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.
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 incubating Escherichia 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 IC(50) values for inhibition of the seven enzymes. The IC(50) 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.
Dierks
E A
EA
Drug Safety and Metabolism, Wyeth-Ayerst Research, Princeton, New Jersey, USA. dierkse@war.wyeth.com
Stams
K R
KR
Lim
H K
HK
Cornelius
G
G
Zhang
H
H
Ball
S E
SE
D002853:Chromatography, Liquid / Q000379:methods*; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D013379:Substrate Specificity
D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System