DMD #19562 Pathways of Carbamazepine Bioactivation In Vitro III. The Role of Human Cytochrome P450 Enzymes in the Formation of 2,3-Dihydroxycarbamazepine

Conversion of the carbamazepine metabolite, 3-hydroxycarbamazepine (3-OHCBZ), to the catechol, 2,3dihydroxycarbamazepine (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 carbamazepineinduced hypersensitivity. Initial in vitro phenotyping studies implicated CYP3A4 as a primary catalyst of 2,3-diOHCBZ formation: 2-hydroxylation of 3-OHCBZ correlated significantly (r≥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 (r=0.893, P<0.001) in the panel of human liver microsomes. Studies with a panel of cDNAexpressed 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 timeand concentration-dependent, but not inhibited by inclusion of glutathione or N-acetylcysteine. CYP3A4, CYP3A5, CYP3A7, CYP2C19 and CYP1A2 converted [C]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 neo-antigen that may play a role in the etiology of carbamazepine-induced idiosyncratic toxicity. This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on May 7, 2008 as DOI: 10.1124/dmd.107.019562 at A PE T Jornals on M arch 1, 2017 dm d.aspurnals.org D ow nladed from