Cytochromes P450, 1A2, and 2C9 are responsible for the human hepatic O-demethylation of R- and S-naproxen.
A preliminary report implicated cytochrome P450 (CYP) 2C9 in the human liver microsomal O-demethylation of S-naproxen, suggesting that this pathway may be suitable for investigation of human hepatic CYP2C9 in vitro. Kinetic and inhibitor studies with human liver microsomes and confirmatory investigations with cDNA-expressed enzymes were undertaken here to define the role of CYP2C9 and other isoforms in the O-demethylation of R- and S-naproxen. All studies utilised a newly developed sensitive and specific HPLC assay that measured the respective O-desmethyl metabolites of R- and S-naproxen in incubations of human liver microsomes and in COS cell lysates. Microsomal R- and S-naproxen O-demethylation kinetics followed Michaelis-Menten kinetics, with respective mean apparent Km values of 123 microM and 143 microM. Sulfaphenazole, a specific inhibitor of CYP2C9, reduced the microsomal O-demethylation of R- and S-naproxen by 43% and 47%, respectively, and the CYP1A2 inhibitor furafylline decreased R- and S-naproxen O-demethylation by 38% and 28%, respectively. R,S-Mephenytoin was a weak inhibitor of R- and S-naproxen O-demethylation, but other CYP isoform specific inhibitors (e.g., coumarin, diethyldithiocarbamate, quinidine, troleandomycin) had little or no effect on these reactions. cDNA-expressed CYP2C9 and CYP1A2 were both shown to O-demethylate R- and S-naproxen. Apparent Km values (92-156 microM) for the reactions catalysed by the recombinant enzymes were similar to those observed for human liver microsomal R- and S-naproxen O-demethylation. The data demonstrate that CYP2C9 and CYP1A2 together account for the majority of human liver R- and S-naproxen O-demethylation, precluding the use of either R- or S-naproxen as a CYP isoform-specific substrate in vitro and in vivo.
Miners
J O
JO
Department of Clinical Pharmacology, Flinders Medical Centre, Bedford Park, Australia.
Coulter
S
S
Tukey
R H
RH
Veronese
M E
ME
Birkett
D J
DJ
D001189:Aryl Hydrocarbon Hydroxylases; D019388:Cytochrome P-450 CYP1A2 / Q000737:chemistry*; D065729:Cytochrome P-450 CYP2C9; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008745:Methylation; D008862:Microsomes, Liver / Q000201:enzymology*; D009288:Naproxen / Q000737:chemistry*; D010090:Oxidoreductases, O-Demethylating / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013237:Stereoisomerism; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000737:chemistry
D065607:Cytochrome P-450 Enzyme Inhibitors; D011994:Recombinant Proteins; D009288:Naproxen; D003577:Cytochrome P-450 Enzyme System; D010090:Oxidoreductases, O-Demethylating; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C504573:CYP1A2 protein, human; D019388:Cytochrome P-450 CYP1A2; D039181:Steroid 16-alpha-Hydroxylase