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

Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, St. Louis Laboratories, St. Louis, Missouri (J.M.H., V.K.); Pfizer Global Research and Development, La Jolla Laboratories, La Jolla, California (M.Z.); University of Washington, Department of Medicinal Chemistry, Seattle, Washington (L.M.B.); and University of Minnesota, Department of Experimental and Clinical Pharmacology, Minneapolis, Minnesota (T.S.T.)

In vitro experiments were conducted to compare k_inact, K_I and inactivation efficiency (k_inact/K_I) 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 (K_s) of 2 µM and an in vitro half-life of 5 min compared with a K_s 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.