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High‐throughput cytochrome P450 (CYP) inhibition screening via a cassette probe‐dosing strategy. VI. Simultaneous evaluation of inhibition potential of drugs on human hepatic isozymes CYP2A6, 3A4, 2C9, 2D6 and 2E1
Author(s) -
Bu HaiZhi,
Magis Lisa,
Knuth Kim,
Teitelbaum Philip
Publication year - 2001
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.290
Subject(s) - chemistry , cyp2a6 , dextromethorphan , chlorzoxazone , pharmacology , chromatography , tolbutamide , cyp3a4 , dosing , microsome , cyp2c9 , cytochrome p450 , cyp2e1 , biochemistry , enzyme , medicine , organic chemistry , endocrinology , diabetes mellitus
The inhibition potential of drugs towards five major human hepatic cytochrome P450 (CYP) isozymes (CYP2A6, 3A4, 2C9, 2D6, and 2E1) was investigated via cassette dosing of the five probe substrates (coumarin, midazolam, tolbutamide, dextromethorphan, and chlorzoxazone) in human liver microsomes using a 96‐well plate format. After microsomal incubations had been terminated with formic acid, the five marker metabolites (7‐hydroxycoumarin, 1′‐hydroxymidazolam, 4‐hydroxytolbutamide, dextrorphan, and 6‐hydroxychlorzoxazone) were simultaneously quantified using direct injection/online guard cartridge extraction/tandem mass spectrometry (DI‐GCE/MS/MS). Several advantages resulted from the use of a short C 18 guard cartridge (4 mm in length) for DI‐GCE/MS/MS, including minimal sample preparation, fast online extraction, short analysis time (2.5 min), and minimal source contamination. In addition, this method demonstrated an inter‐day accuracy range from −8.7 − 7.4% with a precision less than 8.3% for the quantification of all the marker metabolites. The inhibition assay for the five CYP isozymes was evaluated using their known selective inhibitors via individual and cassette dosing of the probe substrates. The IC 50 values measured via cassette dosing were consistent with those observed via individual dosing, which were all in agreement with the reported values. In addition, the validated assay was used to evaluate the inhibitory potential of 23 generic drugs (randomly selected) towards the five CYP isozymes. The results suggest the integration of the cassette dosing strategy and the DI‐GCE/MS/MS method can provide a reliable in vitro approach to screening the inhibitory potential of new chemical entities, with maximal throughput and cost‐effectiveness, in support of drug discovery and development. Copyright © 2001 John Wiley & Sons, Ltd.