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Research ArticleArticle

Validation of Bupropion Hydroxylation as a Selective Marker of Human Cytochrome P450 2B6 Catalytic Activity

Stephanie R. Faucette, Roy L. Hawke, Edward L. Lecluyse, Stacy S. Shord, Bingfang Yan, Ronald M. Laethem and Celeste M. Lindley
Drug Metabolism and Disposition October 2000, 28 (10) 1222-1230;
Stephanie R. Faucette
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Roy L. Hawke
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Edward L. Lecluyse
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Stacy S. Shord
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Bingfang Yan
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Ronald M. Laethem
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Celeste M. Lindley
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Abstract

The purpose of this study was to establish bupropion (BUP) hydroxylation as a selective in vitro marker of cytochrome P450 (CYP) 2B6 catalytic activity. Among a panel of 16 human liver microsomes (HLMs), BUP hydroxylase activity varied 80-fold when assayed at 500 μM substrate and significantly correlated with CYP2B6 blotting density (r2 = 0.99) andS-mephenytoin N-demethylase activity (r2 = 0.98). Kinetic analysis of BUP hydroxylation was performed in a subset of seven HLMs representative of the 80-fold range in activity. Sigmoidal kinetics suggestive of allosteric activation was observed in five HLMs exhibiting low or high activity; the mean apparent Km for BUP hydroxylation in these HLMs (130 μM) was similar to theKm for cDNA-expressed CYP2B6 (156 μM). Nonsaturable, biphasic kinetics was observed in two HLMs exhibiting low activity. Among a panel of cDNA-expressed P450 isoforms, CYP2B6 and CYP2E1 demonstrated the highest rates of BUP hydroxylation at 12 mM BUP (7.0 and 2.4 pmol/min/pmol of P450, respectively). The relative contributions of CYP2B6 and CYP2E1 to BUP hydroxylation were estimated by using immunoinhibitory monoclonal antibodies (MAB) to these enzymes. MAB-2B6 produced 88% maximum inhibition of BUP hydroxylation when assayed at 12 mM BUP in a high activity HLM, whereas MAB-2E1 produced 81% maximum inhibition in a low activity HLM. However, negligible inhibition by MAB-2E1 was observed when low and high activity HLMs were assayed at 500 μM BUP. These results demonstrate selectivity of BUP hydroxylation for CYP2B6 at 500 μM BUP, thereby validating its use as a diagnostic in vitro marker of CYP2B6 catalytic activity.

Footnotes

  • Send reprint requests to: Celeste M. Lindley, Pharm.D., M.S., FCCP, FASHP, Division of Pharmacotherapy, School of Pharmacy, CB #7360, Beard Hall, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360. E-mail: celeste_lindley{at}unc.edu

  • This work was supported in part by a Hollingsworth Faculty Scholarship, awarded to C.M.L. by the School of Pharmacy, University of North Carolina at Chapel Hill, and by a Junior Faculty Development Award, awarded to E.L.L. by the University Research Council, University of North Carolina at Chapel Hill.

  • Abbreviations used are::
    CYP or P450
    cytochrome P450
    7-EFC
    7-ethoxy-4-trifluoromethylcoumarin
    HLM
    human liver microsome
    MAB
    monoclonal antibody
    MAB-2B6
    monoclonal antibody specific to CYP2B6
    MAB-2E1
    monoclonal antibody specific to CYP2E1
    BUP
    bupropion
    • Received June 30, 2000.
    • Accepted July 6, 2000.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 28 (10)
Drug Metabolism and Disposition
Vol. 28, Issue 10
1 Oct 2000
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Research ArticleArticle

Validation of Bupropion Hydroxylation as a Selective Marker of Human Cytochrome P450 2B6 Catalytic Activity

Stephanie R. Faucette, Roy L. Hawke, Edward L. Lecluyse, Stacy S. Shord, Bingfang Yan, Ronald M. Laethem and Celeste M. Lindley
Drug Metabolism and Disposition October 1, 2000, 28 (10) 1222-1230;

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Research ArticleArticle

Validation of Bupropion Hydroxylation as a Selective Marker of Human Cytochrome P450 2B6 Catalytic Activity

Stephanie R. Faucette, Roy L. Hawke, Edward L. Lecluyse, Stacy S. Shord, Bingfang Yan, Ronald M. Laethem and Celeste M. Lindley
Drug Metabolism and Disposition October 1, 2000, 28 (10) 1222-1230;
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