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

The inhibition potential of drugs towards five major human hepatic cytochro me P450 (CYP) isozymes (CYP2A6, 3A4, 2C9, 2D6, and 2E1) was investigated vi a cassette dosing of the five probe substrates (coumarin, midazolam, tolbut amide, dextromethorphan, and chlorzoxazone) in human liver microsomes using a 96-well plate format. After microsomal incubations had been terminated w ith formic acid, the five marker metabolites (7-hydroxycoumarin, 1'-hydroxy midazolam, 4-hydroxytolbutamide, dextrorphan, and 6-hydroxychlorzoxazone) w ere simultaneously quantified using direct injection/online guard cartridge extraction/tandem mass spectrometry (DI-GCE/MS/MS). Several advantages res ulted from the use of a short Cls guard cartridge (4 mm in length) for DI-G CE/MS/MS, including minimal sample preparation, fast online extraction, sho rt analysis time (2.5 min), and minimal source contamination. In addition, this method demonstrated an interday accuracy range from -8.7 - 7.4% with a precision less than 8.3% for the quantification of all the marker metaboli tes. The inhibition assay for the five CYP isozymes was evaluated using the ir known selective inhibitors via individual and cassette dosing of the pro be substrates. The IC50 values measured via cassette dosing were consistent with those observed via individual dosing, which were all in agreement wit h the reported values. In addition, the validated assay was used to evaluat e 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, w ith maximal throughput and cost-effectiveness, in support of drug discovery and development. Copyright (C) 2001 John Wiley & Sons, Ltd.