Searching for new NO-donor aspirin-like molecules: Furoxanylacyl derivatives of salicylic acid and related furazans.

A new group of derivatives of salicylic acid containing NO-donor furoxans, and the related des-NO-furazans, were synthesized and evaluated as new aspirin-like molecules. Their stability was assessed in acid (pH 1) and physiological solutions (pH 7.4), and in human serum. No compound exhibited COX-inhibitory activity against COX-1 and COX-2 isoforms, when tested up to 100µM, respectively, on isolated platelets and on monocytes. Phenylsulfonyl- and cyano-substituted furoxans inhibited platelet aggregation induced by collagen in human platelet-rich plasma, through a cGMP dependent mechanism. Furoxan derivatives displayed cGMP-dependent vasodilator activities, tested on rat aorta strips precontracted with phenylephrine. All products showed anti-inflammatory activity similar to that of ASA, tested on rats by the carrageenan-induced paw edema assay. Unlike ASA, all products showed markedly reduced gastrotoxicity in a rat lesion model.

The hypothalamus-pituitary-adrenal axis does not influence the protective effects of nociceptin/orphanin FQ on the rat gastric mucosa.

The participation of hypothalamus-pituitary-adrenal axis in the gastroprotective effects of nociceptin/orphanin FQ (N/OFQ) has been investigated. Gastric mucosal lesions were induced by intragastric administration of 50% ethanol, 1 ml/rat. Rats received N/OFQ either by the intracerebroventricular (icv) route, at 3 microg/rat, or by the intraperitoneal (ip) route, at 10 microg/kg, 30 min before ethanol administration. The protective effect of icv and ip administered N/OFQ was assessed in adrenalectomized rats and in rats pretreated with the glucocorticoid receptor antagonist, mifepristone, or with the CRF receptor antagonist, alpha-helical CRF(9-41). The damaging effect of ethanol was apparently not influenced by adrenalectomy. N/OFQ markedly reduced macroscopically and histologically assessed gastric mucosal damage. The extent of reduction by N/OFQ was comparable in adrenalectomized and in sham-operated rats, with either icv or ip route of administration. Pretreatment with mifepristone, both icv (80 microg/rat) and ip (10 mg/kg) injected, did not modify the response to icv and ip N/OFQ. Pretreatment with alpha-helical CRF(9-41) (25 microg/rat icv or 250 microg/kg ip), had no effect on the reduction of gastric damage produced by icv or ip N/OFQ. Present findings suggest that the gastroprotective effects of N/OFQ on ethanol-induced damage do not involve the endocrine pathway through the HPA axis.

Searching for new NO-donor aspirin-like molecules: a new class of nitrooxy-acyl derivatives of salicylic acid.

A new class of products in which the phenol group of salicylic acid is linked to alkanoyl moieties bearing nitrooxy functions has been synthesized and studied for their polyvalent actions. The products were stable in acid and neutral media, while they were hydrolyzed in human serum. Their half-lives were dependent upon the structure of alkanoyl moieties. The products showed anti-inflammatory activities similar to aspirin when tested in the carrageenan-induced paw edema assay in the rat. Interestingly, unlike aspirin, they showed reduced or no gastrotoxicity in a lesion model in rats at equimolar doses. A number of them were able to inhibit platelet aggregation induced by collagen in human platelet-rich plasma. All of the products were capable of relaxing rat aortic strips precontracted with phenylephrine in a concentration-dependent manner. Selected members of this new class of nonsteroidal anti-inflammatory drugs might represent possible safer alternatives to aspirin in different clinical settings.

Nociceptin/orphanin FQ prevents gastric damage induced by cold-restraint stress in the rat by acting in the periphery.

The influence of peripheral nociceptin/orphanin FQ (N/OFQ) on cold restraint-induced gastric mucosal damage in the rat was investigated. Exposure to cold-restraint for 3 and 4h caused the formation of hemorrhagic lesions in the glandular portion of the stomach. N/OFQ dose-dependently decreased lesion formation, in the range 0.03-1 microg/kg/h i.p. Its effect was reversed by the selective NOP receptor antagonist [Nphe(1)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-101), 30 microg/kg/h ip. The selective NOP receptor agonist [(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-112), 0.01-0.3 microg/kg/h i.p., similarly reduced lesion formation. Light and scanning electron microscopy confirmed the protective activity of N/OFQ. Cold-restraint stress causes a reduction in mucus content and in adhering mucus layer, partly counteracted by N/OFQ. These results suggest that N/OFQ counteracts acute stress-induced gastric mucosal damage by interacting with NOP receptor and by influencing mucous cell activity.

Epithelial cell proliferation is promoted by the histamine H(3) receptor agonist (R)-alpha-methylhistamine throughout the rat gastrointestinal tract.

The temporal effect of (R)-alpha-methylhistamine on epithelial cell proliferation throughout the rat gastrointestinal tract was investigated. (R)-alpha-methylhistamine was administered at 100 mg/kg orally and the rats were sacrificed 1, 24, 48, 72 and 144 h later. All the animals received 5-bromo-2'-deoxyuridine, (BrdU), 200 mg/kg i.p., 2 h before sacrifice. Gastrointestinal tissue was processed for histology and immunohistochemistry. (R)-alpha-methylhistamine caused a progressive increase in mucosal thickness of gastric fundus, distal small intestine and distal colon. Statistically significant differences from control values were found between 48 and 72 h after (R)-alpha-methylhistamine. (R)-alpha-methylhistamine significantly increased the number of BrdU-positive cells in the gastric fundus and antrum, intermediate and distal small intestine and distal colon. Peak effects were observed between 1 and 24 h after (R)-alpha-methylhistamine administration. Proliferating cell number and mucosal thickness were comparable to those of control rats at 144 h. (R)-alpha-methylhistamine exerts a long lasting growth-promoting effect on the stomach, distal small intestine and distal colon. Present data support a role of histamine H(3) receptors in the normal regulation of cell cycle in epithelial tissue.

Peripheral mechanisms involved in gastric mucosal protection by intracerebroventricular and intraperitoneal nociceptin in rats.

Nociceptin (N/OFQ) exerts multiple effects in the gastrointestinal tract after central or peripheral administration. In the present study, we examined the possible peripheral mechanisms mediating gastric protection by N/OFQ in rats. Gastric mucosal lesions were induced by 50% ethanol (1 ml/rat intragastrically). N/OFQ, administered either intracerebroventricularly (3 microg/rat) or ip (10 microg/kg), significantly reduced macroscopic and histological damage. The protective effect of intracerebroventricular N/OFQ was blocked by atropine, subdiaphragmatic vagotomy, and bretylium. The effect of both central and peripheral N/OFQ was blocked by functional ablation of afferent nerves produced by capsaicin, by the antagonist of calcitonin gene-related peptide, CGRP(8-37), and by the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester. These results indicate that N/OFQ increases gastric mucosal resistance to ethanol by operating both in the central nervous system and in the periphery. Vagal cholinergic and sympathetic pathways mediate the central activity of N/OFQ, whereas vagal nonmuscarinic pathways mediate the peripheral activity of the peptide. The neuronal circuit involving extrinsic sensory neurons, calcitonin gene-related peptide, and nitric oxide is activated by central as well as peripheral N/OFQ. The study provides evidence that N/OFQ contributes to neurally mediated gastric mucosal protection.

Nociceptin/orphanin FQ prevents ethanol-induced gastric lesions in the rat.

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the NOP receptor, exerts a variety of effects on the gastrointestinal tract. The present study was aimed at evaluating the possible implication of N/OFQ in the maintenance of gastric mucosal integrity. N/OFQ was given either centrally or peripherally 30 min prior to intragastric administration (i.g.) of 1 ml/rat of ethanol (either 25% or 50%, v/v), which produces macroscopically visible gastric lesions. Intracerebroventricular (i.c.v.) injection of 2 microg/rat of N/OFQ significantly reduced lesions caused by 50% ethanol, while 1 microg/rat was enough to significantly reduce lesions caused by 25% ethanol. Intracerebroventricular injection of 5 microg/rat of the selective NOP receptor antagonist, UFP-101, completely reversed the protective effect of N/OFQ, 1 or 4 microg/rat against 25% or 50% ethanol, respectively. The intraperitoneal (i.p.) injection of N/OFQ produced a significant reduction of lesions induced by 50% ethanol, the peak effect being observed at 10 microg/kg. Intraperitoneal pretreatment with UFP-101, 120 microg/kg, completely abolished the protective effect of peripherally injected N/OFQ. Therefore, N/OFQ acts both centrally and peripherally as a protective agent against ethanol-induced gastric lesions, and its effect is mediated by NOP receptors.

Nociceptin/orphanin FQ and stress regulate synaptophysin expression in the rat fundic and colonic mucosa.

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the N/OFQ peptide (NOP) receptor, is a neuropeptide regulating gastrointestinal functions. The present study investigated the influence of acute cold-restraint stress and of short- and long-lasting peripheral infusion of N/OFQ on the level of synaptophysin, an exocytotic protein involved in neural plasticity. Exposure to cold-restraint stress for 3h or subcutaneous infusion of N/OFQ, 1 µg/kg/h for 4h, induced a significant increase of the area of synaptophysin-immunoreactive nerve fibers in the fundic mucosa, while prolonged subcutaneous infusion of N/OFQ, 1 µg/kg/h for 52 h and for 14 days, did not modify the synaptophysin-immunostained fibers. In the colonic mucosa stress exposure and subcutaneous infusion of N/OFQ, at any time point considered, had no significant effect on the area of synaptophysin-immunoreactive nerve fibers. Synaptophysin immunoreactive nerve fibers were decreased in knockout rats for the NOP receptor gene both in the fundic and colonic mucosa. Synaptophysin-immunoreactivity was demonstrated in cells located in the basal portion of the fundic mucosa. Our study is the first to show that the N/OFQ/NOP receptor system influences the expression of synaptophysin and hence the process of exocytosis both in nerve terminals and in cells.

Antiinflammatory, gastrosparing, and antiplatelet properties of new NO-donor esters of aspirin.

A new series of NSAIDs in which aspirin is joined by an ester linkage to furoxan moieties, with different ability to release NO, were synthesized and tested for NO-releasing, antiinflammatory, antiaggregatory, and ulcerogenic properties. Related furazan derivatives, aspirin, its propyl ester, and its gamma-nitrooxypropyl ester were taken as references. All the products described present an antiinflammatory trend, maximized in derivatives 12, 16, and 17, they are devoid of acute gastrotoxicity, principally due to their ester nature, and show an antiplatelet activity primarily determined by their ability to release NO. They do not behave as aspirin prodrugs in human serum.

Ligands for histamine H(3) receptors modulate cell proliferation and migration in rat oxyntic mucosa.

1. (R)-alpha-methylhistamine, a selective agonist of histamine H(3) receptors, promotes mucus secretion and increases the number and volume of mucus-secreting cells. The hypothesis that the increased number of mucous cells could reside in an alteration of homeostasis in the gastric epithelium was investigated. 2. (R)-alpha-methylhistamine was administered to rats 1 h (10-100 mg kg(-1) by intragastric and by intraperitoneal route) and 24 h (100 mg kg(-1) by intragastric route) prior to killing. The (S)-isomer of alpha-methylhistamine (55.4 mg kg(-1)), 100 times less potent than the (R)-isomer at H(3) receptors, and the H(3)-receptor agonist FUB 407 (9.14-91.35 mg kg(-1)) were intragrastically administered 1 h prior to killing. The H(1)-receptor antagonist mepyramine (30 mg kg(-1)), the H(2)-receptor antagonist famotidine (3 mg kg(-1)), and the H(3)-receptor antagonists ciproxifan (3 mg kg(-1)) and clobenpropit (30 mg kg(-1)) were intragastrically administered 30 min before (R)-alpha-methylhistamine. Gastric tissue was processed for histology and immunohistochemistry. 3. Within 1 h, (R)-alpha-methylhistamine and FUB 407 dose-dependently increased the number of BrdU-positive cells and of apoptotic cells. (S)-alpha-methylhistamine failed to modify proliferation and apoptosis. The increase in proliferation by (R)-alpha-methylhistamine was reversed by ciproxifan and clobenpropit, but not by mepyramine and famotidine. 4. (R)-alpha-methylhistamine accelerated the differentiation towards pit cells and their outward migration 24 h after its administration. These effects were counteracted by ciproxifan. The apoptosis rate was unaffected at 24 h. 5. These findings reveal a primary role of histamine H(3)-receptor ligands in modulating cell proliferation and migration in rat fundic mucosa.

Water-soluble nitric-oxide-releasing acetylsalicylic acid (ASA) prodrugs.

A series of water-soluble (benzoyloxy)methyl esters of acetylsalicylic acid (ASA), commonly known as aspirin, are described. The new derivatives each have alkyl chains containing a nitric oxide (NO)-releasing nitrooxy group and a solubilizing moiety bonded to the benzoyl ring. The compounds were synthesized and evaluated as ASA prodrugs. After conversion to the appropriate salt, most of the derivatives are solid at room temperature and all possess good water solubility. They are quite stable in acid solution (pH 1) and less stable at physiological pH. In human serum, these compounds are immediately metabolized by esterases, producing a mixture of ASA, salicylic acid (SA), and of the related NO-donor benzoic acids, along with other minor products. Due to ASA release, the prodrugs are capable of inhibiting collagen-induced platelet aggregation of human platelet-rich plasma. Simple NO-donor benzoic acids 3-hydroxy-4-(3-nitrooxypropoxy)benzoic acid (28) and 3-(morpholin-4-ylmethyl)-4-[3-(nitrooxy)propoxy]benzoic acid (48) were also studied as representative models of the whole class of benzoic acids formed following metabolism of the prodrugs in serum. These simplified derivatives did not trigger antiaggregatory activity when tested at 300 µM. Only 28 displays quite potent NO-dependent vasodilatatory action. Further in vivo evaluation of two selected prodrugs, {[2-(acetyloxy)benzoyl]oxy}methyl-3-[(3-[aminopropanoyl)oxy]-4-[3-(nitrooxy)propoxy]benzoate⋅HCl (38) and {[2-(acetyloxy)benzoyl]oxy}methyl 3-(morpholin-4-ylmethyl)-4-[3-(nitrooxy)propoxy]benzoate oxalate (49), revealed that their anti-inflammatory activities are similar to that of ASA when tested in the carrageenan-induced paw edema assay in rats. The gastrotoxicity of the two prodrugs was also determined to be lower than that of ASA in a lesion model in rats. Taken together, these results indicated that these NO-donor ASA prodrugs warrant further investigation for clinical application.

Long-term peripheral infusion of nociceptin/orphanin FQ promotes hyperplasia, activation and migration of mucosal mast cells in the rat gastric fundus.

The endogenous neuropeptide nociceptin/orphanin FQ (N/OFQ) modulates behavioral and gastrointestinal responses to stress. Mucosal mast cells (MMCs) are primary mediators of stress-related responses in the gastrointestinal tract. We investigated the influence of N/OFQ and of the N/OFQ peptide (NOP) receptor antagonist, UFP-101, on MMCs in the rat gastric fundus. N/OFQ was infused subcutaneously for 52 h at 0.1, 1 and 10 µg/kg/h and at 1 µg/kg/h for 4h, 52 h, 7 days and 14 days via Alzet osmotic minipumps. Density of MMCs and connective tissue mast cells (CTMCs) was assessed histochemically and immunohistochemically. Activation and location of MMCs were assessed by transmission electron microscopy. Contacts between MMCs and nerve elements were assessed by double immunofluorescence. N/OFQ (1 µg/kg/h) and UFP-101 (10 and 30 µg/kg/h) were infused subcutaneously in the absence and presence of acute cold-restraint stress and density of MMCs was assessed. Peripheral N/OFQ dose-dependently increased the density of MMCs, while not influencing CTMCs. The increasing effect was maintained up to 14 days following continuous infusion, while after termination of the 4-h infusion, the effect declined rapidly. The peptide promoted the activation of MMCs and their migration from the lamina propria toward the epithelial layer. The association between MMCs and nerve fibers was time-dependently down-regulated following N/OFQ infusion. The stress-induced hyperplasia of MMCs was not influenced by N/OFQ and abolished by UFP-101. UFP-101 alone was ineffective. The present results suggest that endogenous N/OFQ could be considered a potential component of the circuit neuropeptides-mast cells-stress.

Role of nociceptin/orphanin FQ receptors in the decrease of mucosal mast cells caused by acute stress in the rat colon.

AIMS: Mucosal mast cells (MMC) are mediators of the stress responses in the gastrointestinal tract. We examined the effect of acute cold-restraint stress and of the neuropeptide nociceptin/orphanin FQ (N/OFQ), implicated in the modulation of stress responses, on MMC density in the rat colon. MAIN METHODS: Stress was induced by restraining the animals into individual cages at 3°C for 3h. N/OFQ and the selective N/OFQ peptide (NOP) receptor antagonist, UFP-101, were infused subcutaneously via Alzet osmotic minipumps. Segments of the distal colon were collected. MMCs were identified immunohistochemically with a monoclonal antibody to rat mast cell protease (RMCP) II and with a rabbit polyclonal antibody to CD117/c-kit receptor. KEY FINDINGS: Acute stress caused a decrease in the density of MMCs in the rat colonic mucosa. Short-term peripheral infusion of N/OFQ (0.1 to 10 µg/kg/h for 4h) caused a dose-related reduction of MMC density. Peak reduction occurred after the 4-h infusion of N/OFQ, 1 µg/kg/h. Reduction was maintained after the 52-h infusion period and declined following 7 and 14 days of infusion. The infusion of N/OFQ (1µg/kg/h for 4h) in rats exposed to acute stress caused a decrease in MMC density comparable to that obtained with the single treatments. UFP-101, at the doses of 1 and 10 µg/kg/h, which itself had no significant effect on MMC density, when concurrently infused in stress-exposed rats, abolished the stress-induced decrease of MMC density. SIGNIFICANCE: Present results indicate that the peripheral N/OFQ-NOP system is involved in stress-induced reduction of MMC density.

Methods to measure gastric mucosal lesions in the rat.

The maintenance of gastric mucosal integrity is ensured by a dynamic balance between protective and noxious factors. The gastric mucosa has multiple protective mechanisms that allow the mucosa to withstand frequent exposure to potentially damaging agents such as acid and peptic secretions, bacterial products, ingested food, alcoholic beverages, and certain drugs. The imbalance between defensive and aggressive factors is at the basis of the formation of erosions/lesions or ulcerations of the gastric mucosa. The difference between an erosion/lesion and ulceration is that the former is confined to the mucosa, while an ulceration penetrates to the muscularis mucosae. This unit presents two models of acute mucosal lesions induced in the rat by gastrotoxic agents acting through different mechanisms of action. The protocols explain how to measure gastric mucosal lesions by microscopic examination of the stomach by light microscopy and by scanning electron microscopy.

Peripheral infusion of nociceptin/orphanin FQ influences the response of rat gastric and colonic mucosa to repeated stress.

The 17-amino acid peptide nociceptin/orphanin FQ (N/OFQ) plays a role in the regulation of stress responses and of emotional disorders. The objective of this study is to evaluate whether long-term peripheral N/OFQ could dose- and time-dependently influence the responses to repeated cold-restraint stress on the rat gastric and colonic mucosa. Rats were exposed to cold-restraint stress for 3h per day for 1, 2 and 3 consecutive days. N/OFQ was administered at doses of 0.1, 1 and 10 microg/kg/h via Alzet osmotic minipumps. In the gastric fundus, N/OFQ exerted dose-dependent beneficial effects against acute and repeated stress but, after prolonged treatment, became damaging in non-stressed rats. In the distal colon, N/OFQ exerted a protective effect against damage by acute and repeated stress with no influence on epithelial integrity in non-stressed rats. In both regions, the peptide itself dose- and time-dependently reduced intraepithelial mucins. The reduction in mucin content caused by stress was effectively counteracted by N/OFQ, 0.1 microg/kg/h, in the distal colon only. N/OFQ did not modify basal mucosal cell proliferation. The peptide at 0.1 and 1 microg/kg/h had no influence while at 10 microg/kg/h abolished stress-induced increase in cell proliferation. The present results provide evidence that N/OFQ is implicated in the regulation of resting and stress-challenged mucosal integrity and activity of mucin-producing cells.

Nociceptin/orphanin FQ-induced food intake and cocaine amphetamine regulated transcript gene expression in strains derived from rats prone (WOKW) and resistant (Dark Agouti) to metabolic syndrome.

In previous work, we observed that N/OFQ-induced hyperphagia is greater in DA rats, animals resistant to metabolic syndrome, than in WOKW animals, which are prone to this disease. We attributed this difference to the fact that these two strains have different Cart gene sequences and expression. As a preliminary approach to pursue this hypothesis, the present work focused on Cart gene expression by developing from DA and WOKW rats various congenic animals with exchanges of metabolic syndrome-related QTL's of different chromosomes (3, 5, 10 and 16), and analyzing their N/OFQ-induced (2.1, 4.2, and 8.4nmol/rat) food intake in terms of their CART gene expression and N/OFQ hypothalamic immunostaining. Two groupings emerged, the first, with strains 3a, 3b, and 5a with elevated N/OFQ-induced feeding similar to that of the DA rats, and the second, with strains 16 and 10, with lower feeding, like the WOKW rats. There was a perfect correlation between Cart gene expression and N/OFQ-induced feeding data at 30min for the strains DA, 3a, 3b, 5 in the first group, and 16 and WOKW for the second, but not for strain 10. As expected, the strains with low content of Cart gene expression had elevated N/OFQ-induced feeding, but contrary to expectations, strain 10, with the lowest Cart gene expression, exhibited low N/OFQ-induced feeding, on the order of that of the WOKW rats. A comparable trend was observed with N/OFQ hypothalamic immunostaining. This anomaly may be due to other satiety-related factors involved in N/OFQ-induced feeding.

Immunolocalization of histamine H3 receptors on endocrine cells in the rat gastrointestinal tract.

The histamine H3 receptor (H3R) has been identified in the gastrointestinal tract of the rat by immunohistochemistry, using the first validated anti-H3 receptor antibody. Immunoreactivity to H3R was exclusively localized to the endocrine cells scattered in the gastrointestinal mucosa, with positive cells being prominently abundant in the gastric fundus, while they were rarely found in the other regions. In the fundus, positive cells were distributed in the lower half of the mucosa and their number significantly decreased after a 24 h-fasting period. Double-labeling studies were undertaken to identify the H3R-immunoreactive cell types in the fundic and antral mucosa. The H3R-immunoreactive cells were positive for chromogranin A. In the fundus, approximately 90% of cells positive to H3R were also positive to the histamine-forming enzyme, histidine decarboxylase. None of the cells expressing H3R displayed immunoreactivity for gastrin, somatostatin or ghrelin. Location, the influence of food deprivation and colocalization with histidine decarboxylase indicate that H3R positive cells correspond to the enterochromaffin-like cells (ECL).

Morphological features of rat gastric mucosa after acute and chronic treatment with amtolmetin guacyl: comparison with non-selective and COX-2-selective NSAIDs.

BACKGROUND/AIMS: The compound amtolmetin guacyl (AMG) has been characterized in both animal and human studies as a novel non-selective non-steroidal anti-inflammatory drug (NSAID) endowed with lower ulcerogenicity in comparison with traditional NSAIDs due to a unique mechanism of action, namely the increase in endogenous production of gastric nitric oxide. METHODS: Conscious rats were treated either acutely (4 h) or chronically (3 and 14 days) with intragastric AMG (50 and 150 mg/kg), the non-selective NSAID tolmetin (TOL, 30 and 100 mg/kg) or the COX-2-selective NSAID celecoxib (CXIB, 20 and 60 mg/kg). Macroscopically visible and histologic lesions were evaluated. The ultrastructure of mucosal microvasculature was assessed. RESULTS: (1) TOL and CXIB caused quantitatively greater endothelial damage and inflammatory cell infiltration than that induced by AMG; (2) AMG and CXIB, unlike TOL, did not cause epithelial damage after acute or chronic treatment, and (3) gastric lesions induced by TOL underwent adaptation during chronic treatment. CONCLUSION: Endothelial cell damage in the gastric microvasculature is an early event following both non-selective and COX-2-selective inhibitors. The low gastric mucosal toxicity of AMG is confirmed after acute and chronic treatment.