Giuseppe Ronsisvalle

Neuroprotective effects of sigma-1 receptor agonists against beta-amyloid-induced toxicity.

Prolonged exposure of cultured cortical neurons to the residue 25–35 fragment of &bgr;-amyloid protein, in the presence of dizocilpine, an antagonist of the N-methyl-D-aspartate receptor, and of 6,7-dinitroquinoxaline-2,3-dione, an antagonist of &agr;-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors, resulted in the expression of the proapoptotic protein Bax and neuronal death. Beta-amyloid protein(25–35)-induced neuronal death was substantially attenuated by the σ1 receptor agonist 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate. The neuroprotective action of 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate was mimicked by the σ1 ligand methyl (1S,2R)-2-[1-adamantyl(methyl)amino]methyl-1-phenylcyclopropanecarboxylate and was antagonized by the σ1 receptor antagonist N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)-phenyl]-ethylamine monohydrochloride. These results suggest that σ1 receptor agonists might function as neuroprotectant agents in Alzheimers disease.

Analysis of aliphatic amines in air samples by HPLC with electrochemical detection.

A method was developed for the analysis of primary aliphatic amines by high performance liquid chromatography coupled with electrochemical detector. The electrochemical oxidation of aliphatic amines derivatized with 2,5-dihydroxybenzaldehyde was investigated at porous graphite electrodes. The derivatization reactions were performed off-line, before the chromatographic separation. The compounds were separated on a reversed phase column with a methanol-acetonitrile-phosphate buffer and detected setting at an oxidation potential of +0.5 V. The influence of the mobile phase buffer concentration and pH on the detector response was also studied. The derivatization was shown to be quantitative and the response linear between 50 and 200 ng/ml. The method is sensitive, selective and could be applicable for the assay of volatile amines in the field of environmental toxicology and also for biological monitoring after occupational exposure.

Opioid and sigma receptor studies. New developments in the design of selective sigma ligands

New racemic and chiral methyl 2-{[4-(4-chlorophenyl)-4-hydroxypiperi-din-1-yl]methyl}-1-phenylcyclopropanecarboxylate derivatives were synthesized in order to obtain sigma ligands with increased affinity and selectivity compared to (+)-MPCB and haloperidol. The cis-(±)-7 racemic mixture showed a better binding affinity and selectivity than the (±)-8 trans isomers. Between the two cis enantiomers, (+)-7, with configuration (1R,2S), showed a very high affinity and the best selectivity for s1. All compounds synthesized (79) showed a reduced or negligible affinity for opioid and dopaminergic D1 and D2 receptors. Nociceptive in vivo test confirms that (+)-7 (namely MR200), such as non-selective antagonist haloperidol, increased the analgesic effect induced by the k opioid selective ligand U50,488H and reversed the inhibiting effect of (+)-pentazocine on analgesia.

Synthesis and Immunostimulating Activity of A Thioglycolipopeptide Glycomimetic As A Potential Anticancer Vaccine Derived From Tn Antigen

ABSTRACT The Tn epitope is one of the tumor associated O-linked cell surface glycopeptides. It is expressed in over 70% of human epithelial cancers such as lung, colon, stomach and breast carcinomas. The glycosidic linkage of the Tn antigen, between N-acetylgalactosamine and serine or threonine, can be cleaved either chemically or enzymatically in the presence of glycosidases. The latter case is particularly a problem in vivo. Therefore, it would be of great interest to obtain a metabolically stable analogue of the Tn antigen that maintains or improves the immunogenic activity of the latter. The purpose of this work was to synthesize a totally synthetic vaccine using a chemically and metabolically stable glycomimetic of the Tn antigen in which the interglycosidic oxygen was replaced by a sulphur atom (S-Tn). The S-Tn thioglycopeptide was linked to the P3CS immunoadjuvant to obtain the potential S-Tn vaccine. Moreover, we synthesized the natural Tn antigen and derivatized it similarly to obtain the Tn vaccine. Last, we evaluated the immunostimulating activity of the two synthetic potential vaccines in vitro using cultured mouse splenocytes. The S-Tn construct showed immunostimulating activity comparable, in terms of maximal response, to the Tn analogue. Moreover, due to its higher stability the S-Tn construct reached its maximal effect at lower doses compared to the Tn analogue.

Intrastriatal administration of sigma ligands inhibits basal dopamine release in vivo.

In this study, using the new sigma(1/2) (sigma(1/2)) compound MR200, its parent drug haloperidol and the sigma ligand 1,3-di-o-tolylguanidine (DTG), we have investigated the role of striatal sigma receptors in the control of basal dopamine (DA) outflow, by coupling in vitro binding experiments and in vivo microdialysis in the striatum of halothane-anesthetized rats. MR200 with respect to haloperidol, exhibits high affinity for sigma(1) (1.5 nM) and sigma(2) (21.9 nM) receptors, but only negligible affinity for DA receptors. Compared to DTG, MR200 has similar selectivity across neurotransmitter systems, and 46 times higher affinity for sigma(1) receptors. Intrastriatal application of MR200 at 10, but not 0.1 or 1 microM, elicited a pronounced decrease in striatal DA release (-45% of control values). This inhibitory effect was preceded by a transient increase in DA release (+50% over baseline) after 100 microM MR200 administration. DTG at 100, but not 10 microM, significantly reduced DA release (-40%). Haloperidol, whilst increasing DA release at 1 microM, induced a delayed decrease in DA release after 10 microM application. Finally, haloperidol (10 microM) did not modify the inhibitory effect of 10 microM MR200. These results show that striatal sigma receptors control striatal DA release in resting conditions.

A new sigma ligand, (±)-PPCC, antagonizes kappa opioid receptor-mediated antinociceptive effect

The compound (1R,2S/1S,2R)-2-[4-hydroxy-4-phenylpiperidin-1-yl)methyl]-1-(4-methylphenyl) cyclopropanecarboxylate [(+/-)-PPCC] is a ligand with high affinity for sigma (sigma) sites of which the selectivity towards several other receptor systems has been demonstrated. Given the existence of a relationship between the sigma system and the kappa opioid (KOP)-mediated analgesia, to characterize the pharmacological properties of (+/-)-PPCC we analyzed its influence on the analgesic effect of the systemic injected kappa agonist (-)-U-50,488H comparing the effects with those shown by (+)-pentazocine and BD1047. The results demonstrate that the systemic administration of (+/-)-PPCC (1 mg/kg s.c.) does not modify basal tail-flick latency. Pre-treatment with (+/-)-PPCC, at the same dose, significantly decreased the antinociceptive effect of (-)-U-50,488H, analogously to the sigma compounds used. This study confirms that (+/-)-PPCC plays the role of sigma agonist in this model and strengthens the hypothesis of the sigma receptor modulatory role on KOP-mediated analgesia.

Simultaneous determination of amphetamine and one of its metabolites by HPLC with electrochemical detection

A high-performance liquid chromatographic method coupled with electrochemical detector was developed for the separation and quantitation of amphetamine and one of its metabolites, the 4-hydroxynorephedrine. The pre-column derivatisation of these compounds was carried out with 2,5-dihydroxybenzaldehyde as electroactive labelling reagent, in presence of Borohydride Exchange Resin. The new synthetic method developed was fast, clean and high yielding. The analysis was performed in isocratic mode on a reversed phase column 5 microm Hypersil ODS RP-18, 15 cm, using as a mobile phase methanol-NaH(2)PO(4) buffer (50 mM, pH 5.5)(30:70 v/v) containing trietylamine (0.5% v/v) and the products were detected by a porous graphite electrode set at an oxidation potential of +0.6 V. The linearity of response was examined for each derivatised compound and was analysed using solutions in the range 10-40 nmol/ml. The correlation coefficients of the linear regression of the standard curves were greater than 0.99. The method developed in this study was sensitive and very selective. Because of the specificity for primary phenylethylamines, it could be applicable for the assay of other related substances in toxicology and drugs abuse.

Novel Potent and Selective σ Ligands: Evaluation of Their Agonist and Antagonist Properties

Novel enantiomers and diastereoisomers structurally related to σ ligand (+)-MR200 were synthesized to improve σ(1)/σ(2) subtype selectivity. The selective σ(1) ligand (-)-8 showed an antagonist profile determined by phenytoin differential modulation of binding affinity in vitro, confirmed in vivo by an increase of κ opioid analgesia. The σ(2) ligand (-)-9 displayed agonist properties in an in vitro isolated organ bath assay and antiproliferative effects on LNCaP and PC3 prostate cancer cell lines.

Amidinobenzisothiazole derivatives with antidegenerative activity on cartilage

N-(Benzo[d]isothiazol-3-yl)amidines were synthesised and evaluated for their antiinflammatory activity. Encouraging results led us to evaluate these derivatives on the prevention of cartilage destruction in articular disease. Antidegenerative activity was assayed on culture of porcine nasal cartilage and diarthroidal joint human cartilage in the presence of interleukin-1beta (IL-1beta). The amount of glycosaminoglycans (GAGs) and the production of nitric oxide (NO) in the culture medium were determined. The obtained results showed that all the compounds, in the presence of IL-beta, blocked the cartilage breakdown, with different behaviour. The antidegenerative activity is more evident in human cartilage.

Supraspinal injection of Substance P attenuates allodynia and hyperalgesia in a rat model of inflammatory pain.

The neuropeptide Substance P (SP), that has a high affinity for the neurokinin 1 (NK1) receptor, is involved in modulation of pain transmission. Although SP is thought to have excitatory actions and promote nociception in the spinal cord, the peptide induces analgesia at the supraspinal level. The aim of this study was to evaluate the role of supraspinal SP and the NK1 receptor in inflammatory pain induced by injection of carrageenan in the hind paw of the rat. There are two nociceptive behavioral responses associated with this pain state: mechanical allodynia and heat hyperalgesia. Because the NK1 receptor colocalizes with the MOP receptor in supraspinal sites involved in pain modulation, we also decided to study the possible involvement of the opioid system on SP-induced analgesia. We found that treatment with SP, at doses of 3.5, 5 and 7 μg/5 μl/rat i.c.v., clearly showed inhibition of allodynia and hyperalgesia. Pretreatment with the selective NK1 antagonist L-733,060 (10mg/kg i.p.) blocked the SP-induced analgesia, suggesting the involvement of the NK1 receptor. This SP-induced analgesia was significantly reduced by administration of the opioid antagonist naloxone (3mg/kg s.c.). This reduction occurred when SP was administered either before or after the carrageenan injection. These results suggest a significant antinociceptive role for SP and the NK1 receptor in inflammatory pain at the supraspinal level, possibly through the release of endogenous opioids.