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Synergistically enhanced CYP2B6 inducibility between a polymorphic mutation in CYP2B6 promoter and pregnane X receptor activation.

ABSTRACT: CYP2B6 is a highly inducible and polymorphic enzyme involved in the metabolism of an increasing number of clinically important drugs. Significant interindividual variability in CYP2B6 expression has been attributed to either genetic polymorphisms or chemical-mediated induction through the activation of constitutive androstane receptor and/or pregnane X receptor (PXR). It was reported that the -82T?C substitution within the CYP2B6*22 allele creates a functional CCAAT/enhancer-binding protein (C/EBP) binding site and enhances the basal expression of the CYP2B6 gene. Here, we explored whether this polymorphic mutation could affect drug-mediated induction of CYP2B6. Cell-based promoter reporter assays demonstrated that CYP2B6 luciferase activity was synergistically enhanced in the presence of both -82T?C mutation and rifampicin (RIF)-activated PXR. On the other hand, this synergism was attenuated by disrupting the C/EBP binding site or knocking down C/EBP? expression. Mechanistic studies revealed that C/EBP? plays an important role in such synergism by directly interacting with PXR; enhancing RIF-mediated recruitment of PXR to the -82T?C harboring CYP2B6 promoter; and looping the PXR-bound distal phenobarbital-responsive enhancer module toward the proximal C/EBP binding site. Furthermore, the genotype-phenotype association was evaluated in cultured human primary hepatocytes from 44 donors. Interestingly, RIF-mediated induction of CYP2B6 in four -82T/C carriers was higher compared with that in the reference -82T/T homozygotes. Together, our results demonstrate, for the first time, a synergistic interplay between a CYP2B6 polymorphism and PXR-mediated induction, which may contribute to the large individual variations and inducibility of CYP2B6 in humans.

SUBMITTER: Li H

PROVIDER: S-EPMC2981389 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Synergistically enhanced CYP2B6 inducibility between a polymorphic mutation in CYP2B6 promoter and pregnane X receptor activation.

Li Haishan H Ferguson Stephen S SS Wang Hongbing H

Molecular pharmacology 20100712 4

CYP2B6 is a highly inducible and polymorphic enzyme involved in the metabolism of an increasing number of clinically important drugs. Significant interindividual variability in CYP2B6 expression has been attributed to either genetic polymorphisms or chemical-mediated induction through the activation of constitutive androstane receptor and/or pregnane X receptor (PXR). It was reported that the -82T→C substitution within the CYP2B6*22 allele creates a functional CCAAT/enhancer-binding protein (C/E ...[more]

PMID: 20624854

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Project description:The effects of [4'-(6-allyl-methyl-amino-hexyloxy)-2'-fluoro-phenyl]-(4-bromophenyl)-methanone fumarate (Ro 48-8071), an inhibitor of 2,3-oxidosqualene:lanosterol cyclase (cyclase), were evaluated on CYP3A4 and CYP2B6 mRNA content in primary cultured human hepatocytes. In seven hepatocyte culture preparations, 24-h treatment with 3, 10, or 30 microM Ro 48-8071 produced median increases in CYP3A4 mRNA content that were 2.2-, 7.1-, and 8.5-fold greater than untreated control, respectively, and produced increases in CYP2B6 mRNA content that were 3.0-, 4.6-, and 3.4-fold greater than control, respectively. Increases in CYP3A4 immunoreactive protein content were also measured in Ro 48-8071-treated hepatocytes. To evaluate the effects of cyclase inhibitor treatments further, a pregnane X receptor (PXR)-responsive transactivation assay in HepG2 cells was used. Ro 48-8071, trans-N-(4-chlorobenzoyl)-N-methyl-(4-dimethylaminomethylphenyl)-cyclohexylamine (BIBX 79), and 3beta-(2-diethylaminoethoxy)androst-5-en-17-one HCl (U18666A) induced luciferase expression from a PXR-responsive reporter with EC(50)s of 0.113, 0.916, and 0.294 microM, respectively. Treatment of the HepG2 system with (E)N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[(3,3'-bithiophen-5-yl)methoxy]benzenemethanamine (NB-598), an inhibitor of squalene monooxygenase, at concentrations sufficient to achieve cholesterol biosynthesis inhibition significantly inhibited cyclase inhibitor-mediated, but not rifampicin-mediated, reporter induction. Direct treatment of the HepG2 system with 1 to 10 microM squalene 2,3:22,23-dioxide, but not squalene 2,3-oxide, significantly activated PXR-responsive reporter expression. Also, squalene 2,3:22,23-dioxide bound to human PXR in vitro with an IC(50) of 3.35 microM. These data indicate that cyclase inhibitors are capable of producing CYP3A4 and CYP2B6 induction in primary cultured human hepatocytes, and that an endogenous squalene metabolite is a conserved intracrine activator of PXR.

Project description:Efavirenz (more specifically the S-enantiomer) is a cornerstone antiretroviral therapy for treatment of HIV infection. The major primary metabolite is S-8-hydroxyefavirenz, which does not have antiretroviral activity but is neurotoxic. Cytochrome P450 2B6 (CYP2B6) is the major enzyme catalyzing S-8-hydroxyefavirenz formation. CYP2B6 genetics and drug interactions are major determinants of clinical efavirenz disposition and dose adjustment. In addition, as a prototypic CYP2B6 substrate, S-efavirenz and analogs can inform on the structure, activity, catalytic mechanisms, and stereoselectivity of CYP2B6. Metabolism of R-efavirenz by CYP2B6 remains unexplored. This investigation assessed S-efavirenz metabolism by clinically relevant CYP2B6 genetic variants. This investigation also evaluated R-efavirenz hydroxylation by wild-type CYP2B6.1 and CYP2B6 variants. S-Efavirenz 8-hydroxylation by wild-type CYP2B6.1 and variants exhibited positive cooperativity and apparent cooperative substrate inhibition. On the basis of Clmax values, relative activities for S-efavirenz 8-hydroxylation were in the order CYP2B6.4 > CYP2B6.1 ? CYP2B6.5 ? CYP2B6.17 > CYP2B6.6 ? CYP2B6.7 ? CYP2B6.9 ? CYP2B6.19 ? CYP2B6.26; CYP2B6.16 and CYP2B6.18 showed minimal activity. Rates of R-efavirenz metabolism were approximately 1/10 those of S-efavirenz for wild-type CYP2B6.1 and variants. On the basis of Clmax values, there was 14-fold enantioselectivity (S > R-efavirenz) for wild-type CYP2B6.1, and 5- to 22-fold differences for other CYP2B6 variants. These results show that both CYP2B6 516G > T (CYP2B6*6 and CYP2B6*9) and 983T > C (CYP2B6*16 and CYP2B6*18) polymorphisms cause canonical diminishment or loss-of-function variants for S-efavirenz 8-hydroxylation, provide a mechanistic basis for known clinical pharmacogenetic differences in efavirenz disposition, and may predict additional clinically important variant alleles. Efavirenz is the most stereoselective CYP2B6 drug substrate yet identified and may be a useful probe for the CYP2B6 active site and catalytic mechanisms. SIGNIFICANCE STATEMENT: Clinical disposition of the antiretroviral S-efavirenz is affected by CYP2B6 polymorphisms. Expressed CYP2B6 with 516G>T (CYP2B6*6 and CYP2B6*9), and 983T>C (CYP2B6*16 and CYP2B6*18) polymorphisms had a diminishment or loss of function for efavirenz 8-hydroxylation. This provides a mechanistic basis for efavirenz clinical pharmacogenetics and may predict additional clinically important variant alleles. Efavirenz metabolism showed both cooperativity and cooperative substrate inhibition. With greater than 10-fold enantioselectivity (S- vs. R- metabolism), efavirenz is the most stereoselective CYP2B6 drug substrate yet identified. These findings may provide mechanistic insights.

Dataset Information

Synergistically enhanced CYP2B6 inducibility between a polymorphic mutation in CYP2B6 promoter and pregnane X receptor activation.

Publications

Synergistically enhanced CYP2B6 inducibility between a polymorphic mutation in CYP2B6 promoter and pregnane X receptor activation.

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