Description
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. 1. Cytochrome P450: In Vitro Methods and Protocols Zhengyin Yan and Gary W. Caldwell 2. Characteristics of Major Drug Metabolizing Cytochrome P450 Enzymes Kevin Johnson, Dian Su, and Donglu Zhang 3. Quantitative Determination of Cytochrome P450 Using LC/MS/MS Ryan H. Takahashi, Brahim Achour, and Bhagwat Prasad 4. Spectral Quantitation of Total Cytochrome P450 in Microsomes Using a Single-Beam Spectrophotometer David M. Stresser 5. Cytochrome P450 Enzyme Kinetics: Km and Vmax Determinations Using Metabolite Formation and Substrate Depletion Methods Jing Wang 6. Cytochrome P450 Inhibition Assay Using Human Liver Microsomes Jia Jia 7. Evaluation of Time-Dependent Cytochrome P450 Inhibition Using the Area Under the Curve Shift Method Julie Huang 8. Assessing Cytochrome P450 Time Dependent Inhibition (IC50 Shift Assay) Using Both Diluted and Non-Diluted Methods Yu Wang, Lili Xie, and Jing Ni 9. Cytochrome P450 Gene Regulation: Reporter Assays to Assess Pregnane X Receptor (PXR, NR1I2) Activation Marija Pinne and Judy L. Raucy 10. Cytochrome P450 Gene Regulation: Reporter Assays to Assess Aryl Hydrocarbon Receptor (HLHE76, AhR) Activation and Antagonism Marija Pinne and Judy L. Raucy 11. Cytochrome P450 Gene Regulation: Reporter Assays to Assess Direct and Indirect Activation of Constitutive Androstane Receptor (CAR, NR1I3) Marija Pinne and Judy L. Raucy 12. Determination of In Vitro Cytochrome P450 Induction Potential Using Cryopreserved Human Hepatocytes Susan Wong 13. Assessing Cytochrome P450 3A (CYP3A) Induction and Suppression Using Primary Human Hepatocyte Spheroids Jing Wang and Eugene Chen 14. Cytochrome P450-Mediated Metabolic Stability Assay in Liver Microsomes Pasquale Carione 15. Assessment of Cytochrome P450 Metabolic Clearance Using Hepatocyte Suspension Jingwei Cai and Hadil Shalan 16. Cytochrome P450 Knock-Out Assay with Non-Selective Inhibitors Pasquale Carione and Zhengyin Yan 17. Differentiation of Cytochrome P450-Mediated from Non-CYP-Mediated Metabolism: Aldehyde Oxidase and Xanthine Oxidoreductase Kirk D. Kozminski and Michael A. Zientek 18. In Vitro Reaction Phenotyping of Cytochrome P450 Enzymes Susan Wong and Zhengyin Yan 19. Cytochrome P450-Mediated Drug Bioactivation Assay: An Untargeted High Resolution Accurate Mass LC/MS Assay Kevin M. Johnson 20. 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 Yanping Lin, Ying Li, and Gary W. Caldwell 21. 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 John A. Masucci, Feng Liang, Kerem Bingol, Vince Windisch, and Gary W. Caldwell 22. Assessing Amino Acid Concentration Changes as a Result of Drug Dosing and Cytochrome P450 and Non-Cytochrome P450-Mediated Metabolism Kerem Bingol, John A. Masucci, and Gary W. Caldwell
