An automated cocktail method for in vitro assessment of direct and time-dependent inhibition of nine major cytochrome P450 enzymes-application to establishing CYP2C8 inhibitor selectivity

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Kahma , H , Aurinsalo , L , Neuvonen , M , Katajamäki , J , Paludetto , M-N , Viinamäki , J , Launiainen , T , Filppula , A M , Tornio , A , Niemi , M & Backman , J T 2021 , ' An automated cocktail method for in vitro assessment of direct and time-dependent inhibition of nine major cytochrome P450 enzymes-application to establishing CYP2C8 inhibitor selectivity ' , European Journal of Pharmaceutical Sciences , vol. 162 , 105810 . https://doi.org/10.1016/j.ejps.2021.105810

Title: An automated cocktail method for in vitro assessment of direct and time-dependent inhibition of nine major cytochrome P450 enzymes-application to establishing CYP2C8 inhibitor selectivity
Author: Kahma, Helinä; Aurinsalo, Laura; Neuvonen, Mikko; Katajamäki, Jani; Paludetto, Marie-Noelle; Viinamäki, Jenni; Launiainen, Terhi; Filppula, Anne M.; Tornio, Aleksi; Niemi, Mikko; Backman, Janne T.
Contributor organization: INDIVIDRUG - Individualized Drug Therapy
HUSLAB
Medicum
Department of Clinical Pharmacology
University of Helsinki
Faculty of Medicine
Research Programs Unit
Department of Diagnostics and Therapeutics
Janne Backman / Principal Investigator
Clinicum
Date: 2021-07-01
Language: eng
Number of pages: 14
Belongs to series: European Journal of Pharmaceutical Sciences
ISSN: 0928-0987
DOI: https://doi.org/10.1016/j.ejps.2021.105810
URI: http://hdl.handle.net/10138/331373
Abstract: We developed an in vitro high-throughput cocktail assay with nine major drug-metabolizing CYP enzymes, optimized for screening of time-dependent inhibition. The method was applied to determine the selectivity of the time-dependent CYP2C8 inhibitors gemfibrozil 1-O-beta-glucuronide and clopidogrel acyl-beta-D-glucuronide. In vitro incubations with CYP selective probe substrates and pooled human liver microsomes were conducted in 96-well plates with automated liquid handler techniques and metabolite concentrations were measured with quantitative UHPLC-MS/MS analysis. After determination of inter-substrate interactions and Km values for each reaction, probe substrates were divided into cocktails I (tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4/5) and II (coumarin/CYP2A6, S-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2). Time-dependent inhibitors (furafylline/CYP1A2, selegiline/ CYP2A6, clopidogrel/CYP2B6, gemfibrozil 1-O-beta-glucuronide/CYP2C8, tienilic acid/CYP2C9, ticlopidine/ CYP2C19, paroxetine/CYP2D6 and ritonavir/CYP3A) and direct inhibitor (terfenadine/CYP2J2) showed similar inhibition with single substrate and cocktail methods. Established time-dependent inhibitors caused IC50 fold shifts ranging from 2.2 to 30 with the cocktail method. Under time-dependent inhibition conditions, gemfibrozil 1-O-beta-glucuronide was a strong (90% inhibition) and selective (<< 20% inhibition of other CYPs) inhibitor of CYP2C8 at concentrations ranging from 60 to 300 mu M, while the selectivity of clopidogrel acyl-beta-D-glucuronide was limited at concentrations above its IC80 for CYP2C8. The time-dependent IC50 values of these glucuronides for CYP2C8 were 8.1 and 38 mu M, respectively. In conclusion, a reliable cocktail method including the nine most important drug-metabolizing CYP enzymes was developed, optimized and validated for detecting timedependent inhibition. Moreover, gemfibrozil 1-O-beta-glucuronide was established as a selective inhibitor of CYP2C8 for use as a diagnostic inhibitor in in vitro studies.
Subject: Cytochrome P450
Substrate cocktail
Time-dependent inhibition
Drug metabolism
Metabolic interactions
1-O-beta-glucuronide
HUMAN LIVER-MICROSOMES
MECHANISM-BASED INHIBITION
UNSATURATED FATTY-ACIDS
DRUG-DRUG INTERACTIONS
VIVO EXTRAPOLATION
METABOLISM
GEMFIBROZIL
INACTIVATION
ASSAY
CYP3A4
3111 Biomedicine
317 Pharmacy
Peer reviewed: Yes
Rights: cc_by_nc_nd
Usage restriction: openAccess
Self-archived version: publishedVersion


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