Enzyme kinetics in drug metabolism : fundamentals and applications

Dedication
Foreword to the First Edition
Foreword to the Second Edition
Preface
Acknowledgments
Prologue
	Underlying Principles of Enzyme Kinetics
	Crystal Structure, Biochemistry, and Impact on Enzyme Kinetics
	Kinetics of Drug-Metabolizing Enzymes and Transporters
		Modeling Considerations
		Variability in Kinetics
	Case Studies
	References
Contents
Contributors
Part I: Underlying Principles of Enzyme Kinetics
	Chapter 1: Fundamentals of Enzyme Kinetics: Michaelis-Menten and Non-Michaelis-Type (Atypical) Enzyme Kinetics
		1 General Introduction: Activation, Energy, Catalysts, and Enzymes
		2 Introduction to Kinetics
			2.1 Reaction Rates, Rate Constants, and Binding Constants
			2.2 Reaction Order
			2.3 Half-Life
			2.4 Michaelis-Menten Kinetics
				2.4.1 Derivation of the Michaelis-Menten Equation
				2.4.2 Interpretation of Km and Vmax Parameters
				2.4.3 Alternative Approach to Determining Km and Vmax Values
				2.4.4 Practical Considerations in the Determination of Michaelis-Menten Kinetic Parameters
			2.5 Non-Michaelis-Menten (Atypical) Enzyme Kinetics
				2.5.1 Biphasic Kinetics
				2.5.2 Multienzyme Kinetics
				2.5.3 Homotropic Cooperativity
				2.5.4 Heterotropic Cooperativity
				2.5.5 Substrate Inhibition
				2.5.6 Practical Considerations in the Determination of Non-Michaelis-Menten (Atypical) Kinetics
			2.6 Data Analysis
			2.7 Summary
			2.8 Example Experimental Protocol
		3 Questions
		References
	Chapter 2: Reversible Mechanisms of Enzyme Inhibition and Resulting Clinical Significance
		1 Introduction
		2 Mechanisms of Inhibition
			2.1 Competitive Inhibition
			2.2 Noncompetitive Inhibition
			2.3 Mixed-Type Inhibition
			2.4 Uncompetitive Inhibition
		3 In Vitro Methods to Determine the Potential of a Drug To Be a Reversible Inhibitor
			3.1 Assay Materials
			3.2 Procedure
		4 Clinically Significant Reversible Inhibition
		5 Summary
		6 Questions
		References
	Chapter 3: Irreversible Enzyme Inhibition Kinetics and Drug-Drug Interactions
		1 Introduction
		2 Historical Perspective
		3 Direct Evidence in Clinical MBI DDIs
		4 Spectrum of Irreversible Inhibition
		5 Classification of Irreversible Inhibitors
			5.1 MIC Formation
			5.2 Covalent Modification of Apoprotein
			5.3 Heme Destruction
		6 Properties of Irreversible Inhibitors
			6.1 Substrate Protection
			6.2 Irreversibility
			6.3 Inactivator Stoichiometry
			6.4 Inactivation Prior to Release of Active Species
		7 Kinetics of Mechanism-Based Inhibition
		8 In Vitro Methods for Estimating the Extent of Irreversible CYP Inhibition
			8.1 Irreversible Inhibitor Screen: Dilution Method
			8.2 Non-dilution IC50 Shift Assay
			8.3 Dilution IC50 Shift Assay
			8.4 Determination of Enzyme Constants In Vitro
		9 Prediction of Extent of Drug-Drug Interactions In Vivo
			9.1 Theoretical Framework
			9.2 Clinical Application and Predictive Performance
		10 MBI Versus Reversible Inhibition Study Design
		11 Summary
		12 Questions
		References
	Chapter 4: Multienzyme Kinetics and Sequential Metabolism
		1 Introduction
		2 Multiple Enzyme Kinetics
		3 Sequential Metabolism
		4 Summary
		5 Questions for the Reader
		References
	Chapter 5: Consideration of the Unbound Drug Concentration in Enzyme Kinetics
		1 Introduction
		2 Impact of Plasma Protein Binding
			2.1 Plasma Proteins
			2.2 Important Concepts of Plasma Protein Binding
			2.3 Misconceptions of Plasma Protein Binding
			2.4 Case Studies of Plasma Protein Binding
			2.5 Effects of Plasma Protein Binding on In Vitro Metabolism
				2.5.1 Metabolic Stability Assays in the Presence of Exogenous Proteins
				2.5.2 ``Albumin Effect´´ on Glucuronidation and CYP-Mediated Oxidation
				2.5.3 Effects of Plasma Protein Binding on Drug-Drug Interaction (DDI) Studies
			2.6 Methodologies on Plasma Protein Binding Measurement
				2.6.1 Equilibrium Dialysis
				2.6.2 Competitive Equilibrium Dialysis
				2.6.3 Ultrafiltration
				2.6.4 Ultracentrifugation
				2.6.5 Flux Dialysis
				2.6.6 Transil Plasma Protein Binding Assay
		3 Impact of Membrane Permeation
			3.1 Comparison of Intrinsic Clearance between Human Liver Microsomes and Hepatocytes
			3.2 CYP Inhibition Studies with Liver Microsomes, Recombinant Enzymes, and Hepatocytes
		4 Impact of Incubational Binding or Sequestration
			4.1 Partitioning in Membranes
			4.2 Intracellular Sequestration
			4.3 Metabolism-Dependent Trapping
			4.4 Measurement and Prediction of Free Drug Incubational Concentration
				4.4.1 Fraction Unbound in Incubation (fu,inc)
				4.4.2 Fraction unbound of cells (fu,cell)
				4.4.3 In Vitro Unbound Partition Coefficient (Kp,uu)
				4.4.4 Modeling of Intracellular Free Drug Concentration
		5 Fundamental Considerations in Enzyme Kinetics
		6 Summary
		7 Questions
		References
	Chapter 6: Numerical Methods for Modeling Enzyme Kinetics
		1 Introduction: Numerical Methods
		2 Numerical Methods in Pharmacokinetics and Drug Metabolism
			2.1 Michaelis-Menten Kinetics
			2.2 Non-Michaelis-Menten Kinetics
		3 Reversible Inhibition
		4 Time-Dependent Inhibition
			4.1 Kinetics of TDI
			4.2 TDI with MM Kinetics
			4.3 TDI with Non-MM Kinetics
		5 Practical Aspects of Numerical Approaches to Drug Metabolism Kinetics
		6 Summary
		7 Questions
		References
Part II: Crystal Structure, Biochemistry, and Impact on Enzyme Kinetics
	Chapter 7: Crystal Structures of Drug-Metabolizing CYPs
		1 Introduction
		2 Common Structural Features of  CYPs
			2.1 The Transmembrane Anchoring Domain
			2.2 The F and G α-Helices
			2.3 The I-helix
			2.4 The BC-loop/B′-helix
			2.5 The Proximal Surface of  CYP
		3 CYP3A Structures
			3.1 CYP3A4 Structures
			3.2 CYP3A4 Polymorphisms
			3.3 CYP3A5 Structure
		4 CYP2C Structures
		5 CYP2D6 Structures
		6 Summary
		7 Questions
		References
	Chapter 8: The Structure and Mechanism of Drug Transporters
		1 Introduction
			1.1 Transporter Types
			1.2 Drug Transporter Superfamilies
			1.3 Drug Transport Mechanisms
				1.3.1 ``Alternating Access´´ Transport Models
				1.3.2 Other ABC Transporter Mechanisms
			1.4 ABC Transporter Topology
			1.5 ATP Hydrolysis Mechanisms of ABC Transporters
			1.6 ABC Transporters and Their Bacterial Homologs
				1.6.1 Bacterial ABC Transporters
				1.6.2 ABC Transporters in Human Drug Transport
		2 SLC Transporter Superfamily Members That Are Involved in Drug Transport
			2.1 SLC Transporter Folds
				2.1.1 GltPh Fold
				2.1.2 LeuT Fold
				2.1.3 MATE Fold
				2.1.4 MFS Fold
				2.1.5 NhaA Fold
				2.1.6 vcCNT Fold
			2.2 Multidrug and Toxin Extrusion (MATE) Family
				2.2.1 Bacterial MATE Transporters as Models
				2.2.2 Bacterial NorM MATE Transporter Subfamily
				2.2.3 DNA Damage-Inducible Protein F (DinF) MATE Subfamily
		3 Summary and Future Prospects
		4 Questions
		References
Part III: Kinetics of Drug Metabolizing Enzymes and Transporters
	Chapter 9: Enzyme Kinetics of Oxidative Metabolism-Cytochromes P450
		1 Introduction
		2 Cytochrome P450 Enzymology
			2.1 Substrate Binding
			2.2 The Cytochrome P450 Catalytic Cycle
		3 Cytochrome P450 Kinetics
			3.1 Multi-Substrate Active Site Models
			3.2 Non-hyperbolic Saturation Kinetics
			3.3 Inhibition and Activation Kinetics
		4 Practical Considerations and Applications
			4.1 Microsomal Stability Assays and Non-Michaelis-Menten Kinetics
			4.2 CYP Inhibition Assays and Non-Michaelis-Menten Kinetics
		5 Summary
		6 Questions
		References
	Chapter 10: Enzyme Kinetics, Pharmacokinetics, and Inhibition of Aldehyde Oxidase
		1 Introduction
		2 Biochemistry of  AO
		3 Endogenous Substrates
		4 Drug Metabolism by  AO
		5 AO Kinetics
		6 Case Studies of AO-Related Challenges in Drug Discovery and Development
			6.1 Compound Termination Due to High Clearance
			6.2 Compound Termination Due to Toxicity
			6.3 Compound Progression After Mitigation of AO-Related  Risk
		7 AO Inhibition
			7.1 Inhibition Mechanism Studies
			7.2 Structure Inhibition Relationships
			7.3 Irreversible Inhibition
			7.4 Differential AO Inhibition as an In Vitro  Tool
		8 Drug-Drug Interactions
		9 Computational Prediction of Sites and Rates of Metabolism
		10 Current Knowledge and Future Outlook on the Role of AO in Drug Discovery and Development
		11 Summary
		12 Questions
		References
	Chapter 11: Enzyme Kinetics of PAPS-Sulfotransferase
		1 Introduction
		2 Sulfotransferase Enzymes
		3 Properties of Sulfate and Sulfamate Conjugates
		4 Enzyme Catalytic Mechanism
			4.1 Enzymes Involved in the Synthesis of PAPS
			4.2 Structural Features of SULT Enzymes
			4.3 The SULT Catalytic Cycle
		5 Examples of Enzyme Kinetics
		6 Summary
		7 Questions
		References
	Chapter 12: Enzyme Kinetics of Uridine Diphosphate Glucuronosyltransferases (UGTs)
		1 Introduction
		2 Experimental Conditions for In Vitro Glucuronidation
			2.1 Buffer Type, Concentration, and  pH
			2.2 Latency
			2.3 β-Glucuronidase Inhibition
			2.4 Organic Solvents
			2.5 UDP-GlcUA Concentration
			2.6 MgCl2
			2.7 Albumin Effect
		3 Kinetics of Glucuronidation Reactions
			3.1 Bisubstrate Kinetics of Glucuronidation Reactions
			3.2 Single Substrate Kinetics
		4 Assessing Drug-Drug Interactions Arising from UGT Inhibition
			4.1 UGT Reaction Phenotyping
			4.2 UGT Inhibition
		5 In Vitro-In Vivo Extrapolation
		6 Impact of Intrinsic Factors on  UGT
		7 Acyl Glucuronidation: Relevance of Kinetics of Anomerization and Isomerization to Toxicokinetics
		8 Conclusions
		References
	Chapter 13: Principles and Experimental Considerations for In Vitro Transporter Interaction Assays
		1 Introduction
			1.1 Mechanisms of Membrane Transport
			1.2 Kinetics of Transport
			1.3 Localization of Transporters
			1.4 Drug Transporter Families
				1.4.1 ABC Superfamily
				1.4.2 SLCO and SLC Superfamilies
			1.5 Transporter-Mediated Adverse Drug Reactions
		2 Test Compound Considerations
			2.1 Chemical Stability
			2.2 Solubility
			2.3 Analytical Sensitivity
			2.4 Lipophilicity
			2.5 Tolerability
			2.6 Final Thoughts on Test Compound Considerations
		3 Test System Selection
			3.1 Test System Options
			3.2 Relevant Organ Phenotype
				3.2.1 Hepatocyte Uptake in Suspension
				3.2.2 Sandwich-Cultured Hepatobiliary Model
				3.2.3 Caco-2 Cell Monolayer Bidirectional Transport
			3.3 Overexpressing Test Systems
				3.3.1 Efflux Transporter-Transfected Cell Lines
				3.3.2 Uptake Transporter-Transfected Cell Lines
				3.3.3 Vesicle Systems
			3.4 Summary: Final Thoughts on Test System Selection
		References
Part IV: Modeling Considerations
	Chapter 14: Prediction of Drug Clearance from Enzyme and Transporter Kinetics
		1 Introduction
		2 In Vitro Systems
			2.1 Recombinant Enzymes
			2.2 Subcellular Fractions
			2.3 Hepatocytes
			2.4 Vesicles
			2.5 Hepatocyte Coculture Systems
		3 Experimental Considerations
			3.1 Metabolic Clearance
				3.1.1 Importance of Binding Corrections
			3.2 Transporter mediated Clearance Prediction
				3.2.1 Uptake Transporter Clearance
				3.2.2 Efflux Transporter Clearance
					Measurement of Transporter Mediated Biliary Clearance by  SCH
					Bidirectional Cellular Transport Across Monolayer
					Inside Out Membrane Vesicles
			3.3 Effect of Albumin on In Vitro Measurement of Metabolism and Transport
		4 Calculation of In Vivo Intrinsic Clearance (CLint, in vivo)
		5 Extended Clearance Model
		6 Models to Predict Organ Clearance
		7 Improvement in IVIVE
			7.1 Possible Reasons for Clearance Underprediction
			7.2 Inclusion of Extrahepatic Metabolism
				7.2.1 Gut and Gut Wall Metabolism
				7.2.2 Renal Clearance
				7.2.3 Glutathione Conjugation in Extrahepatic Tissues
			7.3 Challenges in Transporter IVIVE
		8 PBPK Modeling
			8.1 Prediction of Intestinal Absorption, Metabolism, and Transport by PBPK Modeling
			8.2 Advantages of PBPK Models
		9 Advanced Systems for Clearance Prediction
		10 Summary and Challenges
		References
	Chapter 15: Systems Biology Approaches to Enzyme Kinetics
		1 Introduction
		2 In Vitro Kinetics Applied to Cellular Systems
			2.1 The Cellular Milieu and Implications on Coupled Systems
			2.2 Other Sources of Cellular Variation
			2.3 Multidrug Resistance Proteins and Variation in Substrate Availability
			2.4 Intracellular Redox State and Variation in Enzyme Activity
		3 Points of Consideration for Construction and Analysis of Network Models
			3.1 Definition of Input and Output
			3.2 Sensitivity Analysis
			3.3 Sensitivity Analysis Example: Redox Cycling of NQO1-Activatable Quinone β-Lapachone
		4 Network Example: Bioactivation of Doxorubicin
			4.1 Step 1: Create a Quantitative In Vitro Network Model from Qualitative In Vitro Data
				4.1.1 Qualitative Information Gathering
				4.1.2 Quantitative Network Model Construction
				4.1.3 Parameter Fitting (In Vitro Model)
				4.1.4 In Vitro Model Testing
			4.2 Step 2: Create a Quantitative Cellular Network Model Based on the Fitted In Vitro Network Model
				4.2.1 Qualitative Information Gathering
				4.2.2 Quantitative Network Model Construction
			4.3 Step 3: Create Cell-Specific Network Models
				4.3.1 Quantitative Information Gathering
				4.3.2 Parameter Fitting (Cellular Model)
				4.3.3 Cellular Model Testing
				4.3.4 Sensitivity Analysis (Cellular Model)
		5 Summary
		References
Part V: Application to the Clinic and Regulatory Considerations
	Chapter 16: Variability in Human In Vitro Enzyme Kinetics
		1 Introduction
		2 Enzyme Sources
			2.1 Methodological Issues with the Collection and Processing of Human Tissue into Microsomes
			2.2 Methodological Issues with the Collection and Processing of Human Tissue into Primary Hepatocytes
			2.3 Applicability of Cell Culture Models for Low-Turnover Compounds
			2.4 The Artificial Nature of Recombinant Human Cytochrome P450 (rCYP) and Purified Reconstituted Systems
		3 Variability Due to Experimental Conditions
			3.1 Variability Introduced by Incubation Media
			3.2 Effect of Organic Solvents on Drug-Metabolizing Enzyme Activity
			3.3 Variability Due to Selection of Different Probe Substrates
			3.4 Nonspecific Binding of Substrates and Inhibitors
			3.5 Variability Introduced by Inappropriate Modeling of Enzyme Kinetic  Data
		4 Variability in Enzyme Kinetics Due to Genetic Polymorphism
			4.1 Genetic Polymorphism of CYP3As and Kinetic Variability
			4.2 Genetic Polymorphism of CYP2C9 and Kinetic Variability
			4.3 Genetic Polymorphism of CYP2D6 and Kinetic Variability
			4.4 Genetic Polymorphism of UGTs and Kinetic Variability
		5 Monte Carlo Simulation as a Tool to Visualize Uncertainty in Pharmacokinetic Predictions Arising from Variability in Experim...
			5.1 Introduction
			5.2 Monte Carlo Simulations to Visualize the Impact of Enzyme Kinetic Variability and Uncertainty
				5.2.1 Monte Carlo Simulations Using the Variability in In Vitro Enzyme Kinetics
		6 Summary
		References
	Chapter 17: Sources of Interindividual Variability
		1 Introduction to Interindividual Variability
		2 Genetic Contributions to Interindividual Variability
			2.1 Mechanisms of Altered Enzyme Function
			2.2 Genetic Basis for Decreased Enzyme Synthesis
			2.3 Genetic Basis for Increased Enzyme Synthesis
			2.4 Genetic Basis for Decreased Enzyme Stability
			2.5 Variation in Regulation by Transcription Factor Genes
			2.6 Genetic Variation Directly Affecting Catalysis
			2.7 Pharmacogenetic Outcomes
		3 Clinically Important Pharmacogenetic Traits
			3.1 CYP2B6
			3.2 CYP2D6
			3.3 CYP2C9
			3.4 CYP2C19
			3.5 CYP3A4
			3.6 CYP3A5
			3.7 UGT1A1
			3.8 UGT1A4
			3.9 ALDH2
			3.10 TPMT
			3.11 DPYD
		4 Impact of Disease and Other Factors
			4.1 Influence of Liver Disease
			4.2 Influence of Kidney Disease
			4.3 Sex-Related Differences
			4.4 Influence of Aging
			4.5 Influence of Pregnancy
			4.6 Other Factors
				4.6.1 Diet
				4.6.2 Microbiome
				4.6.3 Circadian Rhythm/Growth Hormone
				4.6.4 Infection and Inflammation
		5 Summary
		References
	Chapter 18: Ontogeny of Drug-Metabolizing Enzymes
		1 Introduction
		2 Ontogeny of Oxidative, Reductive and Hydrolytic Enzymes
			2.1 Cytochromes P450
				2.1.1 CYP3As
				2.1.2 CYP2D6
				2.1.3 CYP2Cs
				2.1.4 CYP1As
				2.1.5 CYP2E1
				2.1.6 CYP2B6
				2.1.7 CYP2A6
			2.2 Non-cytochrome P450 Enzymes
				2.2.1 Flavin Monooxygenases
				2.2.2 Alcohol and Aldehyde Dehydrogenases
				2.2.3 Esterases and Hydrolases
				2.2.4 AO
		3 Ontogeny of Conjugation Enzymes
			3.1 UGTs
			3.2 SULTs
			3.3 GSTs
			3.4 Methyl Transferases (MTs)
			3.5 NATs
		4 Application of DME Ontogeny Data in Pediatric PBPK Modeling
		5 Mechanism of DME Ontogeny Patterns
		6 Interspecies Differences in Ontogeny of DMEs
			6.1 Mice
			6.2 Rat
			6.3 Other Species
		7 Summary, Challenges and Future Directions
		References
	Chapter 19: How Science Is Driving Regulatory Guidances
		1 Determination of the Enzymes Involved in the Metabolism of a Drug and Their Relative Contribution (fm)
			1.1 In Vitro Metabolism Studies
			1.2 Metabolic Stability Studies
			1.3 Enzyme Kinetics
			1.4 Determination of the Enzyme Contribution Involved in Drug Metabolism to Its Overall Metabolic Clearance
				1.4.1 Recombinant Enzyme Kinetics
				1.4.2 Chemical or Antibody Inhibition Methods
			1.5 Special Considerations
				1.5.1 Low Turnover Drugs
				1.5.2 Drugs with Biphasic Kinetics
				1.5.3 Human Mass Balance Studies
		2 Evaluating DDIs Mediated by CYP Inhibition
			2.1 A General Framework to Evaluate In Vivo Potential of CYP Inhibition
			2.2 Evolution of the Criteria: Unbound vs. Total Concentrations
			2.3 Other Considerations When Applying the Criteria
			2.4 Rank-Order Approach
		3 Evaluating DDIs Mediated by  UGTs
			3.1 DDIs for Drugs as UGT Substrates
			3.2 DDIs for Drugs as UGT Inhibitors
		4 Evaluating DDI Potential of Metabolites
			4.1 DDIs for Metabolites as Substrates
			4.2 DDIs for Metabolites as Inhibitors
		5 Summary
		References
Part VI: Case Studies
	Chapter 20: Case Study 1: Practical Considerations with Experimental Design and Interpretation
		1 Introduction
		2 Understanding Your Enzyme Source
		3 Determination of Linearity
		4 Organic Solvent Concentrations
		5 Substrate Concentrations
		6 Working with Limitations: Lack of Metabolite Standards, Analytical Instrumentation
		7 Unexpected Results: Experimental Error or Atypical Kinetics
		References
	Chapter 21: Case Study 2: Practical Analytical Considerations for Conducting In Vitro Enzyme Kinetic Studies
		1 Practical Considerations for Bioanalysis
			1.1 LLOQ and  ULOQ
			1.2 Linear Versus Quadratic  Fits
			1.3 Analysis Without a Reference Standard
			1.4 Kinetic Constants Without a Reference Standard
			1.5 Linearity Assumptions and Bioanalytical Variability
			1.6 Choice of Buffer
		References
	Chapter 22: Case Study 3: Criticality of High-Quality Curve Fitting-``Getting a Km,app´´ Isn´t as Simple as It May Seem
		1 Introduction
		2 Materials and Methods
		3 Curve Fitting
		4 Notes
		5 Summary
		References
	Chapter 23: Case Study 4: Application of Basic Enzyme Kinetics to Metabolism Studies-Real-Life Examples
		1 Determination of Km and Vmax Values
			1.1 Km and Vmax Are Key Parameters
			1.2 Key Experimental Considerations
			1.3 Is There Anything Else You Need to Know?
		2 Selection of an Appropriate Enzyme System for In Vitro Metabolism Studies
			2.1 Selecting an Enzyme System
			2.2 Using Hepatocytes to Determine Kinetic Parameters
		3 Enzyme Inhibition
			3.1 Assessing DDI Potential
				3.1.1 Reaction Phenotyping Studies
				3.1.2 Is the Type of Inhibition Important?
			3.2 Some Pitfalls to Avoid
		4 Study Design for Metabolite Identification in a Clinical Phase Ia Study
			4.1 Goal of a Metabolite ID Study
			4.2 How Would You Select the Relevant Dose?
			4.3 Selecting Relevant Time Points
			4.4 What In Vitro Data Could You Use to Make Your Decisions Easier?
		References
	Chapter 24: Case Study 5: Predicting the Drug Interaction Potential for Inhibition of CYP2C8 by Montelukast
		1 Background
		2 Montelukast
		3 Estimating Drug Interaction Potential for Montelukast Using In Vitro  Data
		4 Pharmacokinetics of Rosiglitazone and Montelukast
		5 Discussion
		References
	Chapter 25: Case Study 6: Deconvoluting Hyperbilirubinemia-Differentiating Between Hepatotoxicity and Reversible Inhibition of...
		1 Bilirubin Formation and Disposition
		2 Case Study 1: Bilirubin Elevation in Toleration Studies-Inhibition of Metabolism
		3 Case Study 2: Bilirubin Elevation in Dose Toleration Studies-Inhibition of Metabolism and Transport
		4 Questions
		References
	Chapter 26: Case Study 7: Transporters Case Studies-In Vitro Solutions for Translatable Outcomes
		1 Efflux Transporters: Substrate Considerations in the Directional Assay Format
			1.1 Attribute: Low Intrinsic Membrane Permeability
				1.1.1 Fexofenadine
				1.1.2 Rosuvastatin
			1.2 Attribute: High Intrinsic Membrane Permeability
				1.2.1 Prazosin
				1.2.2 Loperamide
			1.3 Attribute: Interactions with Multiple Transporters
				1.3.1 Accurate Substrate Identification with Overlapping Transporter Specificity
				1.3.2 Accurate Substrate Identification When Using Promiscuous Transporter Inhibitors
		2 Uptake Transporters: Experimental Conditions to Accurately Score Substrates
			2.1 Attributes: Drug, Transporter, and Test System Characteristics
				2.1.1 Test Compound Properties
				2.1.2 Transporter Characteristics
				2.1.3 Test System Expression
			2.2 Recommended Solution: Matrix Assay with Multiple Conditions Assessed in Parallel
				2.2.1 Amantadine and Pindolol
				2.2.2 Metformin
		3 Inhibitor Considerations
			3.1 Efflux Transporter Inhibition Assessment
				3.1.1 Attribute: Test System-Dependent Inhibition
					P-gp Inhibition by Verapamil and Ketoconazole
				3.1.2 Attribute: Substrate-Dependent Inhibition
					P-gp Inhibition by Etravirine
			3.2 Uptake Transporter Inhibition Assessment
				3.2.1 Attribute: Substrate-Dependent Inhibition: OATP1B1
				3.2.2 Attribute: Substrate-Dependent Inhibition-OCT1/OCT2
		References
	Chapter 27: Case Study 8: Status of the Structural Mass Action Kinetic Model of P-gp-Mediated Transport Through Confluent Cell...
		1 Introduction
		2 The Structural Mass Action Kinetic Model for P-gp Transport
			2.1 Mass Action Reactions for Transport Across Confluent Cell Monolayers
				2.1.1 P-Gp-Mediated Transport
				2.1.2 Passive Permeability
				2.1.3 Basolateral and Apical Uptake Transporters
			2.2 Data Requirements for Fitting These Mass Action Reaction Kinetic Parameters (KP)
		3 Fitted Transport Data Over  Time
			3.1 Time-Dependent Fits of P-Gp Transport  Data
			3.2 Criteria for Kinetically Requiring Other Transporters for P-Gp Substrates
		4 The Deconvolution of the IC50/Ki Ratio for P-gp
		5 Why Are Uptake Transporters for P-gp Substrates Common in P-gp Expressing Cells?
		6 What Defines the Core Rate Constant for the Time Dependence of Transport Through Confluent Cell Monolayers?
		7 Summary
		8 Questions
		References
	Chapter 28: Case Study 9: Probe-Dependent Binding Explains Lack of CYP2C9 Inactivation by 1-Aminobenzotriazole (ABT)
		1 Cytochrome P450 (CYP) Inhibition
		2 High-Throughput CYP Inhibition Approaches
		3 RapidFire-MS/MS CYP Inhibition Assay
		4 Probe-Dependent CYP2C9 Inhibition
		5 Ineffective CYP2C9 Inhibition by 1-Aminobenzotriazole
		6 Competitive CYP2C9 Inhibition by  ABT
		7 Time-Dependent CYP2C9 Inhibition by  ABT
		8 In Silico Molecular Docking Studies
		9 Summary
		References
	Chapter 29: Case Study 10: A Case to Investigate Acetyl Transferase Kinetics
		1 Introduction
			1.1 Acetyltransferase Family
			1.2 Ping Pong Bi-Bi Enzyme Kinetics
			1.3 NAT Metabolism and Drug Development
				1.3.1 NAT1
				1.3.2 NAT2
		2 Case Studies
			2.1 Case Study 1: Etamicastat
			2.2 Case Study 2: EPZ011652
			2.3 Case Study 3: Unusual Acetyl Metabolites That Are Not NAT Substrates
		3 Summary
		Appendix 1
			Reagents
			Protocol
		References
	Chapter 30: Case Study 11: Considerations for Enzyme Mapping Experiments-Interaction Between the Aldehyde Oxidase Inhibitor Hy...
		1 Purpose of Mapping Enzyme Pathways: Cytochromes P450 and Aldehyde Oxidase
			1.1 Why Is It Important to Define the Metabolic Pathways of a Drug?
			1.2 Cytochrome P450
			1.3 Aldehyde Oxidase
		2 Planning GSH Trapping Experiments
		3 Choosing the Subcellular Fraction
		4 Designing the Experiment
		5 Case Study: Investigating Lapatinib Metabolism by CYP and  AO
		6 Uncovering Mechanism: (Know Your Inhibitor)
		7 Lessons Learned and Remaining Questions
			7.1 Lessons Learned
			7.2 Remaining Questions
		8 Helpful References from the Literature on Hydralazine
		9 Summary
		References
	Chapter 31: Case Study 12: Roadmap to Quantifying Ago2-Mediated siRNA Metabolic Activation Kinetics
		1 Introduction
			1.1 Expanding Concepts of Drug Metabolism Beyond Small Molecules: An siRNA Case Study
			1.2 siRNA Mechanism of Action and the Proposed Mechanism of Ago2-Mediated siRNA Metabolic Activation
		2 Methods
			2.1 Application of High-Resolution LC-MS to Measure siRNA Metabolic Activation
			2.2 Ago2 Incubations with siRNA
				2.2.1 Requirements
				2.2.2 Procedure
			2.3 Solid-Phase Extraction (SPE)
				2.3.1 Requirements
				2.3.2 Procedure
			2.4 LC-MS and Analysis
				2.4.1 Requirements
				2.4.2 Procedure
		3 Results and Discussion
			3.1 Ago2-Mediated Sense-Strand Cleavage: Proof of Concept
			3.2 Ionization Efficiency Correction Approach to Rank Order Molecules and IVIVC
			3.3 Roadmap to Further Evaluation of SS Cleavage
		4 Proposed Application of Numerical Methods to Model siRNA Metabolic Activation Kinetics
			4.1 Modeling and Simulation of siRNA Metabolic Activation
			4.2 Ago2-siRNA Binding Considerations
		5 Summary and Future Considerations
		References
Appendix A:Key Abbreviations
Appendix B:Glossary
Appendix C:Commonly Used Enzyme Kinetics Equations
Chapter 1
Chapter 2
Chapter 3
Chapter 4
Chapter 5
Chapter 6
Chapter 11
Chapter 14
Chapter 17
Chapter 18
Chapter 19
Chapter 22
Chapter 27
Chapter 31
Notes
Epilogue
	Compiled Aha Moments in Enzyme Kinetics: Authors´ Experiences
Index