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

RATIONALE: The development of inhibitors of microsomal prostaglandin (PG)E2 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, PGH2, towards prostacyclin (PGI2). 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 PGE2 biosynthesis, in a concentration-dependent fashion. At 100µM, PGE2 levels were reduced by 66.06 ± 3.30%, associated with a lower extent of TXB2 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 PGE2 biosynthesis with an IC50 value of 3.03 µM (95% CI:0.5-8.75). At 1µM, AF3485 enhanced TXB2 while at higher concentrations, the drug caused a concentration-dependent inhibition of TXB2. 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 PGI2 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 PGI2 production via PPARγ activation, both in vitro and in vivo, which might translate into a protective effect for the cardiovascular system.

The anti-inflammatory agent bindarit acts as a modulator of fatty acid-binding protein 4 in human monocytic cells.

We investigated the cellular and molecular mechanisms by which bindarit, a small indazolic derivative with prominent anti-inflammatory effects, exerts its immunoregulatory activity in lipopolysaccharide (LPS) stimulated human monocytic cells. We found that bindarit differentially regulates the release of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), enhancing the release of IL-8 and reducing that of MCP-1. These effects specifically required a functional interaction between bindarit and fatty acid binding protein 4 (FABP4), a lipid chaperone that couples intracellular lipid mediators to their biological targets and signaling pathways. We further demonstrated that bindarit can directly interact with FABP4 by increasing its expression and nuclear localization, thus impacting on peroxisome proliferator-activated receptor γ (PPARγ) and LPS-dependent kinase signaling. Taken together, these findings suggest a potential key-role of FABP4 in the immunomodulatory activity of bindarit, and extend the spectrum of its possible therapeutic applications to FABP4 modulation.

Identification of potent triazolylpyridine nicotinamide phosphoribosyltransferase (NAMPT) inhibitors bearing a 1,2,3-triazole tail group.

The enzyme nicotinamide phosphoribosyltransferase is both a key intracellular enzyme for NAD biosynthesis (iNAMPT) and an extracellular cytokine (eNAMPT). The relationship between this latter role and the catalytic activity of the enzyme is at present unknown. With the intent of discovering inhibitors specifically able to target eNAMPT, we increased the polarity of MV78 (EC50 = 5.8 nM; IC50 = 3.1 nM), a NAMPT inhibitor previously discovered by us. The replacement of a phenyl ring with a 1,2,3-triazole bearing a protonable N,N-dialkyl methanamine group gave a series of molecules which maintained the inhibition of the enzymatic activity but were unable to cross the plasma membrane and affect cell viability in vitro. Compounds 30b and 30f can therefore be considered as the first experimental/pharmacological tools for scientists that wish to understand the role of the catalytic activity of eNAMPT. Serendipitously, we also discovered a compound (25) which, notwithstanding its high polarity, was able to cross the plasma membrane being cytotoxic, a potent NAMPT inhibitor and effective in reducing growth of triple negative mammary carcinoma in mice. In our hands, 25 lacked retinal and cardiac toxicity, although we observed a lesser toxicity of NAMPT inhibitors in general compared to other reports.

Targeting Serotonin 2A and Adrenergic α1 Receptors for Ocular Antihypertensive Agents: Discovery of 3,4-Dihydropyrazino[1,2-b]indazol-1(2H)-one Derivatives.

Glaucoma affects millions of people worldwide and causes optic nerve damage and blindness. The elevation of the intraocular pressure (IOP) is the main risk factor associated with this pathology, and decreasing IOP is the key therapeutic target of current pharmacological treatments. As potential ocular hypotensive agents, we studied compounds that act on two receptors (serotonin 2A and adrenergic α1 ) linked to the regulation of aqueous humour dynamics. Herein we describe the design, synthesis, and pharmacological profiling of a series of novel bicyclic and tricyclic N2-alkyl-indazole-amide derivatives. This study identified a 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative with potent serotonin 2A receptor antagonism, >100-fold selectivity over other serotonin subtype receptors, and high affinity for the α1 receptor. Moreover, upon local administration, this compound showed superior ocular hypotensive action in vivo relative to the clinically used reference compound timolol.

Identification of Novel Triazole-Based Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors Endowed with Antiproliferative and Antiinflammatory Activity.

Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme involved in the recycling of nicotinamide to maintain adequate NAD levels inside the cells. It has been postulated to be a pharmacological target, as it is overexpressed in cancer cells as well as in inflammatory diseases. We describe the synthesis and characterization of a novel class of one-digit nanomolar NAMPT inhibitors based on in vitro characterization. The most active compound tested, 30c, displayed activity in xenograft and allograft models, strengthening the potential of NAMPT inhibitors as antitumoral drugs. Furthermore, in the present contribution we describe the ability of 30c to significantly improve the outcome of colitis in mice. Given that this is the first report of an effect of NAMPT inhibitors in colitis, this result paves the way for novel applications for this class of compounds.

Hit Optimization of 5-Substituted-N-(piperidin-4-ylmethyl)-1H-indazole-3-carboxamides: Potent Glycogen Synthase Kinase-3 (GSK-3) Inhibitors with in Vivo Activity in Model of Mood Disorders.

Novel treatments for bipolar disorder with improved efficacy and broader spectrum of activity are urgently needed. Glycogen synthase kinase 3ß (GSK-3ß) has been suggested to be a key player in the pathophysiology of bipolar disorder. A series of novel GSK-3ß inhibitors having the common N-[(1-alkylpiperidin-4-yl)methyl]-1H-indazole-3-carboxamide scaffold were prepared taking advantage of an X-ray cocrystal structure of compound 5 with GSK-3ß. We probed different substitutions at the indazole 5-position and at the piperidine-nitrogen to obtain potent ATP-competitive GSK-3ß inhibitors with good cell activity. Among the compounds assessed in the in vivo PK experiments, 14i showed, after i.p. dosing, encouraging plasma PK profile and brain exposure, as well as efficacy in a mouse model of mania. Compound 14i was selected for further in vitro/in vivo pharmacological evaluation, in order to elucidate the use of ATP-competitive GSK-3ß inhibitors as new tools in the development of new treatments for mood disorders.

Pharmacological inhibition of microsomal prostaglandin E synthase-1 suppresses epidermal growth factor receptor-mediated tumor growth and angiogenesis.

BACKGROUND: Blockade of Prostaglandin (PG) E(2) production via deletion of microsomal Prostaglandin E synthase-1 (mPGES-1) gene reduces tumor cell proliferation in vitro and in vivo on xenograft tumors. So far the therapeutic potential of the pharmacological inhibition of mPGES-1 has not been elucidated. PGE(2) promotes epithelial tumor progression via multiple signaling pathways including the epidermal growth factor receptor (EGFR) signaling pathway. METHODOLOGY/PRINCIPAL FINDINGS: Here we evaluated the antitumor activity of AF3485, a compound of a novel family of human mPGES-1 inhibitors, in vitro and in vivo, in mice bearing human A431 xenografts overexpressing EGFR. Treatment of the human cell line A431 with interleukin-1beta (IL-1ß) increased mPGES-1 expression, PGE(2) production and induced EGFR phosphorylation, and vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) expression. AF3485 reduced PGE(2) production, both in quiescent and in cells stimulated by IL-1ß. AF3485 abolished IL-1ß-induced activation of the EGFR, decreasing VEGF and FGF-2 expression, and tumor-mediated endothelial tube formation. In vivo, in A431 xenograft, AF3485, administered sub-chronically, decreased tumor growth, an effect related to inhibition of EGFR signalling, and to tumor microvessel rarefaction. In fact, we observed a decrease of EGFR phosphorylation, and VEGF and FGF-2 expression in tumours explanted from treated mice. CONCLUSION: Our work demonstrates that the pharmacological inhibition of mPGES-1 reduces squamous carcinoma growth by suppressing PGE(2) mediated-EGFR signalling and by impairing tumor associated angiogenesis. These results underscore the potential of mPGES-1 inhibitors as agents capable of controlling tumor growth.

Discovery and pharmacological profile of new 1H-indazole-3-carboxamide and 2H-pyrrolo[3,4-c]quinoline derivatives as selective serotonin 4 receptor ligands.

Since the discovery of the serotonin 4 receptor (5-HT(4)R), a large number of receptor ligands have been studied. The safety concerns and the lack of market success of these ligands have mainly been attributed to their lack of selectivity. In this study we describe the discovery of N-[(4-piperidinyl)methyl]-1H-indazole-3-carboxamide and 4-[(4-piperidinyl)methoxy]-2H-pyrrolo[3,4-c]quinoline derivatives as new 5-HT(4)R ligands endowed with high selectivity over the serotonin 2A receptor and human ether-a-go-go-related gene potassium ion channel. Within these series, two molecules (11 ab and 12 g) were identified as potent and selective 5-HT(4)R antagonists with good in vitro pharmacokinetic properties. These compounds were evaluated for their antinociceptive action in two analgesia animal models. 12 g showed a significant antinociceptive effect in both models and is proposed as an interesting lead compound as a 5-HT(4)R antagonist with analgesic action.

Effects of AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide], a novel inhibitor of human microsomal prostaglandin E synthase-1, on prostanoid biosynthesis in human monocytes in vitro.

Inhibitors of microsomal prostaglandin (PG) E synthase-1 (mPGES-1) are being developed for the relief of pain. Redirection of the PGH(2) substrate to other PG synthases, found both in vitro and in vivo, in mPGES-1 knockout mice, may influence their efficacy and safety. We characterized the contribution of mPGES-1 to PGH(2) metabolism in lipopolysaccharide (LPS)-stimulated isolated human monocytes and whole blood by studying the synthesis of prostanoids [PGE(2), thromboxane (TX)B(2), PGF(2alpha) and 6-keto-PGF(1alpha)] and expression of cyclooxygenase (COX)-isozymes and down-stream synthases in the presence of pharmacological inhibition by the novel mPGES-1 inhibitor AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide]. AF3442 caused a concentration-dependent inhibition of PGE(2) in human recombinant mPGES-1 with an IC(50) of 0.06microM. In LPS-stimulated monocytes, AF3442 caused a concentration-dependent reduction of PGE(2) biosynthesis with an IC(50) of 0.41microM. At 1microM, AF3442 caused maximal selective inhibitory effect of PGE(2) biosynthesis by 61+/-3.3% (mean+/-SEM, P<0.01 versus DMSO vehicle) without significantly affecting other prostanoids (i.e. TXB(2), PGF(2alpha) and 6-keto-PGF(1alpha)). In LPS-stimulated whole blood, AF3442 inhibited in a concentration-dependent fashion inducible PGE(2) biosynthesis with an IC(50) of 29microM. A statistically significant inhibition of mPGES-1 activity was detected at 10 and 100microM (38+/-14%, P<0.05, and 69+/-5%, P<0.01, respectively). Up to 100microM, the other prostanoids were not significantly affected. In conclusion, AF3442 is a selective mPGES-1 inhibitor which reduced monocyte PGE(2) generation also in the presence of plasma proteins. Pharmacological inhibition of mPGES-1 did not translate into redirection of PGH(2) metabolism towards other terminal PG synthases in monocytes. The functional relevance of this observation deserves to be investigated in vivo.