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Research ArticleArticle

VALIDATED ASSAYS FOR HUMAN CYTOCHROME P450 ACTIVITIES

Robert L. Walsky and R. Scott Obach
Drug Metabolism and Disposition June 2004, 32 (6) 647-660; DOI: https://doi.org/10.1124/dmd.32.6.647
Robert L. Walsky
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R. Scott Obach
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Abstract

The measurement of the effect of new chemical entities on human cytochrome P450 marker activities using in vitro experimentation represents an important experimental approach in drug development. In vitro drug interaction data can be used in guiding the design of clinical drug interaction studies, or, when no effect is observed in vitro, the data can be used in place of an in vivo study to claim that no interaction will occur in vivo. To make such a claim, it must be assured that the in vitro experiments are performed with absolute confidence in the methods used and data obtained. To meet this need, 12 semiautomated assays for human P450 marker substrate activities have been developed and validated using approaches described in the GLP (good laboratory practices) as per the code of U.S. Federal Regulations. The assays that were validated are: phenacetin O-deethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), bupropion hydroxylase (CYP2B6), amodiaquine N-deethylase (CYP2C8), diclofenac 4′-hydroxylase and tolbutamide methylhydroxylase (CYP2C9), (S)-mephenytoin 4′-hydroxylase (CYP2C19), dextromethorphan O-demethylase (CYP2D6), chlorzoxazone 6-hydroxylase (CYP2E1), felodipine dehydrogenase, testosterone 6β-hydroxylase, and midazolam 1′-hydroxylase (CYP3A4 and CYP3A5). High-pressure liquid chromatography-tandem mass spectrometry, using stable isotope-labeled internal standards, has been applied as the analytical method. This analytical approach, through its high sensitivity and selectivity, has permitted the use of very low incubation concentrations of microsomal protein (0.01-0.2 mg/ml). Analytical assay accuracy and precision values were excellent. Enzyme kinetic and inhibition parameters obtained using these methods demonstrated high precision and were within the range of values previously reported in the scientific literature. These methods should prove useful in the routine assessments of the potential for new drug candidates to elicit pharmacokinetic drug interactions via inhibition of cytochrome P450 activities.

Footnotes

  • ↵1 Abbreviations used are: P450, cytochrome P450; GLP, good laboratory practices; PPP, 2-phenyl-2-(1-piperdinyl)propane; HPLC, high-pressure liquid chromatography; MS, mass spectrometry; LC-MS/MS, liquid chromatography/tandem mass spectrometry; HLM, human liver microsomes.

    • Received January 9, 2004.
    • Accepted February 23, 2004.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 32 (6)
Drug Metabolism and Disposition
Vol. 32, Issue 6
1 Jun 2004
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Research ArticleArticle

VALIDATED ASSAYS FOR HUMAN CYTOCHROME P450 ACTIVITIES

Robert L. Walsky and R. Scott Obach
Drug Metabolism and Disposition June 1, 2004, 32 (6) 647-660; DOI: https://doi.org/10.1124/dmd.32.6.647

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Research ArticleArticle

VALIDATED ASSAYS FOR HUMAN CYTOCHROME P450 ACTIVITIES

Robert L. Walsky and R. Scott Obach
Drug Metabolism and Disposition June 1, 2004, 32 (6) 647-660; DOI: https://doi.org/10.1124/dmd.32.6.647
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