Pharmacological Characterization of the Microsomal Prostaglandin E(2) Synthase-1 Inhibitor AF3485 In Vitro and In Vivo

RATIONALE: The development of inhibitors of microsomal prostaglandin (PG)E(2) synthase-1 (mPGES-1) was driven by the promise of attaining antiinflammatory agents with a safe cardiovascular profile because of the possible diversion of the accumulated substrate, PGH(2), towards prostacyclin (PGI(2)). OBJECTIVES: We studied the effect of the human mPGES-1 inhibitor, AF3485 (a benzamide derivative) on prostanoid biosynthesis in human whole blood in vitro. To characterize possible off-target effects of the compound, we evaluated: i)the impact of its administration on the systemic biosynthesis of prostanoids in a model of complete Freund's adjuvant (CFA)-induced monoarthritis in rats; ii) the effects on cyclooxygenase (COX)-2 expression and the biosynthesis of prostanoids in human monocytes and human umbilical vein endothelial cells (HUVECs) in vitro. METHODS: Prostanoids were assessed in different cellular models by immunoassays. The effect of the administration of AF3485 (30 and 100 mg/kg,i.p.) or celecoxib (20mg/kg, i.p.), for 3 days, on the urinary levels of enzymatic metabolites of prostanoids, PGE-M, PGI-M, and TX-M were assessed by LC-MS. RESULTS: In LPS-stimulated whole blood, AF3485 inhibited PGE(2) biosynthesis, in a concentration-dependent fashion. At 100μM, PGE(2) levels were reduced by 66.06 ± 3.30%, associated with a lower extent of TXB(2) inhibition (40.56 ± 5.77%). AF3485 administration to CFA-treated rats significantly reduced PGE-M (P < 0.01) and TX-M (P < 0.05) similar to the selective COX-2 inhibitor, celecoxib. In contrast, AF3485 induced a significant (P < 0.05) increase of urinary PGI-M while it was reduced by celecoxib. In LPS-stimulated human monocytes, AF3485 inhibited PGE(2) biosynthesis with an IC(50) value of 3.03 µM (95% CI:0.5–8.75). At 1μM, AF3485 enhanced TXB(2) while at higher concentrations, the drug caused a concentration-dependent inhibition of TXB(2). At 100 μM, maximal inhibition of the two prostanoids was associated with the downregulation of COX-2 protein by 86%. These effects did not involve AMPK pathway activation, IkB stabilization, or PPARγ activation. In HUVEC, AF3485 at 100 μM caused a significant (P < 0.05) induction of COX-2 protein associated with enhanced PGI(2) production. These effects were reversed by the PPARγ antagonist GW9662. CONCLUSIONS: The inhibitor of human mPGES-1 AF3485 is a novel antiinflammatory compound which can also modulate COX-2 induction by inflammatory stimuli. The compound also induces endothelial COX-2-dependent PGI(2) production via PPARγ activation, both in vitro and in vivo, which might translate into a protective effect for the cardiovascular system.