Mechanism-based inactivation of cytochrome P450 2C9 by tienilic acid and (+/-)-suprofen: a comparison of kinetics and probe substrate selection.
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 approximately 9 ml/min/micromol), 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 (approximately 1 ml/min/micromol). 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/micromol) and approximately 3-fold higher (3 ml/min/micromol), 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 microM and an in vitro half-life of 5 min compared with a K(s) of 21 microM 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.
Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, St. Louis Laboratories, St. Louis, Missouri, USA. j.matt.hutzler@pfizer.com
J Matthew
JM
Hutzler
Larissa M
LM
Balogh
Michael
M
Zientek
Vikas
V
Kumar
Timothy S
TS
Tracy
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000493:pharmacokinetics / Q000494:pharmacology*; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors*; D002853:Chromatography, Liquid; D065729:Cytochrome P-450 CYP2C9; D004232:Diuretics / Q000493:pharmacokinetics / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000493:pharmacokinetics / Q000494:pharmacology*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013496:Suprofen / Q000493:pharmacokinetics / Q000494:pharmacology*; D053719:Tandem Mass Spectrometry; D013989:Ticrynafen / Q000493:pharmacokinetics / Q000494:pharmacology*
D000894:Anti-Inflammatory Agents, Non-Steroidal; D004232:Diuretics; D004791:Enzyme Inhibitors; D013496:Suprofen; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D013989:Ticrynafen