Transporters and Drug-Metabolizing Enzymes in Drug Toxicity

Albert P. Li, PhD, is President and CEO of In Vitro ADMET Laboratories. Dr. Li was previously Senior Fellow of Monsanto Company and Research Professor of St. Louis University Medical School. He is Chair of the Scientific Advisory Board for the Institute for Scientific Communication and the editor/co-editor of several books for Wiley, including Drug-Drug Interactions in Pharmaceutical Development (2007) and Enzyme Inhibition in Drug Discovery and Development (2009). He currently has over 180 publications in peer-reviewed journals and frequently serves as chair and speaker in national and international conferences and symposia on drug metabolism and toxicology.<

Preface xix

List of Contributors xxi

Part I Overview 1

1 Overview: Drug Metabolism, Transporter-Mediated Uptake and Efflux, and Drug Toxicity 3
Albert P. Li

1.1 Drug Toxicity as a Challenge in Drug Development 3

1.2 Fate of an Orally Administered Drug 4

1.3 The Multiple Determinant Hypothesis for Idiosyncratic Drug Toxicity 5

1.4 Concluding Remarks 7

1.4.1 A Comprehensive Approach to Safety Evaluation in Drug Development 7

1.4.2 The Dose Makes the Poison – Paracelsus Updated 8

References 8

2 Transporter, Drug Metabolism, and Drug-Induced Liver Injury in Marketed Drugs 11
Minjun Chen, Kristin Ashby, and Yue Wu

2.1 Introduction 11

2.2 Hepatic Metabolism 12

2.2.1 Phase I Metabolism 12

2.2.2 Phase II Metabolism 14

2.3 Reactive Metabolite Formation and Assessment 14

2.3.1 Metabolism and Reactive Metabolites 15

2.3.2 Dose and Reactive Mtabolites 16

2.3.3 Structural Alerts for Avoiding Reactive Metabolites 16

2.3.4 Experimental Approaches for Assessing Reactive Metabolites 18

2.3.4.1 Covalent Binding Assay 18

2.3.4.2 Electrophile Trapping Experiments 18

2.3.4.3 Time Dependent Inactivation of CYP450 Enzymes 19

2.4 Hepatic Transporters 20

2.5 Genetic Variants and Their Impact for Pharmacokinetic Behavior and Safety 24

2.5.1 CYP3A424

2.5.2 CYP3A526

2.5.3 CYP2D626

2.5.4 CYP2C927

2.5.5 CYP2C1927

2.5.6 CYP2B628

2.5.7 UGT1A128

2.5.8 NAT2 28

2.5.9 Hepatic Transporters 29

2.6 Summary 29

Acknowledgment 30

Disclaimer 30

References 30

3 Drug-Metabolism Enzymes and Transporter Activities as Risk Factors of Selected Marketed Drugs Associated with Drug-Induced Fatalities 41
Albert P. Li

3.1 Introduction 41

3.2 Acetaminophen 41

3.2.1 Drug Metabolism and Toxicity 42

3.2.2 Transporters and Toxicity 42

3.2.3 Risk Factors 43

3.3 Cerivastatin 43

3.3.1 Drug Metabolism and Toxicity 43

3.3.2 Transporter and Toxicity 44

3.3.3 Risk Factors 44

3.4 Felbamate 45

3.4.1 Drug Metabolism and Toxicity 45

3.4.2 Transporters and Toxicity 46

3.4.3 Risk Factors 46

3.5 Flucloxacillin 46

3.5.1 Drug Metabolism and Toxicity 46

3.5.2 Transporters and Toxicity 47

3.5.3 Risk Factors 47

3.6 Nefazodone 47

3.6.1 Drug Metabolism and Toxicity 48

3.6.2 Transporters and Toxicity 48

3.6.3 Risk Factors 48

3.7 Obeticholic Acid 49

3.7.1 Drug Metabolism and Toxicity 49

3.7.2 Transporters and Toxicity 50

3.7.3 Risk Factors 50

3.8 Sitaxentan 50

3.8.1 Drug Metabolism and Toxicity 51

3.8.2 Transporters and Toxicity 51

3.8.3 Risk Factors 51

3.9 Sorivudine 52

3.9.1 Drug Metabolism and Toxicity 52

3.9.2 Transporters and Toxicity 52

3.9.3 Risk Factors 52

3.10 Tacrine 52

3.10.1 Drug Metabolism and Toxicity 54

3.10.2 Transporters and Toxicity 54

3.10.3 Risk Factors 54

3.11 Terfenadine 55

3.11.1 Drug Metabolism and Toxicity 55

3.11.2 Transporter and Toxicity 56

3.11.3 Risk Factors 56

3.12 Troglitazone (Rezulin®) 56

3.12.1 Drug Metabolism and Toxicity 57

3.12.2 Transporter and Toxicity 57

3.12.3 Risk Factors 58

3.13 Trovafloxacin 58

3.13.1 Metabolism and Toxicity 59

3.13.2 Transporters and Toxicity 59

3.13.3 Risk Factors 59

3.14 Conclusions 60

References 61

Part II Drug Metabolizing Enzymes and Drug Toxicity 79

4 Drug-Metabolizing Enzymes and Drug Toxicity 81
Albert P. Li

4.1 Introduction 81

4.2 Drug-Metabolism Enzymes Involved in Metabolic Activation and Detoxification 81

4.3 Cytochrome P450 Monooxygenase (CYP) 82

4.3.1 CYP1A 82

4.3.1.1 Metabolic Activation 82

4.3.1.2 Drug Substrates 83

4.3.1.3 Inducers 83

4.3.1.4 Inhibitors 83

4.3.1.5 Individual Variations 83

4.3.1.6 Involvement in Drug Toxicity 83

4.3.2 CYP2A6 84

4.3.2.1 Substrates 84

4.3.2.2 Inducers 84

4.3.2.3 Inhibitors 84

4.3.2.4 Individual Variations 85

4.3.2.5 Involvement in Drug Toxicity 85

4.3.3 CYP2B6 85

4.3.3.1 Substrates 85

4.3.3.2 Inducers 86

4.3.3.3 Inhibitors 86

4.3.3.4 Individual Variations 86

4.3.3.5 Involvement in Drug Toxicity 86

4.3.4 CYP2C8 87

4.3.4.1 Substrates 87

4.3.4.2 Inducers 87

4.3.4.3 Inhibitors 87

4.3.4.4 Individual Variations 88

4.3.4.5 Involvement in Drug Toxicity 88

4.3.5 CYP2C9 88

4.3.5.1 Substrates 88

4.3.5.2 Inducers 88

4.3.5.3 Inhibitors 88

4.3.5.4 Individual Variations 89

4.3.5.5 Involvement in Drug Toxicity 89

4.3.6 CYP2C19 89

4.3.6.1 Substrates 89

4.3.6.2 Inducers 89

4.3.6.3 Inhibitors 89

4.3.6.4 Individual Variations 90

4.3.6.5 Involvement in Drug Toxicity 90

4.3.7 CYP2D6 90

4.3.7.1 Substrates 90

4.3.7.2 Inducers 90

4.3.7.3 Inhibitors 90

4.3.7.4 Individual Variations 90

4.3.7.5 Involvement in Drug Toxicity 91

4.3.8 CYP2E1 91

4.3.8.1 Substrates 91

4.3.8.2 Inducers 91

4.3.8.3 Inhibitors 91

4.3.8.4 Involvement in Drug Toxicity 91

4.3.9 CYP2J2 92

4.3.9.1 Substrates 92

4.3.9.2 Inhibitors 92

4.3.9.3 Inducers 92

4.3.9.4 Individual Variations 92

4.3.9.5 Involvement in Drug Toxicity 92

4.3.10 CYP3A 93

4.3.10.1 Substrates 93

4.3.10.2 Inducers 93

4.3.10.3 Inhibitors 93

4.3.10.4 Individual Variations 93

4.3.10.5 Involvement in Drug Toxicity 94

4.4 Non-P450 Drug-Metabolizing Enzymes 94

4.4.1 Flavin-Containing Monooxygenases (FMOs) 94

4.4.1.1 Substrates 94

4.4.1.2 Inducers 95

4.4.1.3 Inhibitors 95

4.4.1.4 Individual Variations 95

4.4.1.5 Involvement in Drug Toxicity 95

4.4.2 Monoamine Oxidase (MAO) 95

4.4.2.1 Substrates 96

4.4.2.2 Inducers 96

4.4.2.3 Inhibitors 96

4.4.2.4 Individual Variations 96

4.4.2.5 Involvement in Drug Toxicity 96

4.4.3 Alcohol Dehydrogenase (ADH) and Aldehyde Dehydrogenase (ALDH) 97

4.4.3.1 Substrates 97

4.4.3.2 Inducers 97

4.4.3.3 Inhibitors 97

4.4.3.4 Individual Variations 97

4.4.3.5 Involvement in Drug Toxicity 98

4.4.4 Aldehyde Oxidase (AOX) 98

4.4.4.1 Substrates 98

4.4.4.2 Inducers 98

4.4.4.3 Inhibitors 98

4.4.4.4 Individual Variations 99

4.4.4.5 Involvement in drug toxicity 99

4.4.5 Carboxylesterases (CESs) 99

4.4.5.1 Substrates 99

4.4.5.2 Inducers 99

4.4.5.3 Inhibitors 100

4.4.5.4 Individual Variations 100

4.4.5.5 Involvement in Drug Toxicity 100

4.4.6 N-Acetyltransferase (NAT) 100

4.4.6.1 Substrates 100

4.4.6.2 Inducers 100

4.4.6.3 Inhibitors 101

4.4.6.4 Individual Variations 101

4.4.6.5 Involvement in Drug Toxicity 101

4.4.7 Glutathione Transferase (GST) 101

4.4.7.1 Substrates 101

4.4.7.2 Inducers 102

4.4.7.3 Inhibitors 102

4.4.7.4 Individual Variations 102

4.4.7.5 Involvement in Drug Toxicity 102

4.4.8 Methyltransferase (MT) 103

4.4.8.1 Substrates 103

4.4.8.2 Inhibitors 103

4.4.8.3 Individual Variations 103

4.4.8.4 Involvement in Drug Toxicity 103

4.4.9 Uridine Glucuronosyltransferase (UGT) 103

4.4.9.1 Substrates 104

4.4.9.2 Inducers 104

4.4.9.3 Inhibitors 104

4.4.9.4 Individual Variations 104

4.4.9.5 Involvement in Drug Toxicity 104

4.4.10 Sulfotransferase (SULT) 105

4.4.10.1 Substrates 105

4.4.10.2 Inducers 105

4.4.10.3 Inhibitors 106

4.4.10.4 Individual Variations 106

4.4.10.5 Involvement in Drug Toxicity 106

4.5 Conclusions 106

References 107

5 Genetic Polymorphism of Drug-Metabolizing Enzymes and Drug Transporters in Drug Toxicity 139
Ann K. Daly

5.1 Introduction 139

5.2 Drug-Induced Liver Injury 140

5.2.1 Background 140

5.2.2 Polymorphisms Affecting Drug Metabolism and DILI 140

5.2.2.1 Isoniazid 140

5.2.2.2 Diclofenac 146

5.2.2.3 Tolcapone 146

5.2.2.4 Ticlopidine 147

5.2.2.5 Efavirenz 147

5.2.2.6 Troglitazone 147

5.2.3 Polymorphisms Affecting Transporters and DILI 147

5.3 Drug-Induced Skin Injury and Related Hypersensitivity Reactions 149

5.4 Statin-Induced Myopathy 151

5.4.1 Background 151

5.4.2 Cytochromes P450 151

5.4.3 Transporters 152

5.5 Conclusions 154

References 154

6 Acyl Glucuronidation and Acyl-CoA Formation Mechanisms Mediating the Bioactivation and Potential Toxicity of Carboxylic Acid-containing Drugs 167
Mark P. Grillo

6.1 Introduction 167

6.2 Phase II Metabolism 171

6.2.1 Glucuronidation 171

6.2.2 Acyl-CoA Thioester Formation 171

6.3 Chemical Stability of Phase II Metabolites 172

6.3.1 Acyl Glucuronide Instability 172

6.3.2 Acyl-CoA Thioester Stability 175

6.4 Phase II Metabolite Chemical Reactivity 176

6.4.1 Acyl Glucuronide Reactivity with Nucleophiles In vitro 176

6.4.2 Acyl-CoA Thioester Reactivity with Nucleophiles In vitro 180

6.5 Phase II Metabolite-Mediated Covalent Binding 183

6.5.1 Acyl Glucuronide-Mediated covalent Binding to protein 183

6.5.2 Acyl-CoA Thioester-Mediated Covalent Binding to Protein 185

6.6 Phase II Metabolite Prediction of Covalent Binding 187

6.6.1 Prediction of Covalent Binding to Protein by Acyl Glucuronides 187

6.6.2 Prediction of Covalent Binding to Protein by Acyl-CoA Thioesters 189

6.7 Studies Directly Comparing Carboxylic Acid Drug Bioactivation by Acyl Glucuronidation and Acyl-CoA Formation 190

6.8 Prediction of Drug-Induced Liver Injury for Carboxylic Acid Drugs 194

6.9 Conclusions 196

References 197

7 Liquid Chromatography-Mass Spectrometry (LC-MS) Quantification of Reactive Metabolites 207
Qingping Wang and Chuang Lu

7.1 Introduction 207

7.2 LC-MS Methods Using GSH as a Trapping Reagent 209

7.2.1 LC-MS Approaches at Positive Mode Using Constant Neutral Loss (CNL) Scan or Enhanced Product Ion (EPI) Scan 209

7.2.2 LC-MS Approaches at Negative Mode Using Neutral Loss, Pre-Ion Scan (PIS) and XoPI (Extraction of Product Ion) 212

7.2.3 LC-MS Approaches Using Stable Isotopic-GSH 213

7.2.4 LC-MS Approaches Using Combined XoPI and Stable-Isotopic GSH 214

7.2.5 LC-MS Coupled with Software-Assisted Approach 218

7.2.6 Using GSH Derivatives as Trapping Reagents for Detection and Quantitation 219

7.3 Using Other Trapping Reagents 222

7.4 Identification and Characterization of Rearranged GSH Adducts 222

7.5 Strategies for Optimization and Decision Tree 224

7.6 Summary 226

Acknowledgment 227

Abbreviations 227

References 228

8 Human-Based In Vitro Experimental Approaches for the Evaluation of Metabolism-Dependent Drug Toxicity 235
Albert P. Li

8.1 Introduction 235

8.2 Assays for Reactive Metabolites 235

8.2.1 Glutathione Trapping Assay 236

8.2.2 Covalent Binding Assay 236

8.3 Cell-Based Assays for Metabolism-Dependent Toxicity 237

8.4 Primary Human Hepatocyte Assays for Metabolism-Dependent Drug Toxicity 238

8.4.1 In Vitro Screening Assays for Hepatotoxicity 238

8.4.2 Cytotoxic Metabolic Pathway Identification Assay (CMPIA) 238

8.4.3 Metabolic Comparative Cytotoxicity Assay (MCCA) 241

8.4.4 MetMax™ Cryopreserved Human Hepatocytes (MMHH) Metabolic Activation Cytotoxicity Assay (MMACA) 242

8.5 Emerging Hepatocyte Technologies for the Evaluation of Drug Toxicity 242

8.5.1 Human Hepatocytes ROS/ATP Assay for DILI Drugs 242

8.5.2 Long-Term Hepatocyte Cultures 244

8.5.2.1 999Elite™ Long-Term Cultured Human Hepatocytes 244

8.5.2.2 Hepatocyte/Non-Hepatocyte Cocultures 244

8.5.2.3 Human Hepatocyte Spheroids 245

8.5.2.4 Microfluidic 3-Dimensional (3-d) Hepatocyte Cultures 245

8.6 Integrated Discrete Multiple Organ Coculture (IdMOC®) 247

8.7 Conclusion 249

References 251

Part III Drug Transporters and Drug Toxicity 261

9 Mechanism-Based Experimental Models for the Evaluation of BSEP Inhibition and DILI 263
William A. Murphy, Chitra Saran, Paavo Honkakoski, and Kim L.R. Brouwer

9.1 Introduction 263

9.1.1 Drug-Induced Liver Injury 263

9.1.2 Bile Acid Homeostasis and Role of Bile Salt Export Pump 264

9.2 Membrane Vesicles to Study BSEP Inhibition 266

9.2.1 Membrane Vesicle Preparations 267

9.2.2 Membrane Vesicle Assays and Data Interpretation 268

9.3 Sandwich-Cultured Hepatocytes to Study BSEP Inhibition 270

9.3.1 B-CLEAR® Assay 270

9.3.2 Uptake and Efflux Studies with Mechanistic Modeling 273

9.4 Other In Vitro Methods to Study BSEP Inhibition 275

9.5 Computational Methods Used to Predict BSEP Inhibition 277

9.6 In Vitro Models as a Predictor of Clinical DILI 278

9.6.1 The C-DILI™ Assay 278

9.7 Preclinical In Vivo Models for the Evaluation of BSEP/Bsep Inhibition and DILI 279

9.8 In Vivo Clinical Biomarkers of BSEP Inhibition and DILI 282

9.8.1 Serum Bile Acids as Clinical Biomarkers 282

9.8.2 Clinical Biomarkers of DILI 283

9.9 Quantitative Systems Toxicology to Predict DILI 284

9.10 Conclusions 287

Funding Information 287

Conflict of Interest 288

Acknowledgments 288

Reference 288

10 Hepatic Bile Acid Transporters in Drug-Induced Cholestasis 307
Tao Hu and Hongbing Wang

Abbreviations 307

10.1 Introduction 308

10.2 Bile Acid and DIC 308

10.2.1 Bile Acid 309

10.2.1.1 Bile Acid Synthesis 309

10.2.1.2 Bile Acid Transport 310

10.2.2 Cytotoxicity of Bile Acids and DIC 310

10.3 Hepatic Bile Acid Uptake Transporters in DIC 312

10.3.1 Sodium-Taurocholate Cotransporting Polypeptide (NTCP) 312

10.3.1.1 Substrates of NTCP 314

10.3.1.2 Regulation of NTCP 315

10.3.1.3 NTCP and Cholestasis 316

10.3.2 Other Hepatic Bile Acid Uptake Transporters 317

10.4 Hepatic Bile Acid Efflux Transporters in DIC 317

10.4.1 Bile Salt Export Pump (BSEP) 318

10.4.1.1 Substrates of BSEP 318

10.4.1.2 Regulation of BSEP 319

10.4.1.3 Internalization of BSEP 321

10.4.1.4 BSEP and Cholestasis 321

10.4.2 Other Hepatic Bile Acid Efflux Transporters 323

10.4.2.1 MRP2 323

10.4.2.2 MRP3 and MRP4 324

10.5 Bidirectional Bile Acid Transporter OSTα/β 324

10.6 Summary 325

References 326

11 Role of Renal Transporters in Drug–Drug Interactions and Nephrotoxicity 339
Yan Zhang and Donald Miller

11.1 Overview of Renal Transporters 339

11.1.1 Basolateral Transporters 340

11.1.2 Apical Transporters 341

11.2 Renal Transporters and Drug–Drug Interactions 343

11.2.1 Impact on the Pharmacokinetics of Drugs 344

11.2.2 Impact on the Drug PD 350

11.3 Renal Transporters and Nephrotoxicity 352

11.3.1 Nephrotoxicity Unrelated to Drug Transporters 353

11.3.2 Nephrotoxicity Related to Drug Transporters 355

11.4 Biomarkers and Nephrotoxicity 359

11.4.1 Biomarkers for Detecting Glomerular Injury 359

11.4.2 Biomarkers for Drug-Induced Injury to Proximal and Distal Tubules 361

11.5 Conclusion 362

References 365

12 Blood–Brain Barrier Transporters and Central Nervous System Drug Response and Toxicity 377
Donald W. Miller, Stacey Line, Nur A. Safa, and Yan Zhang

12.1 Over-View of the Brain Barriers 377

12.1.1 Blood–Brain Barrier (BBB) 377

12.1.2 Blood–Cerebrospinal Fluid Barrier (BCSFB) 379

12.1.3 CSF as Predictor of Drug Exposure in the Brain 379

12.1.4 Solute Carriers in the BBB 380

12.1.5 Drug Efflux Transporters in the BBB 380

12.2 General Influence of BBB Transporters on Drug Entry into the Brain 383

12.3 BBB-Transporter Effects on CNS Drug Response 386

12.3.1 Influence of Efflux Transporters on Brain Disposition of Drugs 386

12.3.1.1 Anticancer Agents 386

12.3.1.2 Opioids 388

12.3.2 SLCs and BBB Transport of Drugs 391

12.4 Transporter Considerations Influencing CNS Drug Response 391

12.4.1 Transporter Polymorphisms 391

12.4.1.1 P-gp Polymorphism 391

12.4.1.2 BCRP Polymorphism 393

12.4.1.3 SLC Polymorphism 393

12.4.2 Age-Related Alterations in BBB Transporter Function and Drug Response 394

12.4.3 Disease-Dependent Modulation of BBB Transporters and Drug Response 395

12.4.3.1 Inflammation and Pain 395

12.4.3.2 Epilepsy 396

12.4.4 CNS Toxicity Caused by Drug Interactions at the BBB 398

12.5 Conclusions 400

References 401

13 Ototoxicity and Drug Transport in the Cochlea 413
Stefanie Kennon-McGill and Mitchell R. McGill

13.1 Auditory System Anatomy 413

13.1.1 External, Middle, and Inner Ear 413

13.1.1.1 Anatomy of the Inner Ear 414

13.1.1.2 Hair Cell Anatomy 414

13.1.2 Blood–Labyrinth Barrier 415

13.2 Auditory System Physiology 416

13.3 Hearing Loss, Ototoxic Drugs, and Hair Cell Damage 416

13.3.1 Aminoglycosides 417

13.3.2 Platinum Chemotherapeutics 418

13.3.3 Salicylate 419

13.4 Drug Metabolism in the Ear 419

13.4.1 The Importance of Drug Metabolism in the Ear 419

13.4.2 Studies of Drug-Metabolizing Enzymes in Ototoxicity 420

13.4.3 Drug Transporters in the Ear 421

13.5 Conclusion 423

References 423

Part IV Modeling Drug Metabolizing Enzymes-Transporters Interplay for The Prediction of Drug Toxicity 427

14 Application of a PBPK Model Incorporating the Interplay Between Transporters and Drug-Metabolizing Enzymes for the Precise Prediction of Drug Toxicity 429
Kazuya Maeda

14.1 Importance of the Consideration of Intracellular Concentration of Drugs in the Tissue for Estimation of Pharmacological/Toxicological Effects of Drugs 429

14.2 Extended Clearance Concept as a Tool to Explain Theoretically Transporter and Drug-Metabolizing Enzyme Interplay 431

14.3 Theoretical Consideration of the Intracellular Concentration of Drugs in the Tissue 433

14.4 The Benefits of Using a PBPK Model for the Accurate Prediction of Pharmacological/Toxicological Effects of Drugs 436

14.5 VCT to Simulate the Distribution of Clinical Outcomes in a Specific Population with Defined Mean and Variability of Parameters in a PBPK Model 440

14.5.1 VCT of Docetaxel to Estimate the Effects on the Risk of Neutropenia of Genetic Polymorphisms in OATP1B3 and MRP2 442

14.5.2 VCT of Oseltamivir and Its Active Metabolite (Ro 64-0802) to Estimate the Effects on Their Brain Exposure of Genetic Polymorphisms in Multiple Uptake/Efflux Transporters 444

14.5.3 VCT of Irinotecan and Its Metabolites to Estimate the Effects of Genetic Polymorphisms in Multiple Uptake/Efflux Transporters on Irinotecan-Induced Side Effects (Neutropenia, Diarrhea) 447

14.6 Conclusions and Future Perspectives 450

References 451

15 The Extended Clearance Model: A Valuable Tool For Drug-Induced Liver Injury Risk Prediction 455
Birk Poller, Felix Huth, Vlasia Kastrinou-Lampou, Gerd A. Kullak-Ublick, Michael Arand, and Gian Camenisch

15.1 Introduction 455

15.2 Application of the ECM to Estimate Kpuu Liver 457

15.2.1 Introduction to the ECM: Concepts and Application for the Prediction of Hepatic Clearance and Drug–Drug Interactions 457

15.2.2 Concept of Kpuu Liver 460

15.2.3 Estimation of Kpuu Liver from In Vitro Data Using the ECM 461

15.3 Relevant Concentrations for the DILI Risk Assessment 462

15.3.1 Maximum Plasma Concentrations 464

15.3.2 Maximum Hepatic Inlet Concentrations 464

15.3.3 Maximum Intracellular Hepatocyte Concentrations 465

15.4 Assessing the DIC Risk Using ECM-Based Unbound Intrahepatic Concentrations and Accounting for BSEP Inhibition as a Single Mechanism 465

15.5 Assessing the DILI Risk Using the “1/R-Value Model” to Account for the Inhibition of Multiple Pathways 467

15.5.1 ECM-Based 1/R-Value Model 467

15.5.2 1/R vs Safety Margin Relationship 471

15.6 Discussion and Outlook 473

References 475

Index 481