Abstract
Evaluation of the time-dependent inhibition (TDI) of cytochrome P450 (CYP) enzymes is very important for understanding the drug–drug interactions. TDI is usually studied by measuring the half-maximal inhibitory concentration (IC50) shift of a drug candidate after a 30-min incubation with human liver microsomes (HLMs) in the presence and absence of NADPH. There are two main methods to assess the IC50 shift, dilution method, and non-dilution method. In the dilution method, the compound is preincubated for 30 min with a relatively higher concentration of HLMs (with and without NADPH), and the samples are diluted ten-fold before measuring CYP enzyme activity. In the non-diluted method, there is no dilution step between the preincubation and the incubation, and a lower concentration of HLMs can be used throughout the experiment. Here we describe both dilution and non-dilution methods to evaluate the time-dependent inhibition against CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 (midazolam and testosterone as substrate) in human liver microsomes.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Grimm SW et al (2009) The conduct of in vitro studies to address time-dependent inhibition of drug-metabolizing enzymes: a perspective of the pharmaceutical research and manufacturers of America. Drug Metab Dispos 37:1355–1370
Gonzalez FJ, Gelboin HV (1992) Human cytochromes P450: evolution and cDNA-directed expression. Environ Health Perspect 98:81–85
Soars MG, McGinnity DF, Grime K, Riley RJ (2007) The pivotal role of hepatocytes in drug discovery. Chem Biol Interact 168:2–15
Hodgson J (2001) ADMET—turning chemicals into drugs. Nat Biotechnol 19:722–726
Riley RJ (2001) The potential pharmacological and toxicological impact of P450 screening. Curr Opin Drug Discov Devel 4:45–54
White RE (2000) High-throughput screening in drug metabolism and pharmacokinetic support of drug discovery. Annu Rev Pharmacol Toxicol 40:133–157. https://doi.org/10.1146/annurev.pharmtox.40.1.133
Zimmerlin A, Trunzer M, Faller B (2011) CYP3A time-dependent inhibition risk assessment validated with 400 reference drugs. Drug Metab Dispos 39:1039–1046
Guideline on the Investigation of Drug Interactions (2012) CPMP/EWP/560/95/Rev. 1. European Medicines Agency
Guidance for Industry Drug Interaction Studies (2012) Study design, data analysis, implications for dosing, and labeling recommendations. Draft guidance. USFDA
Stresser DM, Mao J, Kenny JR, Jones BC, Grime K (2014) Exploring concepts of in vitro time-dependent CYP inhibition assays. Expert Opin Drug Metab Toxicol 10:157–174
Orr ST et al (2012) Mechanism-based inactivation (MBI) of cytochrome P450 enzymes: structure-activity relationships and discovery strategies to mitigate drug-drug interaction risks. J Med Chem 55:4896–4933
Ghanbari F et al (2006) A critical evaluation of the experimental design of studies of mechanism based enzyme inhibition, with implications for in vitro-in vivo extrapolation. Curr Drug Metab 7:315–334
Kalgutkar AS, Obach RS, Maurer TS (2007) Mechanism-based inactivation of cytochrome P450 enzymes: chemical mechanisms, structure-activity relationships and relationship to clinical drug-drug interactions and idiosyncratic adverse drug reactions. Curr Drug Metab 8:407–447
Riley RJ, Grime K, Weaver R (2007) Time-dependent CYP inhibition. Exp Opin Drug Metabol 3:51–66
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Wang, Y., Xie, L., Ni, J. (2021). Assessing Cytochrome P450 Time-Dependent Inhibition (IC50 Shift Assay) Using Both Diluted and Non-Diluted Methods. In: Yan, Z., Caldwell, G.W. (eds) Cytochrome P450. Methods in Pharmacology and Toxicology. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1542-3_8
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1542-3_8
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1541-6
Online ISBN: 978-1-0716-1542-3
eBook Packages: Springer Protocols