The Chemokine CCL2 Mediates the Seizure-enhancing Effects of Systemic Inflammation.

Epilepsy is a chronic disorder characterized by spontaneous recurrent seizures. Brain inflammation is increasingly recognized as a critical factor for seizure precipitation, but the molecular mediators of such proconvulsant effects are only partly understood. The chemokine CCL2 is one of the most elevated inflammatory mediators in patients with pharmacoresistent epilepsy, but its contribution to seizure generation remains unexplored. Here, we show, for the first time, a crucial role for CCL2 and its receptor CCR2 in seizure control. We imposed a systemic inflammatory challenge via lipopolysaccharide (LPS) administration in mice with mesial temporal lobe epilepsy. We found that LPS dramatically increased seizure frequency and upregulated the expression of many inflammatory proteins, including CCL2. To test the proconvulsant role of CCL2, we administered systemically either a CCL2 transcription inhibitor (bindarit) or a selective antagonist of the CCR2 receptor (RS102895). We found that interference with CCL2 signaling potently suppressed LPS-induced seizures. Intracerebral administration of anti-CCL2 antibodies also abrogated LPS-mediated seizure enhancement in chronically epileptic animals. Our results reveal that CCL2 is a key mediator in the molecular pathways that link peripheral inflammation with neuronal hyperexcitability. SIGNIFICANCE STATEMENT: Substantial evidence points to a role for inflammation in epilepsy, but currently there is little insight as to how inflammatory pathways impact on seizure generation. Here, we examine the molecular mediators linking peripheral inflammation with seizure susceptibility in mice with mesial temporal lobe epilepsy. We show that a systemic inflammatory challenge via lipopolysaccharide administration potently enhances seizure frequency and upregulates the expression of the chemokine CCL2. Remarkably, selective pharmacological interference with CCL2 or its receptor CCR2 suppresses lipopolysaccharide-induced seizure enhancement. Thus, CCL2/CCR2 signaling plays a key role in linking systemic inflammation with seizure susceptibility.

The chemokine (C-C motif) ligand protein synthesis inhibitor bindarit prevents cytoskeletal rearrangement and contraction of human mesangial cells.

Intraglomerular mesangial cells (MCs) maintain structural and functional integrity of renal glomerular microcirculation and homeostasis of mesangial matrix. Following different types of injury, MCs change their phenotype upregulating the expression of α-smooth muscle actin (α-SMA), changing contractile abilities and increasing the production of matrix proteins, chemokines and cytokines. CCL2 is a chemokine known to be involved in the pathogenesis of renal diseases. Its glomerular upregulation correlates with the extent of renal damage. Bindarit is an indazolic derivative endowed with anti-inflammatory activity when tested in experimental diseases. It selectively inhibits the synthesis of inflammatory C-C chemokines including CCL2, CCL7 and CCL8. This work aims to analyse bindarit effects on ET1-, AngII- and TGFß-induced mesangial cell dysfunction. Bindarit significantly reduced AngII-, ET1- and TGFß-induced α-SMA upregulation. In a collagen contraction assay, bindarit reduced AngII-, ET1- and TGFß-induced HRMC contraction. Within 3-6h stimulation, vinculin organization and phosphorylation was significantly impaired by bindarit in AngII-, ET1- and TGFß-stimulated cells without any effect on F-actin distribution. Conversely, p38 phosphorylation was not significantly inhibited by bindarit. Our data strengthen the importance of CCL2 on ET-1, AngII- and TGFß-induced mesangial cell dysfunction, adding new insights into the cellular mechanisms responsible of bindarit protective effects in human MC dysfunction.

The artificial gene Jazz, a transcriptional regulator of utrophin, corrects the dystrophic pathology in mdx mice.

The absence of the cytoskeletal protein dystrophin results in Duchenne muscular dystrophy (DMD). The utrophin protein is the best candidate for dystrophin replacement in DMD patients. To obtain therapeutic levels of utrophin expression in dystrophic muscle, we developed an alternative strategy based on the use of artificial zinc finger transcription factors (ZF ATFs). The ZF ATF 'Jazz' was recently engineered and tested in vivo by generating a transgenic mouse specifically expressing Jazz at the muscular level. To validate the ZF ATF technology for DMD treatment we generated a second mouse model by crossing Jazz-transgenic mice with dystrophin-deficient mdx mice. Here, we show that the artificial Jazz protein restores sarcolemmal integrity and prevents the development of the dystrophic disease in mdx mice. This exclusive animal model establishes the notion that utrophin-based therapy for DMD can be efficiently developed using ZF ATF technology and candidates Jazz as a novel therapeutic molecule for DMD therapy.

The CCL2 synthesis inhibitor bindarit targets cells of the neurovascular unit, and suppresses experimental autoimmune encephalomyelitis.

BACKGROUND: Production of the chemokine CCL2 by cells of the neurovascular unit (NVU) drives critical aspects of neuroinflammation. Suppression of CCL2 therefore holds promise in treating neuroinflammatory disease. Accordingly, we sought to determine if the compound bindarit, which inhibits CCL2 synthesis, could repress the three NVU sources of CCL2 most commonly reported in neuroinflammation--astrocytes, microglia and brain microvascular endothelial cells (BMEC)--as well as modify the clinical course of neuroinflammatory disease. METHODS: The effect of bindarit on CCL2 expression by cultured murine astrocytes, microglia and BMEC was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Bindarit action on mouse brain and spinal cord in vivo was similarly investigated by qRT-PCR following LPS injection in mice. And to further gauge the potential remedial effects of bindarit on neuroinflammatory disease, its impact on the clinical course of experimental autoimmune encephalomyelitis (EAE) in mice was also explored. RESULTS: Bindarit repressed CCL2 expression by all three cultured cells, and antagonized upregulated expression of CCL2 in both brain and spinal cord in vivo following LPS administration. Bindarit also significantly modified the course and severity of clinical EAE, diminished the incidence and onset of disease, and evidenced signs of disease reversal. CONCLUSION: Bindarit was effective in suppressing CCL2 expression by cultured NVU cells as well as brain and spinal cord tissue in vivo. It further modulated the course of clinical EAE in both preventative and therapeutic ways. Collectively, these results suggest that bindarit might prove an effective treatment for neuroinflammatory disease.

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.

Inhibition of in-stent stenosis by oral administration of bindarit in porcine coronary arteries.

OBJECTIVE: We have previously demonstrated that bindarit, a selective inhibitor of monocyte chemotactic proteins (MCPs), is effective in reducing neointimal formation in rodent models of vascular injury by reducing smooth muscle cell proliferation and migration and neointimal macrophage content, effects associated with the inhibition of MCP-1/CCL2 production. The aim of the current study was to evaluate the efficacy of bindarit on in-stent stenosis in the preclinical porcine coronary stent model. METHODS AND RESULTS: One or 2 bare metal stents (Multi-Link Vision, 3.5 mm) were deployed (1:1.2 oversize ratio) in the coronary arteries of 42 pigs (20 bindarit versus 22 controls). Bindarit (50 mg/kg per day) was administered orally from 2 days before stenting until the time of euthanasia at 7 and 28 days. Bindarit caused a significant reduction in neointimal area (39.4%, P<0.001, n=9 group), neointimal thickness (51%, P<0.001), stenosis area (37%, P<0.001), and inflammatory score (40%, P<0.001) compared with control animals, whereas there was no significant difference in the injury score between the 2 groups. Moreover, treatment with bindarit significantly reduced the number of proliferating cells (by 45%, P<0.05; n=6 group) and monocyte/macrophage content (by 55%, P<0.01; n=5-6 group) in stented arteries at day 7 and 28, respectively. These effects were associated with a significant (P<0.05) reduction of MCP-1 plasma levels at day 28. In vitro data showed that bindarit (10-300 µmol/L) reduced tumor necrosis factor-α (50 ng/mL)-induced pig coronary artery smooth muscle cell proliferation and inhibited MCP-1 production. CONCLUSION: Our results show the efficacy of bindarit in the prevention of porcine in-stent stenosis and support further investigation for clinical application of this compound.

Protection from arthritis and myositis in a mouse model of acute chikungunya virus disease by bindarit, an inhibitor of monocyte chemotactic protein-1 synthesis.

Chikungunya virus (CHIKV) is associated with outbreaks of infectious rheumatic disease in humans. Using a mouse model of CHIKV arthritis and myositis, we show that tumor necrosis factor-α, interferon-γ, and monocyte chemotactic protein 1 (MCP-1) were dramatically induced in tissues from infected mice. The same factors were detected in the serum of patients with CHIKV-induced polyarthralgia and polyarthritis, with MCP-1 levels being particularly elevated. Bindarit (MCP inhibitor) treatment ameliorated CHIKV disease in mice. Histological analysis of muscle and joint tissues showed a reduction in inflammatory infiltrate in infected mice treated with bindarit. These results suggest that bindarit may be useful in treating CHIKV-induced arthritides in humans.

Antiproteinuric effect of chemokine C-C motif ligand 2 inhibition in subjects with acute proliferative lupus nephritis.

BACKGROUND/AIMS: To test the role of chemokine C-C motif ligand 2 (CCL2) in the pathogenesis of lupus nephritis (LN), we evaluated the effects of CCL2 inhibition by bindarit therapy in patients with systemic lupus and active renal disease. METHODS: In this proof-of-concept, prospective, randomized, double-blind clinical study, 22 subjects with acute LN were assigned on a 1:1 ratio to 24-week treatment with bindarit (1,200 mg/day) or matching placebo. All subjects were on the same standardized steroid background therapy. Urinary CCL2, urinary albumin excretion (UAE), estimated glomerular filtration rate, time to remission and time to relapse of LN were compared between groups. RESULTS: Urinary CCL2 significantly decreased during bindarit therapy (p = 0.008 vs. baseline) with a reduction that approximated 50% at study end. CCL2 reduction was paralleled by a persistent reduction in UAE that averaged 80% vs. baseline and approximated 90% at study end. Renal function recovery was similar and no difference was found in terms of time to remission and time to relapse of LN between treatment arms. Treatment was safe and well tolerated in all patients. CONCLUSION: In lupus subjects with active nephritis, bindarit significantly reduced albuminuria and urinary CCL2 levels. This study provides the background for longer trials to test renoprotective effect of CCL2 inhibition in LN.

Monocyte chemoattractant proteins mediate myocardial microvascular dysfunction in swine renovascular hypertension.

BACKGROUND: Monocyte chemoattractant proteins (MCPs) play an important role in mediating inflammatory processes. Hypertension (HTN) is associated with inflammation as well as impaired cardiac microcirculatory function and structure, but the contribution of MCPs to these alterations remained unclear. This study tested the hypothesis that MCPs regulate cardiac microvascular function and structure in experimental HTN. METHODS AND RESULTS: Pigs (n=6 per group) were studied after 10 weeks of normal, renovascular HTN, or renovascular HTN+ bindarit (MCPs inhibitor, 50 mg/kg/d PO). Left ventricular (LV) function, myocardial microvascular permeability, and fractional vascular volume were assessed by fast computed tomography before and after adenosine infusion (400 microg/kg/min). Myocardial fibrosis, inflammation, and microvascular remodeling were determined ex vivo. Hypertension was not altered by bindarit, but LV hypertrophy and diastolic function were improved. In response to adenosine, myocardial microvascular permeability increased in HTN (from 0.0083+/-0.0009 to 0.0103+/-0.0011 AU, P=0.038 versus baseline) and fractional vascular volume decreased, whereas both remained unchanged in normal and HTN+bindarit pigs. HTN upregulated endothelin-1 expression, myocardial inflammation, and microvascular wall thickening, which were inhibited by bindarit. CONCLUSIONS: MCPs partly mediate myocardial inflammation, fibrosis, vascular remodeling, and impaired vascular integrity induced by hypertension. Inhibition of MCPs could potentially be a therapeutic target in hypertensive cardiomyopathy.

Role of MCP-1 in endotoxemia and sepsis.

Sepsis is a complex clinical syndrome resulting from a harmful host inflammatory response to infection. Similarly, lipopolysaccharide (LPS) induced endotoxemia is marked by the activation of inflammatory responses, which can lead to shock, multiple organ damage and even death. Inflammatory mediator, chemokines are known to play an important role in the pathogenesis of sepsis and endotoxemia. Monocyte chemoattractant protein (MCP)-1, a prototype of CC chemokines, is a potent chemoattractant and a regulatory mediator involved in a variety of inflammatory diseases. The objective of this study is to investigate the role of MCP-1, by using bindarit, a blocker of MCP-1 synthesis, in murine models of sepsis and endotoxemia. Treatment with bindarit both prophylactically and therapeutically significantly (P<0.05) reduced MCP-1 levels in the lungs and liver in both sepsis and endotoxemia. In addition, prophylactic and therapeutic treatment with bindarit significantly (P<0.05) protected mice against sepsis and endotoxemia, as evidenced by the attenuation in lung and liver myeloperoxidase (MPO) activity, an indicator of neutrophil recruitment. The protective effect of bindarit was further confirmed by histological examination of lung and liver sections. Treatment with bindarit reduced lung and liver injury as indicated by decreased thickening of alveolar and neutrophil infiltration in CLP-induced sepsis and LPS-induced endotoxemia. Considering these results, we propose that anti-MCP-1 strategies may be of potential therapeutic value in the treatment of sepsis and endotoxemia.

CCL2 mediates the circadian response to low dose endotoxin.

The mammalian circadian system is mainly originated in a master oscillator located in the suprachiasmatic nuclei (SCN) in the hypothalamus. Previous reports from our and other groups have shown that the SCN are sensitive to systemic immune activation during the early night, through a mechanism that relies on the action of proinflammatory factors within this structure. Chemokine (C-C motif) ligand 2 (CCL2) is induced in the brain upon peripheral immune activation, and it has been shown to modulate neuronal physiology. In the present work we tested whether CCL2 might be involved in the response of the circadian clock to peripheral endotoxin administration. The CCL2 receptor, C-C chemokine receptor type 2 (CCR2), was detected in the SCN of mice, with higher levels of expression during the early night, when the clock is sensitive to immune activation. Ccl2 was induced in the SCN upon intraperitoneal lipopolysaccharide (LPS) administration. Furthermore, mice receiving an intracerebroventricular (Icv) administration of a CCL2 synthesis inhibitor (Bindarit), showed a reduction LPS-induced circadian phase changes and Icv delivery of CCL2 led to phase delays in the circadian clock. In addition, we tested the possibility that CCL2 might also be involved in the photic regulation of the clock. Icv administration of Bindarit did not modify the effects of light pulses on the circadian clock. In summary, we found that CCL2, acting at the SCN level is important for the circadian effects of immune activation.

A double-blind randomised study to evaluate the efficacy and safety of bindarit in preventing coronary stent restenosis.

AIMS: Bindarit (BND) is a selective inhibitor of monocyte chemotactic protein-1 (MCP-1/CCL2), which plays an important role in generating intimal hyperplasia. Our aim was to explore the efficacy and safety of bindarit in preventing restenosis following percutaneous coronary intervention. METHODS AND RESULTS: A phase II, double-blind, multicentre randomised trial included 148 patients randomised into three arms (BND 600 mg, n=48; BND 1,200 mg, n=49; PLB, n=51). Bindarit was given following PCI and continued for 180 days. Monthly clinical follow-up and six-month coronary angiography were conducted. The primary endpoint was in-segment late loss; the main secondary endpoints were in-stent late loss and major adverse cardiovascular events. Efficacy analysis was carried out on two populations, ITT and PP. There were no significant differences in the baseline characteristics among the three treatment groups. In-segment and in-stent late loss at six months in BND 600, BND 1,200 and PLB were: (ITT 0.54 vs. 0.52 vs. 0.72; p=0.21), (PP 0.46 vs. 0.53 vs. 0.72; p=0.12) and (ITT 0.74 vs. 0.74 vs. 1.05; p=0.01), (PP 0.66 vs. 0.73 vs. 1.06; p=0.003), respectively. The MACE rates at nine months among treatment groups were 20.8% vs. 28.6% vs. 25.5% (p=0.54), respectively. CONCLUSIONS: This was a negative study with the primary endpoint not being met. However, significant reduction in the in-stent late loss suggests that bindarit probably exerts a favourable action on the vessel wall following angioplasty. Bindarit was well tolerated with a compliance rate of over 90%. A larger study utilising a loading dose and targeting a specific patient cohort may demonstrate more significant results.

Effects of MCP-1 inhibition by bindarit therapy in a rat model of polycystic kidney disease.

BACKGROUND/AIMS: Experimental and clinical evidence suggested that monocyte chemoattractant protein-1 (MCP-1/CCL2) has a role in the development of interstitial inflammation and renal failure in polycystic kidney disease (PKD). We investigated whether bindarit, an inhibitor of MCP-1/CCL2 synthesis, could influence the evolution of PKD in PCK rats. METHODS: PCK rats were treated from 5 to 15 weeks of age with vehicle or bindarit. Sprague-Dawley rats served as control. For in vitro studies, murine podocytes were exposed to albumin with or without bindarit. RESULTS: MCP-1 mRNA was upregulated in the kidney of PCK rats and reduced by bindarit. Treatment limited overexpression of MCP-1 protein by epithelial cells of dilated tubules and cysts, and interstitial inflammatory cells. Excessive renal accumulation of monocytes/macrophages was lowered by bindarit by 41%. Serum creatinine slightly increased in PCK rats on vehicle and was similar to controls after bindarit. Kidney and liver cysts were not affected by treatment. Bindarit significantly reduced progressive proteinuria of PCK rats. The antiproteinuric effect was associated with the restoration of the defective nephrin expression in podocytes of PCK rats. Bindarit limited podocyte foot process effacement and ameliorated slit diaphragm frequency. In cultured podocytes, bindarit reduced MCP-1 production in response to albumin and inhibited albumin-induced cytoskeletal remodeling and cell migration. CONCLUSION: This study showed that although bindarit did not prevent renal cyst growth, it limited interstitial inflammation and renal dysfunction and reduced proteinuria in PKD. Thus, bindarit could be considered a therapeutic intervention complementary to therapies specifically acting to block renal cyst growth.

Benzydamine inhibits monocyte migration and MAPK activation induced by chemotactic agonists.

1. The present study was aimed to investigate the effect of benzydamine, an anti-inflammatory drug devoid of activity on arachidonic acid metabolism, on monocyte chemotaxis and to define the possible biochemical correlates of activity. 2. Benzydamine inhibited monocyte chemotaxis in response to three classes of chemoattractants: the prototypic CC-chemokine CCL2 (MCP-1), the microbial product fMLP and the complement cascade component C5a. The effect was dose-dependent with IC50's of 100, 50 and 45 microm for MCP-1/CCL2, fMLP and C5a, respectively. At the dose of 100 microm, the effect resulted in a 50+/-10% inhibition of MCP-1/CCL2-induced chemotaxis and 53+/-6 and 54+/-5% inhibitions of chemotaxis in response of fMLP and C5a, respectively (n=3). 3. Receptor expression as well as calcium fluxes in response to chemoattractants were not affected by benzydamine. 4. Benzydamine strongly inhibited chemoattractant-induced activation of the mitogen-activated protein kinase (MAPK) ERK1/2, and of its upstream activator kinase MEK1/2. ERK1/12 activation in response to chemoattractants was 89-98% inhibited by a 100 microm concentration of benzydamine with an IC50 of 30 microm. 5. Under the same experimental conditions, pretreatment with 100 microm benzydamine caused a 75-89% inhibition of p38 activation (IC50 25 microm). 6. These results indicate that the anti-inflammatory activity of benzydamine is exerted at multiple levels, including monocyte migration to chemotactic factors associated to a blockage of ERK and p38 MAPK pathways.

Acetaminophen down-regulates interleukin-1beta-induced nuclear factor-kappaB nuclear translocation in a human astrocytic cell line.

In previous studies performed to elucidate acetaminophen mechanism of action, we demonstrated that acetaminophen inhibits prostaglandin E2 production by interleukin (IL)-1beta-stimulated T98G human astrocytic cells, without affecting cyclooxygenase-2 enzymatic activity. As this result suggests an effect at transcriptional level, we examined whether the drug interferes with the activation of nuclear factor (NF)-kappaB and STAT3 transcription factors and with SAPK signal transducing factor. Western blot analysis of IkappaBalpha protein in the cytoplasm of IL-1beta-stimulated T98G cells and electrophoretic mobility shift assay (EMSA) on corresponding nuclear extracts indicate that acetaminophen (10-1000 microM) dose-dependently inhibits both IkappaBalpha degradation and NF-kappaB nuclear translocation. In the same cell type neither IL-1beta-dependent SAPK activation nor IL-6-induced STAT3 phosphorylation is affected by the drug. These data indicate that therapeutic concentrations of acetaminophen induce an inhibition of IL-1beta-dependent NF-kappaB nuclear translocation. The selectivity of this effect suggests the existence of an acetaminophen specific activity at transcriptional level that may be one of the mechanisms through which the drug exerts its pharmacological effects.

(E)-[2-(4-Methylsulphonylphenyl)-1-cyclopentenyl-1-methyliden](arylmethyloxy)amines. Methyleneaminoxymethyl (MAOM) analogues of diarylcyclopentenyl cyclooxygenase-2 inhibitors: synthesis and biological properties.

The (E)-[2-(4-Methylsulphonylphenyl)-1-cyclopentenyl-1-methyliden](methyloxy)amine (5) and (arylmethyloxy)amines (6-12) were designed in order to verify the effects on the biological properties of the substitution of an aryl of selective diarylcyclopentenyl cyclooxygenase-2 (COX-2) inhibitors of type 3 with a methyleneaminoxymethyl moiety (MAOMM). Compounds 5-12 were tested in vitro for their inhibitory activity towards COX-1 and COX-2 by measuring prostaglandin E2 (PGE2) production in U937 cell lines and activated J774.2 macrophages, respectively. The compound with the highest in vitro activity towards COX-2 (9) was also assayed in vivo for its antiinflammatory activity by means of the carrageenan-induced paw edema test in rats. Some of the new compounds showed an appreciable in vitro COX-2 inhibitory activity, with IC(50) values in the microM (6,7,9,10,11) range. Compound 9 also exhibited an appreciable in vivo activity (29% inhibition at a dose of 30 mg kg(-1)) when administered intraperitoneally. The structural parameters of 9 were determined by X-ray crystallographic analysis.

Aryl-substituted methyleneaminoxymethyl (MAOM) analogues of diarylcyclopentenyl cyclooxygenase-2 inhibitors: effects of some structural modifications on their biological properties.

The (E)-[2-(4-aminosulfonylphenyl)-1-cyclopentenyl-1-methyliden]-(arylmethyloxy)amines (6a,b), which are the sulfonamidic analogues of the previously described methylsulfonyl derivatives 5a,b, and their corresponding sulfides (7a,b) and sulfoxides (8a,b) were synthesised in order to obtain information about the role played by these different sulphur-containing groups in the cyclooxygenase-2 inhibitory activity of this class of compounds. In addition, other chemical manipulations concerning the oxime-ether substituent of this type of derivatives were affected by preparing compounds 9a,b, which present a methyl group on the oximic carbon of the oxime-ether chain of 5a,b, and compounds 10 and 11, in which the atomic sequence (C=NOCH(2)) of the MAOMM of 8b and 5b, respectively, is inverted. Compounds 6-11 were tested in vitro for their inhibitory activity towards COX-1 and COX-2 by measuring prostaglandin E2 (PGE2) production in U937 cell lines and activated J774.2 macrophages, respectively. Some of the new compounds showed an appreciable in vitro COX-2 inhibitory activity, with IC(50) values in the microM (7a,b, 8a and 9b) or sub-microM (8b) range. This last compound was also assayed in vivo for its antiinflammatory activity by means of the carrageenan-induced paw edema test in rats. No inhibitory effects were detected up to dose of 30 mg kg(-1) orally administered.

Synthesis of heteroaromatic analogues of (2-aryl-1-cyclopentenyl-1-alkylidene)-(arylmethyloxy)amine COX-2 inhibitors: effects on the inhibitory activity of the replacement of the cyclopentene central core with pyrazole, thiophene or isoxazole ring.

Several heteroaromatic analogues of (2-aryl-1-cyclopentenyl-1-alkylidene)-(arylmethyloxy)amine COX-2 inhibitors, in which the cyclopentene moiety was replaced by pyrazole, thiophene or isoxazole ring, were synthesized, in order to verify the influence of the different nature of the central core on the COX inhibitory properties of these kinds of molecules. Among the compounds tested, only the 3-(p-methylsulfonylphenyl) substituted thiophene derivatives 17 and 22, showed a certain COX-2 inhibitory activity, accompanied by an appreciable COX-2 versus COX-1 selectivity. Only one of the 1-(p-methylsulfonylphenyl)pyrazole compounds (16) displayed a modest inhibitory activity towards both type of isoenzymes, while the pyrazole 1-(p-aminosulfonylphenyl) substituted 12 proved to be significantly active only towards COX-1. All the isoxazole derivatives were inactive on both COX isoforms.

Bindarit, inhibitor of CCL2 synthesis, protects neurons against amyloid-ß-induced toxicity.

One of the hallmarks of Alzheimer's disease (AD), the most common age-related neurodegenerative pathology, is the abnormal extracellular deposition of neurotoxic amyloid-ß (Aß) peptides that accumulate in senile plaques. Aß aggregates are toxic to neurons and are thought to contribute to neuronal loss. Evidence indicates that inflammation is involved in the pathophysiology of AD, and activation of glial cells by a variety of factors, including Aß, appears to be a central event. Among molecules produced during inflammation associated with neuronal death, CCL2, also known as monocyte chemotactic protein-1 (MCP-1), seems to be particularly important. Indeed, CCL2 levels are higher in the cerebrospinal fluid of patients with AD than in controls. In the present study, we demonstrated the protective effect of bindarit (which inhibits CCL2 synthesis) against both Aß25-35 and Aß1-42-induced toxicity in primary mixed neural cultures. Bindarit (30-500 µM) reversed cell death induced by Aß in a dose-dependent manner and reduced the transcription and release of CCL2 by astrocytes after Aß treatment, as revealed by qRT-PCR, ELISA, and immunofluorescence staining. Astroglial activation and CCL2 release was induced by ATP released by damaged neurons through interaction with P2X7 receptors present on astrocyte surface. CCL2, interacting with its cognate receptor CCR2, present on neuron surface, strongly contributes to the toxic activity of Aß. Bindarit was able to disconnect this neuro-glial interaction. Our results demonstrate the ability of bindarit to inhibit Aß-induced neuronal death and suggest the potential role of CCL2 inhibitors in the treatment of neuroinflammatory/neurodegenerative diseases.

Monocyte Chemoattractant Protein-1 upregulates GABA-induced current: evidence of modified GABAA subunit composition in cortical neurons from the G93A mouse model of Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder that affects upper and lower motor neurons. Previous evidence has indicated that excitotoxic cell death in ALS may remarkably depend on Cl(-) ion influx through the GABA(A) receptors. In this study we have analysed the effect of Monocyte Chemoattractant Protein-1 (MCP-1), a chemokine expressed to a higher level in ALS patients, on GABAA receptors in cultured cortical neurons from a genetic model of ALS (G93A) and compared with wild type SOD1 (SOD1) and their corresponding non transgenic littermates (Control). By performing electrophysiological experiments we have observed that, in cortical neurons MCP-1 (2-150 ng/ml) induced an enhancement of GABA-evoked currents that was significantly higher in G93A neurons compared to controls. The effect of MCP-1 was not dependent on the activation of its receptor CCR2, while it was blocked by flumazenil, the antagonist of benzodiazepine sites. Analysis of GABAA receptor subunit composition has indicated an altered subunit expression level in G93A cortical neurons compared to controls. Instead, in cultured spinal neurons MCP-1 induced a significant reduction of GABA-evoked currents, also through the benzodiazepine sites, indicating a region-specific mechanism of action. However, no differences were observed in the current reduction between the three neuronal populations. These findings provide the first evidence that MCP-1, acting on benzodiazepine sites, can modulate the GABA-evoked currents, depending on the subunit composition of GABA(A) receptor. In cortical neurons MCP-1 upmodulates the GABA-evoked current and this effect is exacerbated in the mutated neurons. It is reasonable to assume that the higher Cl(-) influx through GABA(A) receptors in the presence of MCP-1 in mutated cortical neurons may induce an excitotoxicity acceleration. Agents able to block the MCP-1 production may then prove useful for ALS treatment.