CYTOCHROME-P450 ISOFORM INHIBITORS AS A TOOL FOR THE INVESTIGATION OFMETABOLIC 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 investiga ted on our human liver microsomes bank (n = 34) and characterized for both K-M and V-max values (n greater than or equal to 3). These metabo lic reactions were: 7-ethoxyresorufin O-deethylation (CYP1A1), phenace tin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), tolbut amide 4-methylhydroxylation (CYP2C9), dextromethorphan O-demethylation (CYP2D6), aniline 4-hydroxylation (CYP2E1) and nifedipine dehydrogena tion (CYP3A4). Literature data-based specific inhibitors were selected and characterized for both their inhibitory constant (K-i) and the in hibition-type toward their specific substrate. Results were as follows : alpha-naphthoflavone (CYP1A2; mixed-type interaction with a K-i = 0. 01 mu M), furafylline (CYP1A2; competitive-type interaction with a K-i = 3 mu M when microsomes were incubated with both furafylline and phe nacetin; noncompetitive-type interaction with a K-i = 0.6 mu M when mi crosomes were preincubated with furafylline and NADPH), pilocarpine (C YP2A6; competitive-type interaction with a K-i = 4 mu M), sulfaphenazo le (CYP2C9; competitive-type interaction with a K-i = 0.3 mu M), quini dine (CYP2D6; competitive-type interaction with a K-i = 0.4 mu M), dia llyldisulfide (CYP2E1; noncompetitive-type interaction with a K-i = 15 0 mu M on an aniline concentration range of 10-60 mu M; competitive-ty pe interaction with a K-i = 100 mu M on an aniline concentration range of 80-2000, mu M) and ketoconazole (CYP3A4; mixed-type interaction wi th a K-i = 0.015 mu M). Once the inhibitors' potency was determined, t he selective effects of these inhibitors were evaluated after incubati on of human hepatic microsomes with isoform-selective substrates in th e presence of the different chemical inhibitors. Up to 10 times the K- i value toward the isoform-selective probe, pilocarpine, sulfaphenazol e, quinidine and ketoconazole exhibited potent inhibitory and specific effects. alpha-Naphthoflavone and furafylline both inhibited phenacet in and 7-ethoxyresorufin O-deethylation processes, a consequence of th e absence of CYP1A1 in noninduced human liver. Diallyidisulfide exhibi ted broad and nonspecific inhibitory effects. When used in their ''win dow of selectivity,'' i.e., up to 10-fold the K-i value, most chemical inhibitors powerfully and specifically inhibited cytochrome P450 isof orm-specific reactions when analyzed at their K-M values.