Cytochrome P450: In Vitro Methods and Protocols -- Characteristics of Major Drug Metabolizing Cytochrome P450 Enzymes -- Quantitative Determination of Cytochrome P450 Using LC/MS/MS -- Spectral Quantitation of Total Cytochrome P450 in Microsomes Using a Single-Beam Spectrophotometer -- Cytochrome P450 Enzyme Kinetics: Km and Vmax Determinations Using Metabolite Formation and Substrate Depletion Methods -- Cytochrome P450 Inhibition Assay Using Human Liver Microsomes -- Evaluation of Time-Dependent Cytochrome P450 Inhibition Using the Area Under the Curve Shift Method -- Assessing Cytochrome P450 Time Dependent Inhibition (IC50 Shift Assay) Using Both Diluted and Non-Diluted Methods -- Cytochrome P450 Gene Regulation: Reporter Assays to Assess Pregnane X Receptor (PXR, NR1I2) Activation -- Cytochrome P450 Gene Regulation: Reporter Assays to Assess Aryl Hydrocarbon Receptor (HLHE76, AhR) Activation and Antagonism -- Cytochrome P450 Gene Regulation: Reporter Assays to Assess Direct and Indirect Activation of Constitutive Androstane Receptor (CAR, NR1I3) -- Determination of In Vitro Cytochrome P450 Induction Potential Using Cryopreserved Human Hepatocytes -- Assessing Cytochrome P450 3A (CYP3A) Induction and Suppression Using Primary Human Hepatocyte Spheroids -- Cytochrome P450-Mediated Metabolic Stability Assay in Liver Microsomes -- Assessment of Cytochrome P450 Metabolic Clearance Using Hepatocyte Suspension -- Cytochrome P450 Knock-Out Assay with Non-Selective Inhibitors -- Differentiation of Cytochrome P450-Mediated from Non-CYP-Mediated Metabolism: Aldehyde Oxidase and Xanthine Oxidoreductase -- In Vitro Reaction Phenotyping of Cytochrome P450 Enzymes -- Cytochrome P450-Mediated Drug Bioactivation Assay: An Untargeted High Resolution Accurate Mass LC/MS Assay -- Simultaneously Assessing Concentration Changes in 20 Biochemical Pathways as a Result of Drug Dosing and Cytochrome P450 and Non-Cytochrome P450-Mediated Metabolism: An Untargeted Metabolomics GC/MS Assay -- Simultaneously Assessing Concentration Changes in 17 Biochemical Pathways as a Result of Drug Dosing and Cytochrome P450 and Non-Cytochrome P450-Mediated Metabolism: A Quasi-Untargeted Metabolomics LC/MS Assay -- Assessing Amino Acid Concentration Changes as a Result of Drug Dosing and Cytochrome P450 and Non-Cytochrome P450-Mediated Metabolism.

This collection explores detailed experimental protocols necessary for setting up a variety of in vitro cytochrome P450 (CYP) assays that are vital in selecting drug candidates in a drug discovery pipeline. Major factors affecting drug metabolism include CYP expression levels, kinetic parameters for individual CYP enzymes, CYP inhibition and induction, time-dependent inhibition (TDI), CYP stability, non-CYP stability, UDP-glucuronosyltransferases (UGT) stability, excretion mechanisms, and drug-drug interactions (DDI), all addressed in this volume. Written for the Methods in Pharmacology and Toxicology series, chapters include helpful background information on the in vitro assay, a list of all the materials, reagents, and equipment necessary to carry out the assay, a step-by-step protocol, notes containing common and unexpected experimental problems in the assay, as well as references containing important supplementary reading. Authoritative and practical, Cytochrome P450: In Vitro Methods and Protocols serves as a key guide for researchers in the area of discovery and development of new medicines. .