Cytochrome P450 isoform inhibitors as a tool for the investigation of metabolic reactions catalyzed by human liver microsomes.
Cytochrome P450 chemical inhibitors are widely used to define the role of individual cytochrome P450 isozyme(s) in a metabolism process. In this study, cytochrome P450 isoform-dependent reactions were investigated on our human liver microsomes bank (n = 34) and characterized for both KM and VMAX values (n > or = 3). These metabolic reactions were: 7-ethoxyresorufin O-deethylation (CYP1A1), phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), tolbutamide 4-methylhydroxylation (CYP2C9), dextromethorphan O-demethylation (CYP2D6), aniline 4-hydroxylation (CYP2E1) and nifedipine dehydrogenation (CYP3A4). Literature data-based specific inhibitors were selected and characterized for both their inhibitory constant (Ki) and the inhibition-type toward their specific substrate. Results were as follows: alpha-naphthoflavone (CYP1A1; mixed-type interaction with a Ki = 0.01 microM), furafylline (CYP1A2; competitive-type interaction with a Ki = 3 microM when microsomes were incubated with both furafylline and phenacetin; noncompetitive-type interaction with a Ki = 0.6 microM when microsomes were preincubated with furafylline and NADPH), pilocarpine (CYP2A6; competitive-type interaction with a Ki = 4 microM), sulfaphenazole (CYP2C9; competitive-type interaction with a Ki = 0.3 microM), quinidine (CYP2D6; competitive-type interaction with a Ki = 0.4 microM, diallyldisulfide (CYP2E1; noncompetitive-type interaction with a Ki = 150 microM on an aniline concentration range of 10-60 microM; competitive-type interaction with a Ki = 100 microM on an aniline concentration range of 80-2000 microM) and ketoconazole (CYP3A4; mixed-type interaction with a Ki = 0.015 microM). Once the inhibitors' potency was determined, the selective effects of these inhibitors were evaluated after incubation of human hepatic microsomes with isoform-selective substrates in the presence of the different chemical inhibitors. Up to 10 times the Ki value toward the isoform-selective probe, pilocarpine, sulfaphenazole, quinidine and ketoconazole exhibited potent inhibitory and specific effects. alpha-Naphthoflavone and furafylline both inhibited phenacetin and 7-ethoxyresorufin O-deethylation processes, a consequence of the absence of CYP1A1 in noninduced human liver. Diallyldisulfide exhibited broad and nonspecific inhibitory effects. When used in their "window of selectivity," ie., up to 10-fold the Ki value, most chemical inhibitors powerfully and specifically inhibited cytochrome P450 isoform-specific reactions when analyzed at their KM values.
Bourrié
M
M
Sanofi Recherche, Department of Preclinical Metabolism and Pharmacokinetics, Montpellier, France.
Meunier
V
V
Berger
Y
Y
Fabre
G
G
D000328:Adult; D000368:Aged; D000498:Allyl Compounds; D065607:Cytochrome P-450 Enzyme Inhibitors; D004220:Disulfides / Q000494:pharmacology; D005260:Female; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D007654:Ketoconazole / Q000494:pharmacology; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D008875:Middle Aged; D010862:Pilocarpine / Q000494:pharmacology; D011802:Quinidine / Q000494:pharmacology; D013426:Sulfaphenazole / Q000494:pharmacology
D000498:Allyl Compounds; D065607:Cytochrome P-450 Enzyme Inhibitors; D004220:Disulfides; D007527:Isoenzymes; D010862:Pilocarpine; D013426:Sulfaphenazole; C028009:diallyl disulfide; D011802:Quinidine; D007654:Ketoconazole