A Pharmacology Primer

Third Edition

A Pharmacology Primer:Theory, Applications, and Methods

Third Edition

Terry P. Kenakin, Ph.D.Principal Research Investigator, Molecular Discovery

GlaxoSmithKline Research and DevelopmentResearch Triangle Park, North Carolina

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Library of Congress Cataloging-in-Publication Data

Kenakin, Terrence P.

A pharmacology primer: theory, applications, and methods / Terry P. Kenakin.

–3rd ed.

p. ; cm.

Includes bibliographical references and index.

ISBN 978-0-12-374585-9 (hardcover : alk. paper) 1. Pharmacology,

Experimental. I. Title.

[DNLM: 1. Receptors, Drug–physiology.

2. Dose-Response Relationship, Drug.

3. Pharmacology–methods. QV 38 k33pg 2009]

RM301.25.K46 2009

6150.7–dc222008040350

British Library Cataloguing in Publication Data

A catalogue record for this book is available from the British Library

ISBN 13: 978-0-12-374585-9

For all information on all Elsevier Academic Press publications

visit our Web site at www.elsevierdirect.com

Printed in China

09 10 9 8 7 6 5 4 3 2 1

As always . . . for Debbie

. . . more ceterum censeo is perhaps necessary in order to rouse pharmacology from its sleep. The sleep is not a naturalone since pharmacology, as judged by its past accomplishments, has no reason for being tired . . .

— Rudolph Bucheim (1820–1879)

I am indebted to GlaxoSmithKline Research and Development for support during the preparation of this book and for

the means and scientific environment to make the science possible.

T.P.K., Research Triangle Park, NC

Contents

Preface xvPreface to the Second Edition xviiPreface to the First Edition xix

1. What Is Pharmacology?

1.1 About This Book 11.2 What Is Pharmacology? 11.3 The Receptor Concept 31.4 Pharmacological Test Systems 41.5 The Nature of Drug Receptors 61.6 Pharmacological Intervention and the

Therapeutic Landscape 71.7 System-Independent Drug Parameters:

Affinity and Efficacy 81.8 What Is Affinity? 101.9 The Langmuir Adsorption Isotherm 121.10 What Is Efficacy? 141.11 Dose-Response Curves 15

1.11.1 Potency and Maximal Response 161.11.2 p-Scales and the Representation

of Potency 171.12 Chapter Summary and Conclusions 181.13 Derivations: Conformational Selection

as a Mechanism of Efficacy 19References 19

2. How Different Tissues Process DrugResponse

2.1 Drug Response as seen through the

“Cellular Veil” 212.2 The Biochemical Nature of Stimulus-

Response Cascades 232.3 The Mathematical Approximation of

Stimulus-Response Mechanisms 262.4 System Effects on Agonist Response:

Full and Partial Agonists 262.5 Differential Cellular Response to

Receptor Stimulus 292.5.1 Choice of Response Pathway 292.5.2 Augmentation or Modulation

of Stimulus Pathway 31

2.5.3 Differences in Receptor Density 322.5.4 Target-Mediated Trafficking of

Stimulus 322.6 Receptor Desensitization and

Tachyphylaxis 352.7 The Measurement of Drug Activity 362.8 Advantages and Disadvantages of

Different Assay Formats 362.9 Drug Concentration as an Independent

Variable 372.9.1 Dissimulation in Drug

Concentration 372.9.2 Free Concentration of Drug 38

2.10 Chapter Summary and Conclusions 392.11 Derivations 39

2.11.1 Series Hyperbolae Can BeModeled by a Single HyperbolicFunction 40

2.11.2 Successive RectangularHyperbolic EquationsNecessarily Lead toAmplification 40

2.11.3 Saturation of Any Step in aStimulus Cascade by TwoAgonists Leads to IdenticalMaximal Final Responses for theTwo Agonists 40

2.11.4 Procedure to Measure of FreeDrug Concentration in theReceptor Compartment 40

References 41

3. Drug-Receptor Theory

3.1 About This Chapter 433.2 Drug-Receptor Theory 443.3 The Use of Mathematical Models in

Pharmacology 453.4 Some Specific Uses of Models in

Pharmacology 453.5 Classical Model of Receptor Function 473.6 The Operational Model of Receptor

Function 483.7 Two-State Theory 493.8 The Ternary Complex Model 50

ix

3.9 The Extended Ternary Model 503.10 Constitutive Receptor Activity 513.11 The Cubic Ternary Complex Model 533.12 Multistate Receptor Models and

Probabilistic Theory 533.13 Chapter Summary and Conclusions 553.14 Derivations 55

3.14.1 Radioligand Binding to ReceptorDimers DemonstratingCooperative Behavior 55

3.14.2 Effect of Variation in an HIV-1Binding Model 56

3.14.3 Derivation of the OperationalModel 57

3.14.4 Operational Model ForcingFunction for Variable Slope 57

3.14.5 Derivation of Two-State Theory 583.14.6 Derivation of the Extended

Ternary Complex Model 583.14.7 Dependence of Constitutive

Activity on Receptor Density 593.14.8 Derivation of the Cubic Ternary

Complex Model 59References 59

4. Pharmacological Assay Formats:Binding

4.1 The Structure of This Chapter 614.2 Binding Theory and Experiment 61

4.2.1 Saturation Binding 634.2.2 Displacement Binding 654.2.3 Kinetic Binding Studies 69

4.3 Complex Binding Phenomena: Agonist

Affinity from Binding Curves 704.4 Experimental Prerequisites for Correct

Application of Binding Techniques 734.4.1 The Effect of Protein

Concentration on BindingCurves 73

4.4.2 The Importance of EquilibrationTime for Equilibrium BetweenTwo Ligands 74

4.5 Chapter Summary and Conclusions 754.6 Derivations 76

4.6.1 Displacement Binding:Competitive Interaction 76

4.6.2 Displacement Binding:Noncompetitive Interaction 76

4.6.3 Displacement of a Radioligandby an Allosteric Antagonist 77

4.6.4 Relationship Between IC50 andKI for Competitive Antagonists 77

4.6.5 Maximal Inhibition of Bindingby an Allosteric Antagonist 77

4.6.6 Relationship Between IC50 andKI for Allosteric Antagonists 77

4.6.7 Two-Stage Binding Reactions 774.6.8 Effect of G-protein Coupling on

Observed Agonist Affinity 784.6.9 Effect of Excess Receptor in

Binding Experiments: SaturationBinding Curve 78

4.6.10 Effect of Excess Receptor inBinding Experiments:Displacement Experiments 78

References 78

5. Agonists: The Measurement of Affinityand Efficacy in Functional Assays

5.1 Functional Pharmacological

Experiments 815.2 The Choice of Functional Assays 825.3 Recombinant Functional Systems 875.4 Functional Experiments: Dissimulation

in Time 885.5 Experiments in Real Time Versus Stop

Time 905.6 The Measurement of Agonist Affinity

in Functional Experiments 915.6.1 Partial Agonists 915.6.2 Full Agonists 95

5.7 Estimates of Relative Efficacy of

Agonists in Functional Experiments 965.8 Chapter Summary and Conclusions 975.9 Derivations 98

5.9.1 Relationship Between the EC50

and Affinity of Agonists 985.9.2 Method of Barlow, Scott, and

Stephenson for Affinity of PartialAgonists 98

5.9.3 Measurement of AgonistAffinity: Method of Furchgott 99

5.9.4 Maximal Response of a PartialAgonist Is Dependent onEfficacy 99

References 100

6. Orthosteric Drug Antagonism

6.1 Introduction 1016.2 Kinetics of Drug-Receptor Interaction 1026.3 Surmountable Competitive

Antagonism 1046.3.1 Schild Analysis 104

x Contents

6.3.2 Patterns of Dose-ResponseCurves That Preclude SchildAnalysis 108

6.3.3 Best Practice for the Use ofSchild Analysis 109

6.3.4 Analyses for Inverse Agonistsin Constitutively ActiveReceptor Systems 110

6.3.5 Analyses for Partial Agonists 1136.3.6 The Method of Lew and

Angus: Nonlinear RegressionalAnalysis 114

6.4 Noncompetitive Antagonism 1166.5 Agonist–Antagonist Hemi-Equilibria 1196.6 Resultant Analysis 1216.7 Chapter Summary and Conclusions 1216.8 Derivations 122

6.8.1 Derivation of the GaddumEquation for CompetitiveAntagonism 123

6.8.2 Derivation of the GaddumEquation for NoncompetitiveAntagonism 123

6.8.3 Derivation of the SchildEquation 123

6.8.4 Functional Effects of an InverseAgonist with the OperationalModel 124

6.8.5 pA2 Measurement for InverseAgonists 124

6.8.6 Functional Effects of a PartialAgonist with the OperationalModel 124

6.8.7 pA2 Measurements for PartialAgonists 125

6.8.8 Method of Stephenson for PartialAgonist Affinity Measurement 125

6.8.9 Derivation of the Method ofGaddum for NoncompetitiveAntagonism 125

6.8.10 Relationship of pA2 and pKB

for Insurmountable OrthostericAntagonism 126

6.8.11 Resultant Analysis 126References 127

7. Allosteric Drug Antagonism

7.1 Introduction 1297.2 The Nature of Receptor Allosterism 1297.3 Properties of Allosteric Modulators 1317.4 Functional Study of Allosteric

Modulators 1367.4.1 Surmountable Allosteric

Modulation (x ¼ 1) 137

7.4.2 Insurmountable AllostericAntagonism (x ¼ 0) 138

7.4.3 Variable Effects on Efficacy(x 6¼ 0) 139

7.5 Measurement of the Potency of

Allosteric Insurmountable Antagonists 1407.6 Methods for Detecting Allosterism 1427.7 Chapter Summary and Conclusions 1447.8 Derivations 144

7.8.1 Allosteric Model of ReceptorActivity 144

7.8.2 Effects of Allosteric Ligands onResponse: Changing Efficacy 145

7.8.3 Schild Analysis for AllostericAntagonists 145

7.8.4 Relationship of pA2 and pKB

for Insurmountable AllostericAntagonism 146

References 146

8. The Process of Drug Discovery

8.1 Pharmacology in Drug Discovery 1498.2 Chemical Sources for Potential Drugs 1498.3 Pharmacodynamics and High-

Throughput Screening 1558.4 Drug Discovery and Development 163

8.4.1 Safety Pharmacology 1688.5 Clinical Testing 1758.6 Chapter Summary and Conclusions 176

References 176

9. Pharmacokinetics

9.1 Introduction 1799.2 Biopharmaceutics 1799.3 The Chemistry of “Druglike” Character 1809.4 Pharmacokinetics 184

9.4.1 Drug Absorption 1849.4.2 Route of Drug Administration 1889.4.3 General Pharmacokinetics 1919.4.4 Metabolism 1939.4.5 Clearance 1949.4.6 Volume of Distribution and

Half Life 1969.4.7 Renal Clearance 2029.4.8 Bioavailability 204

9.5 Nonlinear Pharmacokinetics 2059.6 Multiple Dosing 2069.7 Practical Pharmacokinetics 209

9.7.1 Allometric Scaling 2099.8 Placement of Pharmacokinetic Assays

in Discovery and Development 2119.9 Summary and Conclusions 214

References 214

xiContents

10. Target- and System-BasedStrategies for Drug Discovery

10.1 Some Challenges for Modern Drug

Discovery 21510.2 Target-Based Drug Discovery 216

10.2.1 Target Validation and theUse of Chemical Tools 217

10.2.2 Recombinant Systems 22010.2.3 Defining Biological Targets 221

10.3 Systems-Based Drug Discovery 22410.3.1 Assays in Context 228

10.4 In Vivo Systems, Biomarkers, and

Clinical Feedback 22910.5 Types of Therapeutically Active

Ligands: Polypharmacology 23110.6 Summary and Conclusions 235

References 236

11. “Hit” to Drug: Lead Optimization

11.1 Tracking SAR and Determining

Mechanism of Action: Data Driven

Drug-Based Pharmacology 23911.2 Drug Initiation of Response: Agonism 240

11.2.1 Analysis of Full Agonism 24111.2.2 Quantifying Full Agonist

Potency Ratios 24311.2.3 Analysis of Partial Agonism 24311.2.4 Fitting Data to the Operational

Model 24411.2.5 Affinity-Dependent versus

Efficacy-Dependent AgonistPotency 246

11.2.6 Secondary and Tertiary Testingof Agonists 249

11.3 Inhibition of Agonist Response:Antagonism 25011.3.1 Initial Antagonist Potency:

pIC50 Curves 25011.3.2 Secondary Testing of

Antagonists 25511.3.3 Determining Mode of

Antagonist Action 25711.3.4 Use of the pA2 as a Universal

Determinant of AntagonistPotency 258

11.3.5 Logistics of Analysis ofAntagonism 259

11.4 Summary and Conclusions 26511.5 Derivations 265

11.5.1 System Independence of FullAgonist Potency Ratios:Classical and OperationalModels 268

11.5.2 Model for Allosteric Agonism 26811.5.3 IC50 Correction Factors:

Competitive Antagonists 26911.5.4 Relationship of pA2 and pKB for

Insurmountable OrthostericAntagonism 269

11.5.5 Relationship of pA2 and pKB forInsurmountable AllostericAntagonism 270

11.5.6 Operational Model for PartialAgonist Interaction withAgonist: Variable Slope 270

11.5.7 Operational Model for InverseAgonist Interaction withAgonist: Variable Slope 270

11.5.8 Surmountable AllostericAntagonism: Variable Slope 271

11.5.9 Functional Model for Hemi-Equilibrium Effects: VariableSlope 271

11.5.10 Allosteric Antagonism withChanges in Efficacy: VariableSlope 271

11.5.11 Orthosteric InsurmountableAntagonism: OperationalModel with Variable Slope 271

11.5.12 Allosteric InsurmountableAntagonism: OperationalModel with Variable Slope 272

References 272

12. Statistics and ExperimentalDesign

12.1 Structure of This Chapter 27312.2 Introduction 27312.3 Descriptive Statistics: Comparing

Sample Data 27312.3.1 Gaussian Distribution 27412.3.2 Populations and Samples 27412.3.3 Confidence Intervals 27612.3.4 Paired Data Sets 27712.3.5 One-Way Analysis of Variance 27812.3.6 Two-Way Analysis of Variance 27912.3.7 Regression and Correlation 27912.3.8 Detection of Single versus

Multiple Populations 28112.4. How Consistent Is Experimental Data

with Models? 28212.4.1 Comparison of Data to Models:

Choice of Model 28212.4.2 Curve Fitting: Good Practice 28512.4.3 Outliers and Weighting Data

Points 28512.4.4 Overextrapolation of Data 287

xii Contents

12.4.5 Hypothesis Testing: Exampleswith Dose-Response Curves 289

12.4.6 One Curve or Two? Detectionof Differences in Curves 291

12.4.7 Asymmetrical Dose-ResponseCurves 292

12.4.8 Comparison of Data to LinearModels 293

12.4.9 Is a Given Regression Linear? 29412.4.10 One or More Regression Lines?

Analysis of Covariance 29512.5 Comparison of Samples to “Standard

Values” 29712.5.1 Comparison of Means by Two

Methods or in Two Systems 29812.5.2 Comparing Assays/Methods

with a Range of Ligands 29812.6 Experimental Design and Quality

Control 29912.6.1 Detection of Difference in

Samples 29912.6.2 Power Analysis 300

12.7 Chapter Summary and Conclusions 301References 302

13. Selected Pharmacological Methods

13.1 Binding Experiments 30313.1.1 Saturation Binding 30313.1.2 Displacement Binding 303

13.2 Functional Assays 30513.2.1 Determination of Equiactive

Concentrations on Dose-Response Curves 305

13.2.2 Method of Barlow, Scott, andStephenson for Measurementof the Affinity of a PartialAgonist 307

13.2.3 Method of Furchgott for theMeasurement of the Affinityof a Full Agonist 308

13.2.4 Schild Analysis for theMeasurement of CompetitiveAntagonist Affinity 310

13.2.5 Method of Stephenson forMeasurement of PartialAgonist Affinity 312

13.2.6 Method of Gaddum forMeasurement of Non-competitive Antagonist Affinity 313

13.2.7 Method for Estimating Affinity ofInsurmountable Antagonist(Dextral DisplacementObserved) 315

13.2.8 Resultant Analysis forMeasurement of Affinity ofCompetitive Antagonists withMultiple Properties 316

13.2.9 Measurement of the Affinityand Maximal Allosteric Constantfor Allosteric ModulatorsProducing SurmountableEffects 317

13.2.10 Method for Estimating Affinityof Insurmountable Antagonist(No Dextral DisplacementObserved): Detection ofAllosteric Effect 318

13.2.11 Measurement of pKB forCompetitive Antagonists froma pIC50 320

13.2.12 Kinetics of Antagonist Offset 322

14. Exercises in Pharmacodynamics andPharmacokinetics

14.1 Introduction 32714.2 Agonism 327

14.2.1 Agonism: Structure-ActivityRelationships 327

14.2.2 Prediction of Agonist Effect 32914.2.3 “Super Agonists” 32914.2.4 Atypical Agonists 33014.2.5 Ordering of Affinity and

Efficacy in Agonist Series 33014.2.6 Kinetics of Agonism 33014.2.7 Affinity-Dominant Versus

Efficacy-Dominant Agonists 33114.2.8 Agonist Affinities and

Potencies Do Not Correlate 33414.2.9 Lack of Agonist Effect 33514.2.10 Assay-Specific Agonism 336

14.3 Antagonism 33714.3.1 Antagonist Potency and

Kinetics: Part A 33714.3.2 Antagonist Potency in pIC50

Format (Kinetics Part B) 33914.3.3 Mechanism of Antagonist

Action (Kinetics Part C) 34014.3.4 Mechanism of Antagonist

Action: Curve Patterns 34114.3.5 Mechanism of Action:

Incomplete Antagonism 34114.3.6 pIC50 Mode: Antagonism

Below Basal 34314.3.7 Secondary Effects of

Antagonists 344

xiiiContents

14.3.8 Antagonist Potency VariablyDependent on AgonistConcentration 344

14.4 In Vitro–In Vivo Transitions and

General Discovery 34614.4.1 “Silent Antagonism” 34614.4.2 Loss of Activity 34714.4.3 Marking Relevant Agonism 34814.4.4 In Vitro–In Vivo Correspondence

of Activity 34814.4.5 Divergent Agonist-Dependent

Antagonism 34914.5 SAR Exercises 351

14.5.1 Surrogate Screens 35114.6 Pharmacokinetics 351

14.6.1 Clearance 35114.6.2 Drug–Drug Interactions 35214.6.3 Distribution I 35214.6.4 Distribution II 35214.6.5 Half Life I 35314.6.6 Half Life II 353

14.6.7 Half Life III 35414.6.8 Renal Clearance I 35414.6.9 Renal Clearance II 35414.6.10 Renal Clearance III 35414.6.11 Absorption 35414.6.12 Predictive Pharmacokinetics I 35414.6.13 Predictive Pharmacokinetics II 35514.6.14 Predictive Pharmacokinetics III 35514.6.15 Log D and Pharmacokinetics 355

14.7 Conclusions 355References 355

Appendices: 357A.1 Statistical Tables of Use forAssessing Significant Difference 357A.2 Mathematical Fitting Functions 365Glossary of Pharmacological Terms 371Index 379

xiv Contents

Preface

It has been an interesting experience as an author and

pharmacologist to see the changes that the discipline has

experienced through the drug discovery process. While

the definition of the human genome has undoubtedly

marked pharmacology forever (and advanced it immeasur-

ably), the more we learn, the more we are humbled by nat-

ure’s complexity. With the genome, knowing the road

map is still a long way from completing the journey and

recent experience seems to reinforce the idea that pharma-

cology must be used to understand integrated systems, not

just the pieces they are made of.

This edition incorporates a new trend in drug discov-

ery; namely the consideration of pharmacokinetics and

ADME properties of drugs (absorption, distribution,

metabolism, excretion) early in the process. As prospec-

tive new drugs are tested in more complex systems (with

concomitantly more complex dependent variable values),

the trend in screening is to test fewer compounds of higher

(“druglike”) quality. Finally, this edition also hopefully

fills a previous void whereby the ideas and concepts

discussed can be applied to actual problems in pharma-

cological drug discovery in the form of questions with

accompanying answers. The expanded version now spans

pharmacology from consideration of the independent vari-

able (drug concentration in the form of pharmacokinetics)

to the dependent variable (system-independent measure-

ment of drug activity). As with previous editions, the

emphasis of this book is still on the chemist–biologist inter-

face with special reference to the use of pharmacology by

non-pharmacologists.

Terry Kenakin, Ph.D.Research Triangle Park, NC, 2008

xv

Preface

Preface to the Second Edition

With publication of the human genome has come an ex-

periment in reductionism for drug discovery. With the

evaluation of the number and quality of new drug treatments

from this approach has come a re-evaluation of target-based

versus systems-based strategies. Pharmacology, historically

rooted in systems-based approaches and designed to give

systems-independent measures of drug activity, is suitably

poised to be a major, if not the major, tool in this new

environment of drug discovery.

Compared to the first edition, this book now expands

discussion of tools and ideas revolving around allo-

steric drug action. This is an increasingly therapeutically

relevant subject in pharmacology as new drug screening

utilizes cell function for discovery of new drug entities.

In addition, discussion of system-based approaches, drug

development (pharmacokinetics, therapeutics), sources of

chemicals for new drugs, and elements of translational

medicine have been added. As with the first edition, the

emphasis of this volume is the gaining of understanding

of pharmacology by the nonpharmacologist to the mutual

enrichment of both.

Terry Kenakin, Ph.D.Research Triangle Park, NC, 2006

xvii

Preface

Preface to the First Edition

If scientific disciplines can be said to go in and out of

vogue, pharmacology is exemplary in this regard. The

flourishing of receptor theory in the 1950s, the growth of

biochemical binding technology in the 1970s, and the

present resurgence of interest in defining cellular pheno-

typic sensitivity to drugs have been interspersed with

troughs such as that brought on by the promise of the

human genome and a belief that this genetic road map

may make classical pharmacology redundant. The fallacy

in this belief has been found in experimental data showing

the importance of phenotype over genotype which under-

scores a common finding with roadmaps; They are not

as good as a guide who knows the way. Pharmacology is

now more relevant to the drug discovery process than ever

as the genome furnishes a wealth of new targets to

unravel. Biological science often advances at a rate

defined by the technology of its tools; that is, scientists

cannot see new things in old systems without new eyes.

A veritable explosion in technology coupled with the great

gift of molecular biology have definitely given pharmacol-

ogists new eyes to see.

This book initially began as a series of lectures at

GlaxoSmithKline Research and Development on receptor

pharmacology aimed at increasing the communication

between pharmacologists and chemists. As these lectures

developed it became evident that the concepts were useful

to biologists not specifically trained in pharmacology. In

return, the exchange between the chemists and biologists

furnished new starting points from which to view the phar-

macological concepts. It is hoped that this book will some-

what fill what could be a gap in present biological

sciences, namely the study of dose-response relationships

and how cells react to molecules.

Terry P. Kenakin, Ph.D.Research Triangle Park, 2003

xix