Effects of the CYP2B6*6 allele on catalytic properties and inhibition of CYP2B6 in vitro: Implication for the mechanism of reduced efavirenz metabolism and other CYP2B6 substrates in vivo

Cong Xu, Evan T. Ogburn, Yingying Guo, Zeruesenay Desta

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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 ∼2-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 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 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 μM) than HLMs with CYP2B6*1/*1 genotype (K i = 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.

Original languageEnglish
Pages (from-to)717-725
Number of pages9
JournalDrug Metabolism and Disposition
Volume40
Issue number4
DOIs
StatePublished - Apr 2012

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efavirenz
Alleles
Liver Microsomes
Cytochromes b5
Hydroxylation
Bupropion
Genotype
Proteins
Cytochrome P-450 CYP2B6
In Vitro Techniques

ASJC Scopus subject areas

  • Pharmacology
  • Pharmaceutical Science
  • Medicine(all)

Cite this

@article{777dbe1dce064643965edf782461f042,
title = "Effects of the CYP2B6*6 allele on catalytic properties and inhibition of CYP2B6 in vitro: Implication for the mechanism of reduced efavirenz metabolism and other CYP2B6 substrates in vivo",
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 ∼2-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 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 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 μM) than HLMs with CYP2B6*1/*1 genotype (K i = 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.",
author = "Cong Xu and Ogburn, {Evan T.} and Yingying Guo and Zeruesenay Desta",
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pages = "717--725",
journal = "Drug Metabolism and Disposition",
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TY - JOUR

T1 - Effects of the CYP2B6*6 allele on catalytic properties and inhibition of CYP2B6 in vitro

T2 - Implication for the mechanism of reduced efavirenz metabolism and other CYP2B6 substrates in vivo

AU - Xu, Cong

AU - Ogburn, Evan T.

AU - Guo, Yingying

AU - Desta, Zeruesenay

PY - 2012/4

Y1 - 2012/4

N2 - 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 ∼2-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 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 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 μM) than HLMs with CYP2B6*1/*1 genotype (K i = 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.

AB - 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 ∼2-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 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 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 μM) than HLMs with CYP2B6*1/*1 genotype (K i = 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.

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