Polymorphic Variants of Cytochrome P450 2B6 (CYP2B6.4–CYP2B6.9) Exhibit Altered Rates of Metabolism for Bupropion and Efavirenz: A Charge-Reversal Mutation in the K139E Variant (CYP2B6.8) Impairs Formation of a Functional Cytochrome P450-Reductase Complex

Abstract

In this study, metabolism of bupropion, efavirenz, and 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) by CYP2B6 wild type (CYP2B6.1) and six polymorphic variants (CYP2B6.4 to CYP2B6.9) was investigated in a reconstituted system to gain a better understanding of the effects of the mutations on the catalytic properties of these naturally occurring variants. All six variants were successfully overexpressed in Escherichia coli, including CYP2B6.8 (the K139E variant), which previously could not be overexpressed in mammalian COS-1 cells (J Pharmacol Exp Ther 311: 34 -43, 2004). The steady-state turnover rates for the hydroxylation of bupropion and efavirenz and the O-deethylation of 7-EFC showed that these mutations significantly alter the catalytic activities of CYP2B6. It was found that CYP2B6.6 exhibits 4-and 27-fold increases in the K m values for the hydroxylation of bupropion and efavirenz, respectively, and CYP2B6.8 completely loses its ability to metabolize any of the substrates under normal turnover conditions. However, compared with CYP2B6.1, CYP2B6.8 retains 77% of its 7-EFC O-deethylase activity in the presence of tert-butyl hydroperoxide as an alternative oxidant, indicating that the heme and the active site are catalytically competent. Presteady-state measurements of the rate of electron transfer from NADPH-dependent cytochrome P450 reductase (CPR) to CYP2B6.8 using stopped-flow spectrophotometry revealed that CYP2B6.8 is incapable of accepting electrons from CPR. These observations provide conclusive evidence suggesting that the chargereversal mutation in the K139E variant prevents CYP2B6.8 from forming a functional complex with CPR. Results from this work provide further insights to better understand the genotypephenotype correlation regarding CYP2B6 polymorphisms and drug metabolism.

Nicotine Addiction: Role of the Nicotinic Acetylcholine Receptors Genetic Variability in Knowledge, Prevention and Treatment

“…Indeed, CYP2B6.8 (the K139E variant) is unable to metabolize Bupropion under normal turnover conditions (Zhang et al, 2011). All these data support the evidence that genetic loci contribute to smoking cessation and therapeutic response.…”

Differences in Methadone Metabolism by CYP2B6 Variants

“…In an insect cell system with coexpressed P450 reductase and b 5 , CYP2B6.6 Cl int for bupropion 4-hydroxylation was lower, whereas efavirenz 8-hydroxylation was less but not significantly different from CYP2B6.1 (Xu et al, 2012). CYP2B6.6 activity in other systems (E. coli, Cos-1, Cos-7) was lower toward bupropion and efavirenz (Zhang et al, 2011), and ketamine (Li et al, 2013), but greater for cyclophosphamide (Xie et al, 2003;Ariyoshi et al, 2011;Raccor et al, 2012) and artemether (Honda et al, 2011), and unchanged for selegiline (Watanabe et al, 2010). The present and previous (Gadel et al, 2013) experiments show that methadone is one of the most catalytically diminished substrates for CYP2B6.6.…”

“…CYP2B6.4 results are even more substrate-dependent. In E. coli, Cos-1, and Cos-7 systems, CYP2B6.4 cyclophosphamide 4-hydroxylation was 25% lower than CYP2B6.1 (Ariyoshi et al, 2011;Raccor et al, 2012), the catalytic efficiency for 7-ethoxy-4-trifluoromethylcoumarin, bupropion, and efavirenz was decreased to half, one-third, and unchanged, respectively (Zhang et al, 2011), and artemether and selegiline metabolism was almost doubled (Watanabe et al, 2010;Honda et al, 2011). Methadone metabolism by CYP2B6.4 in the present investigation was greater than by CYP2B6.1.…”

“…CYP2B6.5 activities are variable with other substrates. CYP2B6.5 cyclophosphamide 4-hydroxylation in E. coli, Cos-1, and Cos-7 systems was half that of CYP2B6.1 (Raccor et al, dmd.aspetjournals.org 2012), and artemether and selegiline metabolism was diminished (Watanabe et al, 2010;Honda et al, 2011), whereas 7-ethoxy-4-trifluoromethylcoumarin, bupropion, and efavirenz metabolism was decreased by one-third, half, and increased, respectively, compared with CYP2B6.1 (Zhang et al, 2011;Radloff et al, 2013). CYP2B6.4 results are even more substrate-dependent.…”

“…In a COS-1 system, the activities of CYP2B6.6 and CYP2B6.9, in which 516G.T is the common polymorphism, were both extremely low, but this was attributed to minimal expression (Hofmann et al, 2008). CYP2B6.9 activity toward artemether was somewhat lower than CYP2B6.1 in COS-7 cells (Honda et al, 2011), and toward 7-ethoxy-4-trifluoromethylcoumarin, bupropion, and efavirenz in a reconstituted bacterial expression system (Zhang et al, 2011). The present results suggest that methadone is also one of the more catalytically diminished substrates with CYP2B6.9.…”