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Pathways of Carbamazepine Bioactivation in Vitro. III. The Role of Human Cytochrome P450 Enzymes in the Formation of 2,3-Dihydroxycarbamazepine

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.), Departments of Pediatrics (R.E.P., J.S.L.) and Pharmacology (J.S.L.), University of Missouri-Kansas City, Kansas City, Missouri; Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada (W.L., J.P.U.); and Departments of Cellular & Molecular Pharmacology, Biopharmaceutical Sciences, Pharmaceutical Chemistry and the Liver Center, University of California, San Francisco, San Francisco, California (Y.Q.W., M.A.C.)

Conversion of the carbamazepine metabolite 3-hydroxycarbamazepine (3-OHCBZ) to the catechol 2,3-dihydroxycarbamazepine (2,3-diOHCBZ) followed by subsequent oxidation to a reactive o-quinone species has been proposed as a possible bioactivation pathway in the pathogenesis of carbamazepine-induced hypersensitivity. Initial in vitro phenotyping studies implicated CYP3A4 as a primary catalyst of 2,3-diOHCBZ formation: 2-hydroxylation of 3-OHCBZ correlated significantly (r² 0.929, P < 0.001) with CYP3A4/5 activities in a panel of human liver microsomes (n = 14) and was markedly impaired by CYP3A inhibitors (>80%) but not by inhibitors of other cytochrome P450 enzymes (20%). However, in the presence of troleandomycin, the rate of 2,3-diOHCBZ formation correlated significantly with CYP2C19 activity (r² = 0.893, P < 0.001) in the panel of human liver microsomes. Studies with a panel of cDNA-expressed enzymes revealed that CYP2C19 and CYP3A4 were high (S₅₀ = 30 µM) and low (S₅₀ = 203 µM) affinity enzymes, respectively, for 2,3-diOHCBZ formation and suggested that CYP3A4, but not CYP2C19, might be inactivated by a metabolite formed from 3-OHCBZ. Subsequent experiments demonstrated that preincubation of 3-OHCBZ with human liver microsomes or recombinant CYP3A4 led to decreased CYP3A4 activity, which was both preincubation time- and concentration-dependent, but not inhibited by inclusion of glutathione or N-acetylcysteine. CYP3A4, CYP3A5, CYP3A7, CYP2C19, and CYP1A2 converted [¹⁴C]3-OHCBZ into protein-reactive metabolites, but CYP3A4 was the most catalytically active enzyme. The results of this study suggest that CYP3A4-dependent secondary oxidation of 3-OHCBZ represents a potential carbamazepine bioactivation pathway via formation of reactive metabolites capable of inactivating CYP3A4, potentially generating a neoantigen that may play a role in the etiology of carbamazepine-induced idiosyncratic toxicity.