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Research ArticleArticle

In Vitro Metabolism of Quinidine: The (3S)-3-Hydroxylation of Quinidine Is a Specific Marker Reaction for Cytochrome P-4503A4 Activity in Human Liver Microsomes

Torben Leo Nielsen, Birgitte Buur Rasmussen, Jean-Pierre Flinois, Philippe Beaune and Kim Brøsen
Journal of Pharmacology and Experimental Therapeutics April 1999, 289 (1) 31-37;
Torben Leo Nielsen
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Birgitte Buur Rasmussen
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Jean-Pierre Flinois
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Philippe Beaune
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Kim Brøsen
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Abstract

The aim of this study was to evaluate the (3S)-3-hydroxylation and the N-oxidation of quinidine as biomarkers for cytochrome P-450 (CYP)3A4 activity in human liver microsome preparations. An HPLC method was developed to assay the metabolites (3S)-3-hydroxyquinidine (3-OH-Q) and quinidine N-oxide (Q-N-OX) formed during incubation with microsomes from human liver and from Saccharomyces cerevisiae strains expressing 10 human CYPs. 3-OH-Qformation complied with Michaelis-Menten kinetics (mean values ofVmax and Km: 74.4 nmol/mg/h and 74.2 μM, respectively).Q-N-OX formation followed two-site kinetics with mean values of Vmax,Km andVmax/Km for the low affinity isozyme of 15.9 nmol/mg/h, 76.1 μM and 0.03 ml/mg/h, respectively. 3-OH-Q and Q-N-OXformations were potently inhibited by ketoconazole, itraconazole, and triacetyloleandomycin. Isozyme specific inhibitors of CYP1A2, -2C9, -2C19, -2D6, and -2E1 did not inhibit 3-OH-Q orQ-N-OX formation, withKi values comparable with previously reported values. Statistically significant correlations were observed between CYP3A4 content and formations of 3-OH-Q andQ-N-OX in 12 human liver microsome preparations. Studies with yeast-expressed isozymes revealed that only CYP3A4 actively catalyzed the (3S)-3-hydroxylation. CYP3A4 was the most active enzyme in Q-N-OXformation, but CYP2C9 and 2E1 also catalyzed minor proportions of theN-oxidation. In conclusion, our studies demonstrate that only CYP3A4 is actively involved in the formation of 3-OH-Q. Hence, the (3S)-3-hydroxylation of quinidine is a specific probe for CYP3A4 activity in human liver microsome preparations, whereas the N-oxidation of quinidine is a somewhat less specific marker reaction for CYP3A4 activity, because the presence of a low affinity enzyme is demonstrated by different approaches.

Footnotes

  • Send reprint requests to: Torben Leo Nielsen, Department of Clinical Pharmacology, Institute of Medical Biology, Odense University, Winsløwparken 19, DK-5000 Odense C, Denmark. E-mail:t.nielsen{at}winsloew.ou.dk

  • Abbreviations:
    CYP
    cytochrome P-450
    HL
    human liver
    3-OH-Q
    (3S)-3-hydroxyquinidine
    Q-N-OX
    quinidine N-oxide
    4-CPBG
    4-chlorophenylbiguanide
    [S]
    concentration of substrate
    [I]
    concentration of inhibitor
    IC50
    inhibitor concentration causing a 50% reduction in velocity of a reaction
    n
    Hill coefficient describing sigmoidicity of cooperative binding of an inhibitor
    rs
    correlation coefficient using Spearman’s rank correlation
    • Received May 5, 1998.
    • Accepted October 22, 1998.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 289 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 289, Issue 1
1 Apr 1999
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Research ArticleArticle

In Vitro Metabolism of Quinidine: The (3S)-3-Hydroxylation of Quinidine Is a Specific Marker Reaction for Cytochrome P-4503A4 Activity in Human Liver Microsomes

Torben Leo Nielsen, Birgitte Buur Rasmussen, Jean-Pierre Flinois, Philippe Beaune and Kim Brøsen
Journal of Pharmacology and Experimental Therapeutics April 1, 1999, 289 (1) 31-37;

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Research ArticleArticle

In Vitro Metabolism of Quinidine: The (3S)-3-Hydroxylation of Quinidine Is a Specific Marker Reaction for Cytochrome P-4503A4 Activity in Human Liver Microsomes

Torben Leo Nielsen, Birgitte Buur Rasmussen, Jean-Pierre Flinois, Philippe Beaune and Kim Brøsen
Journal of Pharmacology and Experimental Therapeutics April 1, 1999, 289 (1) 31-37;
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