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
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This work was supported by the National Institutes of Health [Grant GM069753].
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.023358.
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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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