title: [O-methyl 14C]naproxen O-demethylase activity in human liver microsomes: evidence for the involvement of cytochrome P4501A2 and P4502C9/10.
abstract: Cytochrome P450 (CYP) activity in human liver microsomes was measured after the O-demethylation of [O-methyl 14C]naproxen (NAPase). The formation of [14C]formaldehyde in the presence of microsomes was described by an apparent KM(1) and Vmax(1) of 0.16 +/- 0.09 mM and 4.1 +/- 2.8 nmol HCHO/min/mg protein (mean +/- SD; N = 5 different livers), respectively, over a relatively wide naproxen concentration (5-1600 microM) range. With two sets of microsomes, a high KM NAPase component was also detected (mean KM2 = 2.7 mM; mean Vmax2 = 23 nmol HCHO/min/mg). As expected, the O-demethylation of naproxen (0.4 mM) was found to be highly correlated with tolbutamide hydroxylase (TOLase) activity in a panel of human liver microsomes (r = 0.82, p < 0.01, N = 10) and was inhibited (32-54%) by a number of purported CYP2C (CYP2C9/10) inhibitors/substrates (e.g. phenytoin, sulfaphenazole, tienilic acid, tolbutamide, and ibuprofen). Only marginal decreases in activity (< or = 14%) were observed with inhibitors of other CYP proteins. However, NAPase activity was also found to correlate significantly with CYP1A2 [ethoxyresorufin O-deethylase (ERODase)] activity (r = 0.68, p < 0.05, n = 11). In addition, the reaction was inhibited (36-75%, N = 11 different livers) by furafylline (FURA), a CYP1A2-selective mechanism-based inhibitor. The effect of FURA and tienilic acid was additive, leading to 90 +/- 4.2% inhibition of NAPase activity. FURA-inhibited activity also significantly correlated with ERODase activity (r = 0.78, p < 0.01, N = 11), whereas tienilic acid-inhibited activity correlated with TOLase activity (r = 0.63, p < 0.05, N = 10). In human B-lymphoblast microsomes, cDNA-expressed CYP1A2 exhibited relatively high activity (KM = 0.25 mM; Vmax = 24 nmol/min/nmol CYP), when compared with CYP2A6, CYP2D6, CYP2E1, CYP2B6, and CYP3A4. The kinetic parameters for reconstituted purified human liver microsomal CYP2C9 (KM = 0.43 mM; Vmax = 11 nmol/min/nmol CYP) were comparable with those of CYP1A2. It is concluded that the O-demethylation of naproxen (< or = 0.4 mM) is catalyzed by CYP2C subfamily members (CYP2C9/10) and CYP1A2 in human liver microsomes.
authors.affiliation: Drug Metabolism Department, Abbott Laboratories, Abbott Park, IL 60064, USA.
authors.forename: A D
authors.initials: AD
authors.lastname: Rodrigues
authors.forename: M J
authors.initials: MJ
authors.lastname: Kukulka
authors.forename: E M
authors.initials: EM
authors.lastname: Roberts
authors.forename: D
authors.initials: D
authors.lastname: Ouellet
authors.forename: T R
authors.initials: TR
authors.lastname: Rodgers
mesh_terms: D001402:B-Lymphocytes / Q000201:enzymology; D002250:Carbon Radioisotopes; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D009288:Naproxen / Q000378:metabolism; D010090:Oxidoreductases, O-Demethylating / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D002250:Carbon Radioisotopes; D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D009288:Naproxen; D003577:Cytochrome P-450 Enzyme System; D010090:Oxidoreductases, O-Demethylating