title: Major role of human liver microsomal cytochrome P450 2C9 (CYP2C9) in the oxidative metabolism of celecoxib, a novel cyclooxygenase-II inhibitor.
abstract: In vitro studies were conducted to identify the cytochromes P450 (CYP) involved in the oxidative metabolism of celecoxib. The hydroxylation of celecoxib conformed to monophasic Michaelis-Menten kinetics (mean +/- S.D., n = 4 livers, K(m) = 3.8 +/- 0.95 microM, V(max) = 0.70 +/- 0.45 nmol/min/mg protein) in the presence of human liver microsomes, although substrate inhibition was significant at higher celecoxib concentrations. The treatment of a panel of human liver microsomal samples (n = 16 subjects) with antibodies against CYP2C9 and CYP3A4 inhibited the formation of hydroxy celecoxib by 72 to 92% and 0 to 27%, respectively. The presence of both antibodies in the incubation suppressed the activity by 90 to 94%. In addition, the formation of hydroxy celecoxib significantly correlated with CYP2C9-selective tolbutamide methyl hydroxylation (r = 0.92, P <. 001) and CYP3A-selective testosterone 6beta-hydroxylation (r = 0.55, P <.02). In contrast, correlation with activities selective for other forms of CYP was weak (r </= 0.46). Chemical inhibition studies showed that ketoconazole (selective for CYP3A4) and sulfaphenazole (selective for CYP2C9) inhibited the formation of hydroxy celecoxib in a concentration-dependent manner, whereas potent inhibitors selective for other forms of CYP did not show any significant effect over a range of 1 to 10 microM. In agreement, cDNA-expressed CYP2C9 catalyzed the formation of hydroxy celecoxib with an apparent K(m) value (microM) and a V(max) value (pmol/min/pmol recombinant CYP) of 5.9 and 21.7, whereas a higher K(m) value (18.2) and a lower V(max) value (1.42) were obtained with rCYP3A4. It is concluded that methyl hydroxylation of celecoxib is primarily catalyzed by human liver microsomal CYP2C9, although CYP3A4 also plays a role.
authors.affiliation: Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania 19486-0004, USA. cuyue_tang@merck.com
authors.forename: C
authors.initials: C
authors.lastname: Tang
authors.forename: M
authors.initials: M
authors.lastname: Shou
authors.forename: Q
authors.initials: Q
authors.lastname: Mei
authors.forename: T H
authors.initials: TH
authors.lastname: Rushmore
authors.forename: A D
authors.initials: AD
authors.lastname: Rodrigues
mesh_terms: D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases; D001711:Biotransformation; D000068579:Celecoxib; D002851:Chromatography, High Pressure Liquid; D016861:Cyclooxygenase Inhibitors / Q000378:metabolism*; D065729:Cytochrome P-450 CYP2C9; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; D007700:Kinetics; D013058:Mass Spectrometry; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000378:metabolism*; D011720:Pyrazoles; D013056:Spectrophotometry, Ultraviolet; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D013449:Sulfonamides / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D019138:Antibodies, Blocking; D016861:Cyclooxygenase Inhibitors; D010940:Plant Proteins; D011720:Pyrazoles; D013449:Sulfonamides; C111957:cox2 protein, Glycine soja; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; D039181:Steroid 16-alpha-Hydroxylase; C510163:CYP3A4 protein, human; D003576:Electron Transport Complex IV; D000068579:Celecoxib