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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    • "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.
    Drug Metabolism Reviews 10/2013; 46(1). DOI:10.3109/03602532.2013.853078 · 5.36 Impact Factor
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    • "Therapeutically important drugs metabolized primarily by CYP2B6 include the prodrug cyclophosphamide, which is converted to the direct precursor of the cytotoxic metabolites, phosphoramide mustard and acrolein, by 4-hydroxylation (Huang et al., 2000; Roy et al., 2005), the non-nucleoside reverse transcriptase inhibitor (NNRTI), efavirenz, which is 8-hydroxylated to become pharmacologically inactive (Ward et al., 2003; Desta et al., 2007), the atypical antidepressant and smoking cessation agent bupropion, which is converted to pharmacologically active hydroxybupropion (Faucette et al., 2000; Hesse et al., 2000; Turpeinen et al., 2005b), the anesthetics propofol (Court et al., 2001; Oda et al., 2001) and ketamine (Desta et al., 2012), the analgesic pethidine (meperidine; Ramírez et al., 2004); the μ-opioid receptor agonist, methadone (Totah et al., 2008), the antimalarial artemisinin (Svensson and Ashton, 1999; Asimus and Ashton, 2009), among numerous additional metabolic pathways of other drugs, to which CYP2B6 contributes in part, such as the antiretroviral, nevirapine (Erickson et al., 1999), and many others (Turpeinen and Zanger, 2012). Metabolic pathways suitable as probe for CYP2B6 activity include S-mephenytoin N-demethylation (Ko et al., 1998), bupropion hydroxylation (Faucette et al., 2000; Fuhr et al., 2007) and efavirenz, based on in vitro investigations (Ward et al., 2003; Desta et al., 2007). "
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    ABSTRACT: Cytochrome P450 2B6 (CYP2B6) belongs to the minor drug metabolizing P450s in human liver. Expression is highly variable both between individuals and within individuals, owing to non-genetic factors, genetic polymorphisms, inducibility, and irreversible inhibition by many compounds. Drugs metabolized mainly by CYP2B6 include artemisinin, bupropion, cyclophosphamide, efavirenz, ketamine, and methadone. is one of the most polymorphic CYP genes in humans and variants have been shown to affect transcriptional regulation, splicing, mRNA and protein expression, and catalytic activity. Some variants appear to affect several functional levels simultaneously, thus, combined in haplotypes, leading to complex interactions between substrate-dependent and -independent mechanisms. The most common functionally deficient allele is [Q172H, K262R], which occurs at frequencies of 15 to over 60% in different populations. The allele leads to lower expression in liver due to erroneous splicing. Recent investigations suggest that the amino acid changes contribute complex substrate-dependent effects at the activity level, although data from recombinant systems used by different researchers are not well in agreement with each other. Another important variant, [I328T], occurs predominantly in Africans (4-12%) and does not express functional protein. A large number of uncharacterized variants are currently emerging from different ethnicities in the course of the 1000 Genomes Project. The polymorphism is clinically relevant for HIV-infected patients treated with the reverse transcriptase inhibitor efavirenz, but it is increasingly being recognized for other drug substrates. This review summarizes recent advances on the functional and clinical significance of CYP2B6 and its genetic polymorphism, with particular emphasis on the comparison of kinetic data obtained with different substrates for variants expressed in different recombinant expression systems.
    Frontiers in Genetics 03/2013; 4:24. DOI:10.3389/fgene.2013.00024
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    • "In vitro investigations using human liver microsomes and recombinant CYPs suggest that CYP2B6 is the primary CYP enzyme catalyzing the metabolism of bupropion (Faucette et al., 2000; Hesse et al., 2000) and efavirenz (Ward et al., 2003). "
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    ABSTRACT: The effects of sex, ethnicity, and genetic polymorphism on hepatic CYP2B6 expression and activity were previosuly demonstrated in vitro. Race/Ethnic differences in CYP2B6 genotype and phenotype were observed only in women. To identify important covariates associated with inter-individual variation in CYP2B6 activity in vivo, we evaluated these effects in healthy volunteers using bupropion as a CYP2B6 probe substrate. A fixed 150 mg oral sustained-release dose of bupropion was administered to 100 healthy volunteers comprising four sex/ethnicity cohorts (n=25 each): Caucasian males Caucasian, African American and Hispanic females. Blood samples were obtained at 0 and 6 hours post-dose for the measurement of serum bupropion (BU) and hydroxybupropion (HB) concentrations. Whole blood was obtained at baseline for CYP2B6 genotyping. To characterize the relationship between CYP2B6 activity and ethnicity, sex, and genotype when accounting for serum BU concentrations (dose-adjusted log10-transformed), ANCOVA model was fitted in which the dependent variable was CYP2B6 activity represented as the log10-transformed, metabolic ratio of HB to BU concentrations. Several CYP2B6 polymorphisms were associated with CYP2B6 activity. Evidence of dependence of CYP2B6 activity on ethnicity, or genotype-by-ethnicity interactions was not detected in females. These results suggest that CYP2B6 genotype is the most important patient variable for predicting the level of CYP2B6 activity in females, when measured by the metabolism of bupropion. The bupropion metabolic ratio appears to detect known differences in CYP2B6 activity associated with genetic polymorphism, across different ethnic groups. Prospective studies will be needed to validate the use of bupropion as a probe substrate for clinical use.
    Drug metabolism and disposition: the biological fate of chemicals 12/2012; 41(3). DOI:10.1124/dmd.112.048108 · 3.25 Impact Factor
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