Beatrice Severino

PAR1 antagonism protects against experimental liver fibrosis. Role of proteinase receptors in stellate cell activation

In fibroblasts, thrombin induces collagen deposition through activation of a G‐protein–coupled receptor, proteinase‐activated receptor 1 (PAR1). In the current study, we examined whether PAR1 antagonism inhibits hepatic stellate cell (HSC) activation in vitro and whether it protects against fibrosis development in a rodent model of cirrhosis. A rat HSC line was used for in vitro studies whereas cirrhosis was induced by bile duct ligation (BDL). The current results demonstrated that HSCs express PAR1, as well as proteinase‐activated receptors 2 (PAR2) and 4 (PAR4), and that all three PARs were up‐regulated in response to exposure to growth factor in vitro. Exposure to thrombin and to SFLLRN‐(SF)‐NH2, a PAR1 agonist, and GYPGKF (GY)‐NH2, a PAR4 agonist, triggered HSC proliferation and contraction, as well as monocyte chemotactic protein‐1 (MCP‐1) production and collagen I synthesis and release. These effects were inhibited by the PAR1 antagonist. Administration of this antagonist, 1.5 mg/kg/d, to BDL rats reduced liver type I collagen messenger RNA (mRNA) expression and surface collagen by 63%, as measured by quantitative morphometric analysis. Similarly, hepatic and urinary excretion of hydroxyproline was reduced significantly by the PAR1 antagonist. In conclusion, PARs regulates HSC activity; development of PAR antagonists might be a feasible therapeutic strategy for protecting against fibrosis in patients with chronic liver diseases. (HEPATOLOGY 2004;39:365–375.)

Evidence for a protective role played by the Na+/Ca2+ exchanger in cerebral ischemia induced by middle cerebral artery occlusion in male rats ☆

In the present paper, the role played by Na+/Ca2+ exchanger (NCX) in focal cerebral ischemia was investigated. To this aim, permanent middle cerebral artery occlusion (pMCAO) was performed in male rats. The effects on the infarct volume of some inhibitors, such as tyrosine-6 glycosylated form of the exchanger inhibitory peptide (GLU-XIP), benzamil derivative (CB-DMB) and diarylaminopropylamine derivative (bepridil), and of the NCX activator, FeCl3, were examined. FeCl3, CB-DMB, bepridil and GLU-XIP, a modified peptide synthesized in our laboratory in order to facilitate its entrance into the cells through the glucose transporter, were intracerebroventricularly (i.c.v.) infused. FeCl3 (10 microg/kg) was able to reduce the extension of brain infarct volume. This effect was counteracted by the concomitant icv administration of CB-DMB (120 microg/kg). All NCX inhibitors, GLU-XIP, CB-DMB and bepridil, caused a worsening of the brain infarct lesion. These results suggest that a stimulation of NCX activity may help neurons and glial cells that are not irreversibly damaged in the penumbral zone to survive, whereas its pharmacological blockade can compromise their survival.

5-HT1A Receptor: An Old Target as a New Attractive Tool in Drug Discovery from Central Nervous System to Cancer

The serotonin receptor subtype 5-HT(1A) was one of the first serotonin receptor subtypes pharmacologically characterized. This receptor subtype has long been object of intense research and is implicated in the pathogenesis and treatment of anxiety and depressive disorders. In recent years, new chemical entities targeting the 5-HT(1A) receptor (alone or in combination with other molecular targets) have been proposed for novel therapeutic uses in neuroprotection, cognitive impairment, Parkinsons disease, pain treatment, malignant carcinoid syndrome, and prostate cancer. This Perspective compares existing data on expression and signaling activity of the 5-HT(1A) receptor to a ligand with an intrinsic agonist or antagonist profile. Our purpose is also to make a complete overview, useful for underlining the features needed to select a specific pharmacological profile rather than another one. This aspect could be really interesting to consider and justify the 5-HT(1A) receptor as a new attractive target for drug discovery.

Synthesis by microwave irradiation and binding properties of novel 5-HT1A receptor ligands

This work reports the synthesis by microwave irradiation and the binding tests on the 5-HT(1A), 5-HT(2A) and 5-HT(2C) receptors of new substituted piperazines in order to identify selective ligands for 5-HT(1A) subtype receptor. Conventional heating and microwave irradiation of the reactions was compared. Synthesis by microwave irradiation gave the desired compounds in better yields than those obtained by conventional heating. The overall times for the syntheses were considerably reduced. Some resulting active compounds (29 and 39) were characterised by a good selectivity profile for the 5-HT(1A) subtype receptor. The more active compounds were selected and further evaluated for their binding affinities on D(1), D(2) dopaminergic and alpha(1), alpha(2) adrenergic receptors. The compound with higher affinity and selectivity for the 5-HT(1A) over all the considered receptors was the 3-[4-[4-(1,2,3,4-tetrahydronaphthyl)-1-piperazinyl]butan]-benzotriazinone (-)29 (5-HT(1A) K(i)=36 nM, other receptors not active).

Microwave-enhanced solution coupling of the α,α-dialkyl amino acid, Aib☆

Abstract The difficult coupling of α-aminoisobutyric acid (Aib), during the synthesis of dipeptides ( 1 – 6 ), was carried out using PyBOP/HOBt and HBTU/HOBt reagents by application of microwave energy in the presence of solvent. Room temperature, conventional heating (oil bath) and microwave irradiation of the reactions are compared. Synthesis by microwave irradiation gave the desired compounds in higher yields and in shorter reaction times than those obtained by conventional heating or at room temperature.

Synthesis and vasorelaxant activity of new 1,4-benzoxazine derivatives potassium channel openers

As part of a search for new potassium channel openers, the synthesis and vasorelaxant activity of new 1,4-benzoxazine derivatives derived from transformation of the benzopyran skeleton of cromakalim were described. Several new 1,4-benzoxazine derivatives were provided with significant vasorelaxant activity with an overall pharmacological behavior similar to CRK (1f, 1i, 2d, 2e, 2f and 2i).

Synthesis of new 1,2,3-benzotriazin-4-one-arylpiperazine derivatives as 5-HT1A serotonin receptor ligands

A series of novel 1,2,3-benzotriazin-4-one derivatives was prepared and evaluated as ligands for 5-HT receptors. Radioligand binding assays proved that the majority of the novel compounds behaved as good to excellent ligands at the 5-HT1A receptor, some of which were selective with respect 5-HT2A and 5-HT2C receptors. Six analogues (1a, 2a, 2b, 2c, 2e and 2i) were selected and further evaluated for their binding affinities on D1, D2 dopaminergic and alpha1-, alpha2-adrenergic receptors. A o-OCH3 derivative (2e) bound at 5-HT1A sites with subnanomolar affinity (IC50 = 0.059 nM) and shows high selectivity over all considered receptors and may offer a new lead for the development of therapeutically efficacious agents.

A convenient synthesis by microwave heating and pharmacological evaluation of novel benzoyltriazole and saccharine derivatives as 5-HT1A receptor ligands

A series of novel 1,2,3-4-benzoyltriazole and saccharine derivatives were designed and synthesized by microwave heating. They were evaluated on a battery of receptors, including serotonin 5-HT(1A,) 5-HT(2A) and 5-HT(2C), and the most interesting compounds were further evaluated on dopaminergic D(1), D(2) and adrenergic alpha(1), alpha(2) receptors. Conventional and microwave heating of the reactions were compared. Synthesis by microwave heating gave the desired compounds in better yields than those obtained by conventional heating. The overall times for the syntheses were considerably reduced. All compounds displayed moderate affinity for 5-HT(1A) receptor. The most interesting compound 33 showed a high affinity (K(i)=93 nM) which was combined with no affinity on the other receptors considered.

Neurounina-1, a Novel Compound That Increases Na+/Ca2+ Exchanger Activity, Effectively Protects against Stroke Damage

Previous studies have demonstrated that the knockdown or knockout of the three Na+/Ca2+ exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, worsens ischemic brain damage. This suggests that the activation of these antiporters exerts a neuroprotective action against stroke damage. However, drugs able to increase the activity of NCXs are not yet available. We have here succeeded in synthesizing a new compound, named neurounina-1 (7-nitro-5-phenyl-1-(pyrrolidin-1-ylmethyl)-1H-benzo[e][1,4]diazepin-2(3H)-one), provided with an high lipophilicity index and able to increase NCX activity. Ca2+ radiotracer, Fura-2 microfluorimetry, and patch-clamp techniques revealed that neurounina-1 stimulated NCX1 and NCX2 activities with an EC50 in the picomolar to low nanomolar range, whereas it did not affect NCX3 activity. Furthermore, by using chimera strategy and site-directed mutagenesis, three specific molecular determinants of NCX1 responsible for neurounina-1 activity were identified in the α-repeats. Interestingly, NCX3 became responsive to neurounina-1 when both α-repeats were replaced with the corresponding regions of NCX1. In vitro studies showed that 10 nM neurounina-1 reduced cell death of primary cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation. Moreover, in vitro, neurounina-1 also reduced γ-aminobutyric acid (GABA) release, enhanced GABAA currents, and inhibited both glutamate release and N-methyl-d-aspartate receptors. More important, neurounina-1 proved to have a wide therapeutic window in vivo. Indeed, when administered at doses of 0.003 to 30 μg/kg i.p., it was able to reduce the infarct volume of mice subjected to transient middle cerebral artery occlusion even up to 3 to 5 hours after stroke onset. Collectively, the present study shows that neurounina-1 exerts a remarkable neuroprotective effect during stroke and increases NCX1 and NCX2 activities.

Thrombin receptors and their antagonists: an update on the patent literature

Importance of the field: Thrombin plays a central role in cardiovascular inflammation. Most of the cellular responses to thrombin are mediated by cell surface protease-activated receptors (PARs). Several preclinical studies indicate that PARs are potential targets for treating cardiovascular diseases such as thrombosis, atherosclerosis and restenosis. Among PARs, PAR-1 has emerged as an important therapeutic target. Areas covered in this review: This review covers recent advances in the development of thrombin receptors antagonists. It is focused on the search for PAR-1 antagonists as this is at the moment the most promising and attractive target. However, some early promising studies on PAR-3 and -4 antagonists are also reported. What the reader will gain: The review has been written in order to give to the reader hints and references that cover, in our opinion, the most interesting and/or promising approaches in this research field. Take home message: Research on PAR-1 antagonists has finally led to good clinical candidates such as SCH-530348 (Schering-Plough) and E-5555 (Eisai Co.). Clinical trials clearly demonstrate that development of PAR1 antagonists is not only possible but most likely will lead to development of antiplatelet drugs as well as of drugs useful for the treatment of inflammatory, proliferative and neurodegenerative diseases.