[O-METHYL C-14]NAPROXEN O-DEMETHYLASE ACTIVITY IN HUMAN LIVER-MICROSOMES - EVIDENCE FOR THE INVOLVEMENT OF CYTOCHROME P4501A2 AND P4502C9 10/

Cytochrome P450 (CYP) activity in human liver microsomes was measured after the O-demethylation of [O-methyl C-14]naproxen (NAPase). The for mation of [C-14]formaldehyde in the presence of microsomes was describ ed by an apparent K-M(1) and V-max(1) of 0.16 +/- 0.09 mM and 4.1 +/- 2.8 nmol HCHO/min/mg protein (mean +/- SD; N = 5 different livers), re spectively, over a relatively wide naproxen concentration (5-1600 mu M ) range. With two sets of microsomes, a high K-M NAPase component was also detected (mean K-M2 = 2.7 mM; mean V-max2 = 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 i nhibited (32-54%) by a number of purported CYP2C (CYP2C9/10) inhibitor s/substrates (e.g. phenytoin, sulfaphenazole, tienilic acid, tolbutami de, and ibuprofen). Only marginal decreases in activity (less than or equal to 14%) were observed with inhibitors of other CYP proteins. How ever, NAPase activity was also found to correlate significantly with C YP1A2 [ethoxyresorufin O-deethylase (ERODase)] activity (r = 0.68, p < 0.05, n = 11). In addition, the reaction was inhibited (36-75%, N = 1 1 different livers) by furafylline (FURA), a CYP1A2-selective mechanis m-based inhibitor. The effect of FURA and tienilic acid was additive, leading to 90 +/- 4.2% inhibition of NAPase activity. FURA-inhibited a ctivity also significantly correlated with ERODase activity (r = 0.78, p < 0.01, N = 11), whereas tienilic acid-inhibited activity correlate d with TOLase activity (r = 0.63, p < 0.05, N = 10). In human B-lympho blast microsomes, cDNA-expressed CYP1A2 exhibited relatively high acti vity (K-M = 0.25 mM; V-max = 24 nmol/min/nmol CYP), when compared with CYP2A6, CYP2D6, CYP2E1, CYP2B6, and CYP3A4. The kinetic parameters fo r reconstituted purified human liver microsomal CYP2C9 (K-M = 0.43 mM; V-max = 11 nmol/min/nmol CYP) were comparable with those of CYP1A2. I t is concluded that the O-demethylation of naproxen (less than or equa l to 0.4 mM) is catalyzed by CYP2C subfamily members (CYP2C9/10) and C YP1A2 in human liver microsomes.