title: Polymorphic variants (CYP2C9*3 and CYP2C9*5) and the F114L active site mutation of CYP2C9: effect on atypical kinetic metabolism profiles.
abstract: CYP2C9 wild-type protein has been shown to exhibit atypical kinetic profiles of metabolism that may affect in vitro-in vivo predictions made during the drug development process. Previous work suggests a substrate-dependent effect of polymorphic variants of CYP2C9 on the rate of metabolism; however, it is hypothesized that these active site amino acid changes will affect the kinetic profile of a drug's metabolism as well. To this end, the kinetic profiles of three model CYP2C9 substrates (flurbiprofen, naproxen, and piroxicam) were studied using purified CYP2C9*1 (wild-type) and variants involving active site amino acid changes, including the naturally occurring variants CYP2C9*3 (Leu359) and CYP2C9*5 (Glu360) and the man-made mutant CYP2C9 F114L. CYP2C9*1 (wild-type) metabolized each of the three compounds with a distinctive profile reflective of typical hyperbolic (flurbiprofen), biphasic (naproxen), and substrate inhibition (piroxicam) kinetics. CYP2C9*3 metabolism was again hyperbolic for flurbiprofen, of a linear form for naproxen (no saturation noted), and exhibited substrate inhibition with piroxicam. CYP2C9*5-mediated metabolism was hyperbolic for flurbiprofen and piroxicam but linear with respect to naproxen turnover. The F114L mutant exhibited a hyperbolic kinetic profile for flurbiprofen metabolism, a linear profile for naproxen metabolism, and a substrate inhibition kinetic profile for piroxicam metabolism. In all cases except F114L-mediated piroxicam metabolism, turnover decreased and the K(m) generally increased for each allelic variant compared with wild-type enzyme. It seems that the kinetic profile of CYP2C9-mediated metabolism is dependent on both substrate and the CYP2C9 allelic variant, thus having potential ramifications on drug disposition predictions made during the development process.
authors.affiliation: Deparment of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, West Virginia 26506, USA. ttracy@hsc.wvu.edu
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Tracy
authors.forename: J Matthew
authors.initials: JM
authors.lastname: Hutzler
authors.forename: Robert L
authors.initials: RL
authors.lastname: Haining
authors.forename: Allan E
authors.initials: AE
authors.lastname: Rettie
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Hummel
authors.forename: Leslie J
authors.initials: LJ
authors.lastname: Dickmann
mesh_terms: D000483:Alleles; D019943:Amino Acid Substitution; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites / Q000235:genetics; D065729:Cytochrome P-450 CYP2C9; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005480:Flurbiprofen / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009154:Mutation; D009288:Naproxen / Q000737:chemistry / Q000378:metabolism; D010894:Piroxicam / Q000737:chemistry / Q000378:metabolism; D011110:Polymorphism, Genetic; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D010894:Piroxicam; D009288:Naproxen; D005480:Flurbiprofen; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase