Validation of bupropion hydroxylation as a selective marker of human cytochrome P450 2B6 catalytic activity

Division of Pharmacotherapy, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Drug Metabolism and Disposition (Impact Factor: 3.25). 11/2000; 28(10):1222-30.
Source: PubMed


The purpose of this study was to establish bupropion (BUP) hydroxylation as a selective in vitro marker of cytochrome P450 (CYP) 2B6 catalytic activity. Among a panel of 16 human liver microsomes (HLMs), BUP hydroxylase activity varied 80-fold when assayed at 500 microM substrate and significantly correlated with CYP2B6 blotting density (r(2) = 0.99) and S-mephenytoin N-demethylase activity (r(2) = 0.98). Kinetic analysis of BUP hydroxylation was performed in a subset of seven HLMs representative of the 80-fold range in activity. Sigmoidal kinetics suggestive of allosteric activation was observed in five HLMs exhibiting low or high activity; the mean apparent K(m) for BUP hydroxylation in these HLMs (130 microM) was similar to the K(m) for cDNA-expressed CYP2B6 (156 microM). Nonsaturable, biphasic kinetics was observed in two HLMs exhibiting low activity. Among a panel of cDNA-expressed P450 isoforms, CYP2B6 and CYP2E1 demonstrated the highest rates of BUP hydroxylation at 12 mM BUP (7.0 and 2.4 pmol/min/pmol of P450, respectively). The relative contributions of CYP2B6 and CYP2E1 to BUP hydroxylation were estimated by using immunoinhibitory monoclonal antibodies (MAB) to these enzymes. MAB-2B6 produced 88% maximum inhibition of BUP hydroxylation when assayed at 12 mM BUP in a high activity HLM, whereas MAB-2E1 produced 81% maximum inhibition in a low activity HLM. However, negligible inhibition by MAB-2E1 was observed when low and high activity HLMs were assayed at 500 microM BUP. These results demonstrate selectivity of BUP hydroxylation for CYP2B6 at 500 microM BUP, thereby validating its use as a diagnostic in vitro marker of CYP2B6 catalytic activity.

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    • "Overall, direct description of stereoselective urinary excretion of bupropion and metabolites at 48 hours agreed well with a previous (indirect, non-stereoselective) report of urinary recovery at 24 hours (Benowitz et al., 2013) and with findings following administration of C 14 labeled bupropion (Johnston et al., 2002). The enzymes responsible for the oxidative and reductive metabolism of bupropion to form hydroxybupropion, erythro-hydrobupropion, and threohydrobupropion have been investigated extensively (Faucette et al., 2000;Hesse et al., 2000;Faucette et al., 2001;Bondarev et al., 2003;Damaj et al., 2004;Molnari and Myers, 2012;Zhu et al., 2012;Meyer et al., 2013;Skarydova et al., 2014). Metabolite exposure is presumably dependent upon metabolic pathways leading to their formation and elimination. "
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    ABSTRACT: Bupropion is a widely used antidepressant and smoking cessation aid in addition to being one of two FDA recommended probe substrates for evaluation of cytochrome P450 2B6 activity. Racemic bupropion undergoes oxidative and reductive metabolism producing a complex profile of pharmacologically active metabolites with relatively little known about the mechanisms underlying their elimination. An LC-MS/MS assay was developed to simultaneously separate and detect glucuronide metabolites of (R,R)- and (S,S)-hydroxybupropion, (R,R)- and (S,S)-hydrobupropion (threo), and (S,R)- and (R,S)-hydrobupropion (erythro) in human urine and liver subcellular fractions to begin exploring mechanisms underlying enantioselective metabolism and elimination of bupropion metabolites. Human liver microsomal data revealed marked glucuronidation stereoselectivity [Clint, 11.4 versus 4.3 μL/min/mg for the formation of (R,R)- and (S,S)-4-hydroxybupropion glucuronide; and Clmax, 7.7 versus 1.1 μL/min/mg for the formation of (R,R)- and (S,S)-hydrobupropion glucuronide] in concurrence with observed enantioselective urinary elimination of bupropion glucuronide conjugates. Approximately 10% of the administered bupropion dose was recovered in the urine as metabolites with glucuronide metabolites accounting for approximately 40, 15, and 7% of total excreted hydroxybupropion, erythro-hydrobupropion, and threo-hydrobupropion, respectively. Elimination pathways were further characterized using an expressed UGT panel with bupropion enantiomers (both individual and racemic) as substrates. UGT2B7 catalyzed the stereoselective formation of glucuronides of hydroxybupropion, (S,S)-hydrobupropion, (S,R)- and (R,S)-hydrobupropion while UGT1A9 catalyzed the formation of (R,R)-hydrobupropion glucuronide. These data systematically describe the metabolic pathways underlying bupropion metabolite disposition and significantly expand our knowledge of potential contributors to the inter- and intra-individual variability in therapeutic and toxic effects of bupropion in humans.
    Full-text · Article · Jan 2016 · Drug metabolism and disposition: the biological fate of chemicals
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    • "Hydroxybupropion and bupropion were resolved by isocratic, reversed-phase HPLC based on a modification of the method described by Faucette et al. (Faucette et al., 2000) initiated by the addition of an NADPH-generating system, consisting of NADP (1 mM), glucose-6- phosphate (5 mM), and glucose-6-phosphate dehydrogenase (1 U/mL), incubated at 37±0.1°C in a Thermo Forma (Marietta, OH, USA) Benchtop Orbital Shaker and terminated after 30 min by the addition of 50 μl of ice-cold acetonitrile. Protein was precipitated by centrifugation at 10,000 g for 10 min and a direct injection of an aliquot (70 μl) of the supernatant was analyzed by HPLC. "
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    ABSTRACT: Although CYP2B6 catalyzes the biotransformation of many drugs used clinically in children and adults, information regarding the effects of development on CYP2B6 expression and activity are scarce. Utilizing a large panel of human liver samples (201 donors: 24 fetal, 141 pediatric and 36 adult), we quantified CYP2B6 mRNA and protein expression levels, characterized CYP2B6 (bupropion hydroxylase) activity in human liver microsomes (HLMs) and performed an extensive genotype analysis to differentiate CYP2B6 haplotypes so that the impact of genetic variation on these parameters could be assessed. Fetal livers contained extremely low levels of CYP2B6 mRNA relative to post-natal samples and all of the fetal HLMs failed to catalyze bupropion hydroxylation, but fetal CYP2B6 protein levels were not significantly different from post-natal levels. Considerable inter-individual variation in CYP2B6 mRNA expression, protein levels and activity was observed in post-natal HLMs (mRNA, ~40,000-fold; protein, ~300-fold; activity, ~600-fold). The extremely wide range of inter-individual variability in CYP2B6 expression and activity was significantly associated with age (ρ<0.01) following log transformation of the data. Our data suggest that CYP2B6 activity appears as early as the first day of life, increases through infancy, and by 1 year of age, CYP2B6 levels and activity may approach those of adults. Surprisingly, CYP2B6 inter-individual variability was not significantly associated with genetic variation in CYP2B6, nor with differences in gender or ethnicity, suggesting that factors other than these are largely responsible for the wide range of variability in CYP2B6 expression and activity observed among a large group of individuals/samples.
    Full-text · Article · Nov 2015 · Drug metabolism and disposition: the biological fate of chemicals
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    • "The oxidative metabolism of hydroxybupropion is catalyzed by CYP2B6 (Faucette et al., 2000). The reductive metabolism of bupropion has remained uncharacterized for a long time because researchers focused initially on the oxidative pathway of bupropion biotransformation (Chen et al., 2010; Coles & Kharasch, 2007; Faucette et al., 2000, 2001; Hesse et al., 2000, 2004; Kharasch et al., 2008). The first article regarding the reductive metabolism of bupropion was published in 2010 (Wang et al., 2010). "
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    ABSTRACT: Abstract The understanding of drug biotransformation is an important medical topic. The oxidative pathways that involve CYPs have been extensively studied in drug metabolism in contrast to the reductive pathways. This review focuses on drugs that have been reported to be reduced at the carbonyl group in vivo. Although the carbonyl reduction of these drugs is well known, our understanding of the carbonyl reducing enzymes (CRE) that perform these reactions is limited. We have summarized the published data in order to thoroughly describe the reductive metabolism of the selected drugs and to demonstrate the role of carbonyl reduction in the context of their overall metabolism. The number of drugs recognized as substrates for CREs has increased considerably in recent years. Moreover, the importance of carbonyl reduction in the overall metabolism of these drugs is often surprisingly high. Because only limited information is available about the CREs responsible for these reactions, additional research is needed to improve our understanding of the metabolism of drugs undergoing carbonyl reduction. Carbonyl reduction should be investigated during drug development because it can either positively or negatively influence drug efficacy.
    Full-text · Article · Oct 2013 · Drug Metabolism Reviews
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