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The effects of sex, ethnicity, and genetic polymorphism on hepatic CYP2B6 (cytochrome P450 2B6) expression and activity were previously demonstrated in vitro. Race/ethnic differences in CYP2B6 genotype and phenotype were observed only in women. To identify important covariates associated with interindividual variation in CYP2B6 activity in vivo, we evaluated these effects in healthy volunteers using bupropion (Wellbutrin SR GlaxoSmithKline, Research Triangle Park, NC) 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 men and Caucasian, African American, and Hispanic women. Blood samples were obtained at 0 and 6 hours postdose 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 log(10)-transformed), analysis of covariance model was fitted in which the dependent variable was CYP2B6 activity represented as the log(10)-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 women. These results suggest that CYP2B6 genotype is the most important patient variable for predicting the level of CYP2B6 activity in women, 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.

The purpose of this investigation was to evaluate the role of cytochrome P450 (CYP) 3A4 in human liver microsomal bupropion (BUP) hydroxylation. Across the BUP concentration range of 0.075 to 12 mM, cDNA-expressed CYP3A4 demonstrated BUP hydroxylase activity only when incubated with concentrations > or =4 mM. When assayed at 12 mM BUP, cDNA-expressed CYP3A4 catalyzed BUP hydroxylation at a 30-fold lower rate than cDNA-expressed CYP2B6 (0.2 versus 7 pmol/min/pmol of P450). Among a panel of 16 human liver microsomes (HLMs), BUP hydroxylase activity varied 80-fold when assayed at 500 microM and did not strongly correlate with testosterone 6beta-hydroxylase activity when assayed at 250 microM testosterone (r(2) = 0.39), nor with CYP3A4 protein expression. A selective CYP3A4 inhibitor, troleandomycin (TAO), did not significantly alter rates of BUP hydroxylation when assayed in a moderate activity HLM at 10 to 2000 microM BUP, as reflected by a similarity in the kinetic parameters of BUP hydroxylation in the absence or presence of TAO. In addition, the same range of TAO concentrations (0.025-100 microM) that inhibited testosterone 6beta-hydroxylation in a concentration-dependent manner (46-81%) in pooled HLMs produced negligible inhibition (7%) of BUP hydroxylation when assayed at 500 microM BUP. These results suggest that CYP3A4 does not significantly catalyze BUP hydroxylation. Furthermore, these results complement recent data supporting selectivity of BUP hydroxylation for CYP2B6 at 500 microM BUP.

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.