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 Vmax and Km values for 8-hydroxyefavirenz formation and ∼2-fold lower intrinsic clearance (Clint) were noted in expressed CYP2B6.6 protein (−b5) compared with that of CYP2B6.1 protein (−b5); this effect was abolished by Cyt b5. The Vmax and Clint values for 4-hydroxybupropion formation were significantly higher in CYP2B6.6 than in CYP2B6.1 protein, with no difference in Km, whereas coexpression with Cyt b5 reversed the genetic effect on these kinetic parameters. In HLMs, CYP2B6*6/*6 genotype was associated with markedly lower Vmax (and moderate increase in Km) and thus lower Clint 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 (Ki = 1.6 ± 0.8 μM) than HLMs with CYP2B6*1/*1 genotype (Ki = 3.0 ± 1.1 μM). 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.
Footnotes
This work was supported by the National Institutes of Health, National Institute of General Medical Sciences [Grants GM078501, GM078501-04S1, 2R56-GM067308-09A1].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
ABBREVIATIONS:
- SNP
- single nucleotide polymorphism
- HLMs
- human liver microsomes
- P450
- cytochrome P450
- Cyt b5
- cytochrome b5
- HPLC
- high-performance liquid chromatography
- POR
- P450 oxidoreductase
- LC/MS/MS
- liquid chromatography/tandem mass spectrometry
- Clint
- in vitro intrinsic clearance
- CV
- coefficient of variation.
- Received August 17, 2011.
- Accepted January 9, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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