Vol. 30, Issue 11, 1170-1179, November 2002

Pathways of Carbamazepine Bioactivation in Vitro I. Characterization of Human Cytochromes P450 Responsible for the Formation of 2- and 3-Hydroxylated Metabolites

Robin E. Pearce, Gangadhara R. Vakkalagadda, and J. Steven Leeder

Section of Developmental Pharmacology and Experimental Therapeutics, Division of Pediatric Clinical Pharmacology and Medical Toxicology, Children's Mercy Hospitals and Clinics (R.E.P., J.S.L.), and Departments of Pediatrics (J.S.L.), Pharmacology (J.S.L.), and Pharmaceutical Sciences (G.R.V.), University of Missouri-Kansas City, Kansas City, Missouri

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 µM) 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, ~1640 and ~217 µM; apparent V_max values, ~5.71 and ~46.9 pmol/mg of protein/min, respectively). Rates of carbamazepine 2- and 3-hydroxylation correlated strongly with CYP2B6 activity (r  0.757) in a panel of HLMs (n = 8). Carbamazepine 3-hydroxylation also correlated significantly with CYP2C8 activity at a carbamazepine concentration of 30 µM. 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 (~30%), but P450s CYP1A2, 2A6, 2B6, and 3A4 also made significant contributions (~13-18%). 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.