Abstract
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.
Footnotes
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↵2 The NADPH-regenerating system consisting of isocitrate and isocitrate dehydrogenase (ICDH) was added to incubations along with NADPH (1.5 mM) in order to recycle any NADP+ generated in the reaction back into NADPH. The ICDH conversion of isocitrate and NADP+ to oxalosuccinate + NADPH does not require Mn+2 (or Mg+2) ions (Moyle, 1956). However, the subsequent coupled reaction, that is, the formation of α-ketoglutarate from oxalosuccinate, does, and Mn+2 is found to accelerate NADPH formation by stimulating the forward reaction but is not an obligatory cofactor (Moyle, 1956). The Sigma ICDH enzyme used in the experiments described in this manuscript was supplied with Mn+2 ions (manganous sulfate), and the 0.1 M phosphate buffer used in the reaction also contained traces of Mn+2 and Mg+2 ions. For these combined reasons, additional Mg+2 was not included, as is necessary if NADPH is to be generated totally from NADP+.
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This work was supported in part by Grants R01GM58883 (J.S.L.), National Institute of General Medical Sciences, U01H044239 (J.S.L.), National Institutes of Child Health and Human Development, and R01GM44037 (M.A.C.), National Institute of General Medical Sciences.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.019562.
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ABBREVIATIONS: CBZ, carbamazepine; CBZ-IQ, carbamazepine iminoquinone; CBZ-quinone, carbamazepine o-quinone; P450, cytochrome P450; Dex, dexamethasone; 2,3-diOHCBZ, 2,3-dihydroxycarbamazepine; GSH, reduced glutathione; HPLC, high-performance liquid chromatography; 3-OHCBZ, 3-hydroxycarbamazepine; 2-OHIS, 2-hydroxyiminostilbene; MH+, protonated molecular ion; MS, mass spectrometry; NAC, N-acetyl cysteine; PAGE, polyacrylamide gel electrophoresis; RAF, relative activity factor; TAO, troleandomycin.
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The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material. -
↵1 Current affiliation: Biotransformation, Drug Safety and Metabolism, Wyeth Pharmaceuticals, Collegeville, PA.
- Received October 31, 2007.
- Accepted May 5, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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