Cytochrome P450: In Vitro Methods and Protocols (Methods in Pharmacology and Toxicology)

Preface
Contents
Contributors
Chapter 1: Cytochrome P450: In Vitro Methods and Protocols
	1 The Pharmaceutical Industry
		1.1 The Pharmaceutical Process
		1.2 Timelines and Financial Investments in the Pharmaceutical Process
		1.3 DMPK in the Pharmaceutical Process
		1.4 Discovery and Traditional DMPK Assays
	2 Drug Physiology/Pharmacology and DMPK Assays
		2.1 Drug Physiology and ADME Assays
		2.2 Oral Administration: Upper GI Tract
		2.3 Oral Administration: Lower GI Tract
		2.4 Drug Pharmacology and DMPK Assays
		2.5 Oral Bioavailability
		2.6 Half-Life
		2.7 Predicting PK Parameters
		2.8 Toxicokinetics
	3 Strategies for Drug Discovery DMPK Assays
		3.1 Group (1) Assays: CYP and Non-CYP Stability, Inhibition, and Induction
		3.2 Group (2) Assays: CYP Phenotyping and non-CYP Stability
		3.3 Group (3) Assays: CYP Expression Levels, Kinetics, and  TDIs
		3.4 Group (4) Assays: Adverse Drug Reactions
	4 Conclusion and Future Outlook of  DMPK
	References
Chapter 2: Characteristics of Major Drug Metabolizing Cytochrome P450 Enzymes
	1 Introduction to CYP Enzymes: Structure, Function, and Localization
	2 Drug Metabolizing CYP Subfamilies
		2.1 CYP3 Subfamily
		2.2 CYP2 Subfamily
		2.3 CYP1 Subfamily
	3 CYP Metabolic Reactions
		3.1 One CYP: Multiple Reactions
		3.2 Multiple CYPs: Multiple Reactions
		3.3 Multiple CYPs: One Reaction
		3.4 Sequential Reactions: One  CYP
	4 CYP-Mediated Bioactivation
	5 CYP-Mediated  DDI
	6 CYP Polymorphisms
		6.1 CYP2D6
		6.2 CYP2C19
		6.3 CYP2C9
		6.4 CYP3A4
	References
Chapter 3: Quantitative Determination of Cytochrome P450 Using LC-MS/MS
	1 Introduction
	2 Materials and Reagents
		2.1 Sample Preparation
		2.2 Synthetic Peptide Standards
		2.3 Sample Analysis
	3 Methods
		3.1 Tissue Processing and Preparation of Microsomes
		3.2 Proteolytic Digestion of Microsomal Proteins
		3.3 Preparation of Calibration Standards
		3.4 Sample Clean-Up and Addition of Internal Standards
		3.5 Liquid Chromatographic Separation of Peptides
		3.6 MS/MS Measurement of Peptides
		3.7 Data Processing
	4 Notes
	References
Chapter 4: Spectral Quantitation of Total Cytochrome P450 in Microsomes Using a Single-Beam Spectrophotometer
	1 Introduction
	2 Materials and Reagents
		2.1 Reagents
		2.2 Equipment and Consumables
	3 Methods
	4 Notes
	References
Chapter 5: Cytochrome P450 Enzyme Kinetics: Km and Vmax Determinations Using Metabolite Formation and Substrate Depletion Meth...
	1 Introduction
	2 Materials and Reagents
		2.1 Km and Vmax Determination Using Liver Microsome
			2.1.1 Buffers and NADPH Cofactor
			2.1.2 Substrate and Specific Metabolite
			2.1.3 Material for Microsome Incubation
			2.1.4 Solvent
			2.1.5 Consumables
		2.2 Km and Vmax Determination Using Cryopreserved Hepatocyte
			2.2.1 Store Cryopreserved Hepatocyte
			2.2.2 Test Articles Preparation
			2.2.3 Material for Thawing, Counting, and Incubating Cryopreserved Hepatocyte
		2.3 Instrumentation
	3 Methods
		3.1 Metabolite Formation Approach to Determine Km and  Vmax
			3.1.1 Buffer, Cofactor, and Stop Solution
			3.1.2 Performing Serial Dilution of the Substrate
			3.1.3 Microsomal Dilution
			3.1.4 Microsomal Incubation with Substrate
			3.1.5 LC/MS/MS Analysis
			3.1.6 Calculations
		3.2 Substrate Depletion Approach to Determine Km and  Vmax
			3.2.1 Performing the Serial Dilution of the Test Compounds and Preparing Quench Plates
			3.2.2 Prepare Quench Solution and Quench Plates
			3.2.3 Thawing Cryopreserved Suspension Hepatocyte
			3.2.4 Substrate Depletion to Determine Km and  Vmax
			3.2.5 LC/MS/MS
			3.2.6 Calculation
	4 Notes
	References
Chapter 6: Cytochrome P450 Inhibition Assay Using Human Liver Microsomes
	1 Introduction
	2 Materials and Reagents
		2.1 Human Liver Microsomes (HLM)
		2.2 Preparation of 100 mM Phosphate Buffer (PBS, pH 7.4)
		2.3 Preparation of 10 mM NADPH Solution
		2.4 Preparation of Substrates Working Solution
		2.5 Preparation of Inhibitors
		2.6 Internal Standard
	3 Methods
		3.1 Assay preparation
		3.2 Preparation of Working Solutions for Test Compound
		3.3 Preparation of Working Solutions for Standard Inhibitors
		3.4 Procedure for Determination of LC/MS/MS Plate
		3.5 LC/MS/MS Analysis and Data Processing
		3.6 Data Analysis
	4 Notes
	References
Chapter 7: Evaluation of Time-Dependent Cytochrome P450 Inhibition Using the Area Under the Curve Shift Method
	1 Introduction
	2 Materials and Reagents
		2.1 Materials for Incubation
		2.2 Reagents for Incubation
		2.3 Preparation of Test Compound Stock Solution
		2.4 Preparation of Samples for LC/MS/MS
	3 Methods
		3.1 Compound Dilutions
		3.2 Preincubation/Reaction  A
		3.3 Incubation/Reaction  B
		3.4 Termination of Reaction
		3.5 LC/MS/MS
		3.6 Data Analysis and Interpretation
	4 Notes
	References
Chapter 8: Assessing Cytochrome P450 Time-Dependent Inhibition (IC50 Shift Assay) Using Both Diluted and Non-Diluted Methods
	1 Introduction
	2 Materials and Reagents
		2.1 Buffer and Solutions
		2.2 Substrates Stock Solutions
		2.3 CYP Inhibitor Stock Solutions
		2.4 Quenching Solution with Internal Standards (See Note 4)
	3 Methods
		3.1 Non-dilution Method
		3.2 Dilution Method
		3.3 LC/MS/MS Conditions
		3.4 Data Analysis
	4 Notes
	References
Chapter 9: Cytochrome P450 Gene Regulation: Reporter Assays to Assess Pregnane X Receptor (PXR, NR1I2) Activation
	Abbreviations
	1 Introduction
	2 Materials and Reagents
		2.1 Equipment
		2.2 Reagents and Solutions
		2.3 Plasticware
	3 Methods
		3.1 Thawing Cryogenic Vials of DPX2, rPXR, MkPXR, mPXR, dPXR, and ShPXR Cells
		3.2 Identification of Compounds that Activate PXR and Cytotoxicity Assessment
		3.3 Quantitation of PXR Receptor Activation and Interpretation of Results
	4 Notes
	References
Chapter 10: Cytochrome P450 Gene Regulation: Reporter Assays to Assess Aryl Hydrocarbon Receptor (HLHE76, AhR) Activation and ...
	Abbreviations
	1 Introduction
	2 Materials and Reagents
		2.1 Equipment
		2.2 Reagents and Solutions
		2.3 Plasticware
	3 Methods
		3.1 Thawing Cryogenic Vials with 1A2-DRE, MkAhR, rAhR, mAhR, and dAhR Cells
		3.2 Identification of Compounds that Activate AhR and Cytotoxicity Assessment
		3.3 Identification of Compounds that Antagonize Human or Rat AhR and Cytotoxicity Assessment
		3.4 Quantitation of AhR Receptor Activation and Antagonism and Interpretation of Results
	4 Notes
	References
Chapter 11: Cytochrome P450 Gene Regulation: Reporter Assays to Assess Direct and Indirect Activation of Constitutive Androsta...
	Abbreviations
	1 Introduction
	2 Materials and Reagents
		2.1 Equipment
		2.2 Reagents and Solutions
		2.3 Plasticware
	3 Methods
		3.1 Culturing and Splitting of Hepatoma Cells
		3.2 Transfection of Hepatoma Cells
		3.3 Freezing Transiently Transfected CAR Cells
		3.4 Thawing Cryogenic Vials of hCAR1, hCAR3, MkCAR1, MkCAR3, dCAR3, mCAR1, or rCAR3 Cells
		3.5 Identification of Compounds that Activate CAR and Cytotoxicity Assessment
		3.6 Quantitation of CAR Receptor Activation and Interpretation of Results
	4 Notes
	References
Chapter 12: Determination of In Vitro Cytochrome P450 Induction Potential Using Cryopreserved Human Hepatocytes
	1 Introduction
	2 Materials and Reagents
		2.1 Equipment and Consumables
		2.2 Reagents
	3 Methods
		3.1 Cell Culture and Treatment
		3.2 Cellular Morphology and Viability
			3.2.1 Cell Morphology
			3.2.2 Lactate Dehydrogenase (LDH) Assay
			3.2.3 PrestoBlue Viability Assay
		3.3 In Situ CYP Activity Measurements
			3.3.1 CYP Enzyme Activity Assay
			3.3.2 LC-MS/MS Analysis for CYP Activity
			3.3.3 CYP Activity Data Analysis
		3.4 mRNA Measurements
			3.4.1 Branched DNA Signal Amplification
			3.4.2 The Next  Day
			3.4.3 mRNA Data Analysis
			3.4.4 Plate Layout
		3.5 Data Interpretation
			3.5.1 General Consideration
			3.5.2 Special Considerations: EC50, Emax Curves (See Fig. 4)
	4 Notes
	References
Chapter 13: Assessing Cytochrome P450 3A (CYP3A) Induction and Suppression Using Primary Human Hepatocyte Spheroids
	1 Introduction
	2 Materials
		2.1 Spheroids Culture and Treatment
			2.1.1 Material for 3D PHH Spheroids Incubation
			2.1.2 Positive Controls, Probe Substrate, and Regent for RT-PCR
			2.1.3 Consumables
		2.2 Instrument
	3 Methods
		3.1 Prepare Culture Medium and Stop Solution
		3.2 Maintain 3D PHH Spheroids Culture
		3.3 Kinetic Parameter Determination with 3D PHH Spheroids
			3.3.1 Substrate Solution Preparation
			3.3.2 Time and Concentration Linearity and Km Determination
		3.4 Determination of CYP Induction and Suppression with 3D PHH Spheroids
			3.4.1 Incubation Medium Preparation
			3.4.2 Test Articles Preparation
			3.4.3 3D Spheroids Treatment with Positive Control and Test Articles
			3.4.4 Incubation with CYP Probe Substrate and mRNA Measurement
		3.5 LC/MS/MS Analysis for CYP Activity
		3.6 qRT-PCR for mRNA Measurement
		3.7 Calculations
			3.7.1 Calculations Are as Follows
		3.8 Result
			3.8.1 Determination of Kinetic Parameters
			3.8.2 Determination of Emax and EC50 in Activity (Fig. 3) and mRNA (Fig. 4) Values for CYP3A4 Inducer
			3.8.3 Determination of IC50 for CYP3A4 Suppression
	4 Notes
	References
Chapter 14: Cytochrome P450-Mediated Metabolic Stability Assay in Liver Microsomes
	Abbreviations
	1 Introduction
	2 Materials and Reagents
		2.1 Equipment
		2.2 Reagents and Solutions
		2.3 Plasticware
	3 Methods
		3.1 KPi Buffer Preparation
		3.2 NADPH Preparation
		3.3 Test Compound Preparation
		3.4 Liver Microsome Stock Solution Preparation
		3.5 Incubation Setup
		3.6 Sample Workup
		3.7 LC/MS/MS Analysis
		3.8 Data Analysis
	4 Notes
	References
Chapter 15: Assessment of Cytochrome P450 Metabolic Clearance Using Hepatocyte Suspension
	1 Introduction
		1.1 In vitro Hepatic Clearance
		1.2 Existing In Vitro Systems for Metabolic Stability Measurement
		1.3 Overview of Hepatocyte Stability Assay
	2 Materials and Reagents
		2.1 Materials for Hepatocyte Incubations
		2.2 Preparation of Samples for LC/MS/MS Analyses
	3 Methods
		3.1 Metabolic Clearance Screening
		3.2 Metabolic Clearance Definitive
		3.3 LC/MS/MS Analyses
	4 Notes
	References
Chapter 16: Cytochrome P450 Knock-Out Assay with Nonselective Inhibitors
	1 Introduction
	2 Materials and Reagents
		2.1 Equipment
		2.2 Reagents and Solutions
		2.3 Plasticware
	3 Methods
		3.1 1 M 1-Aminobenzotriazole (ABT) stock in DMSO
		3.2 Liver Microsomes
			3.2.1 KPi Buffer Preparation
			3.2.2 Cofactor and Inhibitor Stock Solution Preparations
			3.2.3 Test Compound Working Solution Preparation
			3.2.4 Liver Microsome Stock Solution Preparation
			3.2.5 Microsomal Incubation Protocol
		3.3 Hepatocytes
			3.3.1 Inhibitor Working Solution
			3.3.2 Hepatocyte Thawing and Preparation
			3.3.3 Test Compound Preparation
			3.3.4 Preincubation Setup
		3.4 LC/MS/MS Analysis
		3.5 Data Analysis and Interpretation
	4 Notes
	References
Chapter 17: Differentiation of Cytochrome P450-Mediated from Non-CYP-Mediated Metabolism: Aldehyde Oxidase and Xanthine Oxidor...
	1 Introduction
	2 Materials and Reagents
		2.1 Materials for Assays in Human Liver S9 to Differentiate CYP from Aldehyde Oxidase and Xanthine Oxidoreductase
		2.2 Assays in Human Hepatocytes
		2.3 Liquid Chromatography with Tandem Mass Spectrometry (LC/MS/MS)
	3 Methods
		3.1 Human Liver S9 Incubation Conditions to Identify the Presence or Absence of AO/XOR Metabolism
		3.2 AO/XOR Fraction Metabolized (fm) Determinations
			3.2.1 Determination of fm in Human Liver S9
			3.2.2 Determination of fm in Human Hepatocytes
	4 Notes
	References
Chapter 18: In Vitro Reaction Phenotyping of Cytochrome P450 Enzymes
	1 Introduction
	2 Materials and Reagents
		2.1 Equipment
		2.2 Consumables
		2.3 Reagents and Chemicals
		2.4 Reagents for HLM Incubation
		2.5 Chemicals for LC/MS/MS Analysis
	3 Methods
		3.1 Reaction Phenotyping Using Recombinant  CYPs
		3.2 Reaction Phenotyping Using Pooled Human Liver Microsomes
		3.3 LC/MS/MS Analysis
		3.4 Data Analysis and Interpretation
			3.4.1 Data Analysis
			3.4.2 Sample Data Interpretation
	4 Notes
	References
Chapter 19: Cytochrome P450-Mediated Drug Bioactivation Assay: An Untargeted High Resolution Accurate Mass LC/MS Assay
	1 Introduction
	2 Materials
		2.1 Incubation Reagents
		2.2 LC-MS Analysis
	3 Methods
		3.1 Compound Incubation and Sample Preparation
		3.2 Sample and Data Analysis
	4 Notes
	References
Chapter 20: Simultaneously Assessing Concentration Changes in 20 Biochemical Pathways as a Result of Drug Dosing and Cytochrom...
	1 Introduction
	2 Materials and Reagents
		2.1 Equipment
		2.2 Consumables
		2.3 Chemicals
	3 Methods
		3.1 Regents and Solutions
		3.2 Quality Control
		3.3 Extraction
		3.4 Derivatization
		3.5 Data Collection
		3.6 Data Processing with AMDIS
		3.7 Data Processing with  MPP
		3.8 Statistical Approaches to Screen Potential Biomarkers
		3.9 Network Mapping
	4 Notes
	References
Chapter 21: Simultaneously Assessing Concentration Changes in 17 Biochemical Pathways as a Result of Drug Dosing and Cytochrom...
	1 Introduction
	2 Materials and Reagents
		2.1 Selection of Metabolites
		2.2 Equipment
		2.3 Consumables
		2.4 Chemicals
	3 Methods
		3.1 Sample Preparatory Procedure
		3.2 LC/MS Parameters
		3.3 Data Collection and Processing with Selected Ion Extraction
	4 Notes
	References
Chapter 22: Assessing Amino Acid Concentration Changes as a Result of Drug Dosing and Cytochrome P450 and Non-cytochrome P450-...
	1 Introduction
	2 Materials and Reagents
		2.1 Equipment
		2.2 Consumables
		2.3 Chemicals
	3 Methods
		3.1 Reagents and Solution
		3.2 Quality Control
		3.3 Protein Precipitation
		3.4 NMR Sample Preparation
		3.5 Data Collection
		3.6 Spectrum Processing
		3.7 Metabolite Quantitation
		3.8 Assessing Reproducibility
		3.9 Statistical Analysis
	4 Notes
	References
Index