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Research ArticleArticle

The Role of Human Cytochrome P450 Enzymes in the Formation of 2-Hydroxymetronidazole: CYP2A6 is the High Affinity (Low Km) Catalyst

Robin E. Pearce, Michael Cohen-Wolkowiez, Mario R. Sampson and Gregory L. Kearns
Drug Metabolism and Disposition September 2013, 41 (9) 1686-1694; DOI: https://doi.org/10.1124/dmd.113.052548
Robin E. Pearce
Section of Developmental Pharmacology and Experimental Therapeutics, Division of Pediatric Clinical Pharmacology and Therapeutic Innovation, The Children’s Mercy Hospitals (R.E.P., G.L.K.); Departments of Pediatrics (R.E.P., G.L.K.) and Pharmacology (G.L.K.), University of Missouri-Kansas City, Kansas City, Missouri; Duke Clinical Research Institute (M.C-W., M.R.S.), Department of Pediatrics (M.C-W., M.R.S.), Duke University, Durham, North Carolina; and UNC Eshelman School of Pharmacy (M.R.S), University of North Carolina, Chapel Hill, North Carolina
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Michael Cohen-Wolkowiez
Section of Developmental Pharmacology and Experimental Therapeutics, Division of Pediatric Clinical Pharmacology and Therapeutic Innovation, The Children’s Mercy Hospitals (R.E.P., G.L.K.); Departments of Pediatrics (R.E.P., G.L.K.) and Pharmacology (G.L.K.), University of Missouri-Kansas City, Kansas City, Missouri; Duke Clinical Research Institute (M.C-W., M.R.S.), Department of Pediatrics (M.C-W., M.R.S.), Duke University, Durham, North Carolina; and UNC Eshelman School of Pharmacy (M.R.S), University of North Carolina, Chapel Hill, North Carolina
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Mario R. Sampson
Section of Developmental Pharmacology and Experimental Therapeutics, Division of Pediatric Clinical Pharmacology and Therapeutic Innovation, The Children’s Mercy Hospitals (R.E.P., G.L.K.); Departments of Pediatrics (R.E.P., G.L.K.) and Pharmacology (G.L.K.), University of Missouri-Kansas City, Kansas City, Missouri; Duke Clinical Research Institute (M.C-W., M.R.S.), Department of Pediatrics (M.C-W., M.R.S.), Duke University, Durham, North Carolina; and UNC Eshelman School of Pharmacy (M.R.S), University of North Carolina, Chapel Hill, North Carolina
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Gregory L. Kearns
Section of Developmental Pharmacology and Experimental Therapeutics, Division of Pediatric Clinical Pharmacology and Therapeutic Innovation, The Children’s Mercy Hospitals (R.E.P., G.L.K.); Departments of Pediatrics (R.E.P., G.L.K.) and Pharmacology (G.L.K.), University of Missouri-Kansas City, Kansas City, Missouri; Duke Clinical Research Institute (M.C-W., M.R.S.), Department of Pediatrics (M.C-W., M.R.S.), Duke University, Durham, North Carolina; and UNC Eshelman School of Pharmacy (M.R.S), University of North Carolina, Chapel Hill, North Carolina
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Abstract

Despite metronidazole’s widespread clinical use since the 1960s, the specific enzymes involved in its biotransformation have not been previously identified. Hence, in vitro studies were conducted to identify and characterize the cytochrome P450 enzymes involved in the formation of the major metabolite, 2-hydroxymetronidazole. Formation of 2-hydroxymetronidazole in human liver microsomes was consistent with biphasic, Michaelis-Menten kinetics. Although several cDNA-expressed P450 enzymes catalyzed 2-hydroxymetronidazole formation at a supratherapeutic concentration of metronidazole (2000 μM), at a “therapeutic concentration” of 100 μM only CYPs 2A6, 3A4, 3A5, and 3A7 catalyzed metronidazole 2-hydroxylation at rates substantially greater than control vector, and CYP2A6 catalyzed 2-hydroxymetronidazole formation at rates 6-fold higher than the next most active enzyme. Kinetic studies with these recombinant enzymes revealed that CYP2A6 has a Km = 289 μM which is comparable to the Km for the high-affinity (low-Km) enzyme in human liver microsomes, whereas the Km values for the CYP3A enzymes corresponded with the low-affinity (high-Km) component. The sample-to-sample variation in 2-hydroxymetronidazole formation correlated significantly with CYP2A6 activity (r ≥ 0.970, P < 0.001) at substrate concentrations of 100 and 300 μM. Selective chemical inhibitors of CYP2A6 inhibited metronidazole 2-hydroxylation in a concentration-dependent manner and inhibitory antibodies against CYP2A6 virtually eliminated metronidazole 2-hydroxylation (>99%). Chemical and antibody inhibitors of other P450 enzymes had little or no effect on metronidazole 2-hydroxylation. These results suggest that CYP2A6 is the primary catalyst responsible for the 2-hydroxylation of metronidazole, a reaction that may function as a marker of CYP2A6 activity both in vitro and in vivo.

Footnotes

    • Received April 19, 2013.
    • Accepted June 27, 2013.
  • This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Training Grant T32GM086330] (to M.R.S.).

  • dx.doi.org/10.1124/dmd.113.052548.

  • Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 41 (9)
Drug Metabolism and Disposition
Vol. 41, Issue 9
1 Sep 2013
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Research ArticleArticle

Biotransformation of Metronidazole in Vitro

Robin E. Pearce, Michael Cohen-Wolkowiez, Mario R. Sampson and Gregory L. Kearns
Drug Metabolism and Disposition September 1, 2013, 41 (9) 1686-1694; DOI: https://doi.org/10.1124/dmd.113.052548

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Research ArticleArticle

Biotransformation of Metronidazole in Vitro

Robin E. Pearce, Michael Cohen-Wolkowiez, Mario R. Sampson and Gregory L. Kearns
Drug Metabolism and Disposition September 1, 2013, 41 (9) 1686-1694; DOI: https://doi.org/10.1124/dmd.113.052548
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