INVOLVEMENT OF MULTIPLE CYTOCHROME-P450 ISOFORMS IN NAPROXEN O-DEMETHYLATION

Objective: A series of studies was undertaken to determine the cytochr ome P450 isoform(s) involved in naproxen demethylation and whether thi s included the same isoforms reported to be involved in the metabolism of other NSAIDs. Methods: (S)-Naproxen was incubated with human liver microsomes in the presence of a NADPH-generating system and the forma tion of desmethylnaproxen was measured by high-performance liquid chro matography (HPLC). To further clarify the specific isoforms involved, experiments were conducted with preparations expressing only a single P450 isoform (vaccinia virus-expressed cells and microsomes derived fr om a lymphoblastoid cell line, each transfected with specific P450 cDN As) as well as inhibition studies using human liver microsomes and put ative specific P450 inhibitors. Results: In human liver microsomes (n = 7), desmethylnaproxen formation was observed with a mean k(M) of 92 (21) mu mol.l(-1), V-max of 538 pmol.min(-1).mg(-1) protein and C-int2 (reflective of a second binding site) of 0.36 mu l.min(-1) mg(-1) pro tein. This C-int2 term was added since Eadie-Scatchard analysis sugges ted the involvement of more than one enzyme. Studies using putative sp ecific P450 inhibitors demonstrated inhibition of this reaction by sul faphenazole, (apparent Ki=1.6 mu mol.l(-1)), warfarin (apparent Ki=27 mu mol.l(-1)), piroxicam (apparent Ki=23 mu mol.l(-1)) and tolbutamide (apparent Ki = 128 mu mol.l(-1)). No effect was observed when alpha-n aphthoflavone and troleandomycin were employed as inhibitors, but reac tion with furafylline produced, on average, a maximum inhibition of 23 %. At a naproxen concentration of 150 mu mol.l(-1) formation of desmet hylnaproxen was observed in cells expressing P450 1A2, 2C8, 2C9 and it s allelic variant 2C9R144C. To further characterize these reactions, s aturation kinetics experiments were conducted for the P450s 1A2, 2C8 a nd 2C9. The k(M) and V-max for P450 1A2 were 189.5 mu mol.l(-1) and 7. 3 pmol.min(-1).pmol(-1) P450, respectively. Likewise, estimates of k(M ) and V-max for P450 2C9 were 340.5 mu mol.l(-1) and 41.4 pmol.min(-1) .pmol(-1) P450, respectively. Reliable estimates of k(M) and V-max cou ld not be made for P450 2C8 due to the nonsaturable nature of the proc ess over the concentration range studied. Conclusion: Multiple cytochr ome P450 isoforms (P450 1A2, 2C8 and 2C9) appear to be involved in nap roxen demethylation, although 2C9 appears to be the predominant form.