Abstract
Cytochrome P450 (P450) 2B6 metabolizes a number of clinically relevant drugs and is one of the most highly polymorphic human P450 enzymes, with the Lys262→Arg substitution being especially common in several genetic variants. Therefore, K262R (2B6*4) was created in the CYP2B6dH background (N-terminal-modified and C-terminal His-tagged) and expressed in Escherichia coli. The recombinant CYP2B6dH and K262R were purified and studied to investigate the effect of the Lys262→Arg substitution with six of the most potent drug inhibitors of CYP2B6, namely, clopidogrel, clotrimazole, itraconazole, raloxifene, sertraline, and ticlopidine. K262R showed a >3-fold increase in the Ki values with clopidogrel, itraconazole, and raloxifene and ∼6-fold increase in Ki with sertraline compared with CYP2B6dH. Likewise, K262R showed 2-, 4-, and >20-fold higher Ks values than CYP2B6dH with clopidogrel, sertraline, and itraconazole, respectively. In contrast, when tested with several known type II inhibitors of CYP2B enzymes, K262R showed a 10-fold lower IC50 with 4-(phenyl)pyridine and ∼2-fold lower IC50 with 4-(4-nitrobenzyl)pyridine or 1-(4-phenyl)benzylimidazole than CYP2B6dH. Subsequent analysis predicted possible in vivo drug-drug interactions between the CYP2B6 substrate efavirenz and drug inhibitors clopidogrel, clotrimazole, itraconazole, sertraline, and ticlopidine. Furthermore, Q172H/K262R (2B6*6), which is the most common genetic variant of CYP2B6 harboring K262R, was created in CYP2B6dH, expressed, purified, and characterized for inhibition. Q172H/K262R showed a >6-fold increase in Ki with sertraline and clopidogrel compared with CYP2B6dH. The results suggest that individuals, especially homozygotes, with the 2B6*4 or 2B6*6 allele might be less susceptible to drug interactions resulting from P450 inhibition.
Footnotes
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This work was supported by the National Institutes of Health National Institute of Environmental Health Sciences [Grants ES03619, ES06676].
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Parts of this work were previously presented as follows: Talakad JC, Sun L, Kumar S, and Halpert JR (2008) Characterization of the P450 2B6 genetic variant K262R for temperature stability and drug binding. Experimental Biology-2008; 2008 Apr 2–9; San Diego, CA. American Society for Pharmacology and Experimental Therapeutics, Bethesda, MD.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.023655.
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ABBREVIATIONS: P450, cytochrome P450; DDI, drug-drug interaction; 7-MFC, 7-methoxy-4-(trifluoromethyl)coumarin; CYMAL-5, 5-cyclohexylpentyl-β-d-maltoside; CSM, conserved sequence motif.
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↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
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↵1 Current affiliation: Division of Pharmacology and Toxicology, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri.
- Received July 30, 2008.
- Accepted December 5, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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