EFFECTS OF THE CYTOKINE SYNTHESIS INHIBITOR CGP 47969A ON NITRIC-OXIDE PRODUCTION BY LIPOPOLYSACCHARIDE-STIMULATED J774A.1 MACROPHAGES

CGP 47969A is a novel inhibitor of the biosynthesis of interleukin-1 a nd other cytokines, being developed as an anti-arthritic. The effect o f the compound on lipopolysaccharide (LPS; 1 mu g/ml) stimulated nitri c oxide (NO) production by the mouse macrophage cell line, J774A.1, wa s examined in the present study. CGP 47969A inhibited NO production in a concentration-dependent fashion (0.1-10 mu M; IC50 = 2 mu M) in a 2 4 h assay. Dexamethasone (Dex), which inhibits cytokine and inducible nitric oxide synthase (iNOS) gene transcription, and N-methyl arginine (NMA), a substrate analogue inhibitor of NOS activity, also inhibited NO production in this assay system with IC50 values of approximately 5 nM and 100 mu M, respectively. When iNOS expression was induced by L PS for 24 h, CGP 47969A and Dex did not inhibit NO production, whereas NMA retained activity (IC50 = 40 mu M). In time course experiments, C GP 47969A (10 mu M) Or Dex (1 mu M) were added to J774A.1 cultures at t = 0, 1, 3 or 6 h after LPS. Dex inhibited NO production by 86%, 57%, 35% and 15% at these time points, while CGP 47969A inhibited by 90%, 98%, 89% and 76%. Taken together, the results indicate that CGP 47969A inhibits NO production by an effect similar to the inhibitory effect on cytokine production rather than by inhibition of iNOS enzyme activi ty per se or iNOS gene expression. The ability of CCP 47969A to inhibi t cytokine and NO production may explain its efficacy in animal models of arthritis.