ABSTRACT
Title
Pharmacological characterization of the novel microsomal prostaglandin(PG)E synthase-1 inhibitor AF3485
Authors
L. Di Francesco*, A. Bruno*, E. Ricciotti°, M. Dovizio*, E. Marcantoni*, P. Anzellotti*, B. Garrone§, C. Milanese§, I. Coletta§ and P. Patrignani*
*Dept. of Medicine and Science on Aging, “G. d’Annunzio” University and CeSI, Chieti (Italy)
§R&D Angelini Research Center, Santa Palomba-Pomezia, Rome (Italy)
°University of Pennsylvania, Philadelphia (USA).
*Dept. of Medicine and Science on Aging, “G. d’Annunzio” University and CeSI, Chieti (Italy)
§R&D Angelini Research Center, Santa Palomba-Pomezia, Rome (Italy)
°University of Pennsylvania, Philadelphia (USA).
Abstract
Inhibitors of microsomal prostaglandin(PG)E synthase-1(mPGES-1) are proposed as alternative to NSAIDs to reduce cardiovascular toxicity due to possible redirection of PGH2 substrate to prostacyclin(PGI2) biosynthesis in vivo(Wang et al.,2008). AF3485(a benzamide derivative) is a novel mPGES-1 inhibitor. Here, we investigated the effect of AF3485 on: i) the activity of human and mouse recombinant mPGES-1; ii) prostanoids generated in human monocytes, human umbilical vein endothelial cells(HUVECs) and whole blood in vitro; iii)cyclooxygenase(COX)-2 expression in vitro and iv)prostanoid biosynthesis in vivo in a model of CFA(complete Freund's adjuvant)-induced monoarthritis in rats who received AF3485(30 and 100mg/kg,ip) or celecoxib(20mg/kg,ip) for 3 days. Prostanoids [6-keto-PGF1α (the hydrolysis product of PGI2),PGE2 and TXB2] were assessed by immunoassays and urinary levels of the enzymatic metabolites of PGE2, PGI2, TXB2 and PGD2, i.e. PGE-M, PGI-M, TX-M and PGD-M, respectively, were assessed by HPLC-MS(Wang et al.,2008). COX-2 levels in cells were evaluated by Western Blot.
AF3485 caused a concentration-dependent inhibition of PGE2 generation by human recombinant mPGES-1(IC50:2.55μM), while it caused only a non significant reduction of the mouse recombinant enzyme even at 100μM. In LPS-stimulated human monocytes, AF3485 inhibited PGE2 biosynthesis with an IC50 of 3.4μM. At 1μM, inhibition of PGE2 was associated with enhanced TXB2(by 72%,P<0.05). However, at higher concentrations, AF3485 caused a concentration-dependent inhibition of TXB2. At 100μM, the compound reduced COX-2 levels by 86%. In LPS-stimulated whole blood, AF3485 inhibited PGE2 biosynthesis, in a concentration-dependent fashion. At 100μM of AF3485, both PGE2 and TXB2 were reduced(66.1 and 40.6%,respectively). These results suggest that AF3485 is an inhibitor of mPGES-1 but at higher concentrations, it may affect also monocyte COX-2 expression. To verify the possible role of PPARγ activation in COX-2 down-regulation by AF3485, we compared the effects of an agonist and an antagonist of PPARγ [15-deoxy-Δ12,14-PGJ2 (PGJ2) and GW9662]. Similarly to AF3485, PGJ2(20μM) reduced COX-2 levels and PGE2 generation in LPS-stimulated monocytes. However, GW9662 did not reverse down-regulation of COX-2 caused by PGJ2 or AF3485, suggesting a PPARγ independent mechanism in this phenomenon. We studied the impact of AF3485 on HUVEC COX-2 expression. At 100μM, it caused a significant(P<0.05) induction of COX-2 protein(by 2-fold) associated with enhanced PGI2 generation(by 40%). These effects were reversed by GW9662 in a concentration-dependent fashion.
In order to dissect the modulation of COX-2-dependent prostanoids by AF3485 from its inhibitory effect on mPGES-1, we studied the impact of AF3485 administration to rats on the biosynthesis of prostanoids in vivo. In CFA-treated rats, AF3485, at 100mg/kg but not at 30mg/kg, significantly reduced PGE-M(P<0.01) and TX-M(P<0.05) by 40%. Comparable results were obtained with the COX-2 inhibitor celecoxib. In contrast, AF3485, at 100mg/kg, induced a significant(P<0.05) increase of urinary levels of PGI-M by 50%, while a significant reduction(P<0.01) by 70% was caused by celecoxib. PGD-M levels were not affected both by AF3485 and celecoxib, consistently with a COX-1 origin of the PGD2 metabolite.
In conclusion, AF3485 is a selective inhibitor of human mPGES-1 but at higher concentrations influences COX-2 expression. In LPS-stimulated human monocytes, it down-regulates COX-2-dependent prostanoids by a PPARγ independent mechanism while in endothelial cells it up-regulates COX-2-dependent PGI2 by a PPARγ dependent mechanism. These off-target effects were detected also in vivo in a rat model of arthritis. In fact, despite AF3485 did not affect murine mPGES-1 activity, it reduced the generation of inflammatory PGE2 associated with enhanced endothelial derived generation of vasoprotective PGI2.
AF3485 caused a concentration-dependent inhibition of PGE2 generation by human recombinant mPGES-1(IC50:2.55μM), while it caused only a non significant reduction of the mouse recombinant enzyme even at 100μM. In LPS-stimulated human monocytes, AF3485 inhibited PGE2 biosynthesis with an IC50 of 3.4μM. At 1μM, inhibition of PGE2 was associated with enhanced TXB2(by 72%,P<0.05). However, at higher concentrations, AF3485 caused a concentration-dependent inhibition of TXB2. At 100μM, the compound reduced COX-2 levels by 86%. In LPS-stimulated whole blood, AF3485 inhibited PGE2 biosynthesis, in a concentration-dependent fashion. At 100μM of AF3485, both PGE2 and TXB2 were reduced(66.1 and 40.6%,respectively). These results suggest that AF3485 is an inhibitor of mPGES-1 but at higher concentrations, it may affect also monocyte COX-2 expression. To verify the possible role of PPARγ activation in COX-2 down-regulation by AF3485, we compared the effects of an agonist and an antagonist of PPARγ [15-deoxy-Δ12,14-PGJ2 (PGJ2) and GW9662]. Similarly to AF3485, PGJ2(20μM) reduced COX-2 levels and PGE2 generation in LPS-stimulated monocytes. However, GW9662 did not reverse down-regulation of COX-2 caused by PGJ2 or AF3485, suggesting a PPARγ independent mechanism in this phenomenon. We studied the impact of AF3485 on HUVEC COX-2 expression. At 100μM, it caused a significant(P<0.05) induction of COX-2 protein(by 2-fold) associated with enhanced PGI2 generation(by 40%). These effects were reversed by GW9662 in a concentration-dependent fashion.
In order to dissect the modulation of COX-2-dependent prostanoids by AF3485 from its inhibitory effect on mPGES-1, we studied the impact of AF3485 administration to rats on the biosynthesis of prostanoids in vivo. In CFA-treated rats, AF3485, at 100mg/kg but not at 30mg/kg, significantly reduced PGE-M(P<0.01) and TX-M(P<0.05) by 40%. Comparable results were obtained with the COX-2 inhibitor celecoxib. In contrast, AF3485, at 100mg/kg, induced a significant(P<0.05) increase of urinary levels of PGI-M by 50%, while a significant reduction(P<0.01) by 70% was caused by celecoxib. PGD-M levels were not affected both by AF3485 and celecoxib, consistently with a COX-1 origin of the PGD2 metabolite.
In conclusion, AF3485 is a selective inhibitor of human mPGES-1 but at higher concentrations influences COX-2 expression. In LPS-stimulated human monocytes, it down-regulates COX-2-dependent prostanoids by a PPARγ independent mechanism while in endothelial cells it up-regulates COX-2-dependent PGI2 by a PPARγ dependent mechanism. These off-target effects were detected also in vivo in a rat model of arthritis. In fact, despite AF3485 did not affect murine mPGES-1 activity, it reduced the generation of inflammatory PGE2 associated with enhanced endothelial derived generation of vasoprotective PGI2.