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Research ArticleArticle

Utility of Intersystem Extrapolation Factors in Early Reaction Phenotyping and the Quantitative Extrapolation of Human Liver Microsomal Intrinsic Clearance Using Recombinant Cytochromes P450

Yuan Chen, Liling Liu, Khanh Nguyen and Adrian J. Fretland
Drug Metabolism and Disposition March 2011, 39 (3) 373-382; DOI: https://doi.org/10.1124/dmd.110.035147
Yuan Chen
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Liling Liu
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Khanh Nguyen
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Adrian J. Fretland
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Abstract

Reaction phenotyping using recombinant human cytochromes P450 (P450) has great utility in early discovery. However, to fully realize the advantages of using recombinant expressed P450s, the extrapolation of data from recombinant systems to human liver microsomes (HLM) is required. In this study, intersystem extrapolation factors (ISEFs) were established for CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 using 11 probe substrates, based on substrate depletion and/or metabolite formation kinetics. The ISEF values for CYP2C9, CYP2D6, and CYP3A4 determined using multiple substrates were similar across substrates. When enzyme kinetics of metabolite formation for CYP1A2, 2C9, 2D6, and 3A4 were used, the ISEFs determined were generally within 2-fold of that determined on the basis of substrate depletion. Validation of ISEFs was conducted using 10 marketed drugs by comparing the extrapolated data with published data. The major isoforms responsible for the metabolism were identified, and the contribution of the predominant P450s was similar to that of previously reported data. In addition, phenotyping data from internal compounds, extrapolated using the rhP450-ISEF method, were comparable to those obtained using an HLM-based inhibition assay approach. Moreover, the intrinsic clearance (CLint) calculated from extrapolated rhP450 data correlated well with measured HLM CLint. The ISEF method established in our laboratory provides a convenient tool in early reaction phenotyping for situations in which the HLM-based inhibition approach is limited by low turnover and/or unavailable metabolite formation. Furthermore, this method allows for quantitative extrapolation of HLM intrinsic clearance from rhP450 phenotyping data simultaneously to obtaining the participating metabolizing enzymes.

Footnotes

  • ↵2 Early ADME Department, Roche–Nutley, Nutley, New Jersey.

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

    doi:10.1124/dmd.110.035147.

  • ABBREVIATIONS:

    P450
    cytochrome P450
    rhP450
    recombinant human cytochrome P450
    HLM
    human liver microsomes
    ISEF
    intersystem extrapolation factors
    CLint
    intrinsic clearance
    CLISEF
    ISEF defined with respect to intrinsic clearance CLint
    VISEF
    ISEF defined with respect to intrinsic clearance Vmax
    RMSE
    root mean square error
    MRS
    mean residual sum
    RAF
    relative activity factor.

  • Received June 28, 2010.
  • Accepted December 8, 2010.
  • Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 39 (3)
Drug Metabolism and Disposition
Vol. 39, Issue 3
1 Mar 2011
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Research ArticleArticle

Utility of Intersystem Extrapolation Factors in Early Reaction Phenotyping and the Quantitative Extrapolation of Human Liver Microsomal Intrinsic Clearance Using Recombinant Cytochromes P450

Yuan Chen, Liling Liu, Khanh Nguyen and Adrian J. Fretland
Drug Metabolism and Disposition March 1, 2011, 39 (3) 373-382; DOI: https://doi.org/10.1124/dmd.110.035147

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Research ArticleArticle

Utility of Intersystem Extrapolation Factors in Early Reaction Phenotyping and the Quantitative Extrapolation of Human Liver Microsomal Intrinsic Clearance Using Recombinant Cytochromes P450

Yuan Chen, Liling Liu, Khanh Nguyen and Adrian J. Fretland
Drug Metabolism and Disposition March 1, 2011, 39 (3) 373-382; DOI: https://doi.org/10.1124/dmd.110.035147
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