Effects of theCYP2B6*6Allele on Catalytic Properties and Inhibition of CYP2B6 In Vitro: Implication for the Mechanism of Reduced Efavirenz Metabolism and Other CYP2B6 Substrates In Vivo

Abstract

ABSTRACT:The mechanism by which CYP2B6*6 allele alters drug metabolism in vitro and in vivo is not fully understood. To test the hypothesis that altered substrate binding and/or catalytic properties contribute to its functional consequences, efavirenz 8-hydroxylation and bupropion 4-hydroxylation were determined in CYP2B6.1 and CYP2B6.6 proteins expressed without and with cytochrome b5 (Cyt b5) and in human liver microsomes (HLMs) obtained from liver tissues genotyped for the CYP2B6*6 allele. The susceptibility of the variant protein to inhibition was also tested in HLMs. Significantly higher V max and K m values for 8-hydroxyefavirenz formation and ϳ2-fold lower intrinsic clearance (Cl int ) were noted in expressed CYP2B6.6 protein (؊b5) compared with that of CYP2B6.1 protein (؊b5); this effect was abolished by Cyt b5. The V max and Cl int values for 4-hydroxybupropion formation were significantly higher in CYP2B6.6 than in CYP2B6.1 protein, with no difference in K m , whereas coexpression with Cyt b5 reversed the genetic effect on these kinetic parameters. In HLMs, CYP2B6*6/*6 genotype was associated with markedly lower V max (and moderate increase in K m ) and thus lower Cl int values for efavirenz and bupropion metabolism, but no difference in catalytic properties was noted between CYP2B6*1/*1 and CYP2B6*1/*6 genotypes. Inhibition of efavirenz 8-hydroxylation by voriconazole was significantly greater in HLMs with the CYP2B6*6 allele (K i ‫؍‬ 1.6 ؎ 0.8 M) than HLMs with CYP2B6*1/*1 genotype (K i ‫؍‬ 3.0 ؎ 1.1 M). In conclusion, our data suggest the CYP2B6*6 allele influences metabolic activity by altering substrate binding and catalytic activity in a substrate-and Cyt b5-dependent manner. It may also confer susceptibility to inhibition.

Differences in Methadone Metabolism by CYP2B6 Variants

“…Nonetheless, this did not explain the differences between variants in methadone metabolism. Such varying ratios were also seen previously, with CYP2B6 systems using a single virus for the three proteins (P450, reductase, and b 5 ) (Xu et al, 2012;Gadel et al, 2013). When reductase levels are sufficient for catalytic activity, overexpression of reductase may not be a factor (Nakajima et al, 2002).…”

“…In addition to differences noted above between various expression systems, most have also not included cytochrome b 5 , and the absence or presence of b 5 may influence results. Efavirenz metabolism by both CYP2B6.1 and CYP2B6.6 was unchanged by b 5 , whereas bupropion metabolism by CYP2B6.1 was unchanged by b 5 but metabolism by CYP2B6.6 was diminished (Xu et al, 2012). The present results showed that b 5 increased methadone Cl int by CYP2B6.1 and CYP2B6.4 but not metabolism at therapeutic concentrations.…”

“…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 functional consequences of CYP2B6 allelic variants on catalytic activity in vitro are allele-, substrate-, and expression system-dependent (Turpeinen and Zanger, 2012;Zanger and Klein, 2013). P450 function can also be influenced (or not) by coexpression of cytochrome b 5 (Xu et al, 2012), and allelic variant effects in vivo are additionally influenced by quantitative differences in CYP2B6 protein expression (Turpeinen and Zanger, 2012;Zanger and Klein, 2013). Among the more well studied variants, CYP2B6*4 (785A.G, K262R) is described as causing increased expression and variably increased or decreased enzymatic activity, CYP2B6*5 (1459C.T, R487C) causing decreased expression and increased specific activity, CYP2B6*6 (516G.T, Q172H; 785A.G, K262R) causing markedly reduced expression and substrate-dependent changes in activity, and CYP2B6*18 (983T.C, I328T) having reduced expression and activity (Turpeinen and Zanger, 2012;Zanger and Klein, 2013).…”

Inhibition of Cytochrome P450 2B6 Activity by Voriconazole Profiled Using Efavirenz Disposition in Healthy Volunteers

“…We showed that variants in the CYP2B6 gene lead to reduced EFV metabolism (9,(30)(31)(32)(33). Several studies in HIV-1-infected individuals have demonstrated a strong association between these variants and EFV metabolism (reviewed in references 10 and 34).…”