cyclic-gmp and nitroaspirin

The use of low doses of aspirin on a daily basis has increased greatly in the past 20 years, based on observations that it can significantly reduce the risk of heart attacks and strokes. However, aspirin can also cause severe damage to the stomach. A modified version of aspirin that releases nitric oxide has been developed that seems to offer important advantages over its 103-year-old parent--namely, improved protection for the heart without the unwanted effects on the stomach.

Topics: Animals; Apoptosis; Arteriosclerosis; Aspirin; Caspase Inhibitors; Cell Adhesion; Cyclic GMP; Cytokines; Heart; Humans; Hypertension; Myocardial Infarction; Stroke

Topics: Adenylyl Cyclase Inhibitors; Aspirin; Cyclic GMP; Drug Design; Humans; Natriuretic Agents; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroglycerin; Phosphodiesterase Inhibitors; Piperazines; Purines; Sildenafil Citrate; Sulfhydryl Compounds; Sulfones

Non-steroidal anti-inflammatory drugs (NSAIDs) are used to treat the condition of rheumatoid arthritis, where levels of prostaglandin E2 (PGE2) and granulocyte macrophage-colony stimulating factor (GM-CSF) are elevated in the synovial fluid. NO-NSAIDs are a new class of cyclooxygenase (COX)-inhibitors developed by coupling a nitric oxide (NO)-donating moiety to conventional NSAIDs. We show that, in cytokine-treated synoviocytes (from non-rheumatic patients), NO-naproxen and NO-flurbiprofen like their parent compounds concentration-dependently reduce the levels of PGE2 (an index of COX-2 activity), with a corresponding rise in the release of GM-CSF. Unlike acetylsalicylic acid (ASA), NO-ASA reduces the levels of PGE2, without increasing GM-CSF release, although cell viability is reduced at the highest concentration (1 mM). The effects of NSAIDs and NO-NSAIDs on GM-CSF release were attributable to the PGE2 mediated cyclic (c) AMP pathway because PGE2 reversed the effects of COX blockade. Second, phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX) and Ro-201724 (both of which elevate cAMP levels) decreased GM-CSF release, in the presence of PGE2. Finally, neither sodium nitroprusside nor zaprinast (both of which elevate cGMP levels) affected GM-CSF or PGE2 release. Our findings demonstrate that GM-CSF is regulated by NSAIDs and NO-NSAIDs via inhibition of COX and appears to be mediated via the cAMP pathway. NO-ASA is the exception, because it does not increase GM-CSF release, although at millimolar concentrations cell viability is reduced.

Topics: 1-Methyl-3-isobutylxanthine; 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Blotting, Western; Cells, Cultured; Cyclic AMP; Cyclic GMP; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; Dose-Response Relationship, Drug; Flurbiprofen; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Indomethacin; Membrane Proteins; Naproxen; Nitric Oxide Donors; Nitroprusside; Phosphodiesterase Inhibitors; Prostaglandin-Endoperoxide Synthases; Purinones; Synovial Membrane; Time Factors

A new class of nitric oxide donating aspirin (NO-ASA) drugs may increase the therapeutic impact of aspirin in saphenous vein coronary artery bypass grafting (CABG) not only through the inhibition of thrombosis but also through a reduction of vasospasm and inhibition of vascular smooth muscle cell (VSMC) proliferation (effects that are inhibited by NO but not ASA). In order to test this proposal the effect of three NO-ASA drugs (NCX4040, NCX4050, and NCX4060) on in vitro relaxation and cyclic guanosine monophosphate (cGMP) formation in the human isolated saphenous vein and the proliferation of human VSMCs was investigated.. Saphenous vein segments were obtained from 30 patients undergoing CABG (median age, 59 years; range, 49 to 68). The effect of the NO-ASA adducts, ASA alone, and sodium nitroprusside (NO donor) were investigated on (1) relaxation of phenylephrine-stimulated contraction using an organ bath, (2) cyclic guanosine monophosphate (cGMP) formation using an enzyme-linked immunosorbent assay, and (3) the proliferation of VSMCs derived from saphenous vein using bromo-deoxyuridine (BRDU) incorporation.. All three NO-ASA adducts (at concentrations that inhibited responses by 50% [IC50s] between 1 micromol/L and 100 micromol/L) and nitroprusside (at IC50s between 0.5 and 10 micromol/L) elicited relaxation of isolated human saphenous vein, promoted cGMP formation, and inhibited VSMC proliferation whereas ASA alone (up to 100 micromol/L) had no effect on any variable.. These data indicate that the NO-ASA adducts by virtue of their capacity to release NO and stimulate guanylyl cyclase may be useful not only in the prevention of thrombosis following CABG but also the reduction of saphenous vein graft spasm and neointima formation.

Topics: Aged; Aspirin; Cell Division; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Humans; In Vitro Techniques; L-Lactate Dehydrogenase; Male; Middle Aged; Muscle, Smooth, Vascular; Nitric Oxide Donors; Nitroprusside; Platelet Aggregation Inhibitors; Saphenous Vein; Vasodilation

The aim of this study was to investigate the capacity of the 2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester (NCX 4016), a nitric oxide (NO)-releaser derivative of aspirin, to decrease blood pressure in spontaneously hypertensive rats (SHR) and to counteract the adrenergic vasoconstriction in perfused tail artery of these animals. Oral treatment for 10 consecutive days with NCX 4016 (100 micromol/kg) in SHR and their genetic controls Wistar Kyoto (WKY) rats resulted in a reduction of blood pressure in SHR but not in WKY rats. In SHR, the NCX 4016 treatment increased the serum nitrite/nitrate and diminished the serum thromboxane B2, whereas aspirin did not change blood pressure but abolished the serum thromboxane B2. Perfused tail arteries excised from vehicle-treated SHR exhibited a significant impairment of endothelium-dependent vasorelaxant function. These vessels, prepared from SHR or WKY rats treated orally with NCX 4016 (10, 30 and 100 micromol/kg for 7 consecutive days), revealed a dose-dependent decrease in vasoconstriction in response to transmural nerve stimulation and norepinephrine, whereas aspirin was ineffective. Furthermore, in tail arteries of both SHR and WKY rats treated orally with NCX 4016 (100 micromol/kg for 7 consecutive days), the cGMP increased significantly. In conclusion, NCX 4016, by releasing NO and increasing cGMP in vascular tissue, reduces sympathetic-mediated vasoconstriction in resistance vessels and lowers blood pressure in SHR.

Topics: Animals; Arteries; Aspirin; Blood Pressure; Cyclic GMP; Dose-Response Relationship, Drug; In Vitro Techniques; Nitrates; Nitric Oxide Donors; Nitrites; Perfusion; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Tail; Thromboxane B2; Time Factors; Vasoconstriction

1. The ability of the nitric oxide (NO)-releasing aspirin, NCX 4016, to control vasoconstrictor responses induced by electrical field stimulation (TNS) or by exogenous norepinephrine (NE) was investigated in perfused rat tail artery with intact endothelium. 2. NCX 4016 (25, 50 and 100 microM) dose-dependently antagonized the vasoconstriction caused by TNS (from 0.5 to 64 Hz) and by NE (from 0.01 to 10 microM). The vasorelaxant activity of NCX 4016 (100 microM) in NE-precontracted arteries was concomitant with a marked increase of tissue cyclic GMP (4.9 fold, P<0.001) and was significantly antagonized by the inhibitors of soluble guanylate cyclase, methylene blue and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one. 3. The effect of NCX 4016 was endothelium NO-independent since, in preparations perfused with N(G)-monomethyl-L-arginine (10 microM), this compound prevented the rise in basal perfusion pressure and reversed the accentuation of vasoconstrictor responses caused by NO synthase inhibition. 4. Aspirin-moiety released by NCX 4016 inhibited the 6-keto-PGF(1alpha) formation without interfering with the vasorelaxant activity of NCX 4016, while aspirin (100 microM) was devoid of any activity against vasoconstriction induced by both TNS and NE in perfused rat tail artery. 5. NCX 4016 moderated adrenergic vasoconstriction in perfused rat tail arteries by a direct donation of NO without involving the relaxant factors such as PGI(2) and NO from endothelial cells. 6. The results obtained with NCX 4016 in perfused rat tail artery bears some therapeutical potential in conditions associated with vascular smooth muscle hyperreactivity to adrenergic stimulation.

Topics: Animals; Arteries; Aspirin; Cyclic GMP; Dose-Response Relationship, Drug; Electric Stimulation; Male; Nitric Oxide; Nitric Oxide Synthase; Norepinephrine; omega-N-Methylarginine; Perfusion; Rats; Rats, Wistar; Tail; Vasoconstriction

NCX 4016, a nitro-ester of aspirin endowed with antithrombotic activity, appears to have clinical potential in treating cardiac complications related to coronary insufficiency. This compound has been shown to improve postischemic ventricular dysfunction and to reduce myocardial infarct size in the rabbit. The cardioprotection conferred by NCX 4016 (10, 30, and 100 mg/kg) and aspirin (ASA, 54 mg/kg) was evaluated in anesthetized rats subjected to 30 min of myocardial ischemia followed by 120 min of reperfusion (MI/R). Drugs were given orally for 5 consecutive days. NCX 4016 displayed remarkable cardioprotection in rats subjected to MI/R as was evident in the reduction of ventricular premature beats and in the incidence of ventricular tachycardia and fibrillation; they were reduced dose dependently and correlated with survival of all rats treated with the higher dose of NCX 4016. In these animals, infarct size was restricted proportionally to the dose of NCX 4016 associated with diminution of both plasma creatine phosphokinase and cardiac myeloperoxidase activities. ASA showed only a minor degree of protection against MI/R damage. Rats treated with N(G)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg) demonstrated aggravated myocardial damage in terms of arrhythmias, mortality, and infarct size. Supplementation of nitric oxide (NO) with NCX 4016 (100 mg/kg) greatly reduced the worsening effect caused by L-NAME. The beneficial effects of NCX 4016 appear to derive in large part from the NO moiety, which modulates a number of cellular events leading to inflammation, obstruction of the coronary microcirculation, arrhythmias, and myocardial necrosis.

Topics: Animals; Arrhythmias, Cardiac; Aspirin; Creatine Kinase; Cyclic GMP; Fibrinolytic Agents; Hemodynamics; Male; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion; NG-Nitroarginine Methyl Ester; Peroxidase; Platelet Aggregation Inhibitors; Rats; Rats, Wistar

NCX4016 (2 acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl ester, NicOx S.A., France) is an anti-thrombotic agent, chemically related to acetylsalicylic acid (ASA) and able to release NO. We tested the effects of NCX4016 and ASA on the release of the thromboxane (TX) A(2) metabolite TXB(2), tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), expression and activity of tissue factor (TF) in stimulated, adherent human monocytes. Both ASA and NCX4016 1 - 1000 micromol l(-1) dose-dependently reduced TXB(2) concentration, measured by RIA in the supernatant of 10 microg ml(-1) LPS-stimulated cells. NCX4016 activity was comparable to that of equimolar ASA when incubation lasted 6 h (NCX4016 30 micromol l(-1): -86.0+/-10.1%, NCX4016 300 micromol l(-1): -92.2+/-9.0%, ASA 30 micromol l(-1): -92.3+/-7.5%, ASA 300 micromol l(-1): -97.3+/-1.0%, n=6, M+/-s.d.). Most of the activity of NCX4016 up to 100 micromol l(-1) was prevented by 10 micromol l(-1) ODQ, inhibitor of cyclic GMP. NCX4016 100 - 300 micromol l(-1) reduced TNF-alpha (NCX4016 300 micromol l(-1)=-77.2+/-19.9%, n=6) and IL-6 (NCX4016 300 micromol l(-1): -61.9+/-15.2%, n=6) in LPS stimulated monocytes while ASA had no significant effects. TF activity (NCX4016 300 micromol l(-1): 53.7+/-39.9%, n=4) and immunoreactive TF (NCX4016 300 micromol l(-1): -93.9+/-7.9%, n=7), measured in the supernatant of stimulated cells, were also dose-dependently inhibited by NCX4016 but not by ASA. The present results indicate that NCX4016 inhibits TXA(2) generation as well as cytokine release and TF in human monocytes partly via NO-dependent mechanisms. NCX4016 may have a favourable profile of activities in the clinical setting of athero-thrombosis.

Topics: Aspirin; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibrinolytic Agents; Guanylate Cyclase; Humans; Interleukin-6; Lipopolysaccharides; Monocytes; Nitric Oxide; Oxadiazoles; Quinoxalines; Thromboplastin; Thromboxane B2; Tumor Necrosis Factor-alpha

NO-Aspirin (NCX-4016) releases nitric oxide (NO) in biological systems through as yet unidentified mechanisms. In LLC-PK1 kidney epithelial cells, a 5-h pretreatment with glyceryl trinitrate (GTN, 0.1-1 microM) significantly attenuated the cyclic GMP response to a subsequent challenge with both NO-aspirin or GTN. Similarly, NO-aspirin (10-100 microM) was found to induce tolerance to its own cyclic GMP stimulatory action and to that of GTN. In contrast, cyclic GMP stimulation by the spontaneous NO donor SIN-1, which releases NO independently of enzymatic catalysis, remained unimpaired in cells pretreated with GTN or NO-aspirin. The observed cross-tolerance between NO-aspirin and GTN cells indicates that bioactivation pathways of organic nitrates, which have been shown to involve cytochrome P450, may also be responsible for NO release from NO-aspirin. Prolonged treatment with NO-aspirin causes down-regulation of the cellular cyclic GMP response, suggesting that tolerance may occur during therapy with NO-aspirin.

Topics: Animals; Aspirin; Cell Line; Cyclic GMP; Cyclic N-Oxides; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Epithelial Cells; Free Radical Scavengers; Imidazoles; Kidney; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitroglycerin; Protein Binding; Swine

Caspases, a class of cysteine proteases, modulate apoptosis. Nitric oxide (NO)-releasing nonsteroidal anti-inflammatory drugs (NSAIDs) are a new class of NSAID derivatives with reduced gastrointestinal toxicity. The aim of this study was to investigate whether cysteine endoproteases are involved in the pathogenesis of NSAID gastropathy and are target for NO-aspirin (NCX-4016).. Rats were treated orally with aspirin or equimolar doses of NCX-4016. Caspase activities were measured by fluorometric assay. Apoptosis was quantified by an enzyme-linked immunosorbent assay for histone-associated DNA, DNA ladder on agarose gel, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay. A primary culture of gastric chief cells was used to investigate whether NCX-4016 modulates guanosine 3',5'-cyclic monophosphate (cGMP)-dependent pathways.. Short- and long-term (7 days) aspirin administration resulted in a time- and dose-dependent gastric injury that was associated with apoptosis and caspase up-regulation. Z-VAD.FMK, a pancaspase inhibitor, and NO donors protected from acute damage induced by aspirin. NCX-4016 spared the gastric mucosa and caused caspase inactivation by S-nitrosylation. Inhibition of tumor necrosis factor (TNF)-alpha release or activity by TAPI-2 or anti-TNF-alpha receptor monoclonal antibodies protected against mucosal damage and caspase activation. NCX-4016 protected gastric chief cells from toxicity induced by TNF-alpha by activating cGMP-dependent pathways.. Aspirin administration leads to a TNF-alpha-dependent activation of gastric caspases. NO-aspirin spares the gastric mucosa and inhibits caspase activity through cGMP-dependent and -independent pathways.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Aspirin; Caspase Inhibitors; Caspases; Cyclic GMP; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Gastric Mucosa; Humans; In Situ Nick-End Labeling; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Rats; Salicylates; Time Factors; Tumor Necrosis Factor-alpha; U937 Cells

We studied in vitro the antiplatelet activity of a new nitroderivative chemically related to acetylsalicylic acid: 2 acetoxybenzoate 2-[1-nitroxy-methyl]-phenyl ester (NCX 4016), in order to identify any effects due to the release of nitric oxide and the blockade of cyclo-oxygenase. The effects of scalar doses of NCX 4016 on the early phase of platelet activation, platelet aggregation and thromboxane A2 production were investigated. We observed inhibitory effects of NCX 4016 on platelet adhesion (IC50 = 7.3 x 10(-5) M), platelet cytosolic calcium concentration, assayed by fluorescent probe Fura 2, and the expression of glycoprotein IIb/IIIa (CD41/alpha IIb beta 3) (IC50 = 3.4 x 10(-5) M) and P-selectin (CD62/GMP-140) (IC50 = 4.9 x 10(-5) M) measured by flow cytometry. NCX 4016 also prevented thrombin-induced platelet aggregation (IC50 = 3.9 x 10(-5) M). None of these parameters were affected by acetylsalicylic acid. These inhibitory activities of NCX 4016 were abolished by oxyhaemoglobin and methylene blue. Intracellular cyclic GMP observed during thrombin-induced aggregation was increased by incubation with NCX 4016. These results appear to be attributable to the release of nitric oxide, which activates soluble platelet guanylylcyclase and promotes intracellular cyclic GMP increase. NCX 4016 almost completely inhibited platelet thromboxane A2 production and arachidonic acid-induced platelet aggregation. This also occurred in the presence of oxyhaemoglobin and methylene blue, indicating that its antiplatelet activity can be attributed not only to nitric oxide release but also to cyclo-oxygenase inhibition.

Topics: Adult; Aspirin; Blood Platelets; Calcium; Cyclic GMP; Humans; P-Selectin; Platelet Activation; Platelet Adhesiveness; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Thromboxane A2