Design and synthesis of new bifunctional sigma-1 selective ligands with antioxidant activity.

Herein we report the synthesis of new bifunctional sigma-1 (σ1)-selective ligands with antioxidant activity. To achieve this goal, we combined the structure of lipoic acid, a universal antioxidant, with an appropriate sigma aminic moiety. Ligands 14 and 26 displayed high affinity and selectivity for σ1 receptors (Kiσ1 = 1.8 and 5.5 nM; Kiσ2/σ1 = 354 and 414, respectively). Compound 26 exhibited in vivo antiopioid effects on kappa opioid (KOP) receptor-mediated analgesia. In rat liver and brain mitochondria (RLM, RBM), this compound significantly reduced the swelling and the oxidation of thiol groups induced by calcium ions. Our results demonstrate that the tested compound has protective effects against oxidative stress.

New benzomorphan derivatives of MPCB as MOP and KOP receptor ligands.

There is considerable interest in developing KOP Opioid receptor ligands as clinically useful analgesics. Moreover, compounds with mixed KOP receptor and mu-opioid peptide (MOP) receptor agonist/antagonist properties could have a better therapeutic potential. The benzomorphan-based synthetic ligands MPCB and CCB have been shown to bind KOP receptors with high affinity and selectivity. We report here a series of compounds synthesized to perform structure-affinity relationship (SAR) studies on MPCB. The aim of this study was to optimise KOP receptor-ligand interaction and to modulate MOP receptor selectivity. In the benzylamide analogue of MPCB (compound 9) the presence of a third aromatic nucleus, at an appropriate distance and conformation with respect to aromatic pharmacophoric residues, increased KOP receptor affinity by about 6-fold compared to MPCB (Ki = 35 nM and Ki = 240 nM, respectively). Instead, compound 28 with a tertiary amino group in the nitrogen substituent displayed a comparable KOP receptor affinity (Ki = 179 nM) but also high MOP receptor affinity (Ki = 45 nM). Thus, the present study shows that in benzomorphan-based ligands the presence of different functional groups in the nitrogen substituent, ranging from a positive charged amine to an additional aromatic ring, is able to promote the correct aligment of aromatic pharmacophoric residues with MOP and KOP receptor types. Evaluation of docking simulations of compounds 9 and 28 into the KOP and MOP receptor displayed selective ligand interactions with the important amino acid residues Tyr320 (TMVII) and Trp318 (TMVII), respectively.

TRAIL inhibits angiogenesis stimulated by VEGF expression in human glioblastoma cells.

Tumour growth is tightly related to new blood vessel formation, tissue remodelling and invasiveness capacity. A number of tissular factors fuel the growth of glioblastoma multiforme, the most aggressive brain neoplasm. In fact, gene array analyses demonstrated that the proapoptotic cytokine tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) inhibited mRNA expression of VEGF, along with those of matrix metalloproteinase-2 (MMP-2), its inhibitor tissue inhibitor of matrix metalloproteinases-2 (TIMP-2), as well as the tumour invasiveness-related gene secreted protein acid rich in cysteine (SPARC) in different human glioblastoma cell lines. Particularly, VEGF mRNA and protein expression and release from glioblastoma cells were also inhibited by TRAIL. The latter also exerted antimitogenic effects on human umbilical vein endothelial cells (HUVECs). With the same cells, TRAIL inhibited new vessel formation in the in vitro matrigel model, as well as it exerted powerful inhibition of blood vessel formation induced by an angiogenic cocktail administered in subcutaneous pellets in vivo in the C57 mouse. Moreover, the expression of MMP-2, its inhibitor TIMP-2 and the tumour invasiveness-related protein SPARC were effectively inhibited by TRAIL in glioblastoma cell lines. In conclusion, our data indicate that TRAIL inhibits the orchestra of factors contributing to glioblastoma biological aggressiveness. Thus, the TRAIL system could be regarded as a molecular target to exploit for innovative therapy of this type of tumour.

Different in vitro activity of flurbiprofen and its enantiomers on human articular cartilage.

The 2-arylpropionic acid derivatives or 'profens' are an important group of non-steroidal anti-inflammatory drugs that have been used for the symptomatic treatment of various forms of arthritis. These compounds are chiral and the majority of them are still marketed as racemate although it is known that the (S)- form is the principal effective in the cyclooxygenase inhibition. However, recent findings suggest that certain pharmacological effect of 2-arylpropionic acids cannot be attributed exclusively to the (S)-(+) enantiomer. To obtain further insights into the pharmacological effect of profens, the present study investigated the influence of racemic and pure enantiomers of flurbiprofen on the production of nitric oxide and glycosaminoglycans, key molecules involved in cartilage destruction. The culture of human articular cartilage stimulated by interleukin-1beta (IL-1beta), which plays an important role in the degradation of cartilage, has been established, as a profit experimental model, for reproducing the mechanisms involved in the pathophysiology of arthritic diseases. Our results show that mainly (S)-(+)-flurbiprofen decreases, at therapeutically concentrations, the IL-1beta induced cartilage destruction.

The reproductive system at the neuroendocrine-immune interface: focus on LHRH, estrogens and growth factors in LHRH neuron-glial interactions.

Bidirectional communication between the neuroendocrine and immune systems plays a pivotal role in health and disease. Signals generated by the hypothalamic-pituitary-gonadal (HPG) axis (i.e. luteinizing hormone-releasing hormone, LHRH, and sex steroids) are major players coordinating the development immune system function. Conversely, products generated by immune system activation exert powerful and longlasting effects on HPG axis activity. In the central nervous system (CNS), one chief neuroendocrine-immune (NEI) compartment is represented by the astroglial cell population and its mediators. Of special interest, the major supporting cells of the brain and the thymus, astrocytes and thymic epithelial cells, share a similar origin and a similar set of peptides, transmitters, hormones and cytokines functioning as paracrine/autocrine regulators. This may explain some fundamental analogies in LHRH regulation of both cell types during ontogeny and in adult life. Hence, the neuropeptide LHRH significantly modulates astrocyte and thymic cell development and function. Here we focus this work on LHRH neuron-glial signaling cascades which dictate major changes during LHRH neuronal differentiation and growth as well as in response to hormonal manipulations and pro-inflammatory challenges. The interplay between LHRH, growth factors, estrogens and pro-inflammatory mediators will be discussed, and the potential physiopathological implications of these findings summarized. The overall study highlights the plasticity of this intersystem cross-talk and emphasize neuron-glial interactions as a key regulatory level of neuroendocrine axes activity.

Red wine micronutrients as protective agents in Alzheimer-like induced insult.

Plant extract micronutrients are commonly added to diets for health and prevention of degenerative disease. However, there are barriers to the introduction of these products as antioxidant therapies in counteracting chronic human diseases, probably because the molecular bases of their therapeutic potential are poorly clarified. The present study was designed to evaluate the possible protective effect of combined micronutrients present in black grape skin on toxicity induced by 25-35 beta-amyloid peptid or by serum of Alzheimer's disease patients, in human umbilical vein endothelial cells (HUVECs). The hypothesis was tested by examining the results of lactic dehydrogenase (LDH) release to estimate cytoplasmic membrane breakdown; activity of mitochondrial complexes, reactive oxygen species (ROS) production and malonyl dialdehyde (MDA) levels as markers of oxidative stress induction and COMET assay to evaluate DNA fragmentation. The results demonstrate that black grape skin extract reduces the ROS production, protects the cellular membrane from oxidative damage, and consequently prevents DNA fragmentation. The experimental results suggest that this natural compound may be used to ameliorate the progression of pathology in AD disease therapy.

Neuroprotective activity of chemokines against N-methyl-D-aspartate or beta-amyloid-induced toxicity in culture.

We have examined the effect of various chemokines on neuronal toxicity in culture. In mixed cortical cultures, challenged with a brief pulse of N-methyl-D-aspartate (NMDA, 60 microM, 10 min), chemokines were either present for 2 h preceding the pulse or they were co-applied with NMDA and then kept in the medium for the following 20-24 h. Interleukin-8 (IL-8), regulated on activation of normal T cells expressed and secreted (RANTES) and macrophage/monocyte chemoattractant protein-1 (MCP-1), were neuroprotective under both conditions, whereas stromal cell-derived factor 1alpha (SDF-1alpha) was protective only when applied during and after the NMDA pulse. Mixed or pure neuronal cultures were also exposed for 48 h to a toxic fragment of the beta-amyloid peptide (beta-amyloid peptide-(25-35), 12.5 or 25 microM) in the absence or presence of chemokines. Among a number of chemokines, only RANTES was neuroprotective against beta-amyloid peptide-(25-35)-induced neurotoxicity in both cultures. We conclude that activation of chemokine receptors differentially affects neuronal degeneration induced by excitotoxins or beta-amyloid peptide in cortical cultures.

Central and peripheral involvement of endogenous opioid peptides in gastric protection in stressed rats.

The effects of intraperitoneal and intracerebroventricular administration of the inhibitor of endopeptidase EC 24.11 (enkephalinase), thiorphan, and the synthetic enkephalin analogue [D-Ala2-Met5]enkephalinamide (DALA) were investigated in cold-restraint-stressed rats. Drugs were administered alone or after pretreatment with naloxone or naloxone methiodide given 20 min prior to the drugs. Thiorphan and DALA, administered centrally (4 micrograms i.c.v./rat) or peripherally (400 micrograms/kg), induced a significant gastric protection. Prior treatment with naloxone s.c. (1 mg/kg) inhibited the effect induced by i.c.v. or i.p. injections of thiorphan or DALA. In contrast, s.c. administration of naloxone methiodide (1 mg/kg) did not affect the response induced by central administration of thiorphan or DALA, but was able to prevent that of thiorphan or DALA when they were administered i.p. These data strongly support the hypothesis of a central and peripheral involvement of endogenous opioid peptides in gastric protection in stressed rats.

Prevention of stress-induced gastric ulcers by mu- and delta-opioid agonists in the rat.

The effects of intraperitoneal and intracerebroventricular administration of mu- and delta- selective opioid receptor agonists (DAGO and DPDPE, respectively) on gastric lesions, were investigated in cold-restraint-stressed rats. DAGO and DPDPE, peripherally and centrally administered, induced a significant gastric protection. Naloxone prevented the effects of both opioids whereas naltrindole prevented the gastric protection induced by DPDPE but not that by DAGO. The results suggest that mu- and delta-opioid agonists prevent gastric damage induced by stress through an involvement of both central and peripheral mu- and delta-opioid receptor subtypes.

Effect of indomethacin on opioid-induced gastric protection in cold-restrained stress.

Morphine and the synthetic opioid met-enkephalin analog [D-Ala2, MePhe4, Met(0)5ol] enkephalin (FK 33-824) injected intraperitoneally to rats at doses of 5-20 and 0.5-2 mg/kg respectively showed a protective effect on gastric lesion induced by cold-restraint stress. This protective effect was abolished by pretreatment with indomethacin. This suggests a role for prostaglandins in the protection, induced by opioids of the gastric mucosa against the development of stress-induced ulcers.

Effects of morphine and met-enkephalin analogue on gastric lesions induced by indomethacin.

The effects of morphine HCl and a synthetic met-enkephalin analogue [D-Ala2,MePhe4,Met(O)5ol]enkephalin (FK 33-824) on gastric damage produced by the intraperitoneal administration of indomethacin (10 mg/kg i.p.) have been investigated. Rats intraperitoneally pretreated with morphine HCl (10 mg/kg i.p.) and FK 33-824 (1 mg/kg i.p.) showed a statistically significant reduction both of the number and intensity of lesions induced by indomethacin. This protection was reversed by naloxone HCl (2 mg/kg i.p.). The protective effect was not related to a reduction of gastric secretion since the antisecretory drug cimetidine (25 mg/kg i.p.) and methscopolamine bromide (10 mg/kg i.p.) did not significantly prevent mucosal damage under the same experimental conditions.

Central and peripheral sites of action for the protective effect of opioids of the rat stomach.

Rats exposed to combined cold and restraint exhibited a reduced intensity of gastric damage when pre-treated intraperitoneally with morphine HCl or with the synthetic enkephalin analog [D-Ala2, MePhe4, Met(0)5ol]enkephalin (FK 33-824). Morphine HCl and FK 33-824 prevented some of the indices of the lesion also when injected intracerebroventricularly; morphine methyliodide, quaternary derivative of morphine with does not cross the blood brain barrier, was fully effective, by intraperitoneal route, in preventing the gastric damage. Both peripheral and central mechanisms seem, therefore, involved in the protective effect of opioids on rat gastric mucosa. Morphine HCl and FK 33-824 reduced significantly gastric acid secretion when administered intracerebroventricularly or intraperitoneally; in addition, a concomitant increase of prostaglandin production was observed in rat gastric mucosa after i.p. administration of both opioids. Both these events might contribute to the protective action of opioids on the stomach.

Changes in opiate activity of rat pituitary following acute administration of neuroleptics.

An increase was found in the opiate activity of pituitary extracts obtained rom rats injected with the neuroleptic drugs trifluoperazine and sulpiride. The increase of opiate activity, measured by bioassay, was particularly evident 2 h after the administration of sulpiride. Dexamethasone completely prevented the neuroleptic-induced effect. Trifluoperazine and sulpiride may have affected dopaminergic mechanisms regulating endorphin storage in the pituitary.