Claudio Milanese

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

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

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.

Development and pharmacological characterization of a modified procedure for the measurement of carbonic anhydrase activity

Carbonic anhydrases (CAs) are a family of zinc metalloenzymes of molecular mass 30-60 kDa; seven different isoenzymes belong to this family (Okuyama et al., 1992, Proc Natl Acad Sci USA 89:1315-1319). They may be broadly recognized according to the efficiency with which they catalyze the reversible interconversion of CO2 and HCO3-, and they differ in physicochemical properties, in sensitivity to various inhibitors and in their subcellular localization; cytoplasmic (CA I, CA II, CA III, and CA VII), cell-surface membrane (CA IV), and mitochondrial (CA V) and secretory (CA VI) isoenzymes have been described. Several methods are reported in the literature for the measure of CA enzymatic activity; they may be broadly divided into two categories: those based on the measure of pH variation (pH-stat and colorimetric assays) (Wu et al., 1993, J Ocular Pharm 9:97-108; Maren, 1991, Molec Pharmacol 41:419-426) and the ones in which CO2 production is measured through pCO2 sensors (Botrè and Botrè, 1990, Anal Biochem 185:254-264).

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.

(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.

Adenosine triphosphate affects interleukin -1β release by T98G glioblastoma cells through a purinoceptor-independent mechanism

T98G glioblastoma cells were previously shown to significantly increase interleukin-1beta (IL-1beta) mRNA levels in response to IL-1beta stimulation. This work demonstrates that in such conditions T98G, despite possessing biologically active interleukin converting enzyme, do not release detectable amounts of IL-1beta, even in the presence of 20 mM adenosine triphosphate (ATP). IL-1beta secretion is observed only following concomitant stimulation with 1000 units/ml of IL-1beta and 20 mM ATP. ATP induces a dose-dependent depolarization of T98G plasma membrane, whereas it does not affect Ca(2+) concentration or cell membrane permeability. Our data, together with the observation that the depolarizing effects of ATP are retained after preincubation with 100 microM suramin, an antagonist of P2-purinoceptors, suggest that ATP plays a role in IL-1beta secretion by T98G but its effects do not occur through P2-purinoceptors.