Hydrogen sulfide-releasing aspirin derivative ACS14 exerts strong antithrombotic effects in vitro and in vivo.

OBJECTIVE: Hydrogen sulfide (H(2)S)-releasing NSAIDs exert potent anti-inflammatory effects beyond classical cyclooxygenase inhibition. Here, we compared the platelet inhibitory effects of the H(2)S-releasing aspirin derivative ACS14 with its mother compound aspirin to analyze additional effects on platelets. METHODS AND RESULTS: In platelets of mice fed with ACS14 for 6 days (50 mg/kg per day), not only arachidonic acid-induced platelet aggregation but also ADP-dependent aggregation was decreased, an effect that was not observed with an equimolar dose of aspirin (23 mg/kg per day). ACS14 led to a significantly longer arterial occlusion time after light-dye-induced endothelial injury as well as decreased thrombus formation after ferric chloride-induced injury in the carotid artery. Bleeding time was not prolonged compared with animals treated with equimolar doses of aspirin. In vitro, in human whole blood, ACS14 (25-500 µmol/L) inhibited arachidonic acid-induced platelet aggregation, but compared with aspirin additionally reduced thrombin receptor-activating peptide-, ADP-, and collagen-dependent aggregation. In washed human platelets, ACS14 (500 µmol/L) attenuated αIIbß3 integrin activation and fibrinogen binding and increased intracellular cAMP levels and cAMP-dependent vasodilator-stimulated phosphoprotein (VASP) phosphorylation. CONCLUSIONS: The H(2)S-releasing aspirin derivative ACS14 exerts strong antiaggregatory effects by impairing the activation of the fibrinogen receptor by mechanisms involving increased intracellular cyclic nucleotides. These additional antithrombotic properties result in a more efficient inhibition of thrombus formation in vivo as achieved with aspirin alone.

Organosulfur derivatives of the HDAC inhibitor valproic acid sensitize human lung cancer cell lines to apoptosis and to cisplatin cytotoxicity.

Lung cancer is the leading cause of cancer mortality worldwide and despite efforts made to improve clinical results, continuing poor survival rates indicate that novel therapeutic approaches are needed. Valproic acid (VPA), a short-chain branched fatty acid used mainly for the treatment of epilepsy and bipolar disorder, has been shown to inhibit class I histone deacetylases (HDAC-I), a group of enzymes involved in chromatin remodeling and which are thought to play a role in tumor development. Although evidence of VPA's therapeutic efficacy has also been observed in patients with solid tumors, the very high concentration required to induce antitumor activity limits its clinical usefulness. We used a panel of NSCLC cell lines to evaluate the activity and mechanisms of action of organosulfur valproic acid derivatives, a promising new class of compounds designed to improve the safety and efficacy of the valproic acid molecule and created by coupling it with a hydrogen sulfide (H(2) S)-releasing moiety. Our results highlighted the increased cytotoxic activity of the novel organosulfur derivatives, ACS33 and ACS2, with respect to VPA, starting from low concentrations. In particular, ACS2 exhibited important pro-apoptotic activity triggered by the mitochondrial pathway and also showed anti-invasion potential. Furthermore, our in vitro results identified a highly effective combination schedule of ACS2 and cisplatin capable of inducing a synergistic interaction even when the two drugs were used at low concentrations, which could prove a valid alternative to traditional chemotherapeutic regimens used for advanced lung cancer. Further studies are needed to confirm these preliminary findings.

Effects of hydrogen sulfide-releasing L-DOPA derivatives on glial activation: potential for treating Parkinson disease.

The main lesion in Parkinson disease (PD) is loss of substantia nigra dopaminergic neurons. Levodopa (L-DOPA) is the most widely used therapy, but it does not arrest disease progression. Some possible contributing factors to the continuing neuronal loss are oxidative stress, including oxidation of L-DOPA, and neurotoxins generated by locally activated microglia and astrocytes. A possible method of reducing these factors is to produce L-DOPA hybrid compounds that have antioxidant and antiinflammatory properties. Here we demonstrate the properties of four such L-DOPA hybrids based on coupling L-DOPA to four different hydrogen sulfide-donating compounds. The donors themselves were shown to be capable of conversion by isolated mitochondria to H(2)S or equivalent SH(-) ions. This capability was confirmed by in vivo results, showing a large increase in intracerebral dopamine and glutathione after iv administration in rats. When human microglia, astrocytes, and SH-SY5Y neuroblastoma cells were treated with these donating agents, they all accumulated H(2)S intracellularly as did their derivatives coupled to L-DOPA. The donating agents and the L-DOPA hybrids reduced the release of tumor necrosis factor-alpha, interleukin-6, and nitric oxide from stimulated microglia, astrocytes as well as the THP-1 and U373 cell lines. They also demonstrated a neuroprotective effect by reducing the toxicity of supernatants from these stimulated cells to SH-SY5Y cells. L-DOPA itself was without effect in any of these assays. The H(2)S-releasing L-DOPA hybrid molecules also inhibited MAO B activity. They may be useful for the treatment of PD because of their significant antiinflammatory, antioxidant, and neuroprotective properties.

Physiological and pharmacological features of the novel gasotransmitter: hydrogen sulfide.

Hydrogen sulfide (H(2)S) has been known for hundreds of years because of its poisoning effect. Once the basal bio-production became evident its pathophysiological role started to be investigated in depth. H(2)S is a gas that can be formed by the action of two enzymes, cystathionine gamma-lyase and cystathionine beta-synthase, both involved in the metabolism of cysteine. It has several features in common with the other two well known "gasotransmitters" (nitric oxide and carbon monoxide) in the biological systems. These three gasses share some biological targets; however, they also have dissimilarities. For instance, the three gases target heme-proteins and open K(ATP) channels; H(2)S as NO is an antioxidant, but in contrast to the latter molecule, H(2)S does not directly form radicals. In the last years H(2)S has been implicated in several physiological and pathophysiological processes such as long term synaptic potentiation, vasorelaxation, pro- and anti-inflammatory conditions, cardiac inotropism regulation, cardioprotection, and several other physiological mechanisms. We will focus on the biological role of H(2)S as a molecule able to trigger cell signaling. Our attention will be particularly devoted on the effects in cardiovascular system and in cardioprotection. We will also provide available information on H(2)S-donating drugs which have so far been tested in order to conjugate the beneficial effect of H(2)S with other pharmaceutical properties.

Hydrogen sulfide-releasing NSAIDs attenuate neuroinflammation induced by microglial and astrocytic activation.

Endogenously generated hydrogen sulfide (H(2)S) may have multiple functions in brain. It has been shown that H(2)S attenuates the expression of pro-inflammatory cytokines by lipopolysaccharide (LPS)-activated microglia. Here we demonstrate a neuroprotective effect of NaSH and three H(2)S-releasing compounds, ADT-OH, S-diclofenac, and S-aspirin. When activated by LPS and gamma-interferon, human microglia and THP-1 cells release materials that are toxic to human neuroblastoma SH-SY5Y cells. These phenomena also occur with gamma-interferon-stimulated human astroglia and U118 cells. When these cell types are pretreated with aspirin, diclofenac, NASH, or ADT-OH, the supernatants are significantly less toxic. When they are treated with the NSAID-H(2)S hybrid molecules S-diclofenac and S-aspirin, which are here referred to as S-NSAIDs, there is a significant enhancement of the protection. The effect is concentration and incubation time dependent. Such pretreatment also reduces the release of the proinflammatory mediators TNFalpha, IL-6, and nitric oxide. The H(2)S-releasing compounds are without effect when applied directly to SH-SY5Y cells. These data suggest that hybrid H(2)S releasing compounds have significant antiinflammatory properties and may be candidates for treating neurodegenerative disorders that have a prominent neuroinflammatory component such as Alzheimer disease and Parkinson disease.

New aryldithiolethione derivatives as potent histone deacetylase inhibitors.

A series of dithiolethione derivatives was synthesized and the in vitro HDAC inhibitory activity was tested. The most active compounds, 1 and 2, exhibited an IC(50) in nM range with a strong hyperacetylation of histone H4 in A549 cells. The HDAC inhibitory activity comparable to that of SAHA and the inhibition of A549 cell proliferation suggest that these compounds are worthy of further studies as potential anticancer agents.

Novel dithiolethione-modified nonsteroidal anti-inflammatory drugs in human hepatoma HepG2 and colon LS180 cells.

PURPOSE: Nonsteroidal anti-inflammatory drugs (NSAID) are promising chemopreventive agents against colon and other cancers. However, the molecular basis mediated by NSAIDs for chemoprevention has not been fully elucidated. Environmental carcinogens induce DNA mutation and cellular transformation; therefore, we examined the effect of NSAIDs on carcinogenesis mediated by the aryl hydrocarbon receptor signaling pathway. In this study, we investigated the activities of a new class of NSAIDs containing dithiolethione moieties (S-NSAID) on both arms of carcinogenesis. EXPERIMENTAL DESIGN: We investigated the effects of the S-NSAIDs, S-diclofenac and S-sulindac, on carcinogen activation and detoxification mechanisms in human hepatoma HepG2 and human colonic adenocarcinoma LS180 cells. RESULTS: We found that S-diclofenac and S-sulindac inhibited the activity and expression of the carcinogen activating enzymes, cytochromes P-450 (CYP) CYP1A1, CYP1B1, and CYP1A2. Inhibition was mediated by transcriptional regulation of the aryl hydrocarbon receptor (AhR) pathway. The S-NSAIDs down-regulated carcinogen-induced expression of CYP1A1 heterogeneous nuclear RNA, a measure of transcription rate. Both compounds blocked carcinogen-activated AhR from binding to the xenobiotic responsive element as shown by chromatin immunoprecipitation. S-diclofenac and S-sulindac inhibited carcinogen-induced CYP enzyme activity through direct inhibition as well as through decreased transcriptional activation of the AhR. S-sulindac induced expression of several carcinogen detoxification enzymes of the glutathione cycle including glutathione S-transferase A2, glutamate cysteine ligase catalytic subunit, glutamate cysteine ligase modifier subunit, and glutathione reductase. CONCLUSIONS: These results indicate that S-diclofenac and S-sulindac may serve as effective chemoprevention agents by favorably balancing the equation of carcinogen activation and detoxification mechanisms.

Effect of hydrogen sulphide-donating sildenafil (ACS6) on erectile function and oxidative stress in rabbit isolated corpus cavernosum and in hypertensive rats.

OBJECTIVE To study the effect of the H(2)S-donating derivative of sildenafil (ACS6) compared to sildenafil citrate and sodium hydrosulphide (NaHS) on relaxation, superoxide formation and NADPH oxidase and type 5 phosphodiesterase (PDE5) expression in isolated rabbit cavernosal tissue and smooth muscle cells (CSMCs), and in vivo on indices of oxidative stress induced with buthionine sulphoximine (BSO). MATERIALS AND METHODS Relaxation was studied in an organ bath in response to carbachol and after incubation with interleukin-1beta for 12 h. CSMCs were incubated with tumour-necrosis factor-alpha or the thromboxane A(2) (TXA(2)) analogue, U46619, with or with no sildenafil citrate, ACS6 or NaHS for 16 h. Superoxide formation and the expression of p47(phox) (an active subunit of the NADPH oxidase complex) and PDE5 protein was then assessed using Western blotting. Rats were also treated with BSO (with or with no sildenafil citrate or ACS6) for 7 days; cavernosal cGMP, cAMP, glutathionine and plasma TXA(2) and 8-isoprostane F(2alpha) was measured by enzyme-linked immunosorbent assay. RESULTS ACS6 and sildenafil citrate relaxed cavernosal smooth muscle equipotently; NaHS alone had little effect at up to 100 microm. The formation of superoxide and expression of p47(phox) and PDE5 was reduced by ACS6, sildenafil citrate and NaHS (order of potency: ACS6 > sildenafil citrate > NaHS). The effects of ACS6 were blocked by inhibitors of protein kinase A (PKA) and PKG. In rats treated with BSO, both ASC6 and sildenafil citrate reduced the increased plasma levels of TXA(2) and 8-isoprostane F(2alpha) but increased cGMP, cAMP and glutathionine levels in corpus cavernosum. CONCLUSIONS By virtue of a dual action on PKA and PKG activation, ACS6 not only promotes erection, acutely, but might also have a long-term beneficial effect through inhibition of oxidative stress and downregulation of PDE5.

Exogenous hydrogen sulfide inhibits superoxide formation, NOX-1 expression and Rac1 activity in human vascular smooth muscle cells.

The activity of NADPH oxidase (NOX) is blocked by nitric oxide (NO). Hydrogen sulfide (H(2)S) is also produced by blood vessels. It is reasonable to suggest that H(2)S may have similar actions to NO on NOX. In order to test this hypothesis, the effect of sodium hydrosulfide (NaHS) on O(2)(-) formation, the expression of NOX-1 (a catalytic subunit of NOX) and Rac(1) activity (essential for full NOX activity) in isolated vascular smooth muscle cells (hVSMCs) was investigated. hVSMCs were incubated with the thromboxane A(2) analogue U46619 +/- NaHS for 1 or 16 h, and O(2)(-) formation, NOX-1 expression and Rac(1) activity were assessed. The possible interaction between H(2)S and NO was also studied by using an NO synthase inhibitor, L-NAME, and an NO donor, DETA-NONOate. The role of K(ATP) channels was studied by using glibenclamide. NaHS inhibited O(2)(-) formation following incubation of 1 h (IC(50), 30 nM) and 16 h (IC(50), 20 nM), blocked NOX-1 expression and inhibited Rac(1) activity. These inhibitory effects of NaHS were mediated by the cAMP-protein-kinase-A axis. Exogenous H(2)S prevents NOX-driven intravascular oxidative stress through an a priori inhibition of Rac(1) and downregulation of NOX-1 protein expression, an effect mediated by activation of the adenylylcyclase-cAMP-protein-kinase-G system by H(2)S.

Effect of S-diclofenac, a novel hydrogen sulfide releasing derivative inhibit rat vascular smooth muscle cell proliferation.

S-diclofenac (2-[(2,6-dichlorophenyl) amino] benzene acetic acid 4-(3H-1,2,dithiol-3-thione-5-yl) phenyl ester) is a novel molecule comprising a hydrogen sulfide (H2S)-releasing dithiol-thione moiety attached by an ester linkage to diclofenac. Effect of S-diclofenac (H2S donor) on cell proliferation was investigated on the primary and immortalized rat aortic vascular smooth muscle cells (SMC). Smooth muscle cell proliferation has been considered as a key event in vascular injury in diseases such as atherosclerosis and restenosis after invasive intervention. Clonogenic cell survival assay showed a dose dependent (10-100 microM) decrease in cell survival. Flow cytometric analysis showed that the asynchronized cells are more sensitive than the cells that are synchronized and revealed that the cells in G1 phase are not affected by the treatment of the S-diclofenac. Asynchronized smooth muscle cells treated with the S-diclofenac showed an increase in apoptotic cell death. S-diclofenac treatment also resulted in stabilization of p53 coupled with the induction of downstream proteins such as p21, p53AIP1 and Bax. S-diclofenac did not up-regulate cell levels of the antiapoptotic protein Bcl-2. However, when the cells are synchronized a stimulatory effect of cell growth with the decrease in apoptosis, p53 and p21 was evident. S-diclofenac inhibits smooth muscle cell growth and may play a role in the lesion formation at sites of the vascular injury. The present results suggest that S-diclofenac may be useful for the prevention of smooth muscle cell proliferation in diseases such as vascular obstructive and restenosis.

New sulfurated derivatives of valproic acid with enhanced histone deacetylase inhibitory activity.

One dithiolthione and two new methanethiosulfonate derivatives of valproic acid (VPA) were synthesized and tested in vitro as histone deacetylase (HDAC) inhibitors. The new molecules, as well as their sulfurated moieties, exhibited a much stronger inhibition of HDAC enzymatic and antiproliferative activities and histone hyperacetylation than VPA. ACS 2 is the most interesting compound among the new VPA derivatives and its sulfurated moiety, 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione, also known to be a metabolite of anethole trithione, seems to contribute significantly to its activity. This is the first time that HDAC inhibitory activity is described for dithiolethiones and thiosulfonates.

Addition of nitric oxide through nitric oxide-paracetamol enhances healing rat achilles tendon.

Nitric oxide is an important messenger molecule in many physiological processes. The addition of NO via NO-flurbiprofen enhances the material properties of healing tendon, however, flurbiprofen has a detrimental effect on healing. We asked if NO delivered by a cyclooxygenase 3 inhibitor (paracetamol/acetaminophen) would enhance healing in a rat Achilles tendon healing model. Rats were injected subcutaneously daily with NO-paracetamol, paracetamol or vehicle from two days before surgery to the day of tissue harvesting. Paracetamol had no effect on tendon healing compared with vehicle alone. NO-paracetamol did not change the failure load, but did decrease the water content, enhance the collagen content, reduce the cross-sectional area and improve the ultimate stress of healing tendon compared with paracetamol and vehicle. The collagen organization of the healing tendon in the NO-paracetamol group, as determined by polarized light microscopy, was enhanced. Our data suggests NO-paracetamol increases the total collagen content and enhances organization while decreasing the cross-sectional area of healing rat Achilles tendon and is consistent with human clinical trials where NO has improved the symptoms and signs of tendinopathy.

Anti-inflammatory and gastrointestinal effects of a novel diclofenac derivative.

S-diclofenac (2-[(2,6-dichlorophenyl)amino]benzeneacetic acid 4-(3H-1,2,dithiol-3-thione-5-yl)phenyl ester; ACS 15) is a novel molecule comprising a hydrogen sulfide (H2S)-releasing dithiol-thione moiety attached by an ester linkage to diclofenac. S-diclofenac administration inhibited lipopolysaccharide-induced inflammation (as evidenced by reduced lung and liver myeloperoxidase activity) and caused significantly less gastric toxicity than diclofenac. S-diclofenac did not affect blood pressure or heart rate of the anesthetized rat. S-diclofenac administration downregulated expression of genes encoding enzymes which synthesize nitric oxide, prostanoids, and H2S; reduced plasma IL-1beta/TNF-alpha; and elevated plasma IL-10. Reduced liver NF-kappaB p65 and AP-1/c-fos DNA-binding activity was also observed. These effects were mimicked in large part by a combination of diclofenac plus an H2S-releasing moiety (ADT-OH). Incubation of S-diclofenac (100 microM) with rat plasma or liver homogenate caused a time-dependent release of H2S, which was inhibited by sodium fluoride (10 mM). Administration of S-diclofenac (47.2 micromol/kg, i.p.) to conscious rats significantly increased plasma H2S concentration (at 45 min and 6 h). We propose that H2S release from S-diclofenac in vivo contributes to the observed effects.

NCX-4016 (NO-aspirin) inhibits lipopolysaccharide-induced tissue factor expression in vivo: role of nitric oxide.

BACKGROUND: NCX-4016 is an acetylsalicylic acid (ASA) derivative containing a nitric oxide-releasing moiety. Compared with ASA, NCX-4016 has a broader spectrum of antithrombotic and antiinflammatory activities. We hypothesized that NCX-4016 might inhibit in vivo lipopolysaccharide (LPS)-induced expression of tissue factor (TF). METHODS AND RESULTS: Rats were administered 90 mg/kg NCX-4016 orally for 5 days. Placebo, 50 mg/kg ASA, and 80 mg/kg isosorbide-5-mononitrate (ISMN) were used in control groups. On day 5, rats were injected intraperitoneally with 100 microg/kg LPS and killed 6 hours later. The expression of TF in monocytes was measured by flow cytometry and Western blot analysis. Reverse transcriptase-polymerase chain reaction was performed to assess expression of TF and cyclooxygenase-2 (COX-2) genes. Plasma concentrations of interleukin-1beta and tumor necrosis factor-alpha were measured. Urine samples were collected to evaluate the excretion of the thromboxane metabolite 11-dehydro-thromboxane (TX)B2. Gastric mucosa was inspected. LPS injection was followed by synthesis TF and COX-2 mRNAs in circulating monocytes, which were blunted by NCX-4016 but not by ASA or ISMN. Both NCX-4016 and ISMN reduced TF expression on surface of circulating monocyte. LPS increased the excretion 11-dehydro-TXB2, and this was prevented by NCX-4016 and ASA. Unlike ASA, NCX-4016 reduced plasma interleukin-1beta and tumor necrosis factor-alpha. In addition, NCX-4016 almost completely prevented mucosal damage, whereas ASA increased the extension of gastric lesions in LPS-injected rats. CONCLUSIONS: NCX-4016 prevents monocyte TF expression; this is accompanied by inhibition of TX and cytokine biosynthesis. These additive effects of nitric oxide release and COX inhibition may help explain efficacy and tolerability of NCX-4016.

Co-administration of nitric oxide-aspirin (NCX-4016) and aspirin prevents platelet and monocyte activation and protects against gastric damage induced by aspirin in humans.

OBJECTIVES: The goal of this study was to test the hypothesis that NCX-4016 may have broader anti-inflammatory and antithrombotic effects as well as better gastric tolerability than aspirin in humans. BACKGROUND: NCX-4016 is an aspirin derivative containing a nitric oxide-releasing moiety that prevents platelet activation and modulates tissue factor (TF) expression and cytokine release from lipopolysaccharide (LPS)-stimulated monocytes. METHODS: This was a blind-observer, placebo-controlled, parallel-group study in which 48 healthy subjects were randomized to receive NCX-4016 800 mg twice a day, NCX-4016 800 mg twice a day plus aspirin 325 mg, aspirin 325 mg, or placebo for 21 days. RESULTS: Similar to aspirin alone, NCX-4016 effectively inhibited platelet aggregation induced by 0.6 mmol/ arachidonic acid, clot-stimulated thromboxane (TX) B2 generation in whole blood, and urinary excretion of 11-dehydro-TXB2. Unlike aspirin alone, the administration of NCX-4016 significantly inhibited TF expression in monocytes stimulated ex vivo with 10 micromol/l LPS (determined by flow-cytometry analysis of TF on CD14 positive cells). NCX-4016 also inhibited the rapid TF expression induced in monocytes by a proteinase activated receptor agonist (thrombin receptor activator protein, 2 micromol/l) as well as LPS-induced expression of CD11b . Ex vivo, release of MCP-1 and interleukin-6 were significantly inhibited by NCX-4016, but not by aspirin. NCX-4016 was not associated with gastric damage, and significantly reduced gastric injury when co-administered with aspirin, although both drugs reduced gastric PGE2 production to the same extent. CONCLUSIONS: NCX-4016 is equally effective as aspirin in inhibiting cyclooxygenase activity. However, NCX-4016 causes less gastric damage and prevents monocyte activation. Larger multicenter trials are warranted to establish clinical efficacy and safety of NCX-4016.

Nitroaspirin plus clopidogrel versus aspirin plus clopidogrel against platelet thromboembolism and intimal thickening in mice.

Clopidogrel plus aspirin is the treatment of choice for patients undergoing percutaneous, coronary interventions with stenting, but it does not prevent restenosis. NCX-4016, a nitric oxide-releasing aspirin (nitroaspirin), exerts a wider range of antiplatelet actions compared to aspirin, superior antithrombotic activity and reduces restenosis after arterial injury in animals. The aim of the present study was to compare the combination of nitroaspirin plus clopidogrel with aspirin plus clopidogrel in a model of platelet pulmonary thromboembolism, bleeding and intimal thickening in mice. Drugs were administered orally for 5 days; the antithrombotic effects were evaluated against collagen plus epinephrine-induced pulmonary thromboembolism, the haemorrhagic effects by tail transection bleeding time and the effects on neointima proliferation by histomorphology of photochemically injured femoral arteries. Lung platelet emboli were reduced significantly and more effectively by nitroaspirin plus clopidogrel (-56%, p<0.05 vs control) than by aspirin plus clopidogrel (-26%, p<0.05 vs control). Ex vivo platelet aggregation was inhibited maximally by nitroaspirin plus clopidogrel. Aspirin plus clopidogrel strikingly prolonged the bleeding time while nitroaspirin plus clopidogrel induced a lesser prolongation. Nitroaspirin plus clopidogrel significantly reduced intimal thickening of the femoral artery while aspirin plus clopidogrel was ineffective. Nitroaspirin plus clopidogrel is more effective and less prohaemorrhagic than aspirin plus clopidogrel in mice; provided these data are confirmed in other animal models, nitroaspirin plus clopidogrel may represent a new regimen to be tested in patients undergoing coronary revascularization procedures.

NCX-4016, a nitric oxide-releasing aspirin, protects endothelial cells against apoptosis by modulating mitochondrial function.

We investigated the effect of a nitric oxide (NO)-releasing derivative of aspirin (NCX-4016) on a mitochondria-dependent model of apoptosis in human umbilical endothelial cells (HUVEC). Exposure of HUVEC to staurosporine caused a progressive fall in mitochondrial membrane potential (DeltaPsi(m)) and apoptosis. Exposure to an NO donor, (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO), caused an early (1-3h) hyperpolarization of DeltaPsi(m) and reduction of apoptosis that was followed (at 4-8 h) by an accelerated collapse of DeltaPsi(m) and cell death. In contrast, treatment with NCX-4016, but not with aspirin or a non-NO-releasing analogue of NCX-4016, protected HUVEC against the apoptotic actions of staurosporine for up to 8 h. Confocal microscopy demonstrated that although NCX-4016 released NO in subcellular compartments, DETA-NO caused a generalized increase in cytosolic fluorescence. Exposure to DETA-NO resulted in a rapid and profound inhibition of cell respiration (78.3 +/- 6.4%), whereas NCX-4016 caused a less pronounced reduction in oxygen consumption (43.5 +/- 5.3%). Staurosporine caused a time-dependent activation of proapoptotic caspases. NCX-4016 prevented this activation, whereas DETA-NO failed to inhibit caspase activity. In contrast to DETA-NO, NCX-4016 did not increase mitochondrial oxidative stress. These data demonstrated that NCX-4016 conveys NO directly inside endothelial cells and modulates mitochondrial function.

A nitric oxide-releasing derivative of enalapril, NCX 899, prevents progressive cardiac dysfunction and remodeling in hamsters with heart failure.

Nitric oxide (NO) production is known to be impaired in heart failure. A new compound (NCX 899), a NO-releasing derivative of enalapril was characterized, and its actions were evaluated in Bio 14.6 cardiomyopathic (CM) hamsters with heart failure. The hamsters were randomized to oral treatment for 4 weeks with vehicle (n=11), NCX 899 (NCX, 25 mg/kg, n=10), or enalapril (25 mg/kg, n=10). In the vehicle group, fractional shortening by echocardiography decreased (-23.6+/-2.0%) and LV end-diastolic dimension) increased (+10.9+/-1.0%), whereas fractional shortening increased (+17.5+/-4.4%) in NCX and was unchanged in the enalapril group (both P<0.01 vs. vehicle). End-diastolic dimension decreased only in NCX. LV contractility (LVdP/dt max and Emax) was significantly greater in NCX than in enalapril or vehicle, while relaxation (Tau) was shortened in both NCX and enalapril vs. vehicle. ACE activity was inhibited equally by NCX and enalapril in the CM hamster, and plasma nitrate levels were increased only in NCX (P<0.05 vs. enalapril and vehicle). In aortic strips endothelium-independent relaxation occurred only with NCX. The superior effects of NO-releasing enalapril (NCX) vs. enalapril alone to enhance vascular effects, increase LV contractility and prevent unfavorable remodeling and are consistent with vascular delivery of exogenous NO. NCX 899 may hold promise for the future treatment of heart failure.

NSAIDs increase GM-CSF release by human synoviocytes: comparison with nitric oxide-donating derivatives.

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.

Nitric oxide-releasing aspirin derivative, NCX 4016, promotes reparative angiogenesis and prevents apoptosis and oxidative stress in a mouse model of peripheral ischemia.

BACKGROUND: Recently, nitric oxide (NO) donors have been developed that mimic the physiological intracellular release of NO. We evaluated whether one of these new compounds, consisting of aspirin coupled to an NO-releasing moiety (NCX 4016), would protect limbs from supervening arterial occlusion. METHODS AND RESULTS: Mice were assigned to receive regular chow or chow containing NCX 4016 or aspirin (both at 300 mumol/kg body weight, daily) throughout the 3-week experimental period. One week after randomization, they underwent surgical excision of the left femoral artery. Limb blood flow recovery (laser Doppler flowmetry) was accelerated by NCX 4016 as compared with aspirin or vehicle (P<0.05). In controls, histological analysis revealed a 35% increase in the capillary density of ischemic muscles compared with contralateral ones, indicative of spontaneous angiogenesis. Neovascularization was enhanced by NCX 4016 (91%; P<0.05 versus vehicle), but not by aspirin (51%; P=NS versus vehicle). Furthermore, NCX 4016 reduced endothelial cell (EC) apoptosis (4.3+/-1.0 versus 8.7+/-2.0 in aspirin and 12.6+/-3.3 ECs/1000 cap in vehicle; P<0.05 for either comparison) as well as caspase-3 mRNA levels in ischemic muscles ([caspase-3/GAPDH]*100 = 0.09+/-0.04 versus 2.30+/-0.44 in aspirin and 2.30+/-0.32 in vehicle; P<0.01 for either comparison). Nitrite levels and the ratio of reduced to oxidized glutathione were selectively increased in ischemic muscles by NCX 4016. Vascular endothelial growth factor-A expression was reduced by aspirin, with this effect being blunted by NCX 4016. CONCLUSIONS: Pretreatment with the new oral NO-releasing aspirin derivative stimulates reparative angiogenesis and prevents apoptosis and oxidative stress, thereby alleviating the consequences of supervening arterial occlusion.