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Research ArticleArticle

Mechanism-Based Inactivation of Cytochrome P450 2C9 by Tienilic Acid and (±)-Suprofen: A Comparison of Kinetics and Probe Substrate Selection

J. Matthew Hutzler, Larissa M. Balogh, Michael Zientek, Vikas Kumar and Timothy S. Tracy
Drug Metabolism and Disposition January 2009, 37 (1) 59-65; DOI: https://doi.org/10.1124/dmd.108.023358
J. Matthew Hutzler
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Larissa M. Balogh
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Michael Zientek
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Vikas Kumar
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Timothy S. Tracy
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Abstract

In vitro experiments were conducted to compare kinact, KI and inactivation efficiency (kinact/KI) of cytochrome P450 (P450) 2C9 by tienilic acid and (±)-suprofen using (S)-flurbiprofen, diclofenac, and (S)-warfarin as reporter substrates. Although the inactivation of P450 2C9 by tienilic acid when (S)-flurbiprofen and diclofenac were used as substrates was similar (efficiency of ∼9 ml/min/μmol), the inactivation kinetics were characterized by a sigmoidal profile. (±)-Suprofen inactivation of (S)-flurbiprofen and diclofenac hydroxylation was also described by a sigmoidal profile, although inactivation was markedly less efficient (∼1 ml/min/μmol). In contrast, inactivation of P450 2C9-mediated (S)-warfarin 7-hydroxylation by tienilic acid and (±)-suprofen was best fit to a hyperbolic equation, where inactivation efficiency was moderately higher (10 ml/min/μmol) and ∼3-fold higher (3 ml/min/μmol), respectively, relative to that of the other probe substrates, which argues for careful consideration of reporter substrate when mechanism-based inactivation of P450 2C9 is assessed in vitro. Further investigations into the increased inactivation seen with tienilic acid relative to that with (±)-suprofen revealed that tienilic acid is a higher affinity substrate with a spectral binding affinity constant (Ks) of 2 μM and an in vitro half-life of 5 min compared with a Ks of 21 μM and a 50 min in vitro half-life for (±)-suprofen. Lastly, a close analog of tienilic acid with the carboxylate functionality replaced by an oxirane ring was devoid of inactivation properties, which suggests that an ionic binding interaction with a positively charged residue in the P450 2C9 active site is critical for recognition and mechanism-based inactivation by these close structural analogs.

Footnotes

  • This work was supported by the National Institutes of Health [Grant GM069753].

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

  • doi:10.1124/dmd.108.023358.

  • ABBREVIATIONS: P450, cytochrome P450; DDI, drug-drug interaction; LC-MS/MS, liquid chromatography/tandem mass spectrometry; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate.

    • Received July 12, 2008.
    • Accepted October 3, 2008.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 37 (1)
Drug Metabolism and Disposition
Vol. 37, Issue 1
1 Jan 2009
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Research ArticleArticle

Mechanism-Based Inactivation of Cytochrome P450 2C9 by Tienilic Acid and (±)-Suprofen: A Comparison of Kinetics and Probe Substrate Selection

J. Matthew Hutzler, Larissa M. Balogh, Michael Zientek, Vikas Kumar and Timothy S. Tracy
Drug Metabolism and Disposition January 1, 2009, 37 (1) 59-65; DOI: https://doi.org/10.1124/dmd.108.023358

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Research ArticleArticle

Mechanism-Based Inactivation of Cytochrome P450 2C9 by Tienilic Acid and (±)-Suprofen: A Comparison of Kinetics and Probe Substrate Selection

J. Matthew Hutzler, Larissa M. Balogh, Michael Zientek, Vikas Kumar and Timothy S. Tracy
Drug Metabolism and Disposition January 1, 2009, 37 (1) 59-65; DOI: https://doi.org/10.1124/dmd.108.023358
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