Preface ..................................................... xviii
Dedication ..................................................... xx
Part 1. Introductory Concepts ................................... 1
1. Introduction ................................................. 3
Problems ........................................................ 5
References ...................................................... 5
2. Advantages of Good Drug-like Properties ...................... 6
2.1. Drug-like Properties Are an Integral Part of Drug
Discovery ............................................... 6
2.1.1. Many Properties Are of Interest in
Discovery ........................................ 7
2.1.2. Introduction to the Drug Discovery and
Development Process .............................. 8
2.1.3. Development Attrition is Reduced by
Improving Drug Properties ........................ 9
2.1.4. Poor Drug Properties Also Cause Discovery
Inefficiencies ................................... 9
2.1.5. Marginal Drug Properties Cause
Inefficiencies During Development ............... 10
2.1.6. Poor Properties Can Cause Poor Discovery
Research ........................................ 11
2.2. Changing Emphasis on Properties in Discovery ........... 12
2.3. Property Profiling in Discovery ........................ 14
2.4. Drug-like Property Optimization in Discovery ........... 15
Problems .................................................... 15
References .................................................. 16
3. Barriers to Drug Exposure in Living Systems ................. 17
3.1. Introduction to Barriers ............................... 17
3.2. Drug Dosing ............................................ 18
3.3. Barriers in the Mouth and Stomach ...................... 19
3.4. Gastrointestinal Tract Barriers ........................ 20
3.4.1. Permeation of the Gastrointestinal
Cellular Membrane ............................... 22
3.4.2. Passive Diffusion at the Molecular Level ........ 23
3.4.3. Metabolism in the Intestine ..................... 24
3.4.4. Enzymatic Hydrolysis in the Intestine ........... 24
3.4.5. Absorption Enhancement in the Intestine ......... 26
3.5. Barriers in the Bloodstream ............................ 27
3.5.1. Plasma Enzyme Hydrolysis ........................ 27
3.5.2. Plasma Protein Binding .......................... 27
3.5.3. Red Blood Cell Binding .......................... 28
3.6. Barriers in the Liver .................................. 28
3.6.1. Metabolism ...................................... 29
3.6.2. Biliary Excretion ............................... 29
3.7. Barriers in the Kidney ................................. 29
3.8. Blood-Tissue Barriers .................................. 30
3.9. Tissue Distribution .................................... 30
3.10.Consequences of Chirality on Barriers and
Properties ............................................. 31
3.11.Overview of In Vivo Barriers ........................... 31
Problems .................................................... 32
References .................................................. 33
Part 2. Physicochemical Properties ............................. 35
4. Rules for Rapid Property Profiling from Structure ........... 37
4.1. Lipinski Rules ......................................... 37
4.2. Veber Rules ............................................ 39
4.3. Other Rules ............................................ 39
4.4. Application of Rules for Compound Assessment ........... 39
Problems .................................................... 41
References .................................................. 42
5. Lipophilicity ............................................... 43
5.1. Lipophilicity Fundamentals ............................. 43
5.2. Lipophilicity Effects .................................. 45
5.3. Lipophilicity Case Studies and Structure
Modification ........................................... 46
Problems ....................................................... 47
References ..................................................... 47
6. rKa ......................................................... 48
6.1. rKa, Fundamentals ...................................... 48
6.2. rKa Effects ............................................ 50
6.3. rKa Case Studies ....................................... 50
6.4. Structure Modification Strategies for rKa .............. 54
Problems .................................................... 54
References .................................................. 55
7. Solubility .................................................. 56
7.1. Solubility Fundamentals ................................ 57
7.1.1. Solubility Varies with Structure and
Physical Conditions ............................. 57
7.1.2. Dissolution Rate ................................ 57
7.1.3. Structural Properties Affect Solubility ......... 57
7.1.4. Kinetic and Thermodynamic Solubility ............ 60
7.2. Effects of Solubility .................................. 62
7.2.1. Low Solubility Limits Absorption and
Causes Low Oral Bioavailability ................. 62
7.2.2. Good Solubility is Essential for IV
Formulation ..................................... 63
7.2.3. Acceptance Criteria and Classifications
for Solubility .................................. 63
7.2.4. Molecular Properties for Solubility and
Permeability Often are Opposed .................. 67
7.3. Effects of Physiology on Solubility and
Absorption ............................................. 68
7.3.1. Physiology of the Gastrointestinal Tract ........ 68
7.3.2. Species Differences in Gastrointestinal
Tract ........................................... 68
7.3.3 Food Effect ..................................... 69
7.4. Structure Modification Strategies to Improve
Solubility ............................................. 70
7.4.1. Add Ionizable Groups ............................ 71
7.4.2. Reduce Log P .................................... 73
7.4.3. Add Hydrogen Bonding ............................ 73
7.4.4. Add Polar Group ................................. 74
7.4.5. Reduce Molecular Weight ......................... 74
7.4.6. Out-of-Plane Substitution ....................... 75
7.4.7. Construct a Prodrug ............................. 76
7.5. Strategies for Improving Dissolution Rate .............. 77
7.5.1. Reduce Particle Size ............................ 77
7.5.2. Prepare an Oral Solution ........................ 78
7.5.3. Formulate with Surfactants ...................... 78
7.5.4. Prepare a Salt Form ............................. 78
7.6. Salt Form .............................................. 78
7.6.1. Solubility of Salts ............................. 78
7.6.2. Effect of Salt Form on Absorption and Oral
Bioavailability ................................. 80
7.6.3. Salt Selection .................................. 81
7.6.4. Precautions for Using Salt Forms 82
Problems .................................................... 82
References .................................................. 84
8. Permeability ................................................ 86
8.1. Permeability Fundamentals .............................. 86
8.1.1. Passive Diffusion Permeability .................. 87
8.1.2. Endocytosis Permeability ........................ 89
8.1.3. Active Uptake Permeability ...................... 89
8.1.4. Paracellular Permeability ....................... 89
8.1.5. Efflux Permeability ............................. 89
8.1.6. Combined Permeability ........................... 89
8.2. Permeability Effects ................................... 90
8.2.1. Effect of Permeability on Bioavailability ....... 90
8.2.2. Effect of Permeability on Cell-Based
Activity Assays ................................. 91
8.3. Permeability Structure Modification Strategies ......... 92
8.3.1. Ionizable Group to Non-ionizable Group .......... 92
8.3.2. Add Lipophilicity ............................... 92
8.3.3. Isosteric Replacement of Polar Groups ........... 93
8.3.4. Esterify Carboxylic Acid ........................ 93
8.3.5. Reduce Hydrogen Bonding and Polarity ............ 94
8.3.6. Reduce Size ..................................... 94
8.3.7. Add Nonpolar Side Chain ......................... 96
8.3.8. Prodrug ......................................... 96
Problems .................................................... 97
References .................................................. 98
Part 3. Disposition, Metabolism, and Safety ................... 101
9. Transporters ............................................... 103
9.1. Transporter Fundamentals .............................. 103
9.2. Transporter Effects ................................... 104
9.2.1. Transporters in Intestinal Epithelial
Cells .......................................... 108
9.2.2. Transporters in Liver Hepatocytes .............. 108
9.2.3. Transporters in Kidney Epithelial Cells ........ 110
9.2.4. Transporters in Blood-Brain Barrier
Endothelial Cells .............................. 110
9.2.5. Consequences of Chirality on Transporters ...... 110
9.3. Efflux Transporters ................................... 111
9.3.1. P-glycoprotein (MDR1, ABCB1) [Efflux] .......... 111
9.3.2. Breast Cancer Resistance Protein
(BCRP, ABCG2)[Efflux] .......................... 116
9.3.3. Multidrug Resistance Protein 2 (MRP2,
ABCC2) [Efflux] ................................ 116
9.3.4. Efflux Transporters in the BBB ................. 116
9.4. Uptake Transporters ................................... 117
9.4.1. Organic Anion Transporting Polypeptides
(OATPs, SLCOs) [Uptake] ........................ 117
9.4.2. Di/Tri Peptide Transporters (PEPT1,
PEPT2) [Uptake] ................................ 117
9.4.3. Organic Anion Transporters (OATs)
[Uptake] ....................................... 118
9.4.4. Organic Cation Transporter (OCT) [Uptake] ...... 118
9.4.5. Large Neutral Amino Acid Transporter
(LAT1) [Uptake] ................................ 118
9.4.6. Monocarboxyiic Acid Transporter (MCT1)
[Uptake] ....................................... 118
9.4.7. Other Uptake Transporters ...................... 118
9.4.8. Structure Modification Strategies for
Uptake Transporters ............................ 119
Problems ................................................... 119
References ................................................. 120
10.Blood-Brain Barrier ........................................ 122
10.1.BBB Fundamentals ...................................... 123
10.1.1.BBB Permeation Mechanisms ...................... 124
10.1.2.Brain Distribution Mechanisms .................. 125
10.1.3.Brain-CSF Barrier .............................. 127
10.1.4.Interpreting Data for Brain Penetration ........ 128
10.2.Effects of Brain Penetration .......................... 129
10.3.Structure-BBB Penetration Relationships ............... 130
10.4.Structure Modification Strategies to Improve
Brain Penetration ..................................... 131
10.4.1.Reduce Pgp Efflux .............................. 132
10.4.2.Reduce Hydrogen Bonds .......................... 132
10.4.3.Increase Lipophilicity ......................... 133
10.4.4.Reduce MW ...................................... 133
10.4.5.Replace Carboxylic Acid Groups ................. 133
10.4.6.Add an Intramolecular Hydrogen Bond ............ 133
10.4.7.Modify or Select Structures for
Affinity to Uptake Transporters ................ 133
Problems ................................................... 134
References ................................................. 135
11.Metabolic Stability ........................................ 137
11.1.Metabolic Stability Fundamentals ...................... 138
11.1.1.Phase I Metabolism ............................. 139
11.1.2.Phase II Metabolism ............................ 143
11.2.Metabolic Stability Effects ........................... 145
11.3.Structure Modification Strategies for Phase I
Metabolic Stability ................................... 146
11.3.1.Block Metabolic Site By Adding Fluorine ........ 147
11.3.2.Block Metabolic Site By Adding Other
Blocking Groups ................................ 149
11.3.3.Remove Labile Functional Group ................. 150
11.3.4.Cyclization .................................... 151
11.3.5.Change Ring Size ............................... 151
11.3.6.Change Chirality ............................... 152
11.3.7.Reduce Lipophilicity ........................... 152
11.3.8.Replace Unstable Groups ........................ 153
11.4.Structure Modification Strategies for Phase II
Metabolic Stability ................................... 154
11.4.1.Introduce Electron-Withdrawing Groups
and Steric Hindrance ........................... 154
11.4.2.Change Phenolic Hydroxyl to Cyclic
Urea or Thiourea ............................... 155
11.4.3.Change Phenolic Hydroxyl to Prodrug ............ 155
11.5.Applications of Metabolic Stability Data .............. 156
11.6.Consequences of Chirality on Metabolic
Stability ........................................ 160
11.7.Substrate Specificity of CYP Isozymes ............ 162
11.7.1.CYP 1A2 Substrates ........................ 162
11.7.2.CYP2D6 Substrates ......................... 163
11.7.3.CYP2C9 Substrates ......................... 164
Problems ................................................... 165
References ................................................. 167
12.Plasma Stability ........................................... 169
12.1.Plasma Stability Fundamentals ......................... 169
12.1.1.Consequences of Chirality on Plasma
Stability ...................................... 170
12.2.Effects of Plasma Stability ........................... 170
12.3.Structure Modification Strategies to Improve
Plasma Stability ...................................... 172
12.3.1.Substitute an Amide for an Ester ............... 172
12.3.2.Increase Steric Hindrance ...................... 173
12.3.3.Electron-Withdrawing Groups Decrease
Plasma Stability for Antedrug .................. 173
12.4.Applications of Plasma Stability Data ................. 174
12.4.1.Diagnose Poor In Vivo Performance .............. 174
12.4.2.Alert Teams to a Liability ..................... 174
12.4.3.Prioritize Compounds for In Vivo Animal
Studies ........................................ 174
12.4.4.Prioritize Synthetic Efforts ................... 175
12.4.5.Screening of Prodrugs .......................... 175
12.4.6.Guide Structural Modification .................. 176
Problems ....................................... 176
References ..................................... 177
13.Solution Stability ......................................... 178
13.1.Solution Stability Fundamentals ....................... 178
13.2.Effects of Solution Instability ....................... 180
13.3.Structure Modification Strategies to Improve
Solution Stability .................................... 180
13.3.1.Eliminate or Modify the Unstable Group ......... 180
13.3.2.Add an Electron-Withdrawing Group .............. 181
13.3.3.Isosteric Replacement of Labile
Functional Group ............................... 182
13.3.4.Increase Steric Hindrance ...................... 182
13.4.Applications of Solution Stability Data ............... 183
Problems ................................................... 185
References ................................................. 185
14.Plasma Protein Binding ..................................... 187
14.1.Plasma Protein Binding Fundamentals ................... 187
14.1.1.Consequences of Chirality on PPB ............... 189
14.2.PPB Effects ........................................... 190
14.2.1.Impact of PPB on Distribution .................. 191
14.2.2.Effect of PPB on Clearance ..................... 192
14.2.3.Effect of PPB on Pharmacology .................. 192
14.3.PPB Case Studies ...................................... 193
14.4.Structure Modification Strategies for PPB ............. 193
14.5.Strategy for PPB in Discovery ......................... 194
14.6.Red Blood Cell Binding ................................ 194
Problems ................................................... 194
References ................................................. 195
15.Cytochrome P450 Inhibition ................................. 197
15.1.CYP Inhibition Fundamentals ........................... 197
15.2.Effects of CYP Inhibition ............................. 199
15.3.CYP Inhibition Case Studies ........................... 201
15.3.1.Consequences of Chirality on CYP
Inhibition ..................................... 202
15.4.Structure Modification Strategies to Reduce CYP
Inhibition ............................................ 203
15.5.Reversible and Irreversible CYP Inhibition ............ 204
15.6.Other DDI Issues ...................................... 205
15.6.1.Candidate as Victim to a Metabolism
Inhibition Perpetrator ......................... 205
15.6.2.Candidate as a Victim or Perpetrator at
a Transporter .................................. 206
15.6.3.Candidate as a Victim or Perpetrator of
Metabolic Enzyme Induction ..................... 206
Problems ................................................... 206
References ................................................. 207
16.hERG Blocking .............................................. 209
16.1.hERG Fundamentals ..................................... 209
16.2.hERG Blocking Effects ................................. 211
16.3.hERG Blocking Structure-Activity Relationship ......... 212
16.4.Structure Modification Strategies for hERG ............ 213
Problems ................................................... 213
References ................................................. 214
Additional Reading ......................................... 214
17.Toxicity ................................................... 215
17.1.Toxicity Fundamentals ................................. 216
17.1.1.Toxicity Terms and Mechanisms .................. 217
17.1.2.Toxicity Mechanisms ............................ 217
17.2.Toxicity Case Studies ................................. 221
17.3.Structure Modification Strategies to Improve
Safety ................................................ 222
Problems ................................................... 222
References ................................................. 223
18.Integrity and Purity ....................................... 224
18.1.Fundamentals of Integrity and Purity .................. 224
18.2.Integrity and Purity Effects .......................... 224
18.3.Applications of Integrity and Purity .................. 226
18.3.1.Case Study ..................................... 226
Problems ................................................... 227
References ................................................. 227
19.Pharmacokinetics ........................................... 228
19.1.Introduction to Pharmacokinetics ...................... 228
19.2.PK Parameters ......................................... 229
19.2.1.Volume of Distribution ......................... 229
19.2.2.Area Under the Curve ........................... 231
19.2.3.Clearance ...................................... 231
19.2.4.Half-life ...................................... 233
19.2.5.Bioavailability ................................ 234
19.3.Effects of Plasma Protein Binding on PK
Parameters ............................................ 234
19.4.Tissue Uptake ......................................... 234
19.5.Using PK Data in Drug Discovery ....................... 235
Problems ................................................... 240
References ................................................. 241
20.Lead-like Compounds ........................................ 242
20.1.Lead-likeness ......................................... 242
20.2.Template Conservation ................................. 244
20.3.Triage ................................................ 245
20.4.Fragment-Based Screening .............................. 245
20.5.Lead-like Compounds Conclusions ....................... 247
Problems ................................................... 247
References ................................................. 248
21.Strategies for Integrating Drug-like Properties into
Drug Discovery ............................................. 249
21.1.Assess Drug-like Properties Early ..................... 249
21.2.Rapidly Assess Drug-like Properties for All New
Compounds ............................................. 250
21.3.Develop Structure-Property Relationships .............. 250
21.4.Iterative Parallel Optimization ....................... 251
21.5.Obtain Property Data that Relates Directly to
Structure ............................................. 251
21.6.Apply Property Data to Improve Biological
Experiments ........................................... 252
21.7.Utilize Customized Assays to Answer Specific
Project Questions ..................................... 252
21.8.Diagnose Inadequate Performance in Complex
Systems Using Individual Properties ................... 252
Problems ................................................... 253
References ................................................. 253
Part 4. Methods ............................................... 255
22.Methods for Profiling Drug-like Properties:
General Concepts ........................................... 257
22.1.Property Data Should be Rapidly Available ............. 257
22.2.Use Relevant Assay Conditions ......................... 257
22.3.Evaluate the Cost-to-Benefit Ratio for Assays ......... 257
22.4.Choose an Ensemble of Key Properties to
Evaluate .............................................. 258
22.5.Use Well-Developed Assays ............................. 259
Problems ................................................... 259
References ................................................. 259
23.Lipophilicity Methods ...................................... 260
23.1.In Silico Lipophilicity Methods ....................... 260
23.2.Experimental Lipophilicity Methods .................... 264
23.2.1.Scaled-Down Shake Flask Method for
Lipophilicity .................................. 265
23.2.2.Reversed-Phase HPLC Method for
Lipophilicity .................................. 266
23.2.3.Capillary Electrophoresis Method for
Lipophilicity .................................. 267
23.3.In-Depth Lipophilicity Methods ........................ 267
23.3.1.Shake Flask Method for Lipophilicity ........... 267
23.3.2.pH-Metric Method for Lipophilicity ............. 268
Problems ................................................... 268
References ................................................. 269
24. rKa Methods ............................................... 271
24.1.In Silico rKa Methods ................................. 271
24.2.Experimental rKa Methods .............................. 273
24.2.1.Spectral Gradient Analysis Method for rKa ...... 273
24.2.2.Capillary Electrophoresis Method for rKa ....... 273
24.3.In-Depth rKa Method: pH-Metric ........................ 274
Problems ................................................... 275
References ................................................. 275
25.Solubility Methods ......................................... 276
25.1.Literature Solubility Calculation Methods ............. 276
25.2.Commercial Software for Solubility .................... 277
25.3.Kinetic Solubility Methods ............................ 278
25.3.1.Direct UV Kinetic Solubility Method ............ 278
25.3.2.Nephelometric Kinetic Solubility Method ........ 280
25.3.3.Turbidimetric In Vitro Solubility Method ....... 281
25.3.4.Customized Kinetic Solubility Method ........... 282
25.4 Thermodynamic Solubility Methods ...................... 283
25.4.1.Equilibrium Shake Flask Thermodynamic
Solubility Method .............................. 283
25.4.2.Potentiometric In Vitro Thermodynamic
Solubility Method .............................. 283
25.4.3.Thermodynamic Solubility in Various
Solvents ....................................... 284
Problems ................................................... 285
References ................................................. 285
26.Permeability Methods ....................................... 287
26.1.In Silico Permeability Methods ........................ 287
26.2.In Vitro Permeability Methods ......................... 288
26.2.1.IAM HPLC ....................................... 288
26.2.2.Cell Layer Method for Permeability ............. 288
26.2.3.Artificial Membrane Permeability Assay ......... 292
26.2.4.Comparison of Caco-2 and PAMPA Methods ......... 293
26.3.In Depth Permeability Methods ......................... 294
Problems ................................................... 295
References ................................................. 296
27 Transporter Methods ........................................ 299
27.1.In Silico Transporter Methods ......................... 299
27.2.In Vitro Transporter Methods .......................... 300
27.2.1.Cell Layer Permeability Methods for
Transporters ................................... 300
27.2.2.Uptake Method for Transporters ................. 304
27.2.3.Oocyte Uptake Method for Transporters .......... 304
27.2.4.Inverted Vesicle Assay for Transporters ........ 305
27.2.5.ATPase Assay for ATP Binding Cassette
Transporters ................................... 305
27.2.6.Calcein AM Assay for Pgp Inhibitor ............. 306
27.3.In Vivo Methods for Transporters ...................... 307
27.3.1.Genetic Knockout Animal Experiments for
Transporters ................................... 307
27.3.2.Chemical Knockout Experiments for
Transporters ................................... 307
Problems ................................................... 308
References ................................................. 308
28.Blood-Brain Barrier Methods ................................ 311
28.1.In Silico Methods for BBB ............................. 312
28.1.1.Classification Models .......................... 312
28.1.2.Quantitative Structure-Activity
Relationship Methods ........................... 312
28.1.3.Commercial Software ............................ 313
28.2.In Vitro Methods for BBB .............................. 314
28.2.1.Physicochemical Methods for BBB ................ 314
28.2.2.Cell-based In Vitro Methods [BBB
Permeability] .................................. 317
28.3.In Vivo Methods for BBB ............................... 319
28.3.1.B/P Ratio or Log BB [Brain Distribution] ....... 319
28.3.2.Brain Uptake Index [BBB Permeability] .......... 320
28.3.3.In Situ Perfusion [BBB Permeability,
Log PS, μL/min/g] .............................. 321
28.3.4.Mouse Brain Uptake Assay [BBB
Permeability and Brain Distribution] ........... 322
28.3.5.Microdialysis Method for BBB ................... 323
28.3.6.Cerebrospinal Fluid Method for BBB ............. 324
28.4.Assessment Strategy for Brain Penetration ............. 324
Problems ................................................... 325
References ................................................. 325
29.Metabolic Stability Methods ................................ 329
29.1.In Silico Metabolic Stability Methods ................. 331
29.2.In Vitro Metabolic Stability Methods .................. 331
29.2.1.General Aspects of Metabolic Stability
Methods ........................................ 331
29.2.2.In Vitro Microsomal Assay for Metabolic
Stability ...................................... 335
29.2.3.In Vitro S9 Assay for Metabolic Stability ...... 338
29.2.4.In Vitro Hepatocytes Assay for Metabolic
Stability ...................................... 340
29.2.5.In Vitro Phase II Assay for Metabolic
Stability ...................................... 340
29.2.6.Metabolic Phenotyping .......................... 341
29.2.7.In Vitro Metabolite Structure
Identification ................................. 342
Problems ................................................... 345
References ................................................. 346
30.Plasma Stability Methods ................................... 348
Problems ................................................... 351
References ................................................. 351
31.Solution Stability Methods ................................. 353
31.1.General Method for Solution Stability Assays .......... 353
31.2.Method for Solution Stability in Biological
Assay Media ........................................... 355
31.3.Methods for pH Solution Stability ..................... 356
31.4.Methods for Solution Stability in Simulated
Gastrointestinal Fluids ............................... 356
31.5.Identification of Degradation Products from
Solution Stability Assays ............................. 357
31.6.In-Depth Solution Stability Methods for Late
Stages of Drug Discovery .............................. 357
Problems ................................................... 358
References ................................................. 358
32.CYP Inhibition Methods ..................................... 360
32.1.In Silico CYP Inhibition Methods ...................... 360
32.2.In Vitro CYP Inhibition Methods ....................... 361
32.2.1.Fluorescent Assay for CYP Inhibition ........... 364
32.2.2.Single Substrate HLM Assay for CYP
Inhibition ..................................... 365
32.2.3.Cocktail Substrate HLM Assay for CYP
Inhibition ..................................... 365
32.2.4.Double Cocktail Assay for CYP Inhibition ....... 367
32.3.CYP Inhibition Assessment Strategy .................... 367
Problems ................................................... 368
References ................................................. 368
33.Plasma Protein Binding Methods ............................. 372
33.1.In Silico PPB Methods ................................. 372
33.1.1.Literature In Silico PPB Methods ............... 372
33.1.2.Commercial In Silico PPB Methods ............... 372
33.2.In Vitro PPB Methods .................................. 373
33.2.1.Equilibrium Dialysis Method .................... 373
33.2.2.Ultrafiltration Method ......................... 374
33.2.3.Ultracentrifugation Method ..................... 375
33.2.4.Immobilized Protein High-Performance
Liquid Chromatography Column Method ............ 375
33.2.5.Microdialysis Method ........................... 375
33.2.6.Other PPB Methods .............................. 376
33.3.Red Blood Cell Binding ................................ 376
Problems ................................................... 376
References ................................................. 377
34.hERG Methods ............................................... 378
34.1.In Silico hERG Methods ................................ 379
34.2.In Vitro hERG Methods ................................. 379
34.2.1.Membrane Potential-Sensitive Dye Method
for hERG ....................................... 379
34.2.2.Ligand Binding Method for hERG ................. 381
34.2.3.Rubidium Efflux Method for hERG ................ 381
34.2.4.Patch-Clamp Method for hERG .................... 381
34.2.5.High-Throughput Patch-Clamp Method for
hERG ........................................... 383
34.3.In Vivo hERG Methods .................................. 384
Problems ................................................... 384
References ................................................. 385
35.Toxicity Methods ........................................... 386
35.1.In Silico Toxicity Methods ............................ 387
35.1.1.Knowledge-Based Expert System In Silico
Toxicity Methods ............................... 387
35.1.2.Statistically Based In Silico Toxicity
Methods ........................................ 388
35.2.In Vitro Toxicity Assays .............................. 388
35.2.1.Drug-Drug Interaction .......................... 388
35.2.2.hERG Block Assays .............................. 389
35.2.3.Mutagenicity/Genotoxicity ...................... 389
35.2.4.Cytotoxicity ................................... 391
35.2.5.Teratogenicity: Zebrafish Model ................ 392
35.2.6.Selectivity Screens ............................ 392
35.2.7.Reactivity Screens ............................. 393
35.3.In Vivo Toxicity ...................................... 393
35.3.1.Discovery In Vivo Toxicity ..................... 393
35.3.2.Preclinical and Clinical In Vivo Toxicity ...... 393
35.3.3.Biomarkers of In Vivo Toxic Responses .......... 395
Problems .................................................. 396
References ................................................ 396
36.Integrity and Purity Methods ............................... 399
36.1.Criteria for Integrity and Purity Assays .............. 399
36.2.Samples for Integrity and Purity Profiling ............ 400
36.3.Requirements of Integrity and Purity Profiling
Methods ............................................... 401
36.4.Integrity and Purity Method Advice .................... 401
36.4.1.Sample Preparation ............................. 402
36.4.2.Component Separation ........................... 402
36.4.3.Quantitation ................................... 403
36.4.4.Identity Characterization ...................... 404
36.5.Follow-up on Negative Identity Results ................ 405
36.6.Example Method ........................................ 405
36.7.Method Case Studies ................................... 406
Problems ................................................... 407
References ................................................. 407
37.Pharmacokinetic Methods .................................... 409
37.1.PK Dosing ............................................. 409
37.1.1.Single-Compound Dosing ......................... 409
37.1.2.Cassette Dosing ................................ 409
37.2.PK Sampling and Sample Preparation .................... 410
37.3.Instrumental Analysis ................................. 411
37.4.Example Pharmacokinetic Data .......................... 412
37.5.Tissue Uptake ......................................... 413
Problems ................................................... 414
References ................................................. 414
Part 5. Specific Topics ....................................... 417
38.Diagnosing and Improving Pharmacokinetic Performance ....... 419
38.1.Diagnosing Underlying Property Limitations
from PK Performance ................................... 420
38.1.1.High Clearance After IV Injection .............. 420
38.1.2.Low Oral Bioavailability ....................... 421
38.2.Case Studies on Interpreting Unusual PK
Performance ........................................... 421
38.2.1.PK of CCR5 Antagonist UK-427,857 ............... 421
38.2.2.PK of Triazole Antifungal Voriconazole ......... 422
Problems ................................................... 424
References ................................................. 424
39.Prodrugs ................................................... 426
39.1.Using Prodrugs to Improve Solubility .................. 428
39.2.Prodrugs to Increase Passive Permeability ............. 430
39.2.1.Ester Prodrugs for Carboxylic Acids ............ 431
39.2.2.Ester Prodrugs for Alcohols and Phenols ........ 432
39.2.3.Prodrugs Derived from Nitrogen-Containing
Functional Group ............................... 433
39.3.Transporter-Mediated Prodrugs to Enhance
Intestinal Absorption ................................. 434
39.4.Prodrugs to Reduce Metabolism ......................... 435
39.5.Prodrugs to Target Specific Tissues ................... 436
39.6.Soft Drugs ............................................ 437
Problems ................................................... 437
References ................................................. 438
40.Effects of Properties on Biological Assays ................. 439
40.1.Effects of Insolubility in DMSO ....................... 441
40.2.Dealing with Insolubility in DMSO ..................... 443
40.3.Effects of Insolubility in Aqueous Buffers ............ 443
40.4.Dealing with Insolubility in Aqueous Buffers .......... 445
40.4.1.Modify the Dilution Protocol to Keep
Compounds in Solution .......................... 445
40.4.2.Assess Compound Solubility and
Concentrations ................................. 446
40.4.3.Optimize Assays for Low Solubility
Compounds ...................................... 447
40.4.4.Effects of Permeability in Cell-Based
Assays ......................................... 448
40.4.5.Dealing with Permeability in Cell-Based
Assays ......................................... 448
40.4.6.Effects of Chemical Instability in
Bioassays ...................................... 448
40.4.7.Dealing with Chemical Instability in
Bioassays ...................................... 449
Problems ................................................... 449
References ................................................. 450
41.Formulation ................................................ 453
41.1.Routes of Administration .............................. 454
41.2.Potency Drives Delivery Opportunities ................. 455
41.3.Formulation Strategies ................................ 456
41.4.Practical Guide for Formulation in Drug
Discovery ............................................. 462
41.4.1.Formulation for PK Studies ..................... 463
41.4.2.Formulation for Toxicity Studies ............... 464
41.4.3.Formulation for Pharmacological Activity
Studies ........................................ 464
Problems ................................................... 465
References ................................................. 465
Appendix I Answers to Chapter Problems ...................... 468
Appendix II General References ............................... 492
Appendix III Glossary ......................................... 493
Index ......................................................... 514
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