Abstract
A method for the simultaneous evaluation of the activities of seven major human drug-metabolizing cytochrome P450s (CYP3A4, CYP2D6, CYP2C9, CYP1A2, CYP2C19, CYP2A6, and CYP2C8) was developed. This method uses an in vitro cocktail of specific substrates (midazolam, bufuralol, diclofenac, ethoxyresorufin, S-mephenytoin, coumarin, and paclitaxel) and fast gradient liquid chromatography tandem mass spectrometry. The assay incubation time is 20 min, which is in the linear range for all of the substrates, and the analysis time is 4 min/sample. Substrate specificity was confirmed by incubatingEscherichia coli-expressed enzymes with the cocktail. Potent specific inhibitors of the seven enzymes (ketoconazole, quinidine, sulfaphenazole, tranylcypromine, quercetin, furafylline, and 8-methoxypsoralen) were evaluated in cocktail and individual substrate incubations. Five of these inhibitors were further studied to determine more precise IC50 values for inhibition of the seven enzymes. The IC50 values obtained in both cocktail and individual incubations were in good agreement with published values. This cocktail method offers an efficient, robust way to determine the cytochrome P450 inhibition profile of large numbers of compounds. The enhanced throughput of this method greatly facilitates its use to assess CYP inhibition as a drug candidate selection criterion.
Footnotes
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Send reprint requests to: Elizabeth Dierks, Wyeth-Ayerst Research, CN 8000, Princeton, NJ 08543-8000. E-mail:dierkse{at}war.wyeth.com
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↵1 Portions of this article were previously presented at the 13th International Symposium on Microsomes and Drug Oxidations, Stresa, Italy, July 2000.
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↵2 Current address: Metabolism and Pharmacokinetics, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT 06492-7600.
- Abbreviations used are::
- HPLC
- high-performance liquid chromatography
- CYP
- cytochrome P450
- LC-MS/MS
- liquid chromatography tandem mass spectrometry
- SRM
- selective reaction monitoring
- APCI
- atmospheric pressure chemical ionization
- Received August 14, 2000.
- Accepted October 9, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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A Method for the Simultaneous Evaluation of the Activities of Seven Major Human Drug-Metabolizing Cytochrome P450s Using an in Vitro Cocktail of Probe Substrates and Fast Gradient Liquid Chromatography Tandem Mass Spectrometry
