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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Application of the Relative Activity Factor Approach in Scaling from Heterologously Expressed Cytochromes P450 to Human Liver Microsomes: Studies on Amitriptyline as a Model Substrate

Karthik Venkatakrishnan, Lisa L. von Moltke and David J. Greenblatt
Journal of Pharmacology and Experimental Therapeutics April 2001, 297 (1) 326-337;
Karthik Venkatakrishnan
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Lisa L. von Moltke
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David J. Greenblatt
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Abstract

The relative activity factor (RAF) approach is being increasingly used in the quantitative phenotyping of multienzyme drug biotransformations. Using lymphoblast-expressed cytochromes P450 (CYPs) and the tricyclic antidepressant amitriptyline as a model substrate, we have tested the hypothesis that the human liver microsomal rates of a biotransformation mediated by multiple CYP isoforms can be mathematically reconstructed from the rates of the biotransformation catalyzed by individual recombinant CYPs using the RAF approach, and that the RAF approach can be used for the in vitro-in vivo scaling of pharmacokinetic clearance from in vitro intrinsic clearance measurements in heterologous expression systems. In addition, we have compared the results of two widely used methods of quantitative reaction phenotyping, namely, chemical inhibition studies and the prediction of relative contributions of individual CYP isoforms using the RAF approach. For the pathways of N-demethylation (mediated by CYPs 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and E-10 hydroxylation (mediated by CYPs 2B6, 2D6, and 3A4), the model-predicted biotransformation rates in microsomes from a panel of 12 human livers determined from enzyme kinetic parameters of the recombinant CYPs were similar to, and correlated with the observed rates. The model-predicted clearance viaN-demethylation was 53% lower than the previously reported in vivo pharmacokinetic estimates. Model-predicted relative contributions of individual CYP isoforms to the net biotransformation rate were similar to, and correlated with the fractional decrement in human liver microsomal reaction rates by chemical inhibitors of the respective CYPs, provided the chemical inhibitors used were specific to their target CYP isoforms.

Footnotes

  • Send reprint requests to: David J. Greenblatt, M.D., Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. E-mail: Dj.Greenblatt{at}tufts.edu

  • This work was supported by Grants MH-34223, DA-05258, DA-13209, MH-19924, MH-58435, GM-61834, and RR-00054 from the Department of Health and Human Services. L.L.v.M. was the recipient of a Scientist Development Award (K21-MH-01237) from the National Institute of Mental Health.

  • Abbreviations:
    CYP
    cytochrome P450
    RAF
    relative activity factor
    cDNA
    complementary DNA
    TAO
    troleandomycin
    FDV
    fractional decrement of reaction velocity
    • Received September 14, 2000.
    • Accepted January 3, 2001.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 297 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 297, Issue 1
1 Apr 2001
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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Application of the Relative Activity Factor Approach in Scaling from Heterologously Expressed Cytochromes P450 to Human Liver Microsomes: Studies on Amitriptyline as a Model Substrate

Karthik Venkatakrishnan, Lisa L. von Moltke and David J. Greenblatt
Journal of Pharmacology and Experimental Therapeutics April 1, 2001, 297 (1) 326-337;

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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Application of the Relative Activity Factor Approach in Scaling from Heterologously Expressed Cytochromes P450 to Human Liver Microsomes: Studies on Amitriptyline as a Model Substrate

Karthik Venkatakrishnan, Lisa L. von Moltke and David J. Greenblatt
Journal of Pharmacology and Experimental Therapeutics April 1, 2001, 297 (1) 326-337;
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