Inhibition of Bupropion Metabolism by Selegiline: Mechanism-Based Inactivation of Human CYP2B6 and Characterization of Glutathione and Peptide Adducts

Abstract

ABSTRACT:Selegiline, the R-enantiomer of deprenyl, is used in the treatment of Parkinson's disease. Bupropion, an antidepressant, often used to treat patients in conjunction with selegiline, is metabolized primarily by CYP2B6. The effect of selegiline on the enzymatic activity of human cytochrome CYP2B6 in a reconstituted system and its effect on the metabolism of bupropion were examined. Selegiline was found to be a mechanism-based inactivator of the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation (7-EFC) activity of CYP2B6 as well as bupropion metabolism. The inactivations were time-, concentration-, and NADPH-dependent and were characterized by K I values of 0.14 and 0.6 M, k inact values of 0.022 and 0.029 min ؊1, and t 1/2 values of 31.5 and 24 min, respectively. In standard inhibition assays, selegiline increased the K m of CYP2B6 for bupropion from 10 to 92 M and decreased the k cat by ϳ50%. The reduced carbon-monoxide difference spectrum revealed over a 50% loss in the cytochrome P450 spectrum in the inactivated sample, with no loss in heme, and there was ϳ70% loss in enzyme activity. Trapping of the reactive metabolite using GSH led to the identification of a GSH-selegiline conjugate with a m/z 528 that could be explained by hydroxylation of selegiline followed by the addition of glutathione to the propargyl moiety after oxygenation to form the ketene intermediate. Liquid chromatography-tandem mass spectrometry analysis of the labeled protein following digestion with trypsin revealed the peptide 64 DVFTVHLGPR 73 as the peptide modified by the reactive metabolite of selegiline and the site of adduct formation is Asp64.

Pharmacogenetics of cytochrome P450 2B6 (CYP2B6): advances on polymorphisms, mechanisms, and clinical relevance

“…A comparison of several selective inhibitors revealed that 2-phenyl-2-(1-piperidinyl)propane is probably the most selective CYP2B6 inhibitor in vitro (Walsky and Obach, 2007). Recent in vitro observations identified the progesterone receptor antagonist, mifepristone (RU486; Lin et al, 2009); the anti-Parkinsonian agent selegiline (the R-enantiomer of deprenyl; Sridar et al, 2012), methadone (Amunugama et al, 2012), and tamoxifen (Sridar et al, 2012) as potent mechanism-based inhibitors. In vivo drug–drug interactions have been reported, for example, between thioTEPA and cyclophosphamide (Huitema et al, 2000), clopidogrel and bupropion (Turpeinen et al, 2005a), voriconazole and efavirenz (Liu et al, 2008; Jeong et al, 2009), clopidogrel and efavirenz (Jiang et al, 2012), and between ticlopidine and ketamine (Peltoniemi et al, 2011).…”

Electrochemical generation of selegiline metabolites coupled to mass spectrometry

“…It was also shown that reactive metabolite of selegiline (generated after hydroxylation) can react with glutathione (GSH) and form the corresponding conjugate. As it was shown, this reactive metabolite can also react with proteins [5].…”

Insights into CYP2B6-mediated drug–drug interactions

“…Selegiline is frequently used in the treatment of Parkinson׳s disease. Sridar et al 178 have shown selegiline to be a strong inhibitor of CYP2B6-mediated metabolism of bupropion in vitro , increasing the K m of bupropion from 10 to 92 µmol/L and decreasing the k cat by approximately 50% 178 . This strong inhibition of CYP2B6 by selegiline highlights a serious potential of DDI for combination therapies involving bupropion.…”