Hybrids between H2S-donors and betamethasone 17-valerate or triamcinolone acetonide inhibit mast cell degranulation and promote hyperpolarization of bronchial smooth muscle cells.

Glucocorticoids represent the standard gold treatment of inflammation in asthmatic patients. More recently, H2S has been described to exert positive effect on this disease. Bearing in mind that an improved pharmacological activity and a reduced toxicity can be obtained through hybridization of different molecules, simultaneously modulating multiple targets, we designed and synthesized novel betamethasone 17-valerate and triamcinolone acetonide hybrids with well-known H2S-donor moieties. Synthesized compounds have been evaluated for the potential H2S-releasing profile both in cell-free environment and into the cytosol of bronchial smooth muscle cells (BSMCs). The two hybrids 4b and 5b were investigated by molecular modelling studies and results indicated that the steric accessibility of the isothiocyanate carbon atom can account for their different H2S releasing properties. Furthermore, the most promising derivatives 4b and 5b have been tested for inhibitory effect on mast cell degranulation and for the ability to induce cell membrane hyperpolarization in BSMCs. Significant inhibitory effect on mast cell degranulation was assessed, resulting to reduce ß-hexosaminidase release more efficiently than the corresponding native drugs. Both compounds determined a massive membrane hyperpolarization of BSMCs and proved to be 4-fold more effective compared to reference compound NS1619. These effects represent an enrichment of the pharmacological activity of the native drugs.

A novel FRET peptide assay reveals efficient Helicobacter pylori HtrA inhibition through zinc and copper binding.

Helicobacter pylori (H. pylori) secretes the chaperone and serine protease high temperature requirement A (HtrA) that cleaves gastric epithelial cell surface proteins to disrupt the epithelial integrity and barrier function. First inhibitory lead structures have demonstrated the essential role of HtrA in H. pylori physiology and pathogenesis. Comprehensive drug discovery techniques allowing high-throughput screening are now required to develop effective compounds. Here, we designed a novel fluorescence resonance energy transfer (FRET) peptide derived from a gel-based label-free proteomic approach (direct in-gel profiling of protease specificity) as a valuable substrate for H. pylori HtrA. Since serine proteases are often sensitive to metal ions, we investigated the influence of different divalent ions on the activity of HtrA. We identified Zn++ and Cu++ ions as inhibitors of H. pylori HtrA activity, as monitored by in vitro cleavage experiments using casein or E-cadherin as substrates and in the FRET peptide assay. Putative binding sites for Zn++ and Cu++ were then analyzed in thermal shift and microscale thermophoresis assays. The findings of this study will contribute to the development of novel metal ion-dependent protease inhibitors, which might help to fight bacterial infections.

Synthesis, in vitro and in vivo evaluation of 11C-O-methylated arylpiperazines as potential serotonin 1A (5-HT1A) receptor antagonist radiotracers.

BACKGROUND: Serotonin 1A (5-HT1A) receptors are implicated in the pathogenesis of several psychiatric and neurodegenerative disorders motivating the development of suitable radiotracers for in vivo positron emission tomography (PET) neuroimaging. The gold standard PET imaging agent for this target is [carbonyl-11C]WAY-100635, labeled via a technically challenging multi-step reaction that has limited its widespread use. While several antagonist and agonist-based PET radiotracers for 5-HT 1A receptors have been developed, their clinical translation has been hindered by methodological challenges and/or and non-specific binding. As a result, there is continued interest in the development of new and more selective 5-HT1A PET tracers having a relatively easier and reliable radiosynthesis process for routine production and with favorable metabolism to facilitate tracer-kinetic modeling. The purpose of the current study was to develop and characterize a radioligand with suitable characteristics for imaging 5-HT1A receptors in the brain. The current study reports the in vitro characterization and radiosyntheses of three candidate 5-HT1A receptor antagonists, DF-100 (1), DF-300 (2) and DF-400 (3), to explore their suitability as potential PET radiotracers. RESULTS: Syntheses of 1-3 and corresponding precursors for radiolabeling were achieved from isonicotinic, picolinic acid or picolino nitrile. In vitro binding studies demonstrated nanomolar affinity of the compounds for 5-HT1A receptors. Binding of 1-3 for other biogenic amines, neurotransmitter receptors, and transporters was negligible with the exception of moderate affinities for α1-adrenergic receptors (4-6-fold less potent than that for 5-HT1A receptor). Radioligands [11C]1-3 were efficiently prepared by 11C-O-methylation of the corresponding phenolic precursor in non-decay corrected radiochemical yields of 7-11% with > 99% chemical and radiochemical purities. Dynamic PET studies in rats demonstrated negligible brain uptake of [11C]1 and [11C]2. In contrast, significant brain uptake of [11C]3 was observed with an early peak SUV of 4-5. However, [11C]3 displayed significant off-target binding attributed to α1-adrenergic receptors based on regional distribution (thalamus>hippocampus) and blocking studies. CONCLUSION: Despite efficient radiolabeling, results from PET imaging experiments limit the application of [11C]3 for in vivo quantification of 5-HT1A receptors. Nevertheless, derivatives of compound 3 may provide a scaffold for alternative PET radiotracers with improved selectivity for 5-HT 1A receptors or α1-adrenergic receptors.

New Serotoninergic Ligands Containing Indolic and Methyl Indolic Nuclei: Synthesis and In Vitro Pharmacological Evaluation.

BACKGROUND: Serotonin is an important biogenic amine and is implicated in wideranging physiological and physiopathological processes. Pharmacological manipulation of the serotoninergic system is believed to have a great therapeutic potential. OBJECTIVES: In order to identify selective ligands for 5-HT1A, 5-HT2A and 5-HT2C receptors two series of 4-substituted piperazine derivatives, bearing indolic or methyl indolic nuclei, were synthesized. METHODS: All the compounds, synthesized by standard solution methods, were evaluated for 5- HT1A, 5-HT2A and 5-HT2C receptors. The highest affine and selective compounds have been evaluated also on dopaminergic (D1 and D2) and adrenergic (α1A and α2A) receptors. RESULTS: Several of the newly synthesized molecules showed affinity in the nanomolar range for 5- HT1A, 5-HT2A and 5-HT2C receptors and moderate to no affinity for other relevant receptors (D1, D2, α1A and α2A). CONCLUSION: Compounds 7f and 10a showed a nanomolar affinity towards 5-HT1A with an in vitro pharmacologic profile compatible with antipsychotic drugs.

PCB levels in adipose tissue of dogs from illegal dumping sites in Campania region (Italy).

The aim of the study is to investigate the potential relationship between exposure to PCBs and cancer. In doing so we relied on a sample of dogs coming from a peculiar area of the Campania region (Italy), that has been suffering for illegal waste dumping and open air burning of plastic waste for many years. The latter determined the release of organic and inorganic pollutants, such as the PCBs. By comparing dogs with cancer and healthy dogs, we found much higher PCB concentrations in the former, with a significant difference (p < 0.05) for the non-indicator ∑10NDL-PCB and the DL-PCBs. A regression analysis, controlling for three potentially confounding factors, that are sex, age and weight, confirmed the higher ∑10NDL-PCB concentration in dogs with cancer. Hence, our evidence suggests a potential health hazard for animals and likewise people living in a risky area due to the presence of environmental organic pollutants.

Synthesis and Pharmacological Screening of Pyridopyrimidines as Effective Anti-Diarrheal Agents through the Suppression of Cyclic Nucleotide Accumulation.

The increased levels of cyclic nucleotides (cGMP and cAMP) in enterocytes trigger intracellular mechanisms of ion and fluid secretion into the lumen, causing secretory diarrhea. Twelve novel pyridopyrimidines derived from 5-(3,5-bistrifluoromethylphenyl)-1,3-dimethyl-5,11-dihydro-1H-indeno[2,1 : 5,6]pyrido[2,3-d]pyrimidine-2,4,6-trione (FPIPP) were synthesized and evaluated on intracellular cyclic nucleotide accumulation. All compounds had no effect on either cyclic nucleotide basal levels or on pre-contracted aortic rings. The metabolic activity and viability in T84 cells, assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and the LDH (lactate dehydrogenase) assays, respectively, were not affected by incubation with the compounds (50 µM). Compound VI almost abolished cGMP accumulation (94 % inhibition) induced by STa toxin in T834 cells and significantly reduced (69 %) forskolin-induced cAMP accumulation in Jurkat cells. Compound VI was active in an in vivo model for diarrhea in rabbits. These results prompted us to perform a microscopic histopathological analysis of intestinal tissues, showing that only compound VI preserves the intestine without significant pathological changes and with a decreased inflammatory pattern in comparison to FPIPP. In vitro stability test revealed that compound VI is resistant to oxidation promoted by atmospheric oxygen.

Anti-metastatic Properties of Naproxen-HBTA in a Murine Model of Cutaneous Melanoma.

The beneficial effects of H2S-release and of COXs-inhibition have been exploited in the design of novel anti-inflammatory drugs, the H2S-releasing non-steroidal anti-inflammatory drugs (H2S-NSAIDs), showing promising potential for chemoprevention in cancers. Here, we evaluated the efficacy of a new H2S-releasing derivative of naproxen, named naproxen-4-hydroxybenzodithioate (naproxen-HBTA), in reducing metastatic melanoma features, both in vitro and in vivo. The novel H2S donor has been prepared following a synthetic scheme that provided high yields and purity. In particular, we investigated the effect of naproxen-HBTA in vitro on several metastatic features of human melanoma cells such as proliferation, migration, invasion, and colonies formation and in vivo in a model of cutaneous melanoma. Cell culture studies demonstrated that naproxen-HBTA induced caspase 3-mediated apoptosis and inhibited motility, invasiveness, and focus formation. Finally, daily oral treatment with naproxen-HBTA significantly suppressed melanoma growth and progression in mice. In conclusion, by using this dual approach we propose that the COX-2 and H2S pathways could be regarded as novel therapeutic targets/tools to generate new treatment options based on "combination therapy" for melanoma.

Anxiolytic-like effects of the new arylpiperazine derivatives containing isonicotinic and picolinic nuclei: behavioral and biochemical studies.

Anxiety disorder is a great challenge for modern psychopharmacology. Although a variety of single drugs are used in the treatment of anxiety, it is important to search for new therapeutics with faster onset of action, fewer side effects, and higher efficacy. In this work, we studied the possible anxiolytic action mechanism of two new arylpiperazine derivatives: compounds 4p N-(3-(4-(piperonyl)piperazin-1-yl)propyl)isonicotinamide and 3o N-(2-(4-(pyrimidin-2-yl)piperazin-1-yl)ethyl)picolinamide, focusing on their effects on the GABAergic and 5-HT systems. The elevated plus-maze test (EPM) was used for measuring anxiety. Additionally, in order to elucidate whether the new compounds have impact on the central redox balance, we conducted biochemical studies. In doing so, the relative activity of the enzymes responsible for glutathione metabolism - glutathione peroxidase and reductase (GPx and GR) - was measured. The results of the presented studies confirmed the anxiolytic effects of the new compounds 4p (60 mg/kg) and 3o (7.5 mg/kg), and suggested in the mechanism of their action, direct 5-HT1A receptors' participation and indirect involvement of the GABAergic system. Furthermore, the compounds exerted significant agonistic effect with buspirone (BUS, the 5-HT1A partial agonist, 1 mg/kg i.p.) and diazepam (DZ, the classic benzodiazepine anxiolytic, 0.25 mg/kg s.c.), while WAY 100635 (N-{2-[4-(2-methoxyphenyl)-1-piperazinyl] ethyl}-N-(2-pyridyl) cyclohexanecarboxamide, a selective 5-HT1A antagonist, 0.1 mg/kg s.c.), but not flumazenil (a GABAA -BDZ receptor complex antagonist, 10 mg/kg i.p.) was able to reverse their anxiolytic effects in EPM. A concomitant decrease in GPx by the compound 4p (and to a lesser degree, by compound 3o) further seemed to confirm their anxiolytic and antioxidant activity.

Drawing with oblique coordinates: on a single case.

We describe a patient with right hemisphere damage affected by mild left visuo-spatial neglect and constructional apraxia. During the rehabilitation, he failed to draw a draught-board using horizontal and vertical trajectories, but he performed it successfully using oblique trajectories. These observations suggested an impairment of vertical/horizontal spatial coordinates system. In copying tasks including figure elements in different orientations he drew more accurately components in oblique orientation, whereas failed to reproduce components in horizontal orientation. The patient performed visuospatial perceptual and perceptual-imaginative tasks successfully. From these findings, it is possible to suggest that the oblique coordinate system of reference operates independently of vertical and horizontal coordinate systems in building a complex figure and that, therefore, cardinal orientation do not constitute a reference norm to define oblique orientation, as previously suggested.