Abstract

Conversion of the carbamazepine metabolite, 2-hydroxycarbamazepine, to the potentially reactive species, carbamazepine iminoquinone (CBZ-IQ), has been proposed as a possible bioactivation pathway in the pathogenesis of carbamazepine-induced hypersensitivity. Generation of CBZ-IQ has been proposed to proceed through the intermediate, 2-hydroxyiminostilbene (2-OHIS); however, data suggested that 2-hydroxycarbamazepine is oxidized by cytochromes P450 (P450s) directly to CBZ-IQ, followed by NADPH-mediated reduction to 2-OHIS. In vitro studies were conducted to identify the P450s responsible for converting 2-hydroxycarbamazepine to 2-OHIS and to determine functional consequences of this bioactivation pathway. Formation of 2-OHIS in human liver microsomes (HLMs) was consistent with monophasic, Michaelis-Menten kinetics. The sample-to-sample variation in the rate of 2-OHIS formation correlated significantly (r2 > or = 0.706) with CYP3A4/5 and CYP2B6 activities in a panel of HLMs (n = 10). Studies with a panel of cDNA-expressed enzymes revealed that CYP3A4 preferentially catalyzed 2-OHIS formation; CYP3A4 formed 2-OHIS at a rate >10 times that of other enzymes capable of forming 2-OHIS (CYP1A1, CYP2C19, and CYP3A7). Inhibitors of CYP3A enzymes markedly impaired 2-OHIS formation in HLMs, whereas inhibitors of other P450s resulted in < or = 20% inhibition. Although CYP3A4 was primarily responsible for converting 2-hydroxycarbamazepine to 2-OHIS, neither 2-hydroxycarbamazepine, 2-OHIS, nor CBZ-IQ caused time-dependent inactivation of CYP3A activity. No thiol adducts were formed directly from 2-hydroxycarbamazepine. However, glutathione- and N-acetylcysteine-conjugates were formed with 2-OHIS or CBZ-IQ as substrates. Thus, CYP3A4-dependent secondary oxidation of 2-hydroxycarbamazepine represents a potential carbamazepine bioactivation pathway leading to the formation of thiol-reactive metabolites, intermediates that may play a role in the etiology of idiosyncratic toxicity attributed to carbamazepine.