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Research ArticleArticle

Development of an In Vitro System with Human Liver Microsomes for Phenotyping of CYP2C9 Genetic Polymorphisms with a Mechanism-Based Inactivator

Darcy R. Flora and Timothy S. Tracy
Drug Metabolism and Disposition April 2012, 40 (4) 836-842; DOI: https://doi.org/10.1124/dmd.111.043372
Darcy R. Flora
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Timothy S. Tracy
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Abstract

Polymorphisms in cytochrome P450 enzymes can significantly alter the rate of drug metabolism, as well as the extent of drug-drug interactions. Individuals who homozygotically express the CYP2C9*3 allele (I359L) of CYP2C9 exhibit ∼70 to 80% reductions in the oral clearance of drugs metabolized through this pathway; the reduction in clearance is ∼40 to 50% for heterozygotic individuals. Although these polymorphisms result in a decrease in the activity of individual enzyme molecules, we hypothesized that decreasing the total number of active enzyme molecules in an in vitro system (CYP2C9*1/*1 human liver microsomes) by an equivalent percentage could produce the same net change in overall metabolic capacity. To this end, the selective CYP2C9 mechanism-based inactivator tienilic acid was used to reduce irreversibly the total CYP2C9 activity in human liver microsomes. Tienilic acid concentrations were effectively titrated to produce microsomal preparations with 43 and 73% less activity, mimicking the CYP2C9*1/*3 and CYP2C9*3/*3 genotypes, respectively. With probe substrates specific for other major cytochrome P450 enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6, CYP2E1, and CYP3A4), no apparent changes in the rate of metabolism were noted for these enzymes after the addition of tienilic acid, which suggests that this model is selective for CYP2C9. In lieu of using rare human liver microsomes from CYP2C9*1/*3 and CYP2C9*3/*3 individuals, a tienilic acid-created knockdown in human liver microsomes may be an appropriate in vitro model to determine CYP2C9-mediated metabolism of a given substrate, to determine whether other drug-metabolizing enzymes may compensate for reduced CYP2C9 activity, and to predict the extent of genotype-dependent drug-drug interactions.

Footnotes

  • This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grants GM069753, GM032165].

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

    http://dx.doi.org/10.1124/dmd.111.043372.

  • ABBREVIATIONS:

    HLMs
    human liver microsomes
    P450
    cytochrome P450.

  • Received October 16, 2011.
  • Accepted December 28, 2011.
  • Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 40 (4)
Drug Metabolism and Disposition
Vol. 40, Issue 4
1 Apr 2012
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Research ArticleArticle

TIENILIC ACID-CREATED CYP2C9 GENOTYPE EQUIVALENTS

Darcy R. Flora and Timothy S. Tracy
Drug Metabolism and Disposition April 1, 2012, 40 (4) 836-842; DOI: https://doi.org/10.1124/dmd.111.043372

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Research ArticleArticle

TIENILIC ACID-CREATED CYP2C9 GENOTYPE EQUIVALENTS

Darcy R. Flora and Timothy S. Tracy
Drug Metabolism and Disposition April 1, 2012, 40 (4) 836-842; DOI: https://doi.org/10.1124/dmd.111.043372
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