Mario Pinza

Inhibition of monocyte chemotactic protein-1 synthesis by statins

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-1β. 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.

Synthesis and anti-ulcer activity of new derivatives of glycyrrhetic, oleanolic and ursolic acids

A review is made of the literature describing the structural changes to glycyrrhetic, oleanolic and ursolic acids and their influence on anti-ulcer activity. For the glycyrrhetic acid derivatives some analogues were prepared in which the ketonic group in position 11 was removed and the carboxylic function at position 30 was either intact, reduced to alcohol or transformed into ketone. This first series of compounds suggests the possibility of obtaining compounds devoid of the conjugated ketonic group, maintaining anti-ulcer activity but with reduced or lacking mineralocorticoid activity. Based on these findings, a series of carbenoxolone analogues in the beta-amyrin series of glycyrrhetic and oleanolic acid was prepared. In particular, the delta 9,11 unsaturated compounds 14b and 23b and the 11-methylene derivative 18 present advantages in terms of acute toxicity and mineralocorticoid activity as compared to the reference compound. The derivative 14b in the volunteer showed an increase of gastric PGE2 levels with minor pseudoaldosteronic effect. Among the ursolic acid derivatives, the dihemisuccinate sodium salt 35b demonstrated a good separation between anti-ulcer and mineralocorticoid activities. Nevertheless, kidney and liver toxicity was observed in the monkey thus jeopardizing its further development. Better results were obtained with the uvaol dihemiphthalate sodium salt and the diene analogue 39b. In particular, 38b and 39b showed a potent anti-ulcer activity, 3- to 25-fold higher than carbenoxolone. Furthermore, compound 38b does not show signs of liver toxicity in the monkey.

Mechanism of the inhibitory effect of melatonin on tumor necrosis factor production in vivo and in vitro.

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.

Inhibition of tumor necrosis factor-α (TNF-α)/ TNF-α receptor binding by structural analogues of suramin

Abstract Suramin, a symmetrical polysulfonated urea derivative, promotes the dissociation of trimeric human tumor necrosis factor-α (TNF-α) into biologically inactive subunits and prevents the interaction of TNF-α with its cellular receptors. The aim of this work was to identify compounds structurally related to suramin which inhibit the binding of TNF-α to its receptor. Molecular modeling studies were performed on suramin and TNF-α molecules and likely interaction sites were identified in the docked complex. On this basis, Evans blue, trypan blue, sulfonazo III, beryllon II, and 1,3,6-naphthalenetrisulfonic acid trisodium salt were identified as polysulfonated compounds endowed, to various extents, with the structural characteristics responsible for interaction with TNF-α. N , N -bis(3,5-di- tert -butylphenyl)-3,4,9,10-perylenedicarboximide was used as an unrelated structure. The capacity of these molecules to inhibit the binding of TNF-α with its receptor p55 was tested in vitro by means of a specific immunoenzymatic assay using suramin as reference compound. Evans blue and trypan blue inhibited TNF-α/p55 binding with an ic 50 of 0.75 and 1.00 mM, respectively (suramin ic 50 : 0.65 mM); no effect was observed with the other molecules. Molecular modeling analyses on Evans blue and trypan blue docked into the TNF-α molecule support these experimental results by demonstrating that these compounds share with suramin a similar binding mode to TNF-α. The results of this work provide a new insight into and useful hints for the design of new chemical entities endowed with a potent and selective activity on TNF-α.

Inflammatory molecule release by β-amyloid-treated T98G astrocytoma cells: role of prostaglandins and modulation by paracetamol

Deposition of beta-amyloid in the brain triggers an inflammatory response which accompanies the neuropathologic events of Alzheimers disease and contributes to the destruction of brain tissue. The present study shows that beta-amyloid can stimulate human astrocytoma cells (T98G) to secrete the proinflammatory factors interleukin-6 and prostaglandins. Furthermore, prostaglandins can stimulate T98G to secrete interleukin-6, which in turn triggers the formation of additional prostaglandins. Prostaglandins are, therefore, a key element in the induction and maintenance of a state of chronic inflammation in the brain which may exacerbate the fundamental pathology in Alzheimer patients. Paracetamol (0.01-1000 microM), an unusual analgesic/antipyretic drug which acts preferentially by reducing prostaglandin production within the central nervous system, and indomethacin (0.001-10 microM) caused a clear dose-dependent reduction of prostaglandin E2 production by stimulated T98G cells whereas interleukin-6 release was not affected. These data provide further evidence of the involvement of non-steroidal anti-inflammatory drugs in the inflammatory processes that can be generated by glial cells in intact brain.

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.

Selective Induction of MCP-1 in Human Mesangial Cells by the IL-6/sIL-6R Complex

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-1α (macrophage inflammatory protein 1α). 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-1α. 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.

Radical scavenger activity of bendazac, an anticataract non-steroidal anti-inflammatory agent.

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, bendazacs 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).

Acetaminophen down-regulates interleukin-1β-induced nuclear factor-κB nuclear translocation in a human astrocytic cell line

Abstract In previous studies performed to elucidate acetaminophen mechanism of action, we demonstrated that acetaminophen inhibits prostaglandin E2 production by interleukin (IL)-1β-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)-κB and STAT3 transcription factors and with SAPK signal transducing factor. Western blot analysis of IκBα protein in the cytoplasm of IL-1β-stimulated T98G cells and electrophoretic mobility shift assay (EMSA) on corresponding nuclear extracts indicate that acetaminophen (10–1000 μM) dose-dependently inhibits both IκBα degradation and NF-κB nuclear translocation. In the same cell type neither IL-1β-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-1β-dependent NF-κB 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.

Benzydamine inhibits monocyte migration and MAPK activation induced by chemotactic agonists

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. 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 IC50s of 100, 50 and 45 μM for MCP‐1/CCL2, fMLP and C5a, respectively. At the dose of 100 μM, 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). Receptor expression as well as calcium fluxes in response to chemoattractants were not affected by benzydamine. 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 μM concentration of benzydamine with an IC50 of 30 μM. Under the same experimental conditions, pretreatment with 100 μM benzydamine caused a 75–89% inhibition of p38 activation (IC50 25 μM). 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.