Cytochrome P450 involvement in the biotransformation of cisapride and racemic norcisapride in vitro: Differential activity of individual human CYP3A isoforms

Identification of the human cytochrome P450 (P450) enzymes involved in the metabolism of cisapride and racemic norcisapride [(+/-)-norcisapride] was i nvestigated at 0.1 and 1 muM, concentrations that span the mean plasma C-ma x for cisapride. Formation of norcisapride (Nor), 3-fluoro-4-hydroxycisapri de (3F), and 4-fluoro-2-hydroxycisapride (4F) from cisapride and an unchara cterized metabolite (UNK) from (+/-)-norcisapride in human liver microsomes (HLMs) were consistent with Michaelis-Menten kinetics for a single enzyme (K-m, 6.0,14.3,13.9, and 107 muM; V-max, 1350,696, 568, and 25 pmol/mg of p rotein, respectively). HLMs converted cisapride to Nor at rates that were a t least 3 orders of magnitude greater than those observed for (+/-)-norcisa pride conversion to UNK. The sample-to-sample variation in the rates of Nor , 3F, 4F, and UNK formation correlated strongly (r(2) > 0.796) with CYP3A4/ 5 activity in a panel of HLMs (n=7) and was markedly reduced by ketoconazol e, a potent CYP3A inhibitor. Ketoconazole virtually eliminated (+/-)-norcis apride conversion to UNK (94 +/-0.5%). Studies with 10 cDNA-expressed enzym es revealed that CYP3A4 catalyzed the formation of Nor and 4F at rates >100 times those of non-CYP3A enzymes and >100- and 50-fold higher than CYP3A5 and CYP3A7, respectively. CYP3A4 was the only P450 capable of UNK formation . Therefore, CYP3A4 is the principal P450 enzyme responsible for the conver sion of cisapride to Nor, 3F, and 4F and of (+/-)-norcisapride to UNK. Comp ared with cisapride, factors related to CYP3A4-mediated (+/-)-norcisapride metabolism (e.g., ontogeny of drug-metabolizing enzymes, inhibition, and in duction) should be clinically unimportant due to the apparent lack of depen dence on cytochromes P450 for elimination.