Abstract: Renal artery stenosis (RAS) causes renovascular hypertension and renal damage, which may result from tissue inflammation. We have previously shown that the kidney in RAS exhibits increased expression of monocyte chemoattractant protein (MCP)-1, but its contribution to renal injury remained unknown. This study tested the hypothesis that MCP-1 contributes to renal injury and dysfunction in the stenotic kidney. METHODS: Kidney hemodynamics, function, and endothelial function were quantified in pigs after 10 weeks of experimental RAS (n = 7), RAS supplemented with the MCP-1 inhibitor bindarit (RAS + bindarit, 50 mg/kg/day orally, n = 6), and normal controls (n = 8). Renal inflammation was assessed by the immunoreactivity of MCP-1, its receptor chemotactic cytokine receptor 2, and NFkappaB, and oxidative stress by nicotinamide adenine dinucleotide phosphate-oxidase expression and in-situ superoxide production. Renal microvascular density was evaluated by micro-CT and fibrosis by trichrome staining, collagen-I immunostaining, and hydroxyproline content. RESULTS: After 10 weeks of RAS, blood pressure was similarly elevated in RAS and RAS + bindarit. Compared with normal controls, stenotic RAS kidneys had decreased renal blood flow (5.4 +/- 1.6 vs. 11.4 +/- 1.0 ml/min/kg, P < 0.05) and glomerular filtration rate and impaired endothelial function, which were significantly improved in bindarit-treated RAS pigs (to 8.4 +/- 0.8 ml/min/kg, P < 0.05 vs. RAS). Furthermore, bindarit markedly decreased tubulointerstitial (but not vascular) oxidative stress, inflammation, and fibrosis, and slightly increased renal microvascular density. The impaired renovascular endothelial function, increased oxidative-stress, and fibrosis in the contralateral kidney were also improved by bindarit. CONCLUSION: MCP-1 contributes to functional and structural impairment in the kidney in RAS, mainly in the tubulointerstitial compartment. Its inhibition confers renoprotective effects by blunting renal inflammation and thereby preserving the kidney in chronic RAS.

Abstract: 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 an 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.

Abstract: AIMS: Bindarit is an original compound with peculiar anti-inflammatory activity due to a selective inhibition of a subfamily of inflammatory chemokines, including the monocyte chemotactic proteins MCP-1/CCL2, MCP-3/CCL7, and MCP-2/CCL8. In this study, we investigated the effect of bindarit on neointima formation using two animal models of arterial injury: rat carotid artery balloon angioplasty and wire-induced carotid injury in apolipoprotein E-deficient (apoE(-/-)) mice. METHODS AND RESULTS: Treatment of rats with bindarit (200 mg/kg/day) significantly reduced balloon injury-induced neointima formation by 39% at day 14 without affecting re-endothelialization and reduced the number of medial and neointimal proliferating cells at day 7 by 54 and 30%, respectively. These effects were associated with a significant reduction of MCP-1 levels both in sera and in injured carotid arteries of rats treated with bindarit. In addition, in vitro data showed that bindarit (10-300 microM) reduced rat vascular smooth muscle cell (VSMC) proliferation, migration, and invasion, processes contributing to the injury-induced neointima formation in vivo. Similar results were observed in hypercholesterolaemic apoE(-/-) mice in which bindarit administration resulted in a 42% reduction of the number of proliferating cells at day 7 after carotid injury and in a 47% inhibition of neointima formation at day 28. Analysis of the cellular composition in neointimal lesions of apoE(-/-) mice treated with bindarit showed that the relative content of macrophages and the number of VSMCs were reduced by 66 and 30%, respectively, compared with the control group. CONCLUSION: This study demonstrates that bindarit is effective in reducing neointima formation in both non-hyperlipidaemic and hyperlipidaemic animal models of vascular injury by a direct effect on VSMC proliferation and migration and by reducing neointimal macrophage content. All of these data were associated with the inhibition of MCP-1 production.

Abstract: OBJECTIVE: Alphaviruses such as chikungunya virus, Sindbis virus, o'nyong-nyong virus, Mayaro virus, and Ross River virus (RRV), are commonly associated with arthralgias and overt arthritides worldwide. Understanding the processes by which arthritogenic viruses cause disease is a prerequisite in the quest for better treatments. In this regard, we have recently established that monocyte/macrophages are mediators of alphavirus-induced arthritis in mice. We hypothesized that chemokines associated with monocyte/macrophage recruitment may play an important role in disease. The aim of the present investigations was to determine whether bindarit, an inhibitor of monocyte chemotactic protein (MCP) synthesis, could ameliorate alphavirus-induced rheumatic disease in mice. METHODS: Using our recently developed mouse model of RRV-induced arthritis, which has many characteristics of RRV disease (RRVD) in humans, the effects of bindarit treatment on RRVD in mice were determined via histologic analyses, immunohistochemistry, flow cytometry, real-time polymerase chain reaction analysis, enzyme-linked immunosorbent assay, and electrophoretic mobility shift assay. RESULTS: Bindarit-treated RRV-infected mice developed mild disease and had substantially reduced tissue destruction and inflammatory cell recruitment as compared with untreated RRV-infected mice. The virus load in the tissues was not affected by bindarit treatment. Bindarit exhibited its activity by down-regulating MCPs, which in turn led to inhibition of cell infiltration and lower production of NF-kappaB and tumor necrosis factor alpha, which are involved in mediating tissue damage. CONCLUSION: Our data support the use of inhibitors of MCP production in the treatment of arthritogenic alphavirus syndromes and suggest that bindarit may be useful in treating RRVD and other alphavirus-induced arthritides in humans.

Abstract: 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.

Abstract: Bindarit is an indazolic derivative that is devoid of any immunosuppressive effects and has no effect on arachidonic acid metabolism. However, it has been proved to have anti-inflammatory activity in a number of experimental diseases, including pancreatitis, arthritis, and lupus nephritis. This therapeutic effect has been associated with its ability to interfere selectively with monocyte recruitment, although the underlying molecular mechanisms are unknown. Here we comprehensively examine the effect of bindarit on the chemokine system, and report that in activated monocytes and endothelial cells, it selectively inhibits the production of the monocyte chemotactic protein subfamily of CC inflammatory chemokines (MCP-1/CCL2, MCP-3/CCL7, MCP-2/CCL8). The capacity of bindarit to inhibit the production of a defined set of related CC chemokines by monocytes and endothelial cells likely underlies the anti-inflammatory activity of this agent in disease. The exploitation of the chemokine system as drug target in inflammatory disease has relied mainly on the development of receptor antagonists and blocking antibodies. Here we report on the use of inhibition of synthesis as a potentially viable and selective approach to modify the chemokine system.

Abstract: OBJECTIVE: Chemokines play a fundamental role in trafficking and activation of leukocytes in colonic inflammation. We investigated the ability of bindarit, an inhibitor of monocyte chemoattractant protein-1 (MCP-1/CCL2) synthesis, to inhibit chemokine production by human intestinal epithelial cells (HT-29) and its effect in trinitro-benzene sulfonic acid (TNBS)-induced colitis in mice.MATERIALS AND METHODS: HT-29 cells were incubated with bindarit in the presence of TNF-alpha/IFN-gamma and 24 h later supernatants were collected for MCP-1, IL-8 and RANTES measurement. A 1 mg enema of TNBS was given to BALB/c mice, and bindarit (100 mg/kg) was orally administered twice daily starting from two days before colitis induction. Weight loss, histology, and MCP-1 level and myeloperoxidase (MPO) activity in colon extracts were assessed.RESULTS: In HT-29 cells, bindarit concentration-dependently and selectively inhibited MCP-1 secretion (as well as mRNA expression) primed by TNF-alpha/IFN-gamma. Moreover treatment with bindarit reduced clinical and histopathological severity of TNBS-induced colitis. These effects were associated with significant inhibition of MCP-1 and MPO in colon extracts.CONCLUSIONS: Bindarit exhibits a potent bioactivity in reducing leukocyte infiltration, down-regulating MCP-1 synthesis, and preventing the development of severe colitis in a mice model of TNBS-induced colitis. These observations suggest a potential use of MCP-1 synthesis blockers in intestinal inflammation in humans.

Abstract: Chemokines such as monocyte chemoattractant protein (MCP)-1 are key agonists that attract macrophages to tumors. In melanoma, it has been previously shown that variable levels of MCP-1/CCL2 appear to correlate with infiltrating macrophages and tumor fate, with low to intermediate levels of the chemokine contributing to melanoma development. To work under such conditions, a poorly tumorigenic human melanoma cell line was transfected with an expression vector encoding MCP-1. We found that M2 macrophages are associated to MCP-1+ tumors, triggering a profuse vascular network. To target the protumoral macrophages recruitment and reverting tumor growth promotion, clodronate-laden liposomes (Clod-Lip) or bindarit were administered to melanoma-bearing mice. Macrophage depletion after Clod-Lip treatment induced development of smaller tumors than in untreated mice. Immunohistochemical analysis with an anti-CD31 antibody revealed scarce vascular structures mainly characterized by narrow vascular lights. Pharmacological inhibition of MCP-1 with bindarit also reduced tumor growth and macrophage recruitment, rendering necrotic tumor masses. We suggest that bindarit or Clod-Lip abrogates protumoral-associated macrophages in human melanoma xenografts and could be considered as complementary approaches to antiangiogenic therapy.

Abstract: Chemokines are believed to play a key role in the pathogenesis of acute pancreatitis. We have earlier shown that pancreatic acinar cells produce the chemokine monocyte chemotactic protein (MCP)-1 in response to caerulein hyperstimulation, demonstrating that acinar-derived MCP-1 is an early mediator of inflammation in acute pancreatitis. Blocking chemokine production or action is a major target for pharmacological intervention in a variety of inflammatory diseases, such as acute pancreatitis. 2-Methyl-2-[[1-(phenylmethyl)-1H-indazol-3yl]methoxy]propanoic acid (bindarit) has been shown to preferentially inhibit MCP-1 production in vitro in monocytes and in vivo without affecting the production of the cytokines IL-1, IL-6, or the chemokines IL-8, protein macrophage inflammatory-1alpha, and RANTES. The present study aimed to define the role of MCP-1 in acute pancreatitis with the use of bindarit. In a model of acute pancreatitis induced by caerulein hyperstimulation, prophylactic as well as therapeutic treatment with bindarit significantly reduced MCP-1 levels in the pancreas. Also, this treatment significantly protected mice against acute pancreatitis as evident by attenuated hyperamylasemia neutrophil sequestration in the pancreas (pancreatic MPO activity), and pancreatic acinar cell injury/necrosis on histological examination of pancreas sections.

Abstract: Saliva of bloodfeeding arthropods has been incriminated in facilitating the establishment of parasite in their host. We report on the leukocyte chemoattractive effect of salivary gland homogenate (SGH) from Lutzomyia longipalpis on saliva-induced inflammation in an air pouch model. SGH (0.5 pair/animal) was inoculated in the air pouch formed in the back of BALB/c or C57BL/6 mice. L. longipalpis SGH induced a significant influx of macrophages in BALB/c but not in C57BL/6 mice. SGH-induced cell recruitment reached a peak at 12 h after inoculation and was higher than that induced by the LPS control. This differential cell recruitment in BALB/c mice was directly correlated to an increase in CCL2/MCP-1 expression in the air pouch lining tissue. In fact, treatment with bindarit, an inhibitor of CCL2/MCP-1 synthesis, and also with a specific anti-MCP-1 mAb resulted in drastic reduction of macrophage recruitment and inhibition of CCL2/MCP-1 expression in the lining tissue. CCL2/MCP-1 production was also seen in vitro when J774 murine macrophages were exposed to L. longipalpis SGH. The SGH effect was abrogated by preincubation with serum containing anti-SGH IgG Abs as well as in mice previously sensitized with L. longipalpis bites. Interestingly, the combination of SGH with Leishmania chagasi induced an increased recruitment of neutrophils and macrophages when compared with L. chagasi alone. Taken together these results suggest that SGH not only induces the recruitment of a greater number of macrophages by enhancing CCL2/MCP-1 production but also synergizes with L. chagasi to recruit more inflammatory cells to the site of inoculation.

Abstract: The aim of the present work was to evaluate the effect of bendazac lysine on the human lens epithelial cell line HLE-B3 adhesion to polymethylmethacrylate (PMMA) intraocular lenses (IOLs). After adherence to IOLs, cells were incubated in the presence of the drug for 24 h. The number of cells contained in a 6-mm(2) area was then counted with an inverted phase microscope and adherent cells were distinguished from detached floating cells by focusing through the medium. Results obtained show that bendazac is able to induce a linear dose-dependent inhibition of HLE-B3 adhesiveness to PMMA IOLs. In particular, treatment with bendazac 33, 100 and 300 microM resulted in a 15, 32 and 54% inhibition, respectively. Statistical analysis shows that this effect is significant at 100 microM (p < 0.05) and 300 microM (p < 0.01). The analysis of the effects of bendazac on the viability and on the proliferative capacity of HLE-B3 cells did not show any drug-related toxicity up to the concentration of 400 microM. The present study demonstrates that bendazac lysine is able to inhibit adhesion of lens epithelial cells to PMMA IOLs and suggests the potential beneficial use of this drug in preventing secondary cataract development.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: OBJECTIVE: This study was designed to evaluate therapeutic effects of bindarit, an indazolic derivative able to inhibit monocyte chemoattractant protein-1 (MCP-1) production, in adjuvant induced arthritis in rats. MATERIALS AND METHODS: Arthritis was induced by Freund's complete adjuvant injection. Bindarit was given as a 0.5% medicated diet starting on day 11 after adjuvant injection. The course of arthritis was monitored by sequential paw volume measurement and by radiologic and histologic evaluations. Human osteoblast cell line Saos-2 stimulated with Interleukin-1 (IL-1) was used to assess in vitro bindarit effect on MCP-1 release. In addition, in vivo effects of bindarit on cytokine production were studied in mice injected with lipopolysaccharide (LPS). Immune function studies were performed in mice by evaluating ex vivo antibody response to ovalbumin and splenocytes proliferation to Concanavalin A (Con A). RESULTS: In adjuvant-induced arthritis in rats, bindarit possessed therapeutic activity resulting in a significant inhibition of paw inflammation. Evidence for a disease-modifying activity was also indicated by amelioration of radiologic alterations and by histological evaluation of joints. Additional evidence for beneficial effects in osseous inflammation was provided by an in vitro assay in which bindarit inhibited the release of MCP-1 from IL-1 stimulated osteoblast cells. Moreover, in a murine model of LPS-induced cytokine production bindarit reduced MCP-1 and tumor necrosis factor (TNF)-alpha increase without affecting IL-1 and IL-6 levels. Finally, the drug, given as a 0.5% medicated diet for 14 days, did not affect either anti-ovalbumin serum antibody production or splenocytes proliferative response in mice. CONCLUSIONS: Results obtained indicate that bindarit beneficial effects in experimental arthritis are correlated to MCP-1 and TNF-alpha inhibition and suggest that the control of cytokines and chemokines production can have considerable relevance as regards strategies for the treatment of chronic inflammatory diseases.

Abstract: 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.

Abstract: The beneficial effects of statins on the reduction of cardiovascular events has been partly attributed to their anti-inflammatory properties. In the complex of the different pathogenetic events leading to atherosclerosis, recent data suggest a central role of monocyte chemotactic protein-1 (MCP-1), because mice knock-out for MCP-1 or its receptor CC-chemokine receptor 2 were considerably resistant to plaque formation. In this study we investigated the effect of different statins on in vitro and in vivo production of MCP-1. Lovastatin and simvastatin caused a dose-dependent inhibition of MCP-1 production in peripheral blood mononuclear cells exposed to lipopolysaccharide or inactivated Streptococcus hemoliticus and in human endothelial cells exposed to interleukin-1beta. The addition of mevalonate overrode the inhibitory effect of statins indicating that mevalonate-derived products are important for chemokine production. The in vivo anti-inflammatory effect of statins was investigated using the mouse air-pouch model of local inflammation. Lovastatin and pravastatin were orally administered to mice according to a treatment schedule that significantly inhibited the hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity without affecting total blood cholesterol. At the dose of 10 mg/kg, lovastatin and pravastatin reduced by approximately 50% the lipopolysaccharide-induced leukocytes recruitment and the exudate MCP-1 production. In conclusion, statins, by inhibiting mevalonate-derived products, reduced both in vitro and in vivo the production of chemokines involved in leukocyte migration, and this effect is unrelated to their cholesterol-lowering action.

Abstract: Interleukin (IL) 6, an autocrine growth factor for mesangial cells, and chemokines, which are released from activated mesangial cells and induce leukocyte infiltration, play a critical role in the progression of immune system mediated renal diseases. Since the reciprocal relationship between IL-6 and chemokines in renal inflammation has been barely investigated, we have analyzed whether IL-6 (500 ng/ml), alone or in combination with the soluble form of its receptor (sIL-6R, 200 ng/ml), can induce normal human mesangial cells (NHMC) to release alpha and/or beta chemokines: MCP-1 (monocyte chemoattractant protein 1), IL-8, Rantes (regulated on activation, normal T cell expressed and secreted), and MIP-1alpha (macrophage inflammatory protein 1alpha). Whereas IL-6 or sIL-6R alone were ineffective in inducing significant chemokine release from NHMC, the simultaneous treatment with IL-6 and sIL-6R showed a significant interaction, leading to a strong synergic effect on MCP-1 synthesis and release without exerting any relevant activity on IL-8, Rantes, or MIP-1alpha. Consistently with the unresponsiveness to IL-6, mRNA and protein expression analysis of the two subunits which form the functional IL-6 receptor showed that NHMC express only the gp130 signal-transducing chain and not the subunit-specific IL-6R (gp80). These findings support an unexpected role of the IL-6 system in kidney inflammatory reactions through the selective regulation of monocyte recruitment.

Abstract: Blocking chemokine production or action is a major target for pharmacological intervention in different human diseases. Bindarit (2-methyl-2-[[1-(phenylmethyl)-1H-indazol-3yl]methoxy]propan oic acid) dose-dependently inhibited MCP-1 and TNF-alpha production induced in vitro in monocytes by LPS and Candida albicans. It did not affect the production of the cytokines IL-1, IL-6, or the chemokines IL-8, MIP-1alpha and RANTES. In the air pouch model in mice, oral treatment reduced monocyte recruitment and local MCP-1 production, induced by carrageenan or IL-1 injection. In NZB/W mice, a model of lupus nephritis, oral treatment prolonged survival and delayed the onset of proteinuria. The results presented here show that bindarit is a preferential inhibitor of the production of MCP-1 in vitro and in vivo and suggest that its beneficial effects in models of joint and kidney inflammation are related to its anti-MCP-1 action. It is therefore possible to selectively and differentially regulate chemokines by targeting their production with small synthetic molecules.

Abstract: Melatonin is an antioxidant. Since other antioxidants inhibit the production of tumor necrosis factor (TNF) induced by lipopolysaccharide, we investigated its effect on TNF production in vivo and in vitro and on lethality associated with endotoxic shock. Administration of melatonin to mice (5 mg/kg, s.c., 30 min before or simultaneously with lipopolysaccharide) inhibited serum TNF levels by 50-80% and improved survival of mice treated with a lethal dose of lipopolysaccharide. By studying other, structurally related, indolamines (N-acetyl-5-hydroxytryptamine, 5-methoxytryptamine and 5-hydroxytryptamine) we found a good correlation between antioxidant activity (for which the 5-methoxy group is essential) and the inhibition of TNF production in vivo and in vitro in mononuclear cells. Melatonin did not increase serum corticosterone and did not modify the elevation of serum corticosterone levels by lipopolysaccharide or by interleukin-1. Furthermore, it exerted its inhibitory effect in adrenalectomized or hypophysectomized mice also, indicating that its effect is independent of the hypothalamus-pituitary-adrenal axis.

Abstract: As an alternative to classical immunosuppressants in experimental lupus nephritis, we looked at bindarit, 2-methyl-2-[[1-phenylmethyl)-1H-indazol-3-y1]methoxy]propanoic acid, a novel molecule devoid of immunosuppressive effects, which selectively reduces chronic inflammation in rat adjuvant arthritis. Two groups of NZB/W mice (N = 55 for each group) were given bindarit, (50 mg/kg/day p.o.) or vehicle starting at 2 months of age. Mice were sacrificed at 2, 6, 8 and 10 months or used for survival studies. Bindarit delayed the onset of proteinuria (% proteinuric mice, bindarit vs. vehicle, 6 months: 0 vs. 33% and 8 months: 7% vs. 60%, P < 0.005; 10 months: 53% vs. 80%) and significantly (P < 0.05) protected from renal function impairment (serum BUN, bindarit vs. vehicle: 8 months, 30 +/- 3 vs. 127 +/- 42; 10 months, 53 +/-5 vs. 140 +/- 37 mg/dl). Appearance of anti-DNA antibodies was retarded and survival significantly (P < 0.0001) prolonged by bindarit (% survival, bindarit vs. vehicle: 8 months, 100% vs. 80%; 10 months, 87% vs. 40%; 12 months, 27% vs. 20%). Bindarit significantly limited glomerular hypercellularity, interstitial inflammation and tubular damage. Renal expression of monocyte chemoattractant protein (MCP-1) mRNA (Northern blot) markedly increased (7 - 12-fold in 8- 10-month-old mice vs. 2-month-old) during the progression of nephritis in association with mononuclear cell infiltration. Bindarit completely prevented MCP-1 up-regulation. In another series of experiments, bindarit (0.25% and 0.5% medicated diet, N = 16 for each group) when started at 4.5 months of age in NZB/W mice improved survival in respect to untreated mice (N = 17) in a dose-dependent manner (% survival: 8 months, 94% and 100%, respectively, vs. 47%; 10 months, 75% and 100% vs. 35%; 12 months, 31% and 75% vs. 12%). Survival was even more prolonged when bindarit (0.5% medicated diet) was combined with a low dose of methylprednisolone (1.5 mg/kg i.p.), which that only partially modifies proteinuria and survival of lupus mice, in an additional group of animals (N = 16). Thus, at 14.5 months when all mice given bindarit alone died, 50% of mice on combined therapy were still alive (P < 0.023). Studies are needed to establish whether bindarit may function as a steroid sparing drug in human lupus.

Abstract: OBJECTIVE: The present study was designed to investigate the effects of bindarit on animal survival and renal damage in murine lupus autoimmune disease. METHODS: Female NZB/W mice were used. Bindarit was administered, as a 0.5% medicated diet, starting either before the onset of the pathology or early in the course of the disease, in order to assess the effects of age upon the response. Furthermore, the effects of combined administration of bindarit with low dose i.p. cyclophosphamide bolus were also studied. Proteinuria and anti-dsDNA antibody levels were determined during the course of the study. Renal damage was evaluated by light microscopy. RESULTS: Bindarit markedly prolonged the NZB/W mouse life span (p < 0.001 vs. controls), showing a significant difference even against high dose cyclophosphamide (90 mg/kg ip bolus) chosen as the reference (p < 0.01). Bindarit significantly reduced the degree of renal damage, delayed proteinuria and did not prevent autoantibody development, thus confirming the lack of immunosuppressive activity. CONCLUSION: The present results and other experimental data demonstrating the capacity of the drug to interfere with the inflammatory and immune response cross-talking, indicate that bindarit exerts its action in murine lupus through a novel and original mechanism. These findings, coupled with the evidence that the drug possesses a very safe toxicological profile, suggest that further investigations to assess the potential value of bindarit in the treatment of SLE are warranted.

Abstract: OBJECTIVE: Previous studies have shown that benzydamine (40 mg/kg s.c.) is able to inhibit tumor necrosis factor (TNF) production and to reduce mouse lethality when administered before or concomitantly with LPS. The present study was designed to further investigate benzydamine activity against LPS-induced toxicity in terms of potency and therapeutic effects. METHODS: Female Balb/c mice were used. A dose-response curve of animal lethality versus endotoxin dose was performed (LD50 = 45 micrograms/mouse). Therapeutic effects were studied selecting the dose of LPS to achieve an LD100 (160 micrograms/mouse). Mortality was assessed daily and mice were followed for 8 days. The potential mode of action of therapeutically administered benzydamine was also investigated. TNF alpha and IL-1 beta levels were measured, at 5 h after LPS injection, both in sera and in lungs. Moreover, the drug was assayed in a TNF-dependent cytoxicity test. RESULTS: Benzydamine, administered at 20 mg/kg s.c. simultaneously with the endotoxin, significantly increased LPS LD50 up to 230 micrograms/mouse (p < 0.05). Moreover, the drug significantly protected mice against LPS-induced lethality when administered either 30 min or 4 h after endotoxin injection (p < 0.001). Benzydamine, therapeutically administered at 20 mg/kg s.c., significantly reduced TNF alpha and IL-1 beta production induced by LPS both in serum and lungs and it was shown to inhibit TNF-dependent cytoxicity on L929 cells. CONCLUSIONS: These results clearly demonstrate the therapeutic activity of benzydamine in a simple model of endotoxic shock. Available data confirm the potential role of benzydamine as an anti-cytokine agent and provide suggestions for novel therapeutic applications of this anti-inflammatory drug.

Abstract: Benzydamine is a non-steroidal antiinflammatory drug, devoid of activity on arachidonic acid metabolism, which is extensively used as a topical drug in inflammatory conditions, particularly for the treatment of bacterial vaginosis and Candida albicans-sustained vaginitis. In the present study the effects of benzydamine on the production of several inflammatory cytokines were examined in cultures of Candida albicans-stimulated human mononuclear cells. Benzydamine (6.25-50 microM) inhibited Candida-induced tumor necrosis factor-alpha and, to a lesser extent, interleukin-1 beta production, whereas it did not affect interleukin-6 release. Benzydamine also blocked monocyte chemotactic protein-1 secretion, but it did not affect interleukin-8 production. Unlike benzydamine, ibuprofen and naproxen, two non-steroidal antiinflammatory drugs also used topically, were unable to suppress inflammatory lymphokine production from Candida-activated mononuclear cells. These data suggest that benzydamine may be effective in local Candida infections at least in part by suppressing inflammatory cytokine and monokine production in the vaginal mucosa and consequently decreasing their levels in vaginal secretions.

Abstract: The present study was designed to assess the effect of N,N-dimethyl-3-[(1-benzyl-1H-indazol-3-yl)ossi]-1-propana mine (benzydamine) on in vivo and in vitro production of inflammatory cytokines. Benzydamine inhibited tumour necrosis factor-alpha (TNF-alpha) production in vitro by human lipopolysaccharide-stimulated monocytes with an ED50 of approximately 25 microM (12 donors). Under the same conditions, benzydamine had modest or no effect on production of interleukin (IL-1), IL-6 and IL-8. Inhibition of TNF-alpha production was not restricted to LPS in that similar results were obtained using inactivated streptococci. Inhibition of TNF production was associated with a modest (about 30% at 50 microM, 7 donors) reduction of mRNA. A similar inhibition of TNF-alpha production was also detected with mouse peritoneal macrophages. With mouse cells benzydamine also substantially inhibited IL-1 production in vitro. In vivo treatment with benzydamine (40 mg/kg s.c.) protected mice against LPS lethality. Protection against septic shock was observed when benzydamine was administered before or concomitantly with LPS. Protection against LPS toxicity was associated with a marked reduction of serum levels of TNF-alpha and IL-1 beta, whereas IL-6 was unaffected. Inhibition of inflammatory cytokine production may play a role in the anti-inflammatory activity of benzydamine and provide suggestions for novel therapeutic applications.

Abstract: Oxidative damage to lens components is associated with cataract formation and reactive oxygen species (ROS) overproduction at inflammation sites is thought to lead to the development of inflammatory disorders. Bendazac is a non-steroidal anti-inflammatory drug able to delay the cataractogenic process. Aim of the present study is to characterize, both chemically and biologically, the activity of this anticataract agent as a radical scavenger. Bendazac has been shown to be a strong reacting substrate in a chemical oxidizing system, which mimics a physiological pathway of hydroxy radical generation. In the Fenton-Cier reaction the drug rapidly forms a mixture of hydroxylated derivatives, among which 5-hydroxybendazac, bendazac's main metabolite, being a hydroxy radical scavenger itself. Moreover, by means of a rapid and sensitive flow cytometric method able to determine reactive oxygen intermediate production, bendazac and its 5-hydroxy derivative were shown to inhibit oxidative burst activation in polymorphonuclear neutrophil leukocytes (PMNLs).

Abstract: In the rat, injection of Freund's complete adjuvant was accompanied by a significant increase in concanavalin A (Con A)-reactivity of selected plasma proteins along with an increase in concentrations of selected proteins known as acute phase proteins. We have evaluated the effect of bindarit, (2-[(1-benzyl-indazol-3-yl)methoxy]-2-methyl propionic acid), on the expression of alpha 2-macroglobulin, a known acute-phase protein in the rat. This compound has previously been shown to inhibit heat-induced denaturation of rat serum albumin and to strongly reduce the secondary phase response of adjuvant induced arthritis. Adult rats were induced with chronic inflammation by injection with Freund's complete adjuvant. Bindarit was administered to the chronic inflamed rats as a 0.5% medicated diet. Indomethacin, given by gavage daily at a dose of 1 mg/kg body weight, was used as a reference drug. Qualitative and quantitative changes of Con A-reactive proteins and alpha 2-macroglobulin were examined by lectin- and immuno-blots, and by radioimmunoassay. It was noted that the concentration of alpha 2-macroglobulin increased in rats with adjuvant induced arthritis. The addition of bindarit and indomethacin were able to reduce the concentration of alpha 2-macroglobulin as well as the Con A-reactivity of various proteins to normal level 37 days following treatment. We have also examined the effects of chronic inflammation on the levels of rat clusterin, a testicular and serum glycoprotein related to programmed cell death, tissue regression, and complement cascade reaction; and testibumin, a testicular FSH and testosterone-responsive protein with unknown function. It was noted that chronic inflammation did not induce significant changes in both the clusterin and testibumin concentrations in these experimental groups. The involvement of protein glycosylation and denaturation in the production of new antigenic determinants, their role in the development of chronic inflammatory disease and the potential use of bindarit to investigate the relationship between abnormal glycosylation and autoimmune disease were discussed.

Abstract: Previous studies from this and other laboratories have shown that abnormal glycosylation of several acute-phase proteins can be detected in various pathological conditions including autoimmune diseases. In the present study, we have investigated if abnormal glycosylation is limited to acute-phase proteins. We used the concanavalin A (Con A) blots in conjunction with the peptide mapping techniques to analyze serum samples and cerebrospinal fluids (CSF) obtained from patients with autoimmune diseases: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), mixed connective tissue disease (MCTD), scleroderma (SCL), Sjögren's syndrome (SS), and polymyositis (PM); diseases of probable autoimmune origin: hepatopathies (HP); diseases of suspected autoimmune origin: schizophrenia and Alzheimer's disease (AZ); and conditions not related to autoimmunity: pregnancy (PG) and elevation of the carcinoembryonic antigen (CEA), in comparison to normal donors (NHS). We have micropurified two human proteins; alpha 2-macroglobulin, a non-acute-phase protein and beta-chain of haptoglobin, a known acute-phase protein, from serum samples of individual patients with SLE, RA, MCTD, SCL and SS, and from PG and NHS for analysis. The identity of the purified proteins was confirmed by immunoblots using either monospecific polyclonal or monoclonal antibodies, and by direct N-terminal amino acid sequencing. Peptide maps for each of these proteins were generated using Staphylococcus aureus protease V8, a Glu-C endopeptidase. When the peptide fragments of alpha 2-macroglobulin were resolved by SDS-PAGE and visualized using silver staining, no differences were noted between patient samples and controls. However, when they were examined by lectin blots using Con A, the Con A-reactive fragments increased specifically and significantly in samples derived from patients of SLE, SCL, MCTD, and RA. Similarly when the peptide fragments of the beta-chain of haptoglobin were visualized by silver staining, no differences were noted; however, the Con A reactivity of specific fragments increased in SLE, RA, SCL, and SS patients. Analysis of these results indicated that there has been a selective increase in Con A-reactive fragments in both acute-phase and non-acute-phase proteins in autoimmune conditions. Thus, the study of changes in glycosylation patterns in selected serum proteins may be a valuable diagnostic approach to define the pathophysiology of inflammatory and autoimmune disorders.

Abstract: Induction of arthritis in rats with Freund's complete adjuvant was accompanied by a distinctive alteration of concanavalin A (Con-A) reactivity in their serum proteins in which the concentrations of selected Con-A reactive proteins were significantly higher when compared to healthy rats. To assess if the observed increase in Con-A reactivity of specific serum proteins reflects an increase in carbohydrate moieties in these proteins in addition to an increase in their protein concentrations, a heme binding serum glycoprotein, hemopexin, also an acute phase reactant, was selected as a marker protein. Hemopexin was purified to apparent homogeneity from pools of serum samples derived from rats with yeast induced inflammation, a monospecific polyclonal antibody was prepared and was used for immunoblot analysis. It was noted that the concentration of hemopexin increased in rats with adjuvant induced arthritis; however, its concentration fell to normal levels after administration with a newly synthesized drug, bindarit, (2-[(1-benzyl-indazol-3-yl)methoxy]-2-methyl propionic acid, C19H20N2O3. Hemopexin was micropurified individually from healthy rats, adjuvant induced arthritic rats, and adjuvant arthritic rats treated with bindarit, cleaved with a Glu-C endopeptidase, Staphylococcus aureus protease V8, and the resultant peptide fragments resolved by SDS-PAGE and examined by silver staining, Coomassie blue staining, and lectin blots using Con-A. It was subsequently noted that hemopexin isolated from adjuvant induced arthritic rats showed a significant increase in Con-A reactivity in selected peptide fragments and that such an increase in glycosylation could be reversed to a pattern similar to healthy rats following treatment with bindarit.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Bindarit (or 2-[(1-benzyl-indazol-3-yl)methoxy]-2-methyl propionic acid) reduces heat induced denaturation of bovine and rat serum albumin in vitro (EC50 = 8.5 and 65 micrograms/ml, respectively) and inhibits heat induced serum albumin denaturation after in vivo (12.5-25-50 mg/kg po) administration in rats. To assess the relationship between protein denaturation and the development of chronic inflammatory diseases, the drug (0.5 or 0.12% medicated diet) was studied in comparison with indomethacin (1 mg/kg po daily) in rats injected with complete Freund's adjuvant. Bindarit appeared different from aspirin-like drugs, antiinflammatory steroids and immunosuppressants because it does not reduce primary inflammation of arthritic rats and was shown to be completely inactive on cyclo and lipooxygenase activity in vitro and on immune reactions of mice in vivo. Nevertheless, the drug strongly reduced the development of the secondary phase of adjuvant induced arthritis. The most significant effect of bindarit in this phase was a strong inhibition of serum albumin denaturation in arthritic rats. Assessment of both electrophoretic and quantitative changes suggests that the reduction of albumin during inflammation is due, at least in part, to a denaturation of native albumin, which loses its electrophoretic mobility. The involvement of protein denaturation in the production of new antigenic determinants, their pathogenic relevance in the development of adjuvant arthritis and the possibility that protein stabilization by bindarit could be the mechanism of action of the drug are discussed.

Abstract:

Abstract: The mechanism by which the seminiferous epithelium limits the damaging effects of proteases that are released from degenerating late spermatids does not depend upon protease inhibitors in the systemic circulation since these proteins are excluded from the seminiferous tubule by the blood-testis barrier. The purpose of this study was to identify the major protease inhibitor of the testis and determine its cellular origin. Sertoli cells, the major epithelial component of the seminiferous epithelium, release a protease inhibitor, testicular alpha 2-macroglobulin, in vitro. Immunoprecipitation using [35S]methionine and a monospecific polyclonal antibody prepared against purified testicular alpha 2-macroglobulin establishes that this protein is actively synthesized and secreted by Sertoli cells. Measurements of immunoreactive protease inhibitors in tubular and rete testis fluids collected by micropuncture suggest that alpha 2-macroglobulin rather than alpha 1-antitrypsin is the major protease inhibitor in the seminiferous tubules in vivo. The ability of alpha 2-macroglobulin to inactivate proteases and growth factors such as TGF-beta by a common mechanism suggests that this protein may have a dual function in the testis.

Abstract: A monoclonal antibody, designated A2a18b8, of IgG1 class prepared against human alpha 1-antitrypsin, cross-reacts with alpha 1-antitrypsin in the serum of rat and baboon, but not with alpha 1-antitrypsin in serum of rabbit, pig, hamster, guinea pig, dog, or turtle. We used A2a18b8 in an enzyme-linked immunosorbent assay (ELISA) developed for human alpha 1-antitrypsin. Preliminary ELISA screening of 247 serum samples from patients with various inflammatory disorders indicated that the concentration of a specific epitope(s) on alpha 1-antitrypsin recognized by this monoclonal antibody was increased significantly in patients with active systemic lupus erythematosus, mixed connective tissue disease, and rheumatoid arthritis, but not in patients with sclerodermic disorders or Sjögren's syndrome. Evidently, A2a18b8 has diagnostic value in that it selectively recognizes a specific epitope(s) on alpha 1-antitrypsin that is (are) apparently exposed during selective inflammatory disorders.

Abstract: The time courses of aspirin and salicylate in plasma and ocular tissues of rabbits were investigated after the i.v. administration of aspirin. Unhydrolyzed aspirin rapidly disappears from plasma and many ocular compartments but persists up to 4 hours in aqueous and vitreous humours. Salicylate decreases in plasma follow an exponential kinetics; in aqueous humour and in vascularized tissues the behaviour is similar but with a half-life longer than in plasma. In the cornea, lens and vitreous humour, the concentration of salicylate reaches a peak between 2 and 4 hours, then it decreases very slowly. Our results show that aspirin is protected from the hydrolytic action of plasmatic esterases in aqueous and vitreous humours but is rapidly hydrolyzed in the cornea and lens by local esterases present in these tissues. It is possible that both aspirin and salicylate leave the eye by means of an active transport. Our results also indicate that salicylate can accumulate in the cornea, lens and retina when aspirin is administered repeatedly.

Abstract: Quantitative changes of concanavalin A (Con A)-reactive proteins in serum samples obtained from rats with induced inflammation and from patients with inflammatory and autoimmune diseases were examined by use of lectin blots. Treatment of rats with a single dose of fermented yeast to induce inflammation caused an extensive increase in Con A-reactivity. These changes were time dependent and were similar in both sexes of the animals. When we examined serum samples obtained from patients with various inflammatory disorders for their Con A-reactive proteins as compared with normal donors, we noted that the Con A-reactivity increased in patients with rheumatoid arthritis and systemic lupus erythematosus. Among all the glycoproteins examined by lectin blots with use of Con A, a set of five proteins was selected for detailed analysis by densitometric scanning. These included alpha 2-macroglobulin, P-150, P-95, P-40, and P-35, of Mr 180,000, 150,000, 95,000, 40,000, and 35,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Densitometric scanning analysis of the lectin blots revealed that the Con A-reactivity of these proteins increased during inflammation. Because alpha 2-macroglobulin is not an acute-phase protein in humans, an increase in Con A staining of this protein suggested that altered glycation is associated with autoimmune diseases. Thus, study of changes in Con A-reactive proteins in human sera may facilitate our understanding of the etiology and pathophysiology of autoimmune diseases.