INHIBITION OF COUMARIN 7-HYDROXYLASE ACTIVITY IN HUMAN LIVER-MICROSOMES

Nine organic solvents and 47 commonly used P450 substrates and inhibit ors were examined for their effects on coumarin 7-hydroxylase (CYP2A6) activity in human liver microsomes. Of the nine organic solvents exam ined (final concentration 1%, v/v), only methanol did not inhibit the 7-hydroxylation of coumarin (0.5 to 50 mu M) by human liver microsomes . Dioxane and tetrahydrofuran, which are structurally related to couma rin, were the most inhibitory solvents examined. Although the rates of coumarin 7-hydroxylation varied enormously among nine samples of huma n liver microsomes and cDNA-expressed CYP2A6 (V-max = 179 to 2470 pmol /mg protein/min), the K-m for coumarin 7-hydroxylation was fairly cons tant (ranging from 0.50 to 0.70 mu M). The following chemicals caused little or no inhibition of CYP2A6 as defined by a K-i > 200 mu M: caff eine, chlorzoxazone, cimetidine, dextromethorphan, diazepam, diclofena c, erythromycin, ethinylestradiol, ethynyltestosterone, fluconazole, f urafylline, furfural, hexobarbital, itraconazole, mephenytoin, methima zole, metronidazole, naringenin, naringin, nifedipine, norfloxacin, no rgestrel, orphenadrine, quinidine, papaverine, phenacetin, pyrimethami ne, ranitidine, spironolactone, sulfaphenazole, sulfinpyrazone, testos terone, tolbutamide, troleandomycin, and warfarin. In other words, the se chemicals, at a final concentration of 100 mu M, failed to inhibit CYP2A6 when the concentration of coumarin was equal to K-m (0.50 mu M) . The following chemicals were classified as strong inhibitors of CYP2 A6 (defined by K-i < 200 mu M): clotrimazole, diethyldithiocarbamate, ellipticine, ketoconazole, 8-methoxypsoralen, 4-methylpyrazole, metyra pone, miconazole, alpha-naphthoflavone, nicotine, p-nitrophenol, and t ranylcypromine. The potency with which each chemical inhibited the 7-h ydroxylation of coumarin was independent of which sample of human live r microsomes was studied. One of the most potent inhibitors of coumari n 7-hydroxylase was 8-methoxypsoralen (methoxsalen), which was determi ned to be a mechanism-based inhibitor (suicide substrate) of CYP2A6 (k (inactivation) 0.5 min(-1)). With the exception of 8-methoxypsoralen, preincubation of human liver microsomes and NADPH with the aforementio ned inhibitors did not increase their ability to inhibit CYP2A6. The m ost potent competitive inhibitor of CYP2A6 was tranylcypromine (K-i = 0.04 mu M). Several of the chemicals that strongly inhibited CYP2A6, s uch as ketoconazole and tranylcypromine, are often used with the inten tion of selectively inhibiting human P450 enzymes other than CYP2A6. T he results of this study underscore the need for a systematic evaluati on of the specificity of commonly used P450 inhibitors. (C) 1997 Acade mic Press.