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 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.
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Department of Metabolic Chemistry, Covance Laboratories, Inc., 3301 Kinsman Boulevard, Madison, WI 53704, USA. haizhi.bu@covance.com
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Knuth
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Teitelbaum
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D001189:Aryl Hydrocarbon Hydroxylases; D001711:Biotransformation / Q000187:drug effects*; D002753:Chlorzoxazone / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism; D065637:Cytochrome P-450 CYP2A6; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003915:Dextromethorphan / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D008862:Microsomes, Liver / Q000201:enzymology*; D008874:Midazolam / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors*; D016037:Single-Blind Method; D013048:Specimen Handling; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors*; D013379:Substrate Specificity; D014044:Tolbutamide / Q000378:metabolism
D003374:Coumarins; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D003915:Dextromethorphan; D014044:Tolbutamide; C030123:coumarin; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; C104464:CYP3A protein, human; D065637:Cytochrome P-450 CYP2A6; D051544:Cytochrome P-450 CYP3A; D039181:Steroid 16-alpha-Hydroxylase; D002753:Chlorzoxazone; D008874:Midazolam