Benzydamine plays a role in limiting inflammatory pain induced by neuronal sensitization.

Benzydamine is an active pharmaceutical compound used in the oral care pharmaceutical preparation as NSAID. Beside from its anti-inflammatory action, benzydamine local application effectively reliefs pain showing analgesic and anaesthetic properties. Benzydamine mechanism of action has been characterized on inflammatory cell types and mediators highlighting its capacity to inhibit pro-inflammatory mediators' synthesis and release. On the other hand, the role of benzydamine as neuronal excitability modulator has not yet fully explored. Thus, we studied benzydamine's effect over primary cultured DRG nociceptors excitability and after acute and chronic inflammatory sensitization, as a model to evaluate relative nociceptive response. Benzydamine demonstrated to effectively inhibit neuronal basal excitability reducing its firing frequency and increasing rheobase and afterhyperpolarization amplitude. Its effect was time and dose-dependent. At higher doses, benzydamine induced changes in action potential wavelength, decreasing its height and slightly increasing its duration. Moreover, the compound reduced neuronal acute and chronic inflammatory sensitization. It inhibited neuronal excitability mediated either by an inflammatory cocktail, acidic pH or high external KCl. Notably, higher potency was evidenced under inflammatory sensitized conditions. This effect could be explained either by modulation of inflammatory and/or neuronal sensitizing signalling cascades or by direct modulation of proalgesic and action potential firing initiating ion channels. Apparently, the compound inhibited Nav1.8 channel but had no effect over Kv7.2, Kv7.3, TRPV1 and TRPA1. In conclusion, the obtained results strengthen the analgesic and anti-inflammatory effect of benzydamine, highlighting its mode of action on local pain and inflammatory signalling.

Effect of Sodium Hypochlorite 0.05% on MMP-9 Extracellular Release in Chronic Wounds.

BACKGROUND: In chronic wounds, high concentrations of matrix metalloproteinases (MMPs) can cause excessive proteolysis and slow wound healing. Consequently, restoring a proper MMP balance can help reduce the risk of a chronic wound. An antiseptic solution containing 0.05% sodium hypochlorite (Amukine Med 0.05%, Angelini S.p.A.; hereafter termed NaClO solution) is available on the market. The NaClO solution was proven effective and safe in managing infected skin wounds. To further characterize its activity, this study evaluated the in vitro activity of the NaClO solution on the monocyte release of MMPs. METHODS: Human monocytic THP-1 (ATCC® TIB-202™) cell lines were differentiated into macrophages and treated with different concentrations of NaClO (from 0.05% to 5 × 10-7%). In addition, the THP-1 cell line was stimulated with wound fluid (WF) from patients with active venous leg ulcers in the inflammatory phase. The effect of NaClO (0.025-0.0062%) was also evaluated on healthy human peripheral blood serum samples. The effects of treatments on the gelatinolytic activity of MMP-9 were evaluated by gelatin zymography. The effects on MMPs release were evaluated through the Pro™ Human MMP 9-plex Assay. An exploratory scratch wound healing assay was also performed. RESULTS: The NaClO solution reduced the gelatinolytic activity of MMP-9 and its activated form. The downregulation of MMP-9 gelatinolytic activity was also observed in peripheral blood serum. The MMPs profile showed a reduction in MMP-1 release (p < 0.05) and a slight reduction of the release of MMP-9 and MMP-12 after the treatment with LPS and the NaClO solution. A slight improvement in wound healing was observed after macrophage activation and treatment with the NaClO solution. CONCLUSIONS: The results obtained suggest a possible ability of the NaClO solution to modulate the proteolytic pathways in the wound microenvironment, further characterizing its activity and use in clinical practice during wound care.

Design and validation of neuronal exocytosis blocking peptides as potential novel antiperspirants.

Thermoregulation and heat dissipation by sweat production and evaporation are vital for human survival. However, hyperhidrosis or excessive perspiration might affect people's quality of life by causing discomfort and stress. The prolonged use of classical antiperspirants, anticholinergic medications or botulinum toxin injections for persistent hyperhidrosis might produce diverse side effects that limit their clinical use. Inspired by botox molecular mode of action, we used an in silico molecular modelling approach to design novel peptides to target neuronal acetylcholine exocytosis by interfering with the Snapin-SNARE complex formation. Our exhaustive design rendered the selection of 11 peptides that decreased calcium-dependent vesicle exocytosis in rat DRG neurons, reducing αCGRP release and TRPV1 inflammatory sensitization. The most potent peptides were palmitoylated peptides SPSR38-4.1 and SPSR98-9.1 that significantly suppressed acetylcholine release in vitro in human LAN-2 neuroblastoma cells. Noteworthy, local acute and chronic administration of SPSR38-4.1 peptide significantly decreased, in a dose-dependent manner, pilocarpine-induced sweating in an in vivo mouse model. Taken together, our in silico approach lead to the identification of active peptides able to attenuate excessive sweating by modulating neuronal acetylcholine exocytosis, and identified peptide SPSR38-4.1 as a promising new antihyperhidrosis candidate for clinical development.

α-Glyceryl-phosphoryl-ethanolamine protects human hippocampal neurons from ageing-induced cellular alterations.

Brain ageing has been related to a decrease in cellular metabolism, to an accumulation of misfolded proteins and to an alteration of the lipid membrane composition. These alterations act as contributive aspects of age-related memory decline by reducing membrane excitability and neurotransmitter release. In this sense, precursors of phospholipids (PLs) can restore the physiological composition of cellular membranes and ameliorate the cellular defects associated with brain ageing. In particular, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) have been shown to restore mitochondrial function, reduce the accumulation of amyloid beta (Aß) and, at the same time, provide the amount of acetylcholine needed to reduce memory deficit. Among PL precursors, alpha-glycerylphosphorylethanolamine (GPE) has shown to protect astrocytes from Aß injuries and to slow-down ageing of human neural stem cells. GPE has been evaluated in aged human hippocampal neurons, which are implicated in learning and memory, and constitute a good in vitro model to investigate the beneficial properties of GPE. In order to mimic cellular ageing, the cells have been maintained 21 days in vitro and challenged with GPE. Results of the present paper showed GPE ability to increase PE and PC content, glucose uptake and the activity of the chain respiratory complex I and of the GSK-3ß pathway. Moreover, the nootropic compound showed an increase in the transcriptional/protein levels of neurotrophic and well-being related genes. Finally, GPE counteracted the accumulation of ageing-related misfolded proteins (a-synuclein and tau). Overall, our data underline promising effects of GPE in counteracting cellular alterations related to brain ageing and cognitive decline.

Estimation of brain receptor occupancy for trazodone immediate release and once a day formulations.

Trazodone is approved for the treatment of major depressive disorders, marketed as immediate release (IR), prolonged release, and once a day (OAD) formulation. The different formulations allow different administration schedules and may be useful to facilitate patients' compliance to the antidepressant treatment. A previously verified physiologically-based pharmacokinetic model based on in vitro and in vivo information on trazodone pharmacokinetics was applied, aiming at predicting brain receptor occupancy (RO) after single and repeated dosing of the IR formulation and repeated dosing of the OAD formulation in healthy subjects. Receptors included in the simulations were selected using static calculations of RO based on the maximum unbound brain concentration (Cmax,brain,u ) of trazodone for each formulation and dosing scheme, resulting in 16 receptors being simulated. Seven receptors were simulated for the IR low dose formulation (30 mg), with similar tonset and duration of coverage (range: 0.09-0.25 h and 2.1->24 h, respectively) as well as RO (range: 0.64-0.92) predicted between day 1 and day 7 of dosing. The 16 receptors evaluated for the OAD formulation (300 mg) showed high RO (range: 0.97-0.84 for the receptors also covered by the IR formulation and 0.73-0.48 for the remaining) correlating with affinity and similar duration of time above the target threshold to the IR formulation (range: 2->24 h). The dose-dependent receptor coverage supports the multimodal activity of trazodone, which may further contribute to its fast antidepressant action and effectiveness in controlling different symptoms in depressed patients.

New insights on the arylpiperazinylalkyl pyridazinone ET1 as potent antinociceptive and anti-inflammatory agent.

Pyridazine derivatives, such as arylpiperazinylalkyl pyridazinones, display antinociceptive effects to thermal and chemical stimuli. Here, we extended our previous knowledge on the pharmacological profile of 4-amino-6-methyl-2-(3-(4-(4-methylcyclohexa-1,3-dien-1-yl)piperazin-1-yl)propyl)-5-vinylpyridazin-3(2H)-one, here referred as ET1, paving the way for the comprehension of its complete mechanism of action. To this aim, we have evaluated the mouse behavioural responses in several animal models of pain, the effect of ET1 in the murine model of zymosan-induced paw oedema and air-pouch, assessing the cytokines and the cellular phenotype and finally, an in vitro radioligand binding study was performed on a panel of 30 different receptors. In the formalin test, ET1 reduced both neurogenic and inflammatory phase of nociception induced by the aldehyde. Similarly, ET1 strongly reduced paw licking response in the capsaicin test, the abdominal stretching in the writhing test and the carrageenan-induced thermal hyperalgesia. ET1 also evoked a long-lasting reduction of thermal hyperalgesia. Furthermore, ET1 produced a long-lasting anti-inflammatory effect in the zymosan-induced mouse paw oedema and air-pouch through the selective inhibition of inflammatory monocytes recruitment and the modulation of IL-1ß, IL-6, TNF-α and MCP-1. Binding experiments confirmed an inhibitory effect on adrenergic α1A, α1B and α2A receptors subtypes and, for the first time, a moderate affinity was observed for the following receptors: histamine H1, imidazoline I2, sigma non-opioid intracellular receptor 1 and σ2. These results prompt ET1 as a potent analgesic and anti-inflammatory agent, and support the possibility that it may be suitable for clinical applications in a wide-range of inflammatory-based diseases.

Optimization of Indazole-Based GSK-3 Inhibitors with Mitigated hERG Issue and In Vivo Activity in a Mood Disorder Model.

Bipolar disorders still represent a global unmet medical need and pose a requirement for novel effective treatments. In this respect, glycogen synthase kinase 3ß (GSK-3ß) aberrant activity has been linked to the pathophysiology of several disease conditions, including mood disorders. Therefore, the development of GSK-3ß inhibitors with good in vivo efficacy and safety profile associated with high brain exposure is required. Accordingly, we have previously reported the selective indazole-based GSK-3 inhibitor 1, which showed excellent efficacy in a mouse model of mania. Despite the favorable preclinical profile, analog 1 suffered from activity at the hERG ion channel, which prevented its further progression. Herein, we describe our strategy to improve this off-target liability through modulation of physicochemical properties, such as lipophilicity and basicity. These efforts led to the potent inhibitor 14, which possessed reduced hERG affinity, promising in vitro ADME properties, and was very effective in a mood stabilizer in vivo model.

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.

Antibacterial activity of novel dual bacterial DNA type II topoisomerase inhibitors.

In this study, a drug discovery programme that sought to identify novel dual bacterial topoisomerase II inhibitors (NBTIs) led to the selection of six optimized compounds. In enzymatic assays, the molecules showed equivalent dual-targeting activity against the DNA gyrase and topoisomerase IV enzymes of Staphylococcus aureus and Escherichia coli. Consistently, the compounds demonstrated potent activity in susceptibility tests against various Gram-positive and Gram-negative reference species, including ciprofloxacin-resistant strains. The activity of the compounds against clinical multidrug-resistant isolates of S. aureus, Clostridium difficile, Acinetobacter baumannii, Neisseria gonorrhoeae, E. coli and vancomycin-resistant Enterococcus spp. was also confirmed. Two compounds (1 and 2) were tested in time-kill and post-antibiotic effect (PAE) assays. Compound 1 was bactericidal against all tested reference strains and showed higher activity than ciprofloxacin, and compound 2 showed a prolonged PAE, even against the ciprofloxacin-resistant S. aureus BAA-1720 strain. Spontaneous development of resistance to both compounds was selected for in S. aureus at frequencies comparable to those obtained for quinolones and other NBTIs. S. aureus BAA-1720 mutants resistant to compounds 1 and 2 had single point mutations in gyrA or gyrB outside of the quinolone resistance-determining region (QRDR), confirming the distinct site of action of these NBTIs compared to that of quinolones. Overall, the very good antibacterial activity of the compounds and their optimizable in vitro safety and physicochemical profile may have relevant implications for the development of new broad-spectrum antibiotics.

The Nootropic Drug Α-Glyceryl-Phosphoryl-Ethanolamine Exerts Neuroprotective Effects in Human Hippocampal Cells.

Brain aging involves changes in the lipid membrane composition that lead to a decrease in membrane excitability and neurotransmitter release. These membrane modifications have been identified as contributing factors in age-related memory decline. In this sense, precursors of phospholipids (PLs) can restore the physiological composition of cellular membranes and produce valuable therapeutic effects in brain aging. Among promising drugs, alpha-glycerylphosphorylethanolamine (GPE) has demonstrated protective effects in amyloid-injured astrocytes and in an aging model of human neural stem cells. However, the compound properties on mature neuronal cells remain unexplored. Herein, GPE was tested in human hippocampal neurons, which are involved in learning and memory, and characterized by a functional cholinergic transmission, thus representing a valuable cellular model to explore the beneficial properties of GPE. GPE induced the release of the main membrane phospholipids and of the acetylcholine neurotransmitter. Moreover, the compound reduced lipid peroxidation and enhanced membrane fluidity of human brain cells. GPE counteracted the DNA damage and viability decrease observed in in vitro aged neurons. Among GPE treatment effects, the autophagy was found positively upregulated. Overall, these results confirm the beneficial effects of GPE treatment and suggest the compound as a promising drug to preserve hippocampal neurons and virtually memory performances.

Virtual Screening Approach and Investigation of Structure-Activity Relationships To Discover Novel Bacterial Topoisomerase Inhibitors Targeting Gram-Positive and Gram-Negative Pathogens.

Bacterial resistance is increasing rapidly, requiring urgent identification of new antibacterial drugs that are effective against multidrug-resistant pathogens. Novel bacterial topoisomerase inhibitors (NBTIs) provide a new strategy for investigating the well-validated DNA gyrase and topoisomerase IV targets while preventing cross-resistance issues. On this basis, starting from a virtual screening campaign and subsequent structure-based hit optimization guided by X-ray studies, a novel class of piperazine-like NBTIs with outstanding enzymatic activity against Staphylococcus aureus and Escherichia coli DNA gyrase and topoisomerase IV was identified. Notably, compounds (±)-33, (±)-35, and (±)-36 with potent and balanced multitarget enzymatic profiles exhibited excellent efficacy against selected Gram-positive and Gram-negative pathogens, as well as clinically relevant resistant strains. Overall, the new NBTI chemotype described herein, owing to the broad-spectrum antibacterial activity and favorable in vitro safety profile, might serve as a basis for the development of novel treatments against serious infections.

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.

Proneurogenic Effects of Trazodone in Murine and Human Neural Progenitor Cells.

Several antidepressants increase adult hippocampal neurogenesis (ahNG) in rodents, primates, and, potentially, humans. This effect may at least partially account for their therapeutic activity. The availability of antidepressants whose mechanism of action involves different neurotransmitter receptors represents an opportunity for increasing our knowledge on their distinctive peculiarities and for dissecting the contribution of receptor subtypes in ahNG modulation. The aim of this study was to evaluate, in vitro, the effects of the antidepressant trazodone (TZD) on ahNG by using primary cultures of murine adult hippocampal neural progenitor cells (ahNPCs) and human induced pluripotent stem cell (iPSC)-derived NPCs. We demonstrated that TZD enhances neuronal differentiation of murine as well as human NPCs. TZD is a multimodal antidepressant, which binds with high affinity to 5-HT2a, α1, and 5-HT1a and with lower affinity to 5-HT2c, α2 and 5-HTT. We demonstrated that TZD proneurogenic effects were mediated by 5-HT2a antagonism both in murine and in human NPCs and by 5-HT2c antagonism in murine cells. Moreover NF-κB p50 nuclear translocation appeared to be required for TZD-mediated proneurogenic effects. Interestingly, TZD had no proneurogenic effects in 5-HT depleted ahNPCs. The TDZ bell-shaped dose-response curve suggested additional effects. However, in our model 5-HT1a and α1/α2 receptors had no role in neurogenesis. Overall, our data also demonstrated that serotoninergic neurotransmission may exert both positive and negative effects on neuronal differentiation of ahNPCs in vitro.

Structure-Based Discovery of 1H-Indazole-3-carboxamides as a Novel Structural Class of Human GSK-3 Inhibitors.

An in silico screening procedure was performed to select new inhibitors of glycogen synthase kinase 3ß (GSK-3ß), a serine/threonine protein kinase that in the last two decades has emerged as a key target in drug discovery, having been implicated in multiple cellular processes and linked with the pathogenesis of several diseases. GSK-3ß inhibitors might prove useful as therapeutic compounds in the treatment of conditions associated with elevated levels of enzyme activity, such as type-2 diabetes and neurological disorders, for example, Alzheimer's disease, bipolar disorder, neuronal cell death, stroke, and depression. In this work, virtual screening studies were applied to proprietary compound libraries, and the functional activities of selected compounds were assayed on human GSK-3ß. The in silico screening procedure enabled the identification of eight hit compounds showing pIC50 values ranging from 4.9 to 5.5. X-ray crystallographic studies resulted in a 2.50 Å three-dimensional structure of GSK-3ß complexed with one of the selected compounds, confirming that the inhibitor interacts with the enzyme according to the docking hypothesis. Importantly, molecular docking was able to find a new chemical scaffold for GSK-3ß inhibition, providing grounds for rational structure-based design aimed at further optimization of the initial hits.

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.

A New Acid-oxidizing Solution: Assessment of Its Role on Methicillin-resistant Staphylococcus aureus (MRSA) Biofilm Morphological Changes.

OBJECTIVE: Biofilms represent a key challenge in the treatment of chronic wounds, as they are among the main reasons for delays in chronic wound healing. This in vitro study was aimed at evaluating the activity of a new acid-oxidizing solution (AOS) on biofilm formation. Acid-oxidizing solution contains free chlorine species with stabilized hypochlorous acid in high concentration (> 95%) and is characterized by acidic (pH less than 3) and super-oxidizing (Redox greater than 1000mV) features. MATERIALS AND METHODS: A 3-dimensional in vitro model of reconstructed human epidermis was used to compare the activity of AOS vs 2 reference products (RP) containing betaine and polyhexanide (RP1) and sodium hypochlorite and hypochlorous acid (RP2). Different approaches were used to assess the prevention and eradication of methicillin-resistant Staphyloccocus aureus biofilm by the study products. Xylitol and chlorhexidine were used as positive controls. The activity of the study products on the biofilm structure was evaluated analyzing the ultrastructural modification by scanning electron microscopy, while skin compatibility was assessed on noncolonized tissues measuring the metabolic activity of the cells. RESULTS: In all experiments, AOS showed to be active on the biofilm matrix, modifying its structure and allowing bacterial release from the matrix. In all experiments, no cytotoxicity was observed in the tissues treated with the product suggesting a good compatibility of AOS with skin tissues. Reference product 1 affected the biofilm, suggesting a disruption effect; RP2 was slightly less active than AOS in modifying the biofilm structure. CONCLUSION: Treatment with AOS affects biofilm by modifying its structure and therefore facilitating local bacteria accessibility to bactericidal agents, with consequent potential clinical benefits in the treatment of chronic wounds.

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.

The monocyte chemotactic protein synthesis inhibitor bindarit prevents mesangial cell proliferation and extracellular matrix remodeling.

Glomerular expression of chemotactic protein-1/chemokine (C-C motif) ligand-2 (MCP-1/CCL2) correlates with the degree of renal damage, suggesting a role of this chemokine in the pathogenesis of renal diseases. Bindarit is an original indazolic derivative able to inhibit MCPs synthesis and to significantly decrease MCP-1/CCL2 urinary excretion in patients with Lupus Nephritis, in correlation with reduction in albuminuria. Aim of the present work was to elucidate the effect of MCP-1/CCL2 synthesis inhibition on in vitro models of mesangial cell dysfunction. ET1 (10nM) and AngII (10nM) significantly stimulated MCP-1/CCL2 release by human renal mesangial cells (HRMCs) after 3-12h stimulation. Bindarit (10-300 µM) significantly inhibited MCP-1/CCL2 release in response to both stimuli within 12h. Bindarit also inhibited mRNA MCP-1/CCL2 expression, confirming an effect of the drug at transcriptional level. Bindarit significantly and concentration-dependently inhibited HRMC proliferation, measured as either cell duplication or total DNA/well, and impaired mRNA collagen IV expression, collagen deposition and fibronectin expression induced by AngII and ET1. Exposure of HRMCs to bindarit also impaired MMP2 activation in response to both stimuli, measured by means of gelatin zymography. These data confirm the important role of MCP-1/CCL2 synthesis in mesangial cell dysfunction and support the potential of therapeutic intervention targeting this chemokine in kidney disease.

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.

Targeting monocyte chemotactic protein-1 synthesis with bindarit induces tumor regression in prostate and breast cancer animal models.

Prostate and breast cancer are major causes of death worldwide, mainly due to patient relapse upon disease recurrence through formation of metastases. Chemokines are small proteins with crucial roles in the immune system, and their regulation is finely tuned in early inflammatory responses. They are key molecules during inflammatory processes, and many studies are focusing on their regulatory functions in tumor growth and angiogenesis during metastatic cell seeding and spreading. Bindarit is an anti-inflammatory indazolic derivative that can inhibit the synthesis of MCP-1/CCL2, with a potential inhibitory function in tumor progression and metastasis formation. We show here that in vitro, bindarit can modulate cancer-cell proliferation and migration, mainly through negative regulation of TGF-ß and AKT signaling, and it can impair the NF-κB signaling pathway through enhancing the expression of the NF-κB inhibitor IkB-α. In vivo administration of bindarit results in impaired metastatic disease in prostate cancer xenograft mice (PC-3M-Luc2 cells injected intra-cardially) and impairment of local tumorigenesis in syngeneic Balb/c mice injected under the mammary gland with murine breast cancer cells (4T1-Luc cells). In addition, bindarit treatment significantly decreases the infiltration of tumor-associated macrophages and myeloid-derived suppressor cells in 4T1-Luc primary tumors. Overall, our data indicate that bindarit is a good candidate for new therapies against prostate and breast tumorigenesis, with an action through impairment of inflammatory cell responses during formation of the tumor-stroma niche microenvironment.