Pathways of carbamazepine bioactivation in vitro I. Characterization of human cytochromes P450 responsible for the formation of 2-and 3-hydroxylated metabolites

In vitro studies were conducted to identify the cytochromes P450 (P450s) involved in the formation of 2- and 3-hydroxycarbamazepine, metabolites that may serve as precursors in the formation of protein-reactive metabolites. Human liver microsomes (HLMs) converted carbamazepine (30-300 muM) to 3-hydroxycarbamazepine at rates >25 times those of 2- hydroxycarbamazepine. Both the 2- and 3-hydroxylation of carbamazepine appeared to conform to monophasic Michaelis-Menten kinetics in HLMs (apparent K-m values, similar to1640 and similar to217 muM; apparent V-max values, similar to5.71 and similar to46.9 pmol/mg of protein/min, respectively). Rates of carbamazepine 2- and 3-hydroxylation correlated strongly with CYP2B6 activity (r greater than or equal to 0.757) in a panel of HLMs (n = 8). Carbamazepine 3-hydroxylation also correlated significantly with CYP2C8 activity at a carbamazepine concentration of 30 muM. Formation of 2- and 3-hydroxycarbamazepine did not correlate significantly with any other P450 activities. The chemical inhibitors ketoconazole (CYP3A) and 7-EFC (CYP2B6) inhibited both 2- and 3-hydroxycarbamazepine formation whereas 4-methylpyrazole (CYP2E1) markedly decreased 2- hydroxycarbamazepine formation. Several recombinant P450s catalyzed carbamazepine 2- and 3-hydroxylation, but after adjustment for relative hepatic abundance, CYP3A4 and CYP2B6 appeared to be the major catalysts of carbamazepine 3-hydroxylase activity, and at least five P450s were significant contributors to 2- hydroxycarbamazepine formation; CYP2E1 made the greatest contribution to the Cl-int of carbamazepine 2- hydroxylation (similar to30%), but P450s CYP1A2, 2A6, 2B6, and 3A4 also made significant contributions (similar to13-18%). These results suggest that CYP2B6 and CYP3A4 are largely responsible for the formation of 3-hyrdoxycarbamazepine, whereas multiple P450s (CYP1A2, 2A6, 2B6, 2E1, and 3A4) contributed to 2- hydroxycarbamazepine formation.