Drug-Like Properties (eBook)

Front Cover 1
Drug-Like Properties: Concepts, Structure, Design, and Methods from ADME to Toxicity Optimization 4
Copyright 5
Dedication 6
Contents 8
Preface 20
Preface to Second Edition 20
Preface to First Edition 20
Chapter 1: Introduction 22
1.1. Drug-like Properties in Drug Discovery 22
1.2. Purpose of This Book 23
Problems 23
References 24
Chapter 2: Benefits of Property Assessment and Good Drug-Like Properties 26
2.1. Introduction 26
2.2. Discovery Scientists Optimize Many Properties 26
2.3. Introduction to the Drug Discovery and Development Process 27
2.4. Benefits of Good Drug-like Properties 29
2.4.1. Reduced Development Attrition 29
2.4.2. More Efficient Drug Discovery 29
2.4.3. More Efficient Drug Development 30
2.4.4. Higher Patient Compliance 30
2.4.5. Improved Biological Research in Drug Discovery 30
2.4.6. Enabled Partnerships for Drug Development 31
2.4.7. Human Modeling and Clinical Planning 31
2.4.8. Balance of Properties and Activity 31
2.5. Property Profiling in Drug Discovery 33
2.6. Drug-like Property Optimization in Drug Discovery 33
Problems 33
References 34
Chapter 3: In Vivo Environments Affect Drug Exposure 36
3.1. Introduction 36
3.2. Drug Dosing 37
3.3. Stomach 37
3.3.1. Gastric Acidic Degradation 38
3.4. Intestinal Environment 38
3.4.1. Dissolution Rate 40
3.4.2. Solubility 40
3.4.3. Permeability 40
3.4.4. Intestinal Metabolism 42
3.4.5. Intestinal Enzymatic Hydrolysis 42
3.4.6. Absorption Enhancement in the Intestine 43
3.5. Bloodstream 43
3.5.1. Plasma Enzyme Hydrolysis 44
3.5.2. Plasma Protein Binding 44
3.5.3. Red Blood Cell Binding 44
3.6. Liver 44
3.6.1. Permeation into and out of Hepatocytes 45
3.6.2. Hepatic Metabolism 45
3.6.3. Biliary Extraction 45
3.7. Kidney 45
3.8. Blood-Tissue Barriers 46
3.9. Tissue Distribution 47
3.9.1. Nonspecific Binding in Tissue 47
3.10. Consequences of Chirality 47
3.11. Overview of in vivo Challenges to Drug Exposure 48
Problems 48
References 49
Chapter 4: Prediction Rules for Rapid Property Profiling from Structure 50
4.1. Introduction 50
4.2. General Concepts for Prediction Rules 50
4.3. Rule of 5 51
4.4. Veber Rules 52
4.5. Waring Rules 53
4.6. Golden Triangle 53
4.7. Other Predictive Rules 54
4.8. Application of Rules for Compound Assessment 56
4.9. Applications of Predictive Rules 57
Problems 57
References 59
Chapter 5: Lipophilicity 60
5.1. Lipophilicity Fundamentals 60
5.2. Lipophilicity Effects 61
5.3. Lipophilicity Case Studies and Structure Modification 63
5.3.1. Lipophilicity Modification for Biological Activity 63
5.3.2. Lipophilicity Modification for Pharmacokinetics 64
5.3.3. Lipophilicity Modification for Toxicity 68
Problems 70
References 24
Chapter 6: pKa 72
6.1. pKa Fundamentals 72
6.2. pKa Effects 73
6.2.1. pKa Affects Efficacy 73
6.2.2. pKa Affects Pharmacokinetics 73
6.2.3. pKa Affects Toxicity 74
6.3. pKa Case Studies 74
6.3.1. pKa and Activity Examples 74
6.3.2. pKa and Pharmacokinetics Examples 75
6.4. Structure Modification Strategies for pKa 77
Problems 80
References 80
Chapter 7: Solubility 82
7.1. Introduction 82
7.2. Solubility Fundamentals 82
7.2.1. Solubility Varies with Compound Structure, Form, and Solution Conditions 82
7.2.2. Dissolution Rate 83
7.2.3. Structural Properties Affect Solubility 83
7.2.3.1. Lipophilicity and Crystal Intermolecular Forces Affect Solubility 83
7.2.3.2. Ionizability Greatly Affects Solubility 84
7.2.4. Kinetic and Thermodynamic Solubility 85
7.2.5. Consequences of Chirality on Solubility 87
7.3. Effects of Solubility 87
7.3.1. Low Solubility Affects In Vitro Assays 87
7.3.2. Low Solubility Limits Absorption and Causes Low Oral Bioavailability 87
7.3.3. Good Solubility is Essential for IV Formulation 89
7.3.4. How Much Solubility is Needed? 90
7.3.4.1. Maximum Absorbable Dose 90
7.3.4.2. Biopharmaceutics Classification System 92
7.3.5. Molecular Properties for Solubility and Permeability Are Often Opposed 94
7.4. Effects of Physiology on Solubility and Absorption 94
7.4.1. Physiology of the Gastrointestinal Tract 95
7.4.2. Species Differences in the GI Tract 95
7.4.3. Food Effect 96
7.5. Structure Modification Strategies to Improve Solubility 98
7.5.1. Add Ionizable Groups 99
7.5.2. Reduce log P 101
7.5.3. Add Hydrogen Bonding 102
7.5.4. Add Polar Group 103
7.5.5. Reduce MW 104
7.5.6. Out-of-Plane Substitution 104
7.5.7. Construct a Prodrug 106
7.6. Strategies to Improve Dissolution Rate 106
7.6.1. Reduce Particle Size 106
7.6.2. Prepare an Oral Solution 107
7.6.3. Formulate with Surfactants 107
7.6.4. Prepare a Salt Form 107
7.7. Salt Form 107
7.7.1. Solubility of Salts 108
7.7.2. Effect of Salt Form on Absorption and Oral Bioavailability 109
7.7.3. Salt Selection 110
7.7.4. Precautions of Using Salt Forms 111
7.8. Strategy for Solubility During Drug Discovery 111
Problems 112
References 113
Chapter 8: Permeability 116
8.1. Introduction 116
8.2. Permeability Fundamentals 116
8.2.1. Passive Transcellular Diffusion Permeability 117
8.2.2. Efflux Transport Permeability 120
8.2.3. Uptake Transport Permeability 121
8.2.4. Paracellular Permeability 121
8.2.5. Endocytosis Permeability 121
8.2.6. Net Permeability 121
8.3. Permeability Effects 122
8.3.1. Effect of Permeability on Absorption and Bioavailability 122
8.3.2. Effect of Permeability on Cell-Based Activity Assays 122
8.3.3. Permeation is Involved in Hepatic Clearance 123
8.3.4. Permeation is Involved in Renal Clearance 123
8.3.5. Permeation is Involved in Brain Efficacy 124
8.3.6. Permeation into Tissue Cells is Involved in Efficacy 124
8.4. Permeability Structure Modification Strategies 124
8.4.1. Replace Ionizable Group with Nonionizable Group 124
8.4.2. Add Lipophilicity 125
8.4.3. Isosteric Replacement of Polar Groups 125
8.4.4. Esterify Carboxylic Acid to Form Prodrug 127
8.4.5. Reduce Hydrogen Bonding and Polarity 127
8.4.6. Reduce Molecular Size 128
8.4.7. Add Nonpolar Side Chain 129
8.4.8. Prodrug 129
8.5. Strategy for Permeability 130
Problems 130
References 131
Chapter 9: Transporters 134
9.1. Introduction 134
9.2. Transporter Fundamentals 134
9.3. Transporter Effects 137
9.3.1. Transporters in Intestinal Epithelial Cells 140
9.3.2. Transporters in Liver Hepatocytes 140
9.3.3. Transporters in Kidney Epithelial Cells 140
9.3.4. Transporters in BBB Endothelial Cells 140
9.4. Efflux Transporters 140
9.4.1. P-glycoprotein (MDR1, ABCB1) [Efflux] 141
9.4.1.1. Rules for P-gp Efflux Substrates 142
9.4.1.2. Case Study of P-gp Efflux 142
9.4.1.3. Structure Modification Strategies to Reduce P-gp Efflux 143
9.4.2. Breast Cancer Resistance Protein (BCRP, ABCG2) [Efflux] 145
9.4.3. Multidrug Resistance Protein 2 (MRP2, ABCC2) [Efflux] 146
9.4.4. Efflux Transporters in the BBB 148
9.5. Uptake Transporters 148
9.5.1. Organic Anion Transporting Polypeptide 1A2 for Brain Uptake 148
9.5.2. Organic Anion Transporting Polypeptide 1B1 and 1B3 for Liver Targeting and Clearance Prediction 148
9.5.3. Peptide Transporter 1 (PEPT1) for Intestine Absorption 150
9.5.4. Large Neutral Amino Acid Transporter (LAT1) for Brain Uptake 152
9.5.5. Monocarboxylate Transporter 1 (MCT1) for Oral Absorption 152
9.5.6. Organic Anion Transporters 1 and 3 for Renal Uptake 154
9.5.7. Organic Cation Transporter 2 (OCT2) for Renal Uptake 155
9.5.8. MATE1 and MATE2-K 156
9.5.9. Other Uptake Transporters 156
9.5.10. Structure Modification Strategies for Uptake Transporters 157
Problems 157
References 158
Chapter 10: Blood-Brain Barrier 162
10.1. Introduction 162
10.2. Fundamentals of Brain Exposure 162
10.2.1. Unbound Drug Brain Concentration, Cb,u, and AUCb,u 163
10.2.2. Unbound Brain-Blood Equilibrium Distribution, Kp,uu 163
10.2.3. BBB Permeation Affects Cb,u and Kp,uu 164
10.2.3.1. Rate and Extent of Brain Exposure 165
10.2.3.2. BBB Efflux Transport 166
10.2.3.3. Passive Transcellular Diffusion of BBB Affects Cb,u 166
10.2.3.4. Uptake Transport of BBB Affects Cb,u 166
10.2.3.5. No Fenestrations in BBB 167
10.2.3.6. No Paracellular or Pinocytosis Permeability of BBB 167
10.2.4. ADME Processes Affect Cb,u 167
10.2.5. BCSFB and CSF 167
10.2.5.1. Brain Exposure via BCSFB 167
10.2.5.2. Use of CSF as an ISF Surrogate 167
10.3. Effects of Brain Exposure on Efficacy and Drug Development 168
10.3.1. Effects of BBB Efflux on Human Efficacy 168
10.3.2. Effects of BBB Efflux on Human Clinical Development 168
10.3.3. Effects of Metabolic Clearance on Efficacy 168
10.3.4. Total Brain Exposure is Not Correlated to Efficacy 168
10.4. Structure-Passive Transcellular BBB Permeation Relationships 170
10.5. Structure Modification Strategies to Improve BBB Permeation 171
10.5.1. Reduce P-gp Efflux 172
10.5.2. Reduce Hydrogen Bonds 172
10.5.3. Increase Lipophilicity 172
10.5.4. Reduce Molecular Weight 173
10.5.5. Replace Carboxylic Acid Groups 173
10.5.6. Add an Intramolecular Hydrogen Bond 173
10.5.7. Modify or Select Structures for Affinity to BBB Uptake Transporters 174
10.6. Applications of Brain Exposure 174
10.6.1. Best Practices for Brain Exposure Assessment 174
10.6.2. Scheme for Assessing Brain Exposure 175
10.6.3. Projecting Human Brain Exposure 175
10.6.4. Selecting Candidates for CNS Drug Development 176
10.6.5. Minimizing Brain Exposure for Peripheral Drugs 176
Problems 178
References 179
Chapter 11: Metabolic Stability 182
11.1. Introduction 182
11.2. Metabolic Stability Fundamentals 183
11.2.1. Phase I Metabolism 184
11.2.2. Phase II Metabolism 188
11.3. Metabolic Stability Effects 190
11.4. Structure Modification Strategies for Phase I CYP Metabolic Stability 191
11.4.1. Block Metabolic Site by Adding Fluorine 192
11.4.2. Block Metabolic Site by Adding Other Blocking Groups 194
11.4.3. Remove Labile Functional Group 195
11.4.4. Cyclization 196
11.4.5. Change Ring Size 197
11.4.6. Change Chirality 197
11.4.7. Reduce Lipophilicity 197
11.4.8. Replace Unstable Groups 198
11.5. Structure Modification Strategies for Phase II Metabolic Stability 199
11.5.1. Introduce Electron-Withdrawing Groups and Steric Hindrance 199
11.5.2. Change Phenolic Hydroxyl to Cyclic Urea or Thiourea 200
11.5.3. Change Phenolic Hydroxyl to Prodrug 200
11.6. Applications of Metabolic Stability Data 201
11.7. Consequences of Chirality on Metabolic Stability 204
11.8. Substrate Specificity of CYP Isozymes 206
11.8.1. CYP1A2 Substrates 206
11.8.2. CYP2D6 Substrates 206
11.8.3. CYP2C9 Substrates 207
11.9. Aldehyde Oxidase 209
Problems 212
References 214
Chapter 12: Plasma Stability 216
12.1. Introduction 216
12.2. Plasma Stability Fundamentals 216
12.2.1. Consequences of Chirality on Plasma Stability 216
12.3. Effects of Plasma Instability 217
12.3.1. Pharmacokinetic Effects of Plasma Degradation 217
12.3.2. Degradation of Biological Drugs 217
12.3.3. Bioanalytical Effects of Plasma Degradation 217
12.3.4. Prodrugs and Antedrugs 217
12.4. Structure Modification Strategies to Improve Plasma Stability 219
12.4.1. Substitute an Amide for an Ester 219
12.4.2. Increase Steric Hindrance 220
12.4.3. Electron-Withdrawing Groups Decrease Plasma Stability for Antedrugs 221
12.4.4. Stabilize Biological Drug Candidates 221
12.5. Strategies for Plasma Stability 221
12.5.1. Diagnose Poor In Vivo Performance 221
12.5.2. Alert Teams to Plasma Hydrolysis Liability 222
12.5.3. Prioritize Compounds for in vivo Animal Studies 223
12.5.4. Prioritize Synthetic Efforts 224
12.5.5. Screening of Prodrugs 224
12.5.6. Guide Structural Modification 224
12.5.7. Verify the Plasma Storage Stability of Bioanalytical Samples 225
Problems 226
References 226
Chapter 13: Solution Stability 228
13.1. Introduction 228
13.2. Solution Stability Fundamentals 228
13.2.1. Degradation in Stock Solutions 228
13.2.2. Degradation During In Vitro Biological or ADME Assay 229
13.2.3. Degradation During In Vivo PK, Efficacy, and Toxicity Studies 229
13.2.4. Degradation Reactions 229
13.3. Effects of Solution Instability 230
13.4. Solution Stability Case Studies 230
13.4.1. pH Stability of a ?-Lactam 230
13.4.2. Selecting Conditions for Purification 230
13.4.3. Diagnose Poor In Vitro Bioassay Performance 231
13.4.4. Prioritize Compounds for In Vivo Animal Studies 232
13.4.5. Structure Elucidation of Degradation Products 232
13.5. Structure Modification Strategies to Improve Solution Stability 232
13.5.1. Eliminate or Modify the Unstable Group 232
13.5.2. Add an Electron-Withdrawing Group 234
13.5.3. Isosteric Replacement of Labile Functional Group 235
13.5.4. Increase Steric Hindrance 235
13.6. Applications of Solution Stability in Drug Discovery 236
13.6.1. Obtain an Early Alert About Stability Liabilities 236
13.6.2. Assist Selection of Conditions for Compound Purification Using Knowledge of Instability 236
13.6.3. Develop Structure-Stability Relationships 236
13.6.4. Diagnose Poor In Vitro Bioassay Performance 236
13.6.5. Diagnose Poor In Vivo Performance 236
13.6.6. Prioritize Compounds for In Vivo Animal Studies 236
13.6.7. Elucidate Structures of Degradation Products to Guide Synthetic Optimization 237
13.6.8. Perform Accelerated Stability Studies to Anticipate Development Liabilities 237
Problems 237
References 237
Chapter 14: Plasma and Tissue Binding 240
14.1. Introduction 240
14.2. Drug Binding in Plasma 240
14.3. Drug Binding in Tissue 243
14.4. Free Drug Hypothesis 243
Free Drug Hypothesis 243
14.5. Pharmacokinetics Principles of Oral Drugs Relevant to Drug Binding 245
14.6. The Useful Application of fu 245
14.7. Misconceptions and Unproductive Strategies for PPB 246
14.8. Best Practices Regarding PPB and Tissue Binding 248
Problems 248
References 249
Chapter 15: Cytochrome P450 Inhibition 250
15.1. Introduction 250
15.2. CYP Inhibition Fundamentals 250
15.2.1. Reversible CYP Inhibition 250
15.2.2. Irreversible CYP Inhibition (Mechanism-Based Inhibition) 252
15.3. Effects of CYP Inhibition 254
15.3.1. Drug Candidate as a Perpetrator of Metabolic Inhibition DDI 254
15.3.2. Drug Candidate as Victim of Metabolic Inhibition 256
15.4. CYP Inhibition Case Studies 256
15.4.1. Consequences of Chirality on CYP Inhibition 257
15.5. Structure Modification Strategies to Reduce CYP Inhibition 257
15.6. Other DDIs 260
15.6.1. Drug Candidate as a Victim or Perpetrator of DDI Inhibition of a Non-CYP Metabolic Enzyme 260
15.6.2. Drug Candidate as a Victim or Perpetrator of DDI of a Transporter 260
15.6.3. Drug Candidate as a Victim or Perpetrator of Metabolic Enzyme Induction 260
15.6.4. DDI Caused by Foods and Dietary Supplements 260
15.7. Regulatory Guidance on DDI 260
15.8. Applications of CYP Inhibition 261
Problems 261
References 262
Chapter 16: hERG Blocking 264
16.1. Introduction 264
16.2. hERG Fundamentals 264
16.3. hERG Blocking Effects 266
16.4. hERG Blocking SAR 267
16.5. Structure Modification Strategies for hERG 268
16.6. Applications of hERG Blocking Assessment 270
Problems 270
References 270
Chapter 17: Toxicity 272
17.1. Introduction 272
17.2. Toxicity Fundamentals 272
17.3. Toxic Effect Categories 273
17.3.1. On-Target Effects 274
17.3.2. Off-Target Effects 274
17.3.3. Reactive Metabolites 274
17.4. Examples of Toxicity Effects 277
17.4.1. Metabolic Enzyme Induction 277
17.4.2. Genetic Toxicity and Carcinogenicity 277
17.4.3. Cytotoxicity 277
17.4.4. Teratogenicity 278
17.4.5. Biochemical Profile Changes 278
17.4.6. Phospholipidosis 278
17.5. In Vivo Toxicity 279
17.6. Case Studies of Toxicity in Drug Discovery 279
17.7. Rules for Off-Target Toxicity by Drug Discovery Compounds 280
17.8. Relationship of Cmax to in vivo Toxicity of Drug Discovery Compounds 280
17.9. Structure Modification Strategies to Improve Safety 280
Problems 282
References 282
Chapter 18: Integrity and Purity 284
18.1. Introduction 284
18.2. Fundamentals of Integrity and Purity 284
18.3. Integrity and Purity Effects 284
18.4. Applications of Integrity and Purity 285
18.4.1. Case Study 286
Problems 286
References 286
Chapter 19: Pharmacokinetics 288
19.1. Introduction 288
19.1.1. Reasons to Study PK 288
19.1.2. PK Parameters from Different Dosing Routes 288
19.2. PK Parameters 289
19.2.1. Volume of Distribution (Vd) 289
19.2.2. Area under the Curve (AUC) 291
19.2.3. Clearance (CL) 292
19.2.4. Half-Life (t½) 294
19.2.5. Bioavailability (F) 295
19.3. Tissue Concentration 295
19.4. Using PK Data in Drug Discovery 295
19.5. Relationship of PK to PD 297
19.6. Applications of PK 297
Problems 301
References 302
Chapter 20: Lead Properties 304
20.1. Introduction 304
20.2. Lead-like Properties 304
20.3. Template Property Conservation 305
20.4. Including Properties in Hit Triage 305
20.5. Fragment-based Screening 306
20.6. Ligand LipophilicITY Efficiency 308
20.7. Conclusions 309
Problems 309
References 310
Chapter 21: Strategies for Integrating Drug-Like Properties into Drug Discovery 312
21.1. Introduction 312
21.2. Start Assessing Drug Properties Early to Prioritize Compounds and Plan Structure Modifications 312
21.3. Assess Drug Properties for all New Compounds Rapidly 313
21.4. Develop Structure-Property Relationships 313
21.5. Optimize Activity and Properties in Parallel 313
21.6. Use Single-property Assays to Guide Specific Modifications 313
21.7. Use Complex Property Methods for Decision-making and Human Modeling 314
21.8. Apply Property Data to Improve Biological Experiments 314
21.9. Use Customized Assays to Answer Specific Research Questions 314
21.10. Diagnose the Root Cause of Inadequate Pharmacokinetics 315
21.11. Run in vitro Assays Using Human Materials to Predict Human Performance 315
Problems 315
References 315
Chapter 22: Methods for Profiling Drug-Like Properties: General Concepts 316
22.1. Introduction 316
22.2. It is Valuable for Medicinal Chemists to Understand the ADMET Assays and Collaborate with ADMET Scientists 316
22.3. Choose an Ensemble of Key Properties to Evaluate 316
22.4. Use Relevant Assay Conditions 316
22.5. Property Data Should Be Readily Available 316
22.6. Evaluate the Cost-benefit Ratio for Assays 317
22.7. Use Well Developed Assays that Are Well Validated 317
Problems 318
References 318
Chapter 23: Lipophilicity Methods 320
23.1. In Silico Lipophilicity Methods 320
23.2. Lipophilicity Methods 322
23.2.1. Scaled-Down Shake Flask Method for Lipophilicity 322
23.2.2. Reversed Phase HPLC Method for Lipophilicity 323
23.2.3. CE Method for Lipophilicity 324
23.3. In-Depth Lipophilicity Methods 324
23.3.1. Shake Flask Method for Lipophilicity 324
23.3.2. pH-Metric Method for Lipophilicity 325
Problems 326
References 326
Chapter 24: pKa Methods 328
24.1. Introduction 328
24.2. In Silico pKa Methods 328
24.3. Laboratory pKa Methods 330
24.3.1. 96-Well Microtiter Plate UV Spectroscopy pKa Method 330
24.3.2. Spectral Gradient Analysis Method for pKa 331
24.3.3. Capillary Electrophoresis Method for pKa 332
24.3.4. Definitive pKa Method: pH-Metric 332
24.3.5. Potentiometric Titration Method for pKa 332
Problems 333
References 333
Chapter 25: Solubility Methods 334
25.1. Introduction 334
25.2. Solubility Calculation Estimation 334
25.3. Software for Solubility 334
25.4. Kinetic Solubility Methods 335
25.4.1. Direct UV Kinetic Solubility Method 337
25.4.2. Nephelometric Kinetic Solubility Method 338
25.4.3. Turbidimetric Kinetic Solubility Method 338
25.4.4. Pseudokinetic Solubility Method 339
25.5. Thermodynamic Solubility Methods 339
25.5.1. Equilibrium Shake Flask Thermodynamic Solubility Method 339
25.5.2. Potentiometric Thermodynamic Solubility Method 340
25.5.3. Thermodynamic Solubility in Various Solvents 340
25.6. Customized Solubility Methods 340
25.7. Dissolution Rate Measurement 342
25.8. DMSO Solubility 342
25.9. Commercial CRO Labs Offering Solubility Measurement 342
25.10. Strategy for Solubility Measurement 343
Problems 343
References 344
Chapter 26: Permeability Methods 346
26.1. Introduction 346
26.2. Computational Prediction of Permeability 346
26.2.1. Permeability Prediction Using Structural Property Rules 346
26.2.2. Permeability Prediction Using In Silico Methods 346
26.3. In Vitro Permeability Methods 347
26.3.1. Liposomal Permeability Method 347
26.3.2. IAM High-Performance Liquid Chromatography Permeability Method 347
26.3.3. Caco-2 Monolayer Permeability Method 348
26.3.3.1. General Protocol for Monolayer Permeability 349
26.3.3.2. Additional Considerations for Caco-2 Studies 350
26.3.4. MDCK Monolayer Permeability Method 352
26.3.4.1. MDCKII-LE Monolayer Permeability Method 352
26.3.5. Monolayer Permeability Method with Other Cell Lines 352
26.3.6. PAMPA—Parallel Artificial Membrane Permeability Assay 353
26.3.6.1. General Protocol for PAMPA Permeability 353
26.3.6.2. Additional Considerations for PAMPA Studies 353
26.3.7. Comparison of Caco-2 and PAMPA Methods 354
26.4. In-Depth Permeability Methods 355
26.4.1. Ussing Chamber 355
26.4.2. Cannulated In Vivo Hepatic Portal Vein 355
26.4.3. Perfusion In Vivo Methods 355
26.4.4. In Vivo Pharmacokinetics Method 355
26.5. Applications of Permeability in Drug Discovery 356
Problems 356
References 356
Chapter 27: Transporter Methods 360
27.1. Introduction 360
27.2. In Silico Transporter Methods 360
27.2.1. In Silico Methods for P-gp 360
27.2.2. In Silico Methods for BCRP 360
27.2.3. In Silico Methods for Other Transporters 360
27.3. In Vitro Transporter Methods 361
27.3.1. Bidirectional Cell Monolayer Transwell Permeability Methods for Transporter Studies 361
27.3.1.1. Caco-2 Permeability Method for Transporters 362
27.3.1.2. Transfected Cell Line Permeability Method for Transporters 362
27.3.2. Plated Cell Monolayer Uptake Method for Transporters 364
27.3.3. Cell Suspension Oil Spin Method for Transporters 364
27.3.4. Sandwich-Cultured Hepatocyte Method for Transporters 365
27.3.5. Media Loss Method for Transporters 365
27.3.6. Oocyte Uptake Method for Transporters 366
27.3.7. Inverted Vesicle Assay for Transporters 366
27.3.8. ATPase Assay for ABC Transporters 367
27.3.9. Calcein AM Assay for P-gp Inhibitor 368
27.4. In Vivo Methods for Transporters 369
27.4.1. Genetic Knockout Animal Experiments for Transporters 369
27.4.2. Chemical Knockout Experiments for Transporters 369
Problems 369
References 369
Chapter 28: Blood-Brain Barrier Methods 372
28.1. Introduction 372
28.2. Methods for BBB Permeability 372
28.2.1. Computational and In Silico Methods for BBB Permeability 373
28.2.1.1. Computational Methods for BBB Permeability and Brain Exposure 373
28.2.1.2. In Silico Classification Methods 373
28.2.1.3. QSAR BBB Permeability Methods 373
28.2.1.4. Commercial BBB Permeability Software 373
28.2.2. In Vitro Methods for BBB Permeability 374
28.2.2.1. In Vitro Physicochemical Methods for BBB Permeability 374
28.2.2.2. In Vitro PAMPA-BBB Method for BBB Permeability 375
28.2.2.3. In Vitro DeltalogP Method for BBB Permeability 376
28.2.2.4. In Vitro IAM HPLC Method for BBB Permeability 376
28.2.2.5. In Vitro Surface Activity Method for BBB Permeability 376
28.2.2.6. In Vitro Cell Monolayer Methods for BBB Permeability 377
28.2.2.6.1. In Vitro Microvessel Endothelial Cell Permeability Method for BBB Permeability 377
28.2.2.6.2. In Vitro Caco-2 Method for BBB Permeability 378
28.2.2.6.3. In Vitro MDR1-MDCKII Method for BBB Permeability 378
28.2.2.6.4. In Vitro TR-BBB and TM-BBB Cell Lines for BBB Permeability 378
28.2.3. In Vivo Methods for BBB Permeability 379
28.2.3.1. In Situ Brain Perfusion Method for BBB Permeability 379
28.2.3.2. In Vivo Brain Uptake Index Method for BBB Permeability 380
28.2.3.3. In Vivo Mouse Brain Uptake Method for BBB Permeability and Brain Distribution 380
28.3. Methods for Brain Binding and Distribution 382
28.3.1. In Silico Methods for Brain Binding 382
28.3.2. In Vitro Methods for Brain Binding 382
28.3.2.1. In Vitro Equilibrium Dialysis for Brain Binding 382
28.3.2.2. In Vitro Brain Slice Uptake Method for Brain Binding 383
28.3.2.3. In Vitro Lipid-coated Bead Method for Brain Binding 383
28.3.2.4. In Vitro Ultracentrifugation Brain Homogenate Method for Brain Binding 383
28.3.2.5. In Vitro Microemulsion Retention Factor Method for Brain Binding 383
28.3.3. In Vivo Measurement of Brain Distribution 384
28.3.3.1. In Vivo General Methodology for NeuroPK Studies 384
28.3.3.1.1. Effect of Residual Blood in Brain PK Studies 385
28.3.3.2. In Vivo NeuroPK with Transporter KO Mice 385
28.3.3.3. In Vivo NeuroPK for Cerebrospinal Fluid 385
28.3.3.4. In Vivo Microdialysis Method for Brain Distribution 386
28.3.3.5. In Vivo Imaging for Brain Distribution 386
28.4. Applications of BBB Permeation and Brain Distribution Methods 386
Problems 387
References 387
Chapter 29: Metabolic Stability Methods 392
29.1. Introduction 392
29.2. Metabolic Stability Methods 392
29.3. In Silico Metabolic Stability Methods 393
29.4. In Vitro Metabolic Stability Methods 393
29.4.1. General Aspects of Metabolic Stability Methods 393
29.4.1.1. Metabolic Stability Materials 393
29.4.1.2. Detection Methods for Metabolic Stability 396
29.4.2. In Vitro Microsomal Assay for Metabolic Stability 396
29.4.3. In Vitro S9 Assay for Metabolic Stability 401
29.4.4. In Vitro Hepatocytes Assay for Metabolic Stability 401
29.4.5. In Vitro Phase II Assay for Metabolic Stability 401
29.4.6. Metabolic Reaction Phenotyping 402
29.4.7. In Vitro Metabolite Structure Identification 403
Problems 406
References 406
Chapter 30: Plasma Stability Methods 408
30.1. Introduction 408
30.2. General Protocol for in vitro Plasma Stability 408
30.3. Low-throughput Method for in vitro Plasma Stability 409
30.4. High-throughput Method for in vitro Plasma Stability 409
30.5. Structure Elucidation of Plasma Degradation Products 412
30.6. Strategies for Plasma Stability Measurement 412
Problems 413
References 24
Chapter 31: Solution Stability Methods 414
31.1. Introduction 414
31.2. Methodology for Solution Stability Measurement 414
31.2.1. General Considerations for Drug Discovery Solution Stability Assessment 414
31.2.2. Quenching Problem in Solution Stability Assays 414
31.2.3. An Efficient and Effective Assay Design for Solution Stability 415
31.3. Method for Solution Stability in Biological Assay Media 416
31.4. Example Methods from the Literature for pH Solution Stability 416
31.5. Methods for Solution Stability in Simulated GI Fluids 417
31.6. Identification of Degradation Products from Solution Stability Assays 418
31.7. In-depth Solution Stability Assessment in Late Stage Drug Discovery 418
31.8. Strategy for Solution Stability Assessment 420
Problems 420
References 420
Chapter 32: CYP Inhibition Methods 422
32.1. Introduction 422
32.2. In Silico CYP Inhibition Methods 422
32.3. In Vitro Reversible CYP Inhibition Methods 422
32.3.1. CYP Enzyme Material for CYP Inhibition Methods 423
32.3.2. Probe Substrate Compounds for CYP Inhibition Methods 423
32.3.3. Measurement Techniques for CYP Inhibition Methods 425
32.3.4. Assay Protocols for Reversible CYP Inhibition Methods 425
32.3.4.1. Fluorescent Protocol for Reversible CYP Inhibition 426
32.3.4.2. Single Drug Probe Substrate HLM Protocol for Reversible CYP Inhibition 426
32.3.4.3. Cocktail Drug Probe Substrate HLM Protocol for Reversible CYP Inhibition 427
32.3.4.4. Double Cocktail Assay for Reversible CYP Inhibition 427
32.4. In Vitro Irreversible (TDI) CYP Inhibition Methods 429
32.4.1. Abbreviated TDI Irreversible CYP Inhibition Protocol 429
32.4.2. IC50 Shift TDI CYP Inhibition Protocol 430
32.4.3. In-Depth TDI CYP Inhibition Methods 430
32.5. CYP Inhibition Method Applications 432
Problems 433
References 433
Chapter 33: Plasma and Tissue Binding Methods 436
33.1. Introduction 436
33.2. In Silico Plasma Protein Binding Methods 436
33.2.1. Literature Computational Plasma Protein Binding Methods 436
33.2.2. Commercial In Silico Plasma Protein Binding Methods 436
33.3. In Vitro Binding Methods 437
33.3.1. Equilibrium Dialysis 437
33.3.1.1. Equilibrium Dialysis Method for Plasma 437
33.3.1.2. Equilibrium Dialysis Method for Tissue 438
33.3.1.3. Equilibrium Dialysis Method for Microsomes and Hepatocytes 438
33.3.2. Ultrafiltration Method 438
33.3.3. Ultracentrifugation Method 439
33.3.4. Immobilized Protein HPLC Column Method 439
33.3.5. Microdialysis Method 439
33.3.6. Other Plasma Protein Binding Methods 440
33.4. Red Blood Cell Binding 440
33.5. Contract Research Laboratories for Protein Binding Assays 441
Problems 441
References 441
Chapter 34: hERG Methods 444
34.1. Introduction 444
34.2. In Silico hERG Methods 444
34.3. In Vitro hERG Methods 446
34.3.1. In Vitro Fluorescent Membrane Potential Sensitive Dye Method for hERG 446
34.3.2. In Vitro Radioactive Ligand Binding Method for hERG 447
34.3.3. In Vitro Fluorescence Polarization Ligand Binding Method for hERG 448
34.3.4. In Vitro Rubidium Flux Method for hERG 448
34.3.5. In Vitro Manual Patch-Clamp Method for hERG 448
34.3.6. In Vitro Automated Patch-Clamp Method for hERG 449
34.3.7. In Vitro iPSC Cardiomyocyte Method for hERG 450
34.4. Ex Vivo Methods for hERG Blocking 451
34.4.1. Purkinje Fiber Ex Vivo Method for hERG Blocking 451
34.4.2. Langendorff Perfused Isolated Heart Ex Vivo Method for hERG Blocking 451
34.5. In Vivo Electrocardiography Telemetry for hERG Blocking 451
34.6. Applications of hERG Blocking Methods in Drug Discovery 451
Problems 451
References 452
Chapter 35: Toxicity Methods 454
35.1. Introduction 454
35.2. In Silico Toxicity Methods 454
35.2.1. Knowledge-Based Expert System In Silico Methods for Toxicity 455
35.2.2. Statistics-Based In Silico Methods for Toxicity 455
35.3. In Vitro Toxicity Methods 455
35.3.1. Drug-Drug Interaction Methods 455
35.3.1.1. Metabolic Enzyme Induction Methods 456
35.3.1.1.1. Hepatocyte Method for Metabolic Enzyme Induction 457
35.3.1.1.1.1. In Vitro Measurement of mRNA for Metabolic Enzyme Induction 457
35.3.1.1.1.2. In Vitro Measurement of Enzyme Activity for CYP Induction 457
35.3.1.1.2. In Vitro Nuclear Receptor Activation Methods for CYP Induction 457
35.3.2. In Vitro hERG Blocking Methods 458
35.3.3. In Vitro Genetic Toxicity Methods 458
35.3.3.1. In Vitro Ames Mutagenicity Method 458
35.3.3.2. In Vitro TK Mouse Lymphoma Cell Mutagenicity Method 459
35.3.3.3. In Vitro HPRT Chinese Hamster Ovary Cell Mutagenicity Method 459
35.3.3.4. In Vitro Micronucleus Clastogenicity Method 459
35.3.3.5. In Vitro Comet Clastogenicity Method 459
35.3.3.6. In Vitro GADD45a-GFP Mutagenicity and Clastogenicity Method 460
35.3.4. In Vitro Cytotoxicity Methods 460
35.3.4.1. In Vitro ATP Depletion Cytotoxicity Method 460
35.3.4.2. In Vitro MTT Human Hepatocyte Cytotoxicity Method 460
35.3.4.3. In Vitro LDH Cytotoxicity Method 460
35.3.4.4. In Vitro Neutral Red Cytotoxicity Method 461
35.3.5. In Vitro Embryo Teratogenicity Methods 461
35.3.6. In Vitro Off-Target Selectivity Screens 461
35.3.7. In Vitro Reactive Metabolite Methods 461
35.3.7.1. In Vitro Reactive Metabolite Method Using Glutathione Trapping 461
35.3.7.2. In Vitro Reactive Metabolite Method Using Covalent Protein Binding 461
35.4. In Vivo Toxicity Methods 462
35.4.1. Short-Term In Vivo Toxicity Methods 462
35.4.2. Preclinical and Clinical In Vivo Toxicity Methods 462
35.4.3. In Vivo Toxic Biomarker Methods 463
35.4.3.1. In Vivo Toxicometabonomic Method 463
35.4.3.2. In Vivo Toxicoproteomic Method 464
35.4.3.3. In Vivo Toxicogenomic Method 464
Problems 464
References 464
Chapter 36: Integrity and Purity Methods 468
36.1. Introduction 468
36.2. Samples for Integrity and Purity Profiling 469
36.3. Requirements of Integrity and Purity Profiling Methods 469
36.4. Integrity and Purity Method Characteristics 469
36.4.1. Sample Preparation 470
36.4.2. Sample Component Separation 471
36.4.3. Quantitation 471
36.4.4. Identity Characterization 472
36.5. Follow Up on Negative Identity Results 472
36.6. Example Generic High-Throughput Purity and Integrity Method 473
36.7. Purity and Integrity Case Studies 473
Problems 475
References 475
Chapter 37: Pharmacokinetic Methods 476
37.1. Introduction 476
37.2. Dosing for PK Studies 476
37.2.1. Single Compound Dosing 476
37.2.2. Cassette Dosing 476
37.3. PK Sampling and Sample Preparation 477
37.4. LC/MS/MS Analysis 478
37.5. Advanced PK Studies 479
37.6. Example Pharmacokinetic Data 479
37.7. Tissue Penetration 479
37.8. Unbound Drug Concentration in Plasma or Tissue 481
37.9. Contract Research Laboratories 481
Problems 481
References 482
Chapter 38: Diagnosing and Improving Pharmacokinetic Performance 484
38.1. Introduction 484
38.2. Diagnosing Underlying Property Limitations from PK Performance 485
38.2.1. Diagnosing the Cause of High Clearance or Short Half-Life 485
38.2.2. Diagnosing Cause of Low Oral Bioavailability 485
38.2.3. Diagnosing the Cause of Low AUC or Cmax 486
38.2.4. Diagnosing the Cause of Nonlinear Pharmacokinetics 486
38.3. Case Studies on Diagnosing Unfavorable PK Behavior 486
38.3.1. Pharmacokinetics of CCR5 antagonist UK-427,857 486
38.3.2. Pharmacokinetics of Triazole Antifungal Voriconazole 487
38.3.3. Optimization of a PDE5 Inhibitor 489
Problems 490
References 490
Chapter 39: Prodrugs 492
39.1. Introduction 492
39.2. Prodrug Design Differs with the ADME Process and Administration Route 494
39.3. Using Prodrugs to Improve Solubility 494
39.4. Prodrugs to Increase Passive Permeability 497
39.4.1. Ester Prodrugs for Carboxylic Acids 497
39.4.2. Ester Prodrugs for Alcohols and Phenols 499
39.4.3. Prodrugs Derived from Nitrogen-Containing Functional Group 500
39.5. Transporter-Mediated Prodrugs to Enhance Intestinal Absorption 500
39.6. Prodrugs to Reduce Metabolism 503
39.7. Prodrugs to Target Specific Tissues 504
39.8. Soft Drugs 504
Problems 505
References 505
Chapter 40: Effects of Properties on Biological Assays 508
40.1. Introduction 508
40.2. Effects of Insolubility IN DMSO 510
40.3. Dealing with Insolubility in DMSO 511
40.4. Effects of Insolubility in Aqueous Buffers 511
40.5. Dealing with Insolubility in Aqueous Buffers 513
40.5.1. Modify the Dilution Protocol to Keep Compounds in Solution 513
40.5.2. Assess Compound Solubility and Concentrations 513
40.5.3. Optimize Assays for Low-Solubility Compounds 514
40.5.4. Effects of Permeability in Cell-Based Assays 514
40.5.5. Dealing with Permeability in Cell-Based Assays 515
40.5.6. Effects of Chemical Instability in Bioassays 515
40.5.7. Dealing with Chemical Instability in Bioassays 515
Problems 515
References 516
Chapter 41: Formulation 518
41.1. Introduction 518
41.2. Routes of Administration 518
41.2.1. Oral (PO) 519
41.2.2. Intravenous (IV) 519
41.2.3. Intraperitoneal (IP) 519
41.2.4. Subcutaneous (SC) 520
41.2.5. Intramuscular (IM) 520
41.3. Potency Drives Delivery Opportunities 520
41.4. Formulation Strategies 520
41.4.1. Adjust pH of Dosing Solution 521
41.4.2. Use Co-Solvent 521
41.4.3. Utilize Surfactants 522
41.4.4. Lipid-Based Formulation 523
41.4.5. Drug Complexation 525
41.4.6. Solid Dispersions 526
41.4.7. Particle Size Reduction 526
41.5. Practical Guide for Formulation in Drug Discovery 528
41.5.1. Formulation for PK Studies 528
41.5.2. Formulation for Toxicity Studies 529
41.5.3. Formulation for Pharmacological Activity Studies 529
Problems 529
Answers 530
References 530
Appendix I: Answers to Chapter Problems 532
Chapter 1—Introduction 532
Chapter 2—Benefits of Property Assessment and Good Drug-like Properties 532
Chapter 3—In Vivo Environments Affect Drug Exposure 532
Chapter 4—Prediction Rules for Rapid Property Profiling From Structure 533
Chapter 5—Lipophilicity 534
Chapter 6—pKa 535
Chapter 7—Solubility 535
Chapter 8—Permeability 536
Chapter 9—Transporters 537
Chapter 10—Blood-Brain Barrier 537
Chapter 11—Metabolic Stability 537
Chapter 12—Plasma Stability 538
Chapter 13—Solution Stability 539
Chapter 14—Plasma Protein Binding 539
Chapter 15—Cytochrome P450 Inhibition 540
Chapter 16—hERG Blocking 540
Chapter 17—Toxicity 541
Chapter 18—Purity and Integrity 541
Chapter 19—Pharmacokinetics 541
Chapter 20—Lead Properties 542
Chapter 21—Strategies for Integrating Drug-like Properties into Drug Discovery 542
Chapter 22—Methods for Profiling Drug-like Properties: General Concepts 542
Chapter 23—Lipophilicity Methods 543
Chapter 24—pKa Methods 543
Chapter 25—Solubility Methods 543
Chapter 26—Permeability Methods 544
Chapter 27—Transporter Methods 544
Chapter 28—Blood-Brain Barrier Methods 545
Chapter 29—Metabolic Stability Methods 545
Chapter 30—Plasma Stability Methods 545
Chapter 31—Solution Stability Methods 546
Chapter 32—CYP Inhibition Methods 546
Chapter 33—Plasma Tissue Binding Methods 546
Chapter 34—hERG Methods 546
Chapter 35—Toxicity Methods 547
Chapter 36—Integrity and Purity Methods 547
Chapter 37—Pharmacokinetic Methods 547
Chapter 38—Diagnosing and Improving Pharmacokinetic Performance 548
Chapter 39—Prodrugs 548
Chapter 40—Effects of Properties on Biological Assays 549
Chapter 41—Formulation 550
Appendix II 552
General Reference Books 552
Appendix III: Glossary 554
Index 572
Back Cover 582