AUTHOR=Di Francesco Luigia , Bruno Annalisa , Ricciotti Emanuela , Tacconelli Stefania , Dovizio Melania , Guillem-Llobat Paloma , Alisi Maria Alessandra , Garrone Beatrice , Coletta Isabella , Mangano Giorgina , Milanese Claudio , FitzGerald Garret A. , Patrignani Paola 

TITLE=Pharmacological Characterization of the Microsomal Prostaglandin E2 Synthase-1 Inhibitor AF3485 In Vitro and In Vivo

JOURNAL=Frontiers in Pharmacology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2020.00374

DOI=10.3389/fphar.2020.00374

ISSN=1663-9812

ABSTRACT=<sec><title>Rationale</title><p>The development of inhibitors of microsomal prostaglandin (PG)E<sub>2</sub> 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<sub>2</sub>, towards prostacyclin (PGI<sub>2</sub>).</p></sec><sec><title>Objectives</title><p>We studied the effect of the human mPGES-1 inhibitor, AF3485 (a benzamide derivative) on prostanoid biosynthesis in human whole blood <italic>in vitro</italic>. 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) <italic>in vitro</italic>.</p></sec><sec><title>Methods</title><p>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.</p></sec><sec><title>Results</title><p>In LPS-stimulated whole blood, AF3485 inhibited PGE<sub>2</sub> biosynthesis, in a concentration-dependent fashion. At 100μM, PGE<sub>2</sub> levels were reduced by 66.06 ± 3.30%, associated with a lower extent of TXB<sub>2</sub> inhibition (40.56 ± 5.77%). AF3485 administration to CFA-treated rats significantly reduced PGE-M (P &lt; 0.01) and TX-M (P &lt; 0.05) similar to the selective COX-2 inhibitor, celecoxib. In contrast, AF3485 induced a significant (P &lt; 0.05) increase of urinary PGI-M while it was reduced by celecoxib. In LPS-stimulated human monocytes, AF3485 inhibited PGE<sub>2</sub> biosynthesis with an IC<sub>50</sub> value of 3.03 µM (95% CI:0.5–8.75). At 1μM, AF3485 enhanced TXB<sub>2</sub> while at higher concentrations, the drug caused a concentration-dependent inhibition of TXB<sub>2</sub>. 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 &lt; 0.05) induction of COX-2 protein associated with enhanced PGI<sub>2</sub> production. These effects were reversed by the PPARγ antagonist GW9662.</p></sec><sec><title>Conclusions</title><p>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<sub>2</sub> production <italic>via</italic> PPARγ activation, both <italic>in vitro</italic> and <italic>in vivo</italic>, which might translate into a protective effect for the cardiovascular system.</p></sec>