Design of Respiratory Function Safety Studies 8 90
8.10 Behavior and Physiological Function: Primary Observation (Irwin) Test in
Rodents 8 92
Basic Protocol: Primary Observation (Irwin) Test in the Rat 8 92
Alternate Protocol: Primary Observation (Irwin) Test in the Mouse 8 95
Appendices
1 Reagents and Solutions A1 1
2 Nomenclature and Usage A2 1
2A Enzyme Nomenclature Web Sites A2 1
2B Receptor Nomenclature Web Sites A2 2
3 Standard Techniques A3 1
3A Assays for Total Protein A3 1
Basic Protocol 1: Hartree Lowry Assay for Quantitation of Total Protein A3 2
Basic Protocol 2: Bicinchoninic Acid (BCA) Assay for Quantitation of
Total Protein A3 3
Basic Protocol 3: Ultraviolet Absorption to Measure Total Protein A3 3
Basic Protocol 4: Coomassie Dye Binding Assay (Bradford Assay) to
Measure Total Protein A3 4
Basic Protocol 5: Trichloroacetic Acid Precipitation of Protein Samples A3 5
Contents Short Protocols in Pharmacology and Drug Discovery
xiii
3B Protein Analysis by SDS PAGE and Detection by Coomassie Blue or
Silver Staining A3 5
Electricity and Electrophoresis A3 6
Basic Protocol: Denaturing (SDS) Discontinuous Gel Electrophoresis:
Laemmli Gel Method A3 7
Staining Proteins in Gels A3 10
Support Protocol 1: Coomassie Blue Staining A3 10
Support Protocol 2: Silver Staining A3 11
4 Selected Suppliers of Reagents and Equipment A4 1
References 1
Index
Short Protocols in Pharmacology and Drug Discovery Contents
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CONTENTS
Preface xv
Contributors xxi
1 Introduction to Drug Discovery and Development
1.1 Overview of Drug Discovery and Development 1 1
Pharmacology 1 2
Medicinal Chemistry 1 4
Drug Discovery 1 4
Drug Targets 1 6
Lead Discovery 1 10
Compound Sources 1 10
Biologicals, Antisense, and RNA Interference 1 13
Computational Chemistry 1 14
High Throughput Screening 1 14
Hit to Lead 1 15
Lead Optimization 1 16
Structure Activity Relationships (SARs) 1 16
Drug Metabolism and Pharmacokinetics 1 18
Preclinical Development 1 21
2 Receptor Characterization
2.1 Receptors as Drug Targets 2 1
Receptor Classification and Nomenclature 2 3
Receptor Structure and Motifs 2 3
Receptor Ligands 2 7
Constitutively Active Receptors 2 8
Ligand Receptor Interactions 2 9
Orphan Receptors 2 12
Neurotransmitters, Neurohormones, and Neuromodulators 2 13
Allosteric Ligands 2 14
Human Recombinant Receptors 2 14
Receptor Mutations and Chimeras 2 16
Assessing Receptor Function 2 17
2.2 Receptor Theory and Function 2 20
Origins of Receptor Theory 2 20
Occupancy Theory 2 20
Two State Theory 2 31
Ternary Complex Model of Receptors 2 36
Summary 2 49
23 Practical Aspects of Radioligand Binding 2 49
Fundamentals of Radioligand Binding Assays 2 51
Guidelines for Establishing a Radioligand Binding Assay 2 55
Analysis of Binding Data 2 64
Troubleshooting 2 85
2.4 Measurement of Allosteric Interactions at G Protein Coupled Receptors
Using Radioligand Binding 2 85
Overview of Receptor Allosterism 2 85
Protocols for the Detection of Allosteric Interactions Using Radioligand
Binding 2 94
Basic Protocol 1: Measurement of Allosteric Modulation of Radioligand
Binding Using Saturation Experiments 2 94
Contents Short Protocols in Pharmacology and Drug Discovery
iii
Basic Protocol 2: Measurement of Allosteric Modulation of Radioligand
Binding: Inhibition or Potentiation Experiments 2 97
Basic Protocol 3: Measurement of Allosteric Modulation of Radioligand
Binding to Cloned Receptors in Membranes: Dissociation Kinetic
Studies Using Isotopic Dilution 2 99
Support Protocol 1: Data Analysis of Saturation Binding Experiments 2 101
Support Protocol 2: Data Analysis of Equilibrium Inhibition (or
Potentiation) Experiments 2 103
Support Protocol 3: Data Analysis of Inhibition (or Potentiation)
Experiments Under Nonequilibrium Conditions 2 106
Support Protocol 4: Data Analysis of Dissociation Kinetic Experiments 2 108
Extensions of the Allosteric Model 2 110
2.5 Characterization of Opioid Receptor Binding and Function 2 110
Basic Protocol 1: Measurement of Opiod Receptor Binding to Cloned
Receptors in Membranes 2 110
Alternate Protocol 1: Measurement of Opioid Receptor Binding in Tissue
Membrane Homogenates 2 113
Basic Protocol 2: Measurement of Binding to Cloned Nociceptin/Orphanin FQ
(ORL1) Receptors in Membranes 2 114
Alternate Protocol 2: Flashplate Scintillation Proximity Assay to Measure
ORL1 Receptor Binding 2 115
Basic Protocol 3: Testing Inhibitors of Opioid Receptor Ligand Binding 2 115
Functional Assays 2 116
Basic Protocol 4: Measurement of Opiod Receptor Mediated ["SJGTP^S
Binding Using Basic Flashplates 2 118
Alternate Protocol 3: Measurement of Opioid Receptor Mediated [35S]GTP7S
Binding Using Wheat Germ Agglutinin (WGA) Coated Scintillation
Proximity Assay (SPA) Beads 2 120
Alternate Protocol 4: Measurement of Opioid Receptor Mediated [35S]GTP yS
Binding Using Filtration 2 120
Support Protocol 1: Saponin Enhancement of Opioid Receptor Mediated
[35S]GTP yS Binding 2 121
Basic Protocol 5: Testing Compounds for Functional Activity at Opioid
Receptors Using [35 S]GTP y S Binding 2 122
Support Protocol 2: Data Analysis 2 123
2.6 Characterization of GABAA Receptor Binding 2 133
Strategic Planning 2 133
Basic Protocol: Benzodiazepine Binding in Brain Membranes 2 135
Support Protocol: Preparation of Membranes 2 137
Alternate Protocol 1: Benzodiazepine Binding to All Diazepam Sensitive
GABAA Receptors 2 137
Alternate Protocol 2: Benzodiazepine Binding to Diazepam Sensitive GABAA
Receptor Suptype Containing the al Subunit 2 138
Alternate Protocol 3: Benzodiazepine Binding to Diazepam Sensitive GABAA
Receptor Subtypes Containing the a5 Subunit 2 138
Alternate Protocol 4: Benzodiazepine Binding to All Diazepam Sensitive and
Diazepam Insensitive G AB AA Receptors 2 138
Alternate Protocol 5: Benzodiazepine Binding to All Diazepam Insensitive
GABAA Receptors 2 139
2.7 GABA Transporters Assays 2 144
Basic Protocol: Measurement of GABA Uptake in Transiently Transfected Cell
Lines 2 144
Alternate Protocol: Batch Transfection of Cell Lines for GABA Uptake Assay 2 150
3 Enzyme Assays
3.1 Overview of Signal Transduction 3 2
Protein Functional Modules 3 3
Intracellular Signaling Proteins 3 6
Short Protocols in Pharmacology and Drug Discovery Contents
3.2 Adenylyl and Guanylyl Cyclase Assays 3 22
Basic Protocol 1: Measurement of Adenylyl Cyclase Activity In Vitro 3 22
Support Protocol 1: Preparation and Calibration of Dowex Columns 3 24
Support Protocol 2: Preparation and Calibration of Alumina Columns 3 25
Support Protocol 3: Tissue Preparation 3 26
Support Protocol 4: Cell Preparation 3 26
Basic Protocol 2: Measurement of Guanylyl Cyclase Activity In Vitro 3 27
3.3 Assays for Cyclic Nucleotide Specific Phosphodiesterases (Families 1 to 5) 3 30
Basic Protocol 1: Measurement of PDE1 Activity In Vitro Using Radiolabeled
Substrate and Alumina Acid Columns 3 31
Support Protocol 1: Production of PDE Enzyme in 5/9 Cells 3 34
Support Protocol 2: Production of PDE Enzyme in Transfected Cells 3 34
Support Protocol 3: Titration of PDE 1 Enzyme to Determine Enzyme Working
Concentration 3 34
Support Protocol 4: Determination of KM of PDE1 3 35
Alternate Protocol 1: PDE2 Enzyme Inhibition Assay Using Radiolabeled
Substrate and Alumina Acid Column 3 36
Alternate Protocol 2: PDE3 Enzyme Inhibition Assay Using Radiolabeled
Substrate and Alumina Acid Columns 3 36
Alternate Protocol 3: PDE4 Enzyme Inhibition Assay Using Radiolabeled
Substrate and Alumina Acid Columns 3 36
Alternate Protocol 4: PDE5 Enzyme Inhibition Assay Using Radiolabeled
Substrate And Alumina Acid Columns 3 36
Basic Protocol 2: Measurement of PDE1 Inhibition In Vitro Using Fluorescence
Methods 3 37
3.4 Phosphoinositide Turnover 3 39
Phosphoinositide Signaling Pathways 3 39
Experimental Strategies 3 42
Assays of Receptor Stimulated Phosphoinositide Turnover 3 44
Basic Protocol 1: Incorporation of [3H]Inositol into Phospholipids in
Tissue Slices and Measurement of [3H]Inositol Phosphates 3 44
Alternate Protocol 1: Separation of Individual [3H]Inositol
Polyphosphates 3 45
Basic Protocol 2: [3H]Mvo Inositol Prelabeling Assay in Cultured Cells 3 46
Support Protocol 1: Purification of [3H]Afyo Inositol 3 47
Support Protocol 2: Preparation of Chloride Form Dowex AG1X8
Columns 3 47
Support Protocol 3: Preparation of Formate Form Dowex AG1X8 Columns 3 48
Basic Protocol 3: Estimation of Inositol (1,4,5) Trisphosphate in Tissue
Slices by Mass Assay 3 48
Support Protocol 4: Preparation of Tissue Slices 3 49
Alternate Protocol 2: Inositol (1,5,5) Trisphosphate Mass Assay in
Cultured Cells 3 50
Support Protocol 5: Preparation of Inositol Trisphosphate Binding Protein 3 50
3.5 Nitric Oxide Synthase Assays 3 51
Basic Protocol: Arginine to Citrulline Conversion Assay for Measuring NOS
Activity 3 52
Support Protocol 1: Preparation of NOS Containing Tissue Extracts 3 54
Support Protocol 2: One Step Partial Purification of NOS 3 55
Support Protocol 3: Preparation of NOS From Cultured Cells 3 55
Support Protocol 4: Purification of [3H]Arginine by Thin Layer
Chromatography 3 56
3.6 HIV Protease Assays 3 57
Basic Protocol: Fluorometric Screening to Identify Inhibitors of HIV Protease 3 57
Support Protocol 1: Assay of HIV Protease to Determine Working Enzyme
Concentration 3 60
Support Protocol 2: Data Analysis 3 61
Contents Short Protocols in Pharmacology and Drug Discovery
v
3.7 Topoisomerase Assays 3 62
Basic Protocol 1: Assay of Topoisomerase I Activity 3 63
Basic Protocol 2: Assay of Topoisomerase II Activity 3 64
Support Protocol: Preparation of Mammalian Cell Extracts for Assaying
Topoisomerase Activity 3 64
Basic Protocol 3: In Vivo Determination of Topoisomerase Covalent
Complexes Using the K+/SDS Assay 3 65
Basic Protocol 4: Determination of DNA Cleavage by Purified Topoisomerase I 3 67
Basic Protocol 5: In Vitro Quantitative Determination of Topoisomerase II
Cleavage Using the K+/SDS Assay 3 69
Alternate Protocol: Gel Electrophoresis Determination of Topoisomerase II
Cleavage 3 70
3.8 Cytochrome P450 Assays 3 71
Basic Protocol 1: Evaluation of a Test Compound as an Inhibitor of CYP1A2
Activity Using Phenacetin 3 74
Basic Protocol 2: Evaluation of a Test Compound as an Inhibitor of CYP2A6
Activity Using Coumarin 3 76
Basic Protocol 3: Evaluation of a Test Compound as an Inhibitor of CYP2B6
and CYP2C19 Activities Using S Mephenytoin 3 78
Basic Protocol 4: Evaluation of a Test Compound as an Inhibitor of CYP2C8
Activity Using Paclitaxel 3 79
Basic Protocol 5: Evaluation of a Test Compound as an Inhibitor of CYP2C9
Activity Using Tolbutamide 3 80
Basic Protocol 6: Evaluation of a Test Compound as an Inhibitor of CYP2D6
Activity Using Dextromethorphan 3 82
Basic Protocol 7: Evaluation of a Test Compound as an Inhibitor of CYP2E1
Activity Using Chlorzoxazone 3 83
Basic Protocol 8: Evaluation of a Test Compound as an Inhibitor of CYP3A4
Activity Using Testosterone 3 85
Alternate Protocol 1: High Throughput Screening for CYP Inhibition Using
7 Benzyloxyquinoline 3 86
Alternate Protocol 2: High Throughput Screening for CYP Inhibition Using
Testosterone 3 87
Alternate Protocol 3: Evaluate a Test Compound as a Time Dependent
Inhibitor of CYP Activity 3 90
4 Isolated Tissues
4.1 Overview of Length Tension Relationships in Isolated Tissue 4 1
Isometric Contraction 4 3
Isotonic Recording 4 6
Auxotonic Recording 4 7
Conclusions 4 9
4.2 Isolated Cardiac Muscle Assays 4 9
Basic Protocol 4 9
4.3 Isolated Blood Vessel Assays 4 13
Basic Protocol 1: Ring Preparations of Blood Vessels 4 13
Alternate Protocol: Spiral Strip Preparations of Blood Vessels 4 14
Basic Protocol 2: Perfused Preparations of Blood Vessels 4 16
4.4 a Adrenoceptor Assays 4 18
Basic Protocol 1: Evaluation of a,A Adrenoceptors in Isolated Vas Deferens 4 18
Support Protocol: Setup of Tissue Bath Apparatus for Adrenoceptor Studies 4 20
Alternate Protocol 1: Evaluation of aiA Adrenoceptors in Canine Prostate 4 20
Alternate Protocol 2: Evaluation of x1B Adrenoceptors in Rat Spleen 4 23
Alternate Protocol 3: Evaluation of ot1D Adrenoceptors in Isolated Rat Aorta 4 23
Alternate Protocol 4: Evaluation of a2A Adrenoceptors in Isolated Vas
Deferens by Electrical Stimulation 4 24
Basic Protocol 2: Evaluation of 012 Adrenoceptors by Superfusion of
Radiolabeled Isolated Tissues 4 26
Short Protocols in Pharmacology and Drug Discovery Contents
vi
4.5 B Adrenoceptor Assays 4 29
Basic Protocol 1: (Ji Adrenoceptors: Guinea Pig Left Atria 4 29
Alternate Protocol: Bi Adrenoceptors: Guinea Pig Right Atria 4 31
Basic Protocol 2: B2 Adrenoceptors: Guinea Pig Trachea 4 33
Basic Protocol 3: S2 Adrenoceptors: Rat Uterus 4 35
Basic Protocol 4: Measuring B Adrenoceptor Stimulated Lipolytic Activity 4 36
Support Protocol 1: Isolation and Culture of Primary Preadipocytes and
Adipocytes 4 39
Support Protocol 2: Measuring Adipocyte Differentiation by Nile Red
Staining or Triglyceride Accumulation 4 42
4.6 PI (Adenosine) Purinoceptor Assays 4 43
Basic Protocol: Measurement of Tissue Responsiveness to PI Purinoceptor
Agonists 4 43
Support Protocol 1: Preparation of Guinea Pig Left Atrium 4 46
Support Protocol 2: Preparation of Porcine Left Circumflex Coronary Artery 4 46
Support Protocol 3: Preparation of Guinea Pig Aorta 4 47
Support Protocol 4: Quantitation of Responses 4 47
4.7 Opioid Receptor Assays 4 48
Basic Protocol 1: Measurement of Opioid Activity in the Guinea Pig Ileum 4 48
Basic Protocol 2: Measurement of Opioid Activity in the Mouse Vas Deferens 4 56
4.8 Nicotinic Acetylcholine Receptor Assays 4 62
Basic Protocol 1: The Innervated Guinea Pig Tracheal/Esophageal Isolated
Tissue Preparation to Study Receptors on Autonomic Nerves 4 62
Alternate Protocol 1: Nicotinic Agonist Mediated Effects: Isolated Guinea Pig
Trachealis and Ileum 4 70
Basic Protocol 2: Nicotinic Receptors in Striated Muscle: The Isolated
Diaphragm/Phrenic Nerve Preparation 4 71
Alternate Protocol 2: Nicotinic Receptors in Striated Muscle: The Isolated,
Innervated Guinea Pig Esophagus 4 72
4.9 Muscarinic Receptor Assays 4 72
Basic Protocol 1: Muscarinic Mi Receptors: Canine Saphenous Vein 4 72
Basic Protocol 2: Muscarinic M2 Receptors: Guinea Pig Paced Left Atrium 4 74
Basic Protocol 3: Muscarinic M3 Receptors: Rat Urinary Bladder 4 76
Alternate Protocol 1: Muscarinic M3 Receptors: Guinea Pig Trachea 4 77
Alternate Protocol 2: Muscarinic M3 Receptors: Guinea Pig Ileum 4 78
Basic Protocol 4: Muscarinic M4 Receptors: Rabbit Anococcygeus 4 78
4.10 Histamine Receptor Assays 4 81
Basic Protocol 1: Histamine Hi Receptors: Guinea Pig Ileum 4 81
Basic Protocol 2: Histamine H2 Receptors: Guinea Pig Right Atrium 4 83
Basic Protocol 3: Histamine H? Receptors: Electrically Stimulated Guinea Pig
Ileum 4 85
4.11 Prostanoid Receptor Assays 4 87
Basic Protocol 1: Evaluation of Prostanoid DP Receptor Activity in Human
Washed Platelets 4 87
Basic Protocol 2: Evaluation of Prostanoid EPi Receptors in Guinea Pig
Isolated Trachea (GPT) 4 89
Basic Protocol 3: Evaluation of Prostanoid EP2 Receptor Activity in Isolated
Ring Preparations from the Rabbit Ear Artery 4 92
Basic Protocol 4: Evaluation of Prostanoid EP3 Receptors in Guinea Pig
Isolated Vas Deferens (GPVD) 4 94
Alternate Protocol 1: Evaluation of Prostanoid EP3 Receptors in Guinea Pig
Isolated Aorta (GPA) 4 96
Basic Protocol 5: Evaluation of Prostanoid EP4 Receptor Activity in Isolated
Ring Preparations from the Rabbit Saphenous Vein 4 97
Basic Protocol 6: Evaluation of Prostanoid FP Receptors in Mouse Uterus 4 100
Alternate Protocol 2: Evaluation of Prostanoid FP Receptors in Cat and Dog
Isolated Iris Sphincter (CIS/DIS) 4 102
Contents Short Protocols in Pharmacology and Drug Discovery
vii
Basic Protocol 7: Evaluation of Prostanoid IP Receptors in Human Isolated
Pulmonary Artery (HPA) 4 104
Alternate Protocol 3: Evaluation of Prostanoid IP Receptors in Rat Isolated
Vagus Nerve (RVN) 4 105
Basic Protocol 8: Evaluation of TP Receptor Activity in Human Washed
Platelets 4 107
Alternate Protocol 4: Evaluation of TP Receptor Activity in Isolated Ring
Preparations from the Rabbit Saphenous Vein 4 109
4.12 Cysteinyl Leukotriene Receptor Assays 4 112
Basic Protocol: Measurement of CysLT Induced Contractions in Guinea Pig
Trachea 4 113
Support Protocol: Storage and Spectroscopic Quantification of CysLT Solutions 4 119
4.13 5 Hydroxytryptamine Receptor Assays 4 121
Basic Protocol 1: Inhibition of the 5 HTiA Receptor Mediated Twitch
Response in Guinea Pig Transmurally Stimulated Ileum 4 121
Basic Protocol 2: 5 HTiB Receptor Mediated Contraction of Rat Caudal Artery 4 124
Basic Protocol 3: 5 HTiD Receptor Mediated Relaxation of Precontracted
Guinea Pig Jugular Vein 4 127
Alternate Protocol: 5 HTiD Receptor Mediated Contraction of Rabbit Cerebral
Arteries 4 129
Basic Protocol 4: 5 HT2A Receptor Mediated Contraction of Rabbit Thoracic
Aorta 4 131
Basic Protocol 5: 5 HT2B Receptor Mediated Contraction of the Rat Stomach
Fundus 4 132
Basic Protocol 6: 5 HT2C Receptor Mediated Contraction of the Rat Jugular
Vein 4 134
Basic Protocol 7: 5 HTj Receptor Mediated Contraction of Guinea Pig Heal
Smooth Muscle 4 135
Basic Protocol 8: 5 HT4 Receptor Mediated Relaxation of Rat Esophageal
Tunica Muscularis Mucosa 4 137
Basic Protocol 9: 5 HT7 Receptor Mediated Relaxation of the
Endothelium Denuded, Precontracted Rabbit Jugular Vein 4 139
5 Animal Models of Disease
5.1 Models of Cardiovascular Disease: Measurement of Arrhythmogenic/
Antiarrhythmic Activity in the Guinea Pig and Antihypertensive Activity in
the Conscious Rat 5 2
Basic Protocol 1: Measuring Digoxin Induced Arrhythmias in the Guinea Pig 5 2
Basic Protocol 2: Measuring Antihypertensive Activity in the Instrumented
Conscious Rat 5 3
5.2 Models of Pulmonary Disease: Acute and Chronic Allergic Asthma in the
Monkey and Acute and Chronic Viral Pulmonitis in the Mouse 5 5
Basic Protocol 1: Inhaled Allergen Induced Acute Responses in Primates 5 5
Support Protocol 1: Processing of Bronchoalveolar Lavage (BAL) Samples 5 8
Basic Protocol 2: Repeated Inhaled Allergen Induced Airway
Hyperresponsiveness and Eosinophilia in Primates as a Model of Chronic
Asthma 5 8
Basic Protocol 3: Lung Inflammation and Dysfunction Associated with
Respiratory Syncytial Virus (RSV) Infection in Mice 5 9
Support Protocol 2: Preparation of High Titer Respiratory Syncytial Virus
(RSV) Stocks 5 13
53 Models of Pulmonary Disease: Guinea Pig Models of Asthma 5 14
Strategic Planning 5 15
Basic Protocol 1: Measurements of Pulmonary Insufflation Pressure in
Anesthetized, Paralyzed, Mechanically Ventilated Guinea Pigs 5 16
Alternate Protocol 1: Measurement of Reflex Tracheal Contractions in
Anesthetized, Paralyzed, Mechanically Ventilated Guinea Pigs 5 21
Basic Protocol 2: Induction of Airways Hyperresponsiveness in Guinea Pigs
Through Active Sensitization and Allergen Challenge 5 24
Short Protocols in Pharmacology and Drug Discovery Contents
viii
Basic Protocol 3: Measurement of Cough Reflexes in Conscious Guinea Pigs 5 25
Alternate Protocol 2: Measurement of Cough Reflexes in Anesthetized Guinea
Pigs 5 26
Basic Protocol 4: Measurement of Inflammatory Cell Influx into the Airways by
Bronchoalveolar Lavage (BAL) 5 27
Basic Protocol 5: Quantification of Plasma Extravasation 5 28
5.4 Models of Inflammation: Carrageenan Induced Paw Edema in the Rat 5 29
Basic Protocol 5 29
5.5 Models of Inflammation: Adjuvant Induced Arthritis in the Rat 5 30
Basic Protocol 5 30
5.6 Models of Inflammation: Carrageenan Air Pouch in the Rat 5 32
Basic Protocol 5 32
5.7 Models of Pain: Hot Plate and Formalin Test in Rodents 5 33
Basic Protocol 1: Measurement of Acute Pain Using the Hot Plate Test 5 33
Basic Protocol 2: Measurement of Persistent Pain Using the Formalin Test 5 34
5.8 Models of Pain: Neuropathic Pain in the Rat 5 37
Basic Protocol 1: The Chronic Constriction Injury (CCI) Model of Neuropathic
Pain 5 37
Basic Protocol 2: The Partial Sciatic Ligation (PSL) Model of Neuropathic
Pain 5 38
Basic Protocol 3: The Spinal Nerve Ligation (SNL) Model of Neuropathic
Pain 5 39
Support Protocol 1: Behavioral Assays: Heat Hyperalgesia 5 39
Support Protocol 2: Behavior Assays: Mechano Hyperalgesia 5 40
Support Protocol 3: Behavioral Assays: Mechano Allodynia 5 41
Support Protocol 4: Behavioral Assays: Cold Allodynia 5 42
5.9 Models of Visceral Pain: Colorectal Distension (CRD) 5 42
Basic Protocol 1: EMG Electrode Implantation in the Rat 5 43
Alternate Protocol 1: EMG Electrode Implantation in the Mouse 5 44
Basic Protocol 2: Preparing Rats for CRD 5 45
Alternate Protocol 2: Preparing Mice for CRD 5 46
Basic Protocol 3: CRD Testing Paradigms 5 48
5.10 Models of Pain: Postoperative Pain 5 50
Basic Protocol 5 50
5.11 Models of Affective Illness: Forced Swimming and Tail Suspension Tests in
Rodents 5 53
Basic Protocol: Behavioral Despair Test in the Rat 5 53
Alternate Protocol 1: Forced Swimming Test in the Mouse 5 54
Alternate Protocol 2: Tail Suspension Test in Mice 5 55
5.12 Models of Affective Illness (Depression): Chronic Mild Stress 5 56
Basic Protocol 5 56
5.13 Models of Schizophrenia: Phencyclidine Disruption of Prepulse Inhibition
(PPI) of Startle in Rats 5 58
Strategic Planning: Experimental Materials 5 58
Basic Protocol: Prepulse Inhibition (PPI) of Startle in Rats 5 60
5.14 Models of Schizophrenia: Amphetamine Induced Hyperactivity and
Stereotypies in Mice 5 61
Basic Protocol 1: Antagonism of Amphetamine Induced Hyperactivity in Mice 5 61
Basic Protocol 2: Antagonism of Amphetamine Induced Stereotypies in Mice 5 63
5.15 Models of Anxiety Disorder: Stress Induced Hyperthermia (SIH) in
Group Housed Mice 5 64
Basic Protocol 5 64
5.16 Models of Anxiety Disorder: Ultrasonic Vocalizations of Isolated Rat Pups 5 69
Basic Protocol 5 69
Contents Short Protocols in Pharmacology and Drug Discovery
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5.17 Models of Urogenital Dysfunction: Benign Prostatic Hyperplasia (BPH) 5 74
Basic Protocol 1: Measurement of Prostatic Pressure and Blood Pressure in the
Anesthetized Dog 5 74
Alternate Protocol: Hypogastric Nerve Stimulation to Produce Rises in Prostate
Pressure in the Anesthetized Dog 5 76
Basic Protocol 2: Measurement of Prostatic Pressure and Blood Pressure in the
Conscious Dog 5 77
Support Protocol: Measurement of Postural Reflexes in Conscious Dogs 5 79
5.18 Models of Urogenital Dysfunction: Urinary Urge Incontinence (UUI) 5 81
Basic Protocol: Measurement of Pelvic Nerve Stimulated Bladder Contraction
and Cardiovascular Function in the Anesthetized Dog 5 81
Alternate Protocol: Measurement of Pelvic Nerve Stimulated Bladder
Contraction and Cardiovascular Function in the Anesthetized Rat 5 84
Support Protocol: Measurement of Parasympathetic Nerve Induced Salivary
Secretion from the Submandibular Gland of the Dog 5 85
5.19 Models of Urogenital Dysfunction: Volume Evoked Micturition Reflex
(VEMR) in the Conscious Rat 5 87
Basic Protocol 5 87
5.20 Models of Urogenital Dysfunction: Erectile Dysfunction 5 90
Basic Protocol 1: Direct Measurement of Intracavernous and Intra Arterial
Pressures in the Anesthetized Rat 5 90
Alternate Protocol: Direct Measurement of Intracavernous and Intra Arterial
Pressures in the Anesthetized Mouse During Erections 5 100
Basic Protocol 2: Direct Measurement of Intracavernous Pressure in the
Conscious and Freely Moving Rat During Erections 5 100
5.21 Models of Dementia: Delayed Alternation in Aged Rats 5 104
Basic Protocol: Acquisition of an Operant Delayed Alternation Task in Young
and Aged Rats 5 104
Alternate Protocol: Stabilized Performance of an Operant Delayed Alternation
Task in Young and Aged Rats 5 106
5.22 Models of Dementia: Spatial Discrimination Water Maze Test in
Septal Lesioned Rats 5 107
Basic Protocol 5 107
5.23 Models for Eating Disorders: Dietary Hyperphagia and Anorexia Nervosa 5 111
Strategic Planning 5 111
Basic Protocol 1: Induction of Dietary Hyperphagia 5 112
Basic Protocol 2: Induction of Anorexia Nervosa 5 115
5.24 Models of Epilepsy: Electroshock and Chemical Induced Convulsions in the
Mouse 5 118
Basic Protocol 1: Anticonvulsant Determination Using Electroshock Induced
Convulsions in the Mouse 5 118
Alternate Protocol 1: Anticonvulsant Determination by the Method of Limits
Using Electroshock Induced Convulsions in the Mouse 5 119
Basic Protocol 2: Proconvulsant/Anticonvulsant Determination Using
Pentylenetetrazol Induced Clonk and Tonic Convulsions in the Mouse 5 120
Alternate Protocol 2: Anticonvulsant Determination Using
Pentylenetetrazol Induced Clonic Convulsions in the Mouse 5 122
Basic Protocol 3: Anticonvulsant Determination Using a Timed Intravenous
Infusion of Pentylenetetrazol to Induce Convulsions in the Mouse 5 122
Basic Protocol 4: Anticonvulsant Determination Using Bicuculline, Picrotoxin,
or Strychnine Induced Convulsions in the Mouse 5 124
5.25 In Vivo Cancer Models: Three Mouse Models Used in Experimental
Therapeutics 5 126
Strategic Planning 5 126
Basic Protocol 1: Intravenously Implanted Murine P388 Leukemia 5 126
Basic Protocol 2: Subcutaneously Implanted Murine B16 Melanoma 5 128
Basic Protocol 3: Two Examples of Subcutaneously Implanted Human Tumor
Xenografts, LX 1 (Lung) and MX 1 (Breast) Tumors 5 130
Short Protocols in Pharmacology and Drug Discovery Contents
x
6 Pharmacokinetics
6.1 Overview of Pharmacokinetics 6 1
Basic Protocol 1: Compound Administration in Rats 6 2
Support Protocol 1: Methoxyflurane Anesthesia of the Rat 6 4
Basic Protocol 2: Blood Collection in Rats 6 5
Basic Protocol 3: Excreta Collection in Rats 6 7
Basic Protocol 4: Tissue Collection in Rats 6 8
Support Protocol 2: Sample Processing: Radiolabeled Compound 6 12
Support Protocol 3: Sample Processing: Nonradiolabeled Compounds 6 13
Support Protocol 4: Data Calculation 6 14
6.2 Estimating Intestinal Mucosal Permeation of Compounds Using Caco 2 Cell
Monolayers 6 15
Basic Protocol 1: Measurement of the Apparent Permeability Coefficient of a
Compound Across Caco 2 Cell Monolayers 6 16
Support Protocol 1: Testing Caco 2 Monolayer Integrity: Mannitol Flux
Experiment 6 17
Support Protocol 2: Growing Caco 2 Cell Monolayers on Filters 6 21
Support Protocol 3: Starting and Maintaining Caco 2 Cells in Culture 6 22
6.3 Determination of Compound Binding to Plasma Proteins 6 24
Basic Protocol 1: Determination of Plasma Protein Binding by Ultrafiltration 6 25
Basic Protocol 2: Determination of Plasma Protein Binding by Traditional
Equilibrium Dialysis 6 26
Alternate Protocol: Determination of Plasma Protein Binding by 96 Well Plate
Equilibrium Dialysis 6 30
6.4 Determination of the Stability of Drugs in Plasma 6 31
Basic Protocol 6 31
6.5 In Vitro Drug Metabolism Using Liver Microsomes 6 34
Basic Protocol: Measurement of Compound Metabolic Stability Using Liver
Microsomes 6 34
Alternate Protocol: Higher Throughput Screening for Metabolic Stability 6 35
Support Protocol 1: Preparation of Microsomes from Rat Liver 6 37
Support Protocol 2: Determination of Cytochrome P450 (CYP) Content in
Microsomes 6 38
6.6 Hepatic Clearance and Drug Metabolism Using Isolated Perfused Rat Liver 6 39
Basic Protocol: Analysis of Hepatic Clearance Using IPRL 6 39
Support Protocol 1: Viability Evaluation 6 43
Support Protocol 2: Measuring LDH Activity 6 44
7 Drug Discovery Technologies
7.1 Overview of Chemical Diversity 7 2
High Throughput Screening (HTS) 7 3
Natural Products and the Screening of Biodiversity 7 3
Modification of Existing Structures 7 6
Rationally Planned Approaches 7 7
Identification and Exploitation of Basic Pharmacophores 7 8
Combinatorial Chemistry 7 9
Evolutionary Chemistry 7 10
Orphan Ligands and Orphan Receptors 7 10
7.2 Overview on High Throughput Screening 7 22
High Throughput Screening 7 22
Precedents 7 23
Disease Targets 7 23
Compound Libraries 7 24
Contents Short Protocols in Pharmacology and Drug Discovery
xi
Combinatorial Chemistry 7 25
Natural Products 7 26
Compound Storage and Handling 7 27
Mother and Daughter Plates 7 28
Barcodes 7 29
Assay Formats 7 29
Assay Design 7 31
Classical Receptor and Enzyme Assays 7 32
Homogeneous Radioactive Bioassays 7 33
Fluorescence 7 34
Luminescence 7 37
Reporter Gene Assays 7 37
Melanophore Technology 7 38
Cell Based Assays for Ion Channel Targets 7 38
Assay Robustness 7 39
Robotic Versus Automated Workstations 7 39
Hit to Lead Process 7 41
Pharmacological and Pharmaceutical Profiling 7 41
Data Evaluation 7 43
Is It Worth the Effort? 7 43
Outlook and Further Directions 7 44
7.3 Testing for Inverse Agonism with Constitutive Receptor Systems 7 44
GPCR Constitutive Activity 7 44
The Influence of Ligands on Constitutive GPCR Systems 7 49
Optimal Conditions for Constitutively Active Systems 7 51
Advantages and Disadvantages of Constitutively Active GPCR Assay Systems 7 55
Conclusions 7 57
7.4 Overview On the Use of Therapeutic Antibodies in Drug Discovery 7 58
History of Therapeutic Antibodies 7 58
Basic Antibody Structure 7 58
Technologies for Generation of Therapeutic Human Antibodies 7 60
Design Opportunities for an Antibody Therapeutic 7 65
Immunogenicity 7 68
Expression Technologies 7 69
How Do Therapeutic Antibodies Compare with Traditional Drugs? 7 69
Summary 7 73
8 Safety Pharmacology/Toxicology
8.1 Overview of Safety Pharmacology 8 2
Terminology 8 2
Safety Pharmacology Versus Toxicology 8 3
Comparison of Japanese and ICH Guidelines 8 3
CNS Safety Pharmacology 8 4
Cardiovascular Safety Pharmacology 8 5
Respiratory Safety Pharmacology 8 6
Timing of Safety Pharmacology Studies and Good Laboratory Practice 8 6
Ethical and Animal Welfare Issues 8 7
Statistical Evaluation 8 7
Conclusions 8 8
8.2 Gastrointestinal Tract: Gastrointestinal Ulceration in the Rat 8 8
Basic Protocol 8 8
8.3 Gastrointestinal Tract: Intestinal Transit and Ulcerogenic Activity in the Rat 8 10
Basic Protocol 1: Measurement of Intestinal Transit in the Rat 8 10
Basic Protocol 2: Measurement of Ulcerogenic Activity in the Rat 8 11
Short Protocols in Pharmacology and Drug Discovery Contents
yii
8.4 Toxicology in the Drug Discovery and Development Process 8 12
New Chemical Entities (NCEs; Synthetic Organic Chemicals) 8 13
New Biological Entities (NBEs) 8 17
Models of Drug Effect 8 19
Incorporation of Toxicokinetics into the Toxicity Profile 8 23
Regulatory Guidelines for Toxicology Profiles 8 27
Special Issues for Biotechnology Derived NBEs 8 40
IND Safety Reports 8 45
Summary and Future Approaches 8 45
8.5 Abuse Liability: Place Preference Test in Rodents 8 47
Basic Protocol 1: Place Preference Test in Rats 8 47
Basic Protocol 2: Place Preference Test in Mice 8 49
8.6 Abuse Liability: Self Administration in Monkeys 8 51
Strategic Planning 8 52
Basic Protocol: Self Administration Training and Testing 8 55
Support Protocol 1: Catheter Implantation 8 57
Support Protocol 2: Determination of Catheter Patency Using the Methohexital
Test 8 58
8.7 Vigilance Controlled Quantified EEG in Safety Pharmacology 8 59
Strategic Planning 8 59
Basic Protocol 1: Electrode Implantation 8 63
Basic Protocol 2: Vigilance Controlled Quantified EEG in Implanted Rats 8 67
Support Protocol: Cable Construction 8 70
8.8 The Action Potential of the Purkinje Fiber: An In Vitro Model for Evaluation
of the Proarrhythmic Potential of Cardiac and Noncardiac Drugs 8 70
Basic Protocol: Measurement of Changes in the Action Potential Parameters in
the Purkinje Fiber During Superfusion with a Test Compound 8 71
Alternate Protocol 1: Evaluation of the Proarrhythmic Risk of a Test Compound
on Epinephrine Facilitated Early Afterdepolarizations 8 76
Alternate Protocol 2: Evaluation of the Proarrhythmic Risk of a Test Compound
on the Occurrence of Delayed Afterdepolarizations and/or Triggered
Activities 8 79
8.9 Respiratory Function Assessment in Safety Pharmacology 8 80
Functional Disorders of the Respiratory System 8 84
Techniques for Measuring Changes in Respiratory Function 8 85
Design of Respiratory Function Safety Studies 8 90
8.10 Behavior and Physiological Function: Primary Observation (Irwin) Test in
Rodents 8 92
Basic Protocol: Primary Observation (Irwin) Test in the Rat 8 92
Alternate Protocol: Primary Observation (Irwin) Test in the Mouse 8 95
Appendices
1 Reagents and Solutions A1 1
2 Nomenclature and Usage A2 1
2A Enzyme Nomenclature Web Sites A2 1
2B Receptor Nomenclature Web Sites A2 2
3 Standard Techniques A3 1
3A Assays for Total Protein A3 1
Basic Protocol 1: Hartree Lowry Assay for Quantitation of Total Protein A3 2
Basic Protocol 2: Bicinchoninic Acid (BCA) Assay for Quantitation of
Total Protein A3 3
Basic Protocol 3: Ultraviolet Absorption to Measure Total Protein A3 3
Basic Protocol 4: Coomassie Dye Binding Assay (Bradford Assay) to
Measure Total Protein A3 4
Basic Protocol 5: Trichloroacetic Acid Precipitation of Protein Samples A3 5
Contents Short Protocols in Pharmacology and Drug Discovery
xiii
3B Protein Analysis by SDS PAGE and Detection by Coomassie Blue or
Silver Staining A3 5
Electricity and Electrophoresis A3 6
Basic Protocol: Denaturing (SDS) Discontinuous Gel Electrophoresis:
Laemmli Gel Method A3 7
Staining Proteins in Gels A3 10
Support Protocol 1: Coomassie Blue Staining A3 10
Support Protocol 2: Silver Staining A3 11
4 Selected Suppliers of Reagents and Equipment A4 1
References 1
Index
Short Protocols in Pharmacology and Drug Discovery Contents |
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| spelling | Short protocols in pharmacology and drug discovery a compendium of methods from current protocols in pharmacology editorial board, S. J. Enna (editor-in-chief) ; Michael Williams (editor-in-chief) ; John W. Ferkany ; Terry Kenakin ; Roger D. Porsolt Hoboken, N.J. Wiley 2007 Getr. Zählung txt rdacontent n rdamedia nc rdacarrier Includes bibliography and index Introduction to drug discovery and development -- Receptor binding -- Enzyme assays -- Isolated tissues -- Animal models of disease -- Pharmacokinetics -- Drug discovery technologies -- Safety pharmacology/toxicology Pharmacology / Laboratory manuals Drugs / Design / Laboratory manuals Drug Design Laboratory Manuals Drugs Design Laboratory manuals Pharmacology Laboratory Manuals Pharmacology Laboratory manuals Methode (DE-588)4038971-6 gnd rswk-swf Pharmakologie (DE-588)4045687-0 gnd rswk-swf Pharmakologie (DE-588)4045687-0 s Methode (DE-588)4038971-6 s DE-604 Enna, Salvatore Joseph 1944- Sonstige (DE-588)140120351 oth Williams, Michael Sonstige oth http://www.loc.gov/catdir/toc/ecip072/2006033930.html lizenzfrei Inhaltsverzeichnis HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=015655583&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Short protocols in pharmacology and drug discovery a compendium of methods from current protocols in pharmacology Pharmacology / Laboratory manuals Drugs / Design / Laboratory manuals Drug Design Laboratory Manuals Drugs Design Laboratory manuals Pharmacology Laboratory Manuals Pharmacology Laboratory manuals Methode (DE-588)4038971-6 gnd Pharmakologie (DE-588)4045687-0 gnd |
| subject_GND | (DE-588)4038971-6 (DE-588)4045687-0 |
| title | Short protocols in pharmacology and drug discovery a compendium of methods from current protocols in pharmacology |
| title_auth | Short protocols in pharmacology and drug discovery a compendium of methods from current protocols in pharmacology |
| title_exact_search | Short protocols in pharmacology and drug discovery a compendium of methods from current protocols in pharmacology |
| title_exact_search_txtP | Short protocols in pharmacology and drug discovery a compendium of methods from current protocols in pharmacology |
| title_full | Short protocols in pharmacology and drug discovery a compendium of methods from current protocols in pharmacology editorial board, S. J. Enna (editor-in-chief) ; Michael Williams (editor-in-chief) ; John W. Ferkany ; Terry Kenakin ; Roger D. Porsolt |
| title_fullStr | Short protocols in pharmacology and drug discovery a compendium of methods from current protocols in pharmacology editorial board, S. J. Enna (editor-in-chief) ; Michael Williams (editor-in-chief) ; John W. Ferkany ; Terry Kenakin ; Roger D. Porsolt |
| title_full_unstemmed | Short protocols in pharmacology and drug discovery a compendium of methods from current protocols in pharmacology editorial board, S. J. Enna (editor-in-chief) ; Michael Williams (editor-in-chief) ; John W. Ferkany ; Terry Kenakin ; Roger D. Porsolt |
| title_short | Short protocols in pharmacology and drug discovery |
| title_sort | short protocols in pharmacology and drug discovery a compendium of methods from current protocols in pharmacology |
| title_sub | a compendium of methods from current protocols in pharmacology |
| topic | Pharmacology / Laboratory manuals Drugs / Design / Laboratory manuals Drug Design Laboratory Manuals Drugs Design Laboratory manuals Pharmacology Laboratory Manuals Pharmacology Laboratory manuals Methode (DE-588)4038971-6 gnd Pharmakologie (DE-588)4045687-0 gnd |
| topic_facet | Pharmacology / Laboratory manuals Drugs / Design / Laboratory manuals Drug Design Laboratory Manuals Drugs Design Laboratory manuals Pharmacology Laboratory Manuals Pharmacology Laboratory manuals Methode Pharmakologie |
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