Alterations of intestinal motor responsiveness in a model of mild mesenteric ischemia/reperfusion in rats.

In this study we investigate the changes in intestinal motor responsiveness after mild mesenteric ischemia/reperfusion in anaesthetized rats. Motor responsiveness to pharmacological/electrical stimulation was studied in isolated ileum excised from sham-operated rats or animals which underwent occlusion of superior mesenteric artery (1 h) plus interruption of collateral blood flow and reperfusion for 0, 24, 72 h. Only 24 h reperfusion resulted in a significant suppression in acetylcholine induced contractile response and in indomethacin induced relaxation. In the presence of adrenergic and cholinergic blockade a greater relaxant response to field stimulation (trains 10 s every min, 120 mA, 1 ms and 10 Hz) was unmasked in all groups except 24 h reperfused rats. Such effect was sensitive to N(G)-Nitro-L-arginine methyl ester (NOS unselective inhibitor) and the proteolytic enzyme alpha-chymotrypsin but resistant to aminoguanidine (iNOS selective inhibitor). In conclusion, in this rat model, intestinal mild ischemia/24 h reperfusion induces reversible changes in enteric motility attributable to a decrease in eicosanoids, nitric oxide and neuropeptides availability.

Synthesis and in vitro pharmacology of novel heterocyclic muscarinic ligands.

A set of novel heterocyclic ligands (7a-9a, 7b-9b, and 9c) structurally related to oxotremorine 2 was designed, synthesized, and tested at muscarinic receptor subtypes. In the binding experiments at cloned hm1-5, the presence of the 2-methylimidazole/2-methyl-3-alkylimidazolium moiety in place of the pyrrolidine ring revealed, in derivatives 8a, 8b, and 9c, a moderate selectivity for some receptor subtypes. The functional in vitro assays yielded results that correlated closely to binding data. In general, on passing from agonists bearing the pyrrolidine moiety to their analogues carrying the 2-methylimidazole function, the overall pharmacological efficacy profile is shifted from agonism toward partial agonism. The insertion of the 2-methyl-3-alkylimidazolium moiety advances the effect such that the compounds are pure antagonists. Quite similarly, chiral 3-oxo-Delta(2)-isoxazoline (+/-)-10 behaved as a weak antagonist unable to discriminate the different muscarinic receptor subtypes.

Protective effect of Foeniculum vulgare essential oil and anethole in an experimental model of thrombosis.

In a previous screening work, Foeniculum vulgare essential oil emerged from a pool of 24 essential oils for its antiplatelet properties and its ability to destabilize the retraction of the coagulum. In the present work the main component of the oil, anethole, tested in guinea pig plasma was as potent as fennel oil in inhibiting arachidonic acid-, collagen-, ADP- and U46619-induced aggregation (IC(50) from 4 to 147 microg ml(-1)). It also prevented thrombin-induced clot retraction at concentrations similar to fennel oil. The essential oil and anethole, tested in rat aorta with or without endothelium, displayed comparable NO-independent vasorelaxant activity at antiplatelet concentrations which have been proved to be free from cytotoxic effects in vitro. In vivo, both F. vulgare essential oil and anethole orally administered in a subacute treatment to mice (30 mg kg(-1)day(-1) for 5 days) showed significant antithrombotic activity preventing the paralysis induced by collagen-epinephrine intravenous injection (70% and 83% protection, respectively). At the antithrombotic dosage they were free from prohemorrhagic side effect at variance with acetylsalicylic acid used as reference drug. Furthermore, both F. vulgare essential oil and anethole (100 mg kg(-1) oral administration) provided significant protection toward ethanol induced gastric lesions in rats. In conclusion, these results demonstrate for F. vulgare essential oil, and its main component anethole, a safe antithrombotic activity that seems due to their broad spectrum antiplatelet activity, clot destabilizing effect and vasorelaxant action.

Evidence for the involvement of 5-HT2A receptors in mild mesenteric ischemia/reperfusion dysfunctions in mice.

In this study, the involvement of 5-HT2A receptors on mesenteric ischemia-reperfusion injury was examined in mice. Intestinal ischemia produced by 45 min occlusion of superior mesenteric artery was followed by 24h reperfusion (I/R). The 5-HT2A selective antagonist, ketanserin (0.5 mgkg(-1)) or the 5-HT2A agonist DOI (0.25 mgkg(-1)) was intravenously administered before ischemia and 8h after the beginning of reperfusion. The effects were compared with those obtained in sham operated animals (S). Ketanserin prevented the upper gastrointestinal transit delay induced by I/R (P<0.01), protected intestine from leukocyte recruitment as indicated by jejunal myeloperoxidase activity (P<0.05) and reverted Evans Blue extravasation elicited by I/R in lung, colon and jejunum (P<0.05). On the other hand, 5-HT2A activation by DOI mimicked the effects of I/R in S mice prolonging small intestine transit (P<0.05) and enhancing neutrophil accumulation in jejunal tissues (P<0.05). Furthermore, the reduction of ADP-induced platelet aggregation in plasma of I/R mice was prevented by ketanserin treatment. All together, these findings support the critical involvement of 5-HT2A receptor subtype in mediating the damage induced by mesenteric I/R in mice.

Protective effect of proteinase-activated receptor 2 activation on motility impairment and tissue damage induced by intestinal ischemia/reperfusion in rodents.

We hypothesized that proteinase-activated receptor-2 (PAR(2)) modulates intestinal injuries induced by ischemia/reperfusion. Ischemia (1 hour) plus reperfusion (6 hours) significantly delayed gastrointestinal transit (GIT) compared with sham operation. Intraduodenal injection of PAR(2)-activating peptide SLIGRL-NH(2) significantly accelerated transit in ischemia/reperfusion but not in sham-operated rats. GIT was significantly delayed in ischemia/reperfusion and sham-operated PAR(2)(-/-) mice compared with PAR(2)(+/+). SLIGRL-NH(2) significantly accelerated transit in ischemia/reperfusion in PAR(2)(+/+) but not in PAR(2)(-/-) mice. Prevention of mast cell degranulation with cromolyn, ablation of visceral afferents with capsaicin, and antagonism of calcitonin gene-related peptide (CGRP) and neurokinin-1 receptors with CGRP(8-37) and RP67580, respectively, abolished the SLIGRL-NH(2)-induced stimulatory effect on transit in ischemia/reperfusion. Tissue damage was significantly reduced by SLIGRL-NH(2); this effect was not observed in cromolyn-, capsaicin-, or RP67580-treated rats but was detected following CGRP(8-37). Intestinal PAR(2) mRNA levels were not affected by SLIGRL-NH(2) in ischemia/reperfusion. We propose that PAR(2) modulates GIT and tissue damage in intestinal ischemia/reperfusion by a mechanism dependent on mast cells and visceral afferents. PAR(2) effect on transit might be mediated by CGRP and substance P, whereas the effect on tissue damage appears to involve substance P but not CGRP. PAR(2) might be a signaling system in the neuroimmune communication in intestinal ischemia/reperfusion.

Discovery of histamine H3 receptor antagonistic property of simple imidazole-free derivatives: Preliminary pharmacological investigation.

The histamine H3 receptor subtype negatively modulates the release of various neurotransmitters such as histamine, glutamate, norepinephrine, acetylcholine and many others mainly in the CNS and H3 antagonists have been developed to treat central diseases characterized by neurotransmission disturbance such as schizophrenia, memory/learning and sleep disorders. In search for non-imidazole histamine H3 receptor antagonists, currently indicated as a promising class of H3 blockers, a series of simple alkylpiperidine derivatives has been studied to attain a preliminary pharmacological profile. The compounds were characterized in vitro in terms of binding affinity, antagonistic potency and selectivity at rodent H3 receptors. The imidazole-free derivatives possessed moderate to pronounced antagonistic potency at guinea-pig ileal H3 receptor consistent with binding affinity at rat brain H3 receptors and showed a favourable receptor selectivity profile. For the compound 5, with the highest affinity at rat H3 receptors, comparable values were calculated in binding (pKi = 8.35) and functional (pA2 = 8.22) assays in SK-N-MC cells stably expressing human H3 receptors. These findings indicate to extend the investigation to pharmacokinetic property and central effects to gain deeper knowledge on the pharmacological potential of this compound.

The selective inhibition of inducible nitric oxide synthase prevents intestinal ischemia-reperfusion injury in mice.

Nitric oxide (NO) involvement in intestinal ischemia-reperfusion (I/R) injury has been widely suggested but its protective or detrimental role remains still question of debate. Here, we examine the impact of supplementation or inhibition of NO availability on intestinal dysmotility and inflammation caused by mesenteric I/R in mice. Ischemia 45min and reperfusion 24h were performed by superior mesenteric artery occlusion in female Swiss mice. Saline-treated sham-operated (S) or normal mice without surgery (N) served as controls. Drugs were subcutaneously injected 0, 4, 8, and 18 h after ischemia. Upper gastrointestinal transit (GIT, estimated through black marker gavage), intestinal myeloperoxidase activity (MPO), intestinal malondialdehyde levels (MDA), Evans blue extravasation (EB), intestinal histological damage, and mean arterial pressure (MAP) were considered. In I/R mice, GIT was significantly delayed compared to S and N groups; MPO activity and EB extravasation enhanced, whereas MDA levels did not change. Compared to N and S groups, in I/R mice selective iNOS inhibitor P-BIT significantly prevented motor, MPO and EB changes; putative iNOS inhibitor aminoguanidine significantly counteracted GIT delay but not neutrophil recruitment and the increase in vascular permeability; NOS inhibitor l-NAME and NO precursor l-arginine were scarcely or no effective. Furthermore, in S mice aminoguanidine caused a significant increase of MPO activity reverted by H(1) histamine receptor antagonist pre-treatment. Unlike P-BIT, aminoguanidine and l-NAME injection increased MAP. These findings confirm a detrimental role for iNOS-derived NO overproduction during reperfusion. Aminoguanidine-associated neutrophil recruitment suggests that this drug could act through mechanisms additional to iNOS inhibition involving both eNOS blockade, as indicated by its hemodynamic effects, and indirect activation of H(1) histamine receptors.

Synthesis, antiplatelet and antithrombotic activities of new 2-substituted benzopyrano[4,3-d]pyrimidin-4-cycloamines and 4-amino/cycloamino-benzopyrano[4,3-d]pyrimidin-5-ones.

Atherothrombotic coronary artery disease, associated with deep vein thrombosis, is one of the most common causes of death worldwide. Recently, antiplatelet combination therapy using agents with different mechanisms of action, such as aspirin, dipyridamole, and thienopyridines, seems to be an attractive preventive approach. Moreover, several large, randomized clinical trials support combination therapy with aspirin plus warfarin in high-risk patients with atherosclerotic heart disease. Our research on the benzopyrano[4,3-d]pyrimidine system gave rise to the synthesis of a large number of compounds endowed with in vitro anti-aggregating activity. Several SAR considerations suggest that the benzopyranopyrimidine system is an appropriate scaffold to obtain molecules that are able to act simultaneously in different pathways of aggregation. Now, we report the synthesis of new 2-substituted benzopyrano[4,3-d]pyrimidin-4-cycloamines and 4-amino/cycloamino-benzopyrano[4,3-d]pyrimidin-5-ones and the results of the pharmacological study on haemostasis. Some tested compounds showed a large-spectrum antiplatelet activity in vitro, and are more potent than aspirin as antithrombotics in vivo but, at variance with aspirin, they do not increase bleeding. This paper describes novel antithrombotic compounds with an interesting pharmacological profile and a potentially attractive benefit/risk ratio, with their mechanism of action generally, but not exclusively, dependent on antiplatelet activity, deserving further investigations.

New insights into the pharmacological properties of potent antiplatelet 2-amino-benzo[d]isothiazol-3-one derivatives.

A series of 2-amino-benzo[d]isothiazol-3-one derivatives (1-8), previously described as in vitro potent antiaggregatory agents endowed with spasmolytic properties, was evaluated for in vitro antiplatelet activity in guinea-pig platelet rich plasma and for in vivo effects on experimental thrombosis and bleeding time in mice. All the 2-amino-benzo[d]isothiazol-3-one derivatives 1-8 were more potent antiplatelets against collagen than acetylsalicylic acid and, unlike this drug, strongly inhibited thromboxane agonist U46619-induced aggregation. Subacutely administered (5mgkg(-1) day i.p. for 5 days), compounds 6 and 7 protected mice from collagen/epinephrine induced pulmonary thromboembolism at about 20-fold lower doses than acetylsalicylic acid and they prolonged bleeding time like the most part of the other derivatives. The potent antithrombotic activity was coupled with the absence of any lethal and ulcerogenic effect up to 200mgkg(-1) os.

Synthesis and biological evaluation of GABA derivatives able to cross the blood-brain barrier in rats.

Two new GABA derivatives, 1 and 2, were synthesized and tested for their capacity to display CNS activity, which was assessed by determining the effects on the duration of pentobarbital-induced hypnosis in rats. Compound 1, peripherally injected, significantly prolonged the hypnosis time, a typical GABA-mimetic effect, while both intracerebroventricular and intravenous administration of compound 2 surprisingly shortened the hypnotic effect in an atropine-sensitive way. The study was extended also to compounds 1a, 1b and 2a, putative oxidative/hydrolytic metabolites of 1 and 2.

Synthesis, biological activity, QSAR and QSPR study of 2-aminobenzimidazole derivatives as potent H3-antagonists.

We report the design, synthesis, QSPR and QSAR of a new class of H(3)-antagonists, having a 2-aminobenzimidazole moiety connected to the 4(5) position of an imidazole ring through di- or tri-methylene chains. Eleven substituents, selected by experimental design to obtain broad and non-correlated variation in their lipophilic, electronic and steric properties, were introduced at the 5(6) position of the benzimidazole nucleus. The compounds were tested for their H(3)-receptor affinity, by displacement of [(3)H]-(R)-alpha-methylhistamine ([(3)H]-RAMHA) binding to rat brain membranes (pK(i)), for intrinsic activity, evaluating their effect on [(35)S]GTPgammaS binding to rat brain membranes, and for H(3)-antagonist potency, on electrically stimulated guinea-pig ileum (pK(B)). The pK(i) values of the derivatives with longer chain (5a-k) ranged over 2 orders of magnitude, with the 5(6)-methoxy derivative 5d endowed with sub-nanomolar affinity (pK(i)=9.37). The series having two methylene groups in the chain spacer (4a-k), showing a small variation in affinity, revealed to be somewhat insensitive to ring substitution. Lipophilicity (log P) and basicity (pK(a)) of the newly synthesized compounds were measured and related to receptor affinity in a QSAR study. Multiple regression analysis (MRA) showed an approximate parabolic dependence of pK(i) on log P, while an additional electronic effect of the substituents on benzimidazole tautomerism is suspected.