Hydrogen sulphide-releasing diclofenac derivatives inhibit breast cancer-induced osteoclastogenesis in vitro and prevent osteolysis ex vivo.

BACKGROUND AND PURPOSE: Hydrogen sulphide (H(2)S) and prostaglandins are both involved in inflammation, cancer and bone turnover, and non-steroidal anti-inflammatory drugs (NSAIDs) and H(2)S donors exhibit anti-inflammatory and anti-tumour properties. H(2)S-releasing diclofenac (S-DCF) derivatives are a novel class of NSAIDs combining the properties of a H(2)S donor with those of a conventional NSAID. EXPERIMENTAL APPROACH: We studied the effects of the S-DCF derivatives ACS15 and ACS32 on osteoclast and osteoblast differentiation and activity in vitro, human and mouse breast cancer cells support for osteoclast formation and signalling in vitro, and osteolysis ex vivo. KEY RESULTS: The S-diclofenac derivatives ACS15 and ACS32 inhibited the increase in osteoclast formation induced by human MDA-MB-231 and MCF-7 and mouse 4T1 breast cancer cells without affecting breast cancer cell viability. Conditioned media from human MDA-MB-231 cells enhanced IκB phosphorylation and osteoclast formation and these effects were significantly inhibited following treatment by ACS15 and ACS32, whereas the parent compound diclofenac had no effects. ACS15 and ACS32 inhibited receptor activator of NFκB ligand-induced osteoclast formation and resorption, and caused caspase-3 activation and apoptosis in mature osteoclasts via a mechanism dependent on IKK/NFκB inhibition. In calvaria organ culture, human MDA-MB-231 cells caused osteolysis, and this effect was completely prevented following treatment with ACS15 and ACS32. CONCLUSIONS AND IMPLICATIONS: S-diclofenac derivatives inhibit osteoclast formation and activity, suppress breast cancer cell support for osteoclastogenesis and prevent osteolysis. This suggests that H(2)S-releasing diclofenac derivatives exhibit anti-resorptive properties, which might be of clinical value in the treatment of osteolytic bone disease.

H2S releasing aspirin protects amyloid beta induced cell toxicity in BV-2 microglial cells.

ß-Amyloid (Aß) plaques are characteristic hallmarks of Alzheimer's disease. In the present study, we examined the neuroprotective effects of S-aspirin, a hydrogen sulfide (H(2)S)-releasing aspirin, on Aß-induced cell toxicity. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay showed that S-aspirin, but not aspirin, significantly increased cell viability in BV-2 microglial cells, indicating that S-aspirin may protect cells against injury via releasing H(2)S. S-aspirin at 2.5-10 µM significantly increased cell viability and decreased lactate dehydrogenase release in Aß-treated BV-2 microglial cells. Western blotting analysis showed that S-aspirin suppressed the protein expression levels of cyclooxygenase-2 and growth arrest DNA damage (GADD). These data suggest that S-aspirin may protect microglial cells by inhibition of Aß-induced inflammation and cell cycle re-entry. To study whether S-aspirin can protect mitochondria function, mitochondria membrane potential was measured with molecular probe JC-1. It was found that S-aspirin protected mitochondria from Aß-induced loss of mitochondrial member potential. (ΔΨm). In addition, S-aspirin also prevented Aß-induced activation of p38-mitogen activated protein kinase (MAPK). In conclusion, our results suggest that S-aspirin may protect microglial injury via inhibition of inflammation, prevention of mitochondria function, and stimulation of cell growth via stimulating p38-MAPK pathway. Our study may suggest that S-aspirin may have potential therapeutic value for the treatment of Alzheimer's disease.

Dithiolethione compounds inhibit Akt signaling in human breast and lung cancer cells by increasing PP2A activity.

The chemopreventative effects of dithiolethione compounds are attributed to their activation of antioxidant response elements (AREs) by reacting with the Nrf2/Keap1 protein complex. In this study, we show antiproliferative effects of the dithiolethione compound ACS-1 in human cancer cell lines (A549 and MDA-MB-231) by increasing the activity of the tumor suppressor protein phoshatase 2A (PP2A). ACS-1 inhibited epidermal growth factor (EGF)-induced cellular proliferation in a concentration- and time-dependent manner. Akt activation, as determined by serine-473 phosphorylation, was inhibited by ACS-1 in cells stimulated with either EGF or fibronectin. Furthermore, ACS-1 inhibited mammalian target of rapamycin signaling and decreased c-myc protein levels. ACS-1 did not proximally alter EGF receptor or integrin signaling, but caused a concentration-dependent increase in PP2A activity. The effect of ACS-1 on Akt activation was not observed in the presence of the PP2A inhibitor okadaic acid. ACS-1 effects on PP2A activity were independent of ARE activation and cAMP formation. In addition to ACS-1, other dithiolethione compounds showed similar effects in reducing Akt activation, suggesting that this class of compounds may have other effects beyond chemoprevention.

H2S-donating sildenafil (ACS6) inhibits superoxide formation and gp91phox expression in arterial endothelial cells: role of protein kinases A and G.

BACKGROUND AND PURPOSE: Superoxide (O(2)(*-)), derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, is associated with acute respiratory distress syndrome (ARDS). NADPH oxidase activity and expression are blocked by nitric oxide (NO) and sildenafil. As another gas, hydrogen sulphide (H(2)S) is formed by blood vessels, the effect of sodium hydrosulphide (NaHS) and the H(2)S-donating derivative of sildenafil, ACS6, on O(2)(*-) formation and the expression of gp91(phox) (a catalytic subunit of NADPH oxidase) in porcine pulmonary arterial endothelial cells (PAECs) was investigated. EXPERIMENTAL APPROACH: PAECs were incubated with 10 ng mL(-1) tumour necrosis factor-alpha (TNFalpha) (+/-NaHS or ACS6), both of which released H(2)S, for 2 h or 16 h. O(2)(*-) was measured. Expression of gp91(phox) was measured by western blotting and the role of cyclic AMP (cAMP) and/or cyclic GMP was assessed using protein kinase inhibitors. KEY RESULTS: After either 2- or 16-h incubations, O(2)(*-) formation by PAECs was inhibited by NaHS or ACS6, with IC(50) values of about 10 nM and less than 1 nM, respectively. Both 100 nM NaHS and 1 nM ACS6 completely inhibited gp91(phox) expression induced by TNFalpha. The effects of NaHS were blocked by the inhibition of protein kinase A (PKA), but not PKG, and not by the inhibition of guanylyl cyclase. Effects of ACS6 were blocked by inhibition of both PKA and PKG. Both NaHS and ACS6 augmented cAMP formation. CONCLUSION AND IMPLICATIONS: H(2)S inhibited O(2)(*-) formation and upregulation of NADPH oxidase in PAECs through the adenylyl cyclase-PKA pathway. ACS6 may be effective in treating ARDS through both elevation of cAMP and inhibition of phosphodiesterase type 5 activity.

The hydrogen sulphide-releasing derivative of diclofenac protects against ischaemia-reperfusion injury in the isolated rabbit heart.

BACKGROUND AND PURPOSE: Hydrogen sulphide (H(2)S) is an endogenous gaseous mediator active in the multilevel regulation of pathophysiological functions in mammalian cardiovascular tissues. EXPERIMENTAL APPROACH: This study investigated the pharmacological activity of a new H(2)S-releasing derivative of diclofenac, S-diclofenac (2-[(2,6-dichlorophenyl)amino]benzeneacetic acid 4-(3H-1,2-dithiole-3-thione-5-yl)-phenyl ester) in the isolated rabbit heart submitted to low-flow ischaemia-reperfusion damage. KEY RESULTS: S-diclofenac (3, 10 and 30 microM), despite inhibiting prostacyclin generation by cardiac tissues, achieved dose-dependent normalization of coronary perfusion pressure, reducing left ventricular contracture during ischaemia and improving left ventricular developed pressure and +/-dP/dt(max) at reperfusion. Creatine kinase and lactate dehydrogenase activities in heart perfusates were significantly reduced during reperfusion. These effects were accompanied by substantial release of reduced glutathione (GSH), indicating that the H(2)S moiety may have up-regulated cysteine transport. The anti-ischaemic activities of S-diclofenac and the H(2)S-donor sodium hydro sulphide (NaHS) were partially prevented by the K(ATP) channel antagonist glibenclamide, suggesting a mechanism similar to H(2)S-induced cardioprotection in metabolic ischaemic preconditioning. Perfusion with the nitric oxide (NO) synthase inhibitor N(G)-monomethyl-L-arginine worsened the myocardial ischaemia-reperfusion damage, but this was dose-dependently prevented by S-diclofenac and NaHS, suggesting that the released H(2)S may have overcome NO deficiency. CONCLUSION AND IMPLICATIONS: These data show that S-diclofenac had marked anti-ischaemic activity in ischaemic-reperfused rabbit hearts despite inhibition of prostaglandin generation. Increased GSH formation leading to activation of K(ATP) channels may have contributed to this beneficial effect. The pharmacological profile of S-diclofenac and its anti-inflammatory activity, with diminished gastrointestinal side effects, offer therapeutic applications in cardiovascular disease.

Modulation of angiogenesis by dithiolethione-modified NSAIDs and valproic acid.

BACKGROUND AND PURPOSE: Angiogenesis involves multiple signaling pathways that must be considered when developing agents to modulate pathological angiogenesis. Because both cyclooxygenase inhibitors and dithioles have demonstrated anti-angiogenic properties, we investigated the activities of a new class of anti-inflammatory drugs containing dithiolethione moieties (S-NSAIDs) and S-valproate. EXPERIMENTAL APPROACH: Anti-angiogenic activities of S-NSAIDS, S-valproate, and the respective parent compounds were assessed using umbilical vein endothelial cells, muscle and tumor tissue explant angiogenesis assays, and developmental angiogenesis in Fli:EGFP transgenic zebrafish embryos. KEY RESULTS: Dithiolethione derivatives of diclofenac, valproate, and sulindac inhibited endothelial cell proliferation and induced Ser(78) phosphorylation of hsp27, a known molecular target of anti-angiogenic signaling. The parent drugs lacked this activity, but dithiolethiones were active at comparable concentrations. Although dithiolethiones can potentially release hydrogen sulphide, NaSH did not reproduce some activities of the S-NSAIDs, indicating that the dithioles regulate angiogenesis through mechanisms other than release of H(2)S. In contrast to the parent drugs, S-NSAIDs, S-valproate, NaSH, and dithiolethiones were potent inhibitors of angiogenic responses in muscle and HT29 tumor explants assessed by 3-dimensional collagen matrix assays. Dithiolethiones and valproic acid were also potent inhibitors of developmental angiogenesis in zebrafish embryos, but the S-NSAIDs, remarkably, lacked this activity. CONCLUSIONS AND IMPLICATION: S-NSAIDs and S-valproate have potent anti-angiogenic activities mediated by their dithiole moieties. The novel properties of S-NSAIDs and S-valproate to inhibit pathological versus developmental angiogenesis suggest that these agents may have a role in cancer treatment.

Ultrastructural investigations on protective effects of NCX 4016 (nitroaspirin) on macrovascular endothelium in diabetic Wistar rats.

The effect of a nitric oxide-donating aspirin derivative, 2-acetoxy-benzoate 3-(nitroxy-methyl)phenyl ester (NCX 4016), and aspirin on the aortic endothelium of diabetic rats was investigated by using scanning and transmission electron microscopy. Control and streptozotocin-treated rats were used. Metabolic control was assessed by measuring blood and urine metabolites, and 24-h urine volume. The ultrastructural study was performed after 7 weeks of diabetes and 6 weeks of therapy. Streptozotocin treatment induced a persistent hyperglycemia which was not influenced by the pharmacological treatments. Values of blood metabolites were in line with the diabetic status. Both scanning and transmission electron microscopy revealed that aortic endothelium was severely damaged in all diabetic rats except for the NCX 4016 treated ones. Our data document the protective effects of NCX 4016 on the vascular endothelium of diabetic rats. Since aspirin had no protective action, NCX 4016 may have exerted its beneficial action by releasing nitric oxide.

The flurbiprofen derivatives HCT1026 and HCT1027 inhibit bone resorption by a mechanism independent of COX inhibition and nitric oxide production.

Prostaglandins and nitric oxide both modulate bone resorption and bone formation. We previously reported that a nitrosylated derivative of flurbiprofen, termed HCT1026, exerted inhibitory effects on osteoclastic bone resorption, which could not be reproduced by combining the parent compound with nitric oxide (NO) donors. The aim of this study was to investigate the mechanism by which HCT1026 inhibits bone resorption. We compared the effects of flurbiprofen and HCT1026 on osteoclast and osteoblast activity with those of HCT1027--an analogue of HCT1026, which lacks an NO-donating moiety. We found that HCT1026 and HCT1027 inhibited bone resorption in interleukin (IL)-1-stimulated murine osteoblast-bone marrow cocultures, with half-maximal effects (IC50) at 20 +/- 5 microM for HCT1026 and 25 +/- 6 microM for HCT1027 compared with 399 +/- 25 microM for flurbiprofen (P < 0.0001). These differences were unrelated to cyclooxygenase (COX) inhibition since HCT1026 and HCT1027 were about seven to eight times less potent than flurbiprofen at inhibiting COX-1 activity and half as potent at inhibiting COX-2 activity. Further studies showed that HCT1026 and HCT1027 activated caspase-3 in rabbit osteoclasts and promoted osteoclast apoptosis, as assessed by nuclear morphology and TUNEL assays. We conclude that HCT1026 and HCT1027 inhibit osteoclast formation and activity by a mechanism that is independent of NO production and COX inhibition. This raises the possibility that both compounds interact with a novel molecular target expressed on osteoclasts to promote apoptosis and inhibit bone resorption. This demonstrates that HCT1026 and derivatives could represent a novel class of antiresorptive drugs with therapeutic value in the treatment of bone diseases associated with accelerated bone loss due to osteoclast activation.

NO-aspirin: mechanism of action and gastrointestinal safety.

Nitric oxide-releasing aspirins are new chemical entities obtained by adding a nitric oxide-releasing moiety to aspirin. NCX-4016 is the prototype of this family of molecules. NCX-4016 consists of the parent molecule (aspirin) linked to a 'spacer' via an ester linkage, which is in turn connected to a nitric oxide-releasing moiety. Both aspirin and nitric oxide moieties of NCX-4016 contribute to its effectiveness, the latter occurring via both cyclic guanosyl monophosphate-dependent and -independent mechanisms. In vitro studies have shown that NCX-4016 inhibits platelet aggregation induced by aspirin-sensitive (arachidonic acid) and aspirin-insensitive (thrombin) agonist. In contrast to aspirin, NCX-4016 exerts a multilevel regulation of inflammatory target, including caspase-1 and NF-kappaB. This broad spectrum of activities translates to an increased potency of this drug in modulating cardiovascular inflammation. Human studies have shown, that while nitric oxide-aspirin maintains its anti-thrombotic activity, it spares the gastrointestinal tract. Indeed, a 7-day course of NCX-4016 results in 90% reduction of gastric damage caused by equimolar doses of aspirin. Further studies are ongoing to define whether this superior anti-inflammatory and anti-thrombotic profile translates in clinical benefits in patients with cardiovascular diseases.

The effects of sham and full spinalization on the antinociceptive effects of NCX-701 (nitroparacetamol) in monoarthritic rats.

Nitric oxide (NO)-releasing NSAIDs have been shown to be safer and more potent as antinociceptive and anti-inflammatory agents than their parent compounds. NCX-701 (nitroparacetamol), in contrast to paracetamol, is an effective antinociceptive drug in normal animals but their effectiveness in monoarthritis has not been compared. We have now investigated this question by comparing the antinociceptive effects of i.v. NCX-701 and paracetamol in monoarthritic rats under alpha-chloralose anesthesia. The influence of spinalization on the effects of NCX-701 was also studied. NCX-701 and paracetamol were equipotent in reducing single motor unit responses to noxious mechanical stimulation, ID50s of 320+/-1.2 and 305+/-1.2 micromol/kg, respectively. The mechanism of action seems to be different since NCX-701, but not paracetamol, reduced wind-up. This effect suggests a central action, probably within the spinal cord. Sham spinalization reduced the effect of NCX-701 on nociceptive responses drastically. In spinalized animals, however, the effect was similar to that observed in intact animals, indicating a strong effect of NCX-701 at spinal sites, which counterbalances the decrease in the activity induced by the surgery. We conclude that NCX-701 is an effective antinociceptive drug in arthritic animals, with a mechanism of action located in the spinal cord, and different to that of paracetamol.

Addition of nitric oxide via nitroflurbiprofen enhances the material properties of early healing of young rat Achilles tendons.

OBJECTIVE AND DESIGN: To determine if the addition of nitric oxide (NO) via nitroflurbiprofen (NO-flurbiprofen) would enhance rat Achilles tendon healing. MATERIALS AND METHODS: Sixty-five male Sprague-Dawley rats were randomly divided into NO-flurbiprofen, flurbiprofen and vehicle groups, given drugs or vehicle subcutaneously, and their right Achilles tendon divided. Histological assessment was carried out at day 5, 10, and 15 post-operation. Healing tendon biomechanical properties and hydroxyproline content were measured at day 10. RESULTS: The healing Achilles tendon from the NO-flurbiprofen and flurbiprofen groups showed a better organization of extracellular collagenous matrix than that from the vehicle group. Flurbiprofen and NO-flurbiprofen decreased healing tendon cross-sectional area by 30% and 20%. This reduction was accompanied by a decreased failure load in the flurbiprofen group, but not the NO-flurbiprofen group. NO-flubiprofen prevented the reduction of body weight gain observed in the flubiprofen group. CONCLUSION: Both flurbiprofen and NO-flurbiprofen promoted better collagen reorganization during tendon healing. NO-flurbiprofen further improved tendon healing by increasing tendon stress and reducing the side effects (body weight loss) of flurbiprofen. The enhanced tendon healing by NO-flurbiprofen is likely due to the release of NO from the compound.

Effects of non-steroidal anti-inflammatory drugs on cyclo-oxygenase and lipoxygenase activity in whole blood from aspirin-sensitive asthmatics vs healthy donors.

1. Cyclo-oxygenase (COX) and lipoxygenase (LO) share a common substrate, arachidonic acid. Aspirin and related drugs inhibit COX activity. In a subset of patients with asthma aspirin induces clinical symptoms associated with increased levels of certain LO products, a phenomenon known as aspirin-sensitive asthma. The pharmacological pathways regulating such responses are not known. 2. Here COX-1 and LO activity were measured respectively by the formation of thromboxane B(2) (TXB(2)) or leukotrienes (LT) C(4), D(4) and E(4) in whole blood stimulated with A23187. COX-2 activity was measured by the formation of prostaglandin E(2) (PGE(2)) in blood stimulated with lipopolysaccharide (LPS) for 18 h. 3. No differences in the levels of COX-1, COX-2 or LO products or the potency of drugs were found in blood from aspirin sensitive vs aspirin tolerant patients. Aspirin, indomethacin and nimesulide inhibited COX-1 activity, without altering LO activity. Indomethacin, nimesulide and the COX-2 selective inhibitor DFP [5,5-dimethyl-3-(2-isopropoxy)-4-(4-methanesulfonylphenyl)-2(5H)-furanone] inhibited COX-2 activity. NO-aspirin, like aspirin inhibited COX-1 activity in blood from both groups. However, NO-aspirin also reduced LO activity in the blood from both patient groups. Sodium salicylate was an ineffective inhibitor of COX-1, COX-2 or LO activity in blood from both aspirin-sensitive and tolerant patients. 4. Thus, when COX activity in the blood of aspirin-sensitive asthmatics is blocked there is no associated increase in LO products. Moreover, NO-aspirin, unlike other NSAIDs tested, inhibited LO activity in the blood from both aspirin sensitive and aspirin tolerant individuals. This suggests that NO-aspirin may be better tolerated than aspirin by aspirin-sensitive asthmatics.

Nitric-oxide releasing molecules: a new class of drugs with several major indications.

Nitric oxide (NO) deficiency has been implicated in many pathological and physiological processes within the mammalian body providing a plausible biologic basis for the use of NO replacement therapy in these conditions. Exogenous NO sources may hopefully constitute a powerful way to supplement NO when the body cannot generate enough for normal biological functions. This theory has opened up the possibility of designing new drugs that are capable of delivering NO into tissues and the bloodstream in a sustained and controlled manner. This objective has been reached by grafting an organic nitrate structure onto existing molecules with various spacers such as aliphatic or aromatic chain, with different degree of complexity. This approach has led to the synthesis of several new chemical entities in various pharmacological classes, whose profile seems to challenge the parent drug not only on the basis of new pharmacological properties but also on a better toxicological and safety profile. In this article, general aspects on NO and NO donors are reviewed. Major focus is placed upon recent developments of novel NO donors, NO releasing device(s) as well as innovative improvements to conventional NO donors. Several examples are given in some important therapeutic indications such as cardiovascular diseases (NO-aspirin), pain and inflammation (NO-paracetamol), osteoporosis and urinary incontinence (NO flurbiprofen with aliphatic spacer), Alzheimer s disease (NO-flurbiprofen with anti-oxidant spacer), respiratory disorders (NO-steroids).

Inhibition of bone resorption in vitro and prevention of ovariectomy-induced bone loss in vivo by flurbiprofen nitroxybutylester (HCT1026).

OBJECTIVE: Inhibitors of prostaglandin production, such as nonsteroidal antiinflammatory drugs (NSAIDs), and pharmacologic nitric oxide (NO) donors, such as organic nitrates, have been suggested to protect against bone loss in both humans and experimental animals. Recently, a new class of nitrosylated NSAID (known as NO-NSAIDs) has been developed, which combines the properties of a NO donor with those of a cyclooxygenase (COX) inhibitor. This study investigated the effects of one of these compounds, flurbiprofen nitroxybutylester (HCT1026), on bone metabolism in vitro and in vivo. METHODS: The effects of HCT1026 on osteoclast formation and resorption were determined in vitro using cocultures of primary mouse osteoblasts and osteoclasts. The effect of HCT1026 in vivo was assessed using a mouse model of ovariectomy-induced bone loss. RESULTS: HCT1026 was significantly more efficacious than the parent compound, flurbiprofen, at inhibiting osteoclast formation and bone resorption in vitro, and these effects could not be reproduced by combinations of flurbiprofen with a variety of NO donors. Studies in vivo showed that HCT1026 protected against ovariectomy-induced bone loss by inhibiting osteoclastic bone resorption, whereas flurbiprofen at similar concentrations was ineffective. CONCLUSION: These data indicate that HCT1026 is a potent inhibitor of bone resorption in vitro and protects against ovariectomy-induced bone loss in vivo by a novel mechanism that appears to be distinct from its NO donor properties and from its inhibitory effects on COX activity. We conclude that HCT1026 may be of clinical value in the prevention and treatment of inflammatory diseases such as rheumatoid arthritis, which are characterized by joint inflammation as well as periarticular and systemic bone loss.

NO-mesalamine protects colonic epithelial cells against apoptotic damage induced by proinflammatory cytokines.

The activation of a self-amplifying cascade of caspases, of which caspase-8 is the apical protease, mediates Fas-, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-, and TNF-alpha-induced apoptosis in colon cell lines. Nitric oxide (NO) protects from apoptosis induced by Fas and TNF-alpha. We examined whether NCX-456, an NO-releasing derivative of mesalamine, protects colon epithelial cells from cytokine-induced apoptosis. Caco-2 and HT-29 cell lines express death factor receptors and are driven to apoptosis in response to incubation with Fas-agonistic antibody, TNF-alpha/interferon-gamma, and TRAIL. The two novel observations reported here are that 1) cotreatment of cells with NCX-456, but not mesalamine, resulted in concentration-dependent protection against death factor-induced apoptosis and inhibition of caspase activity, and 2) exposure to dithiothreitol, an agent that effectively removes NO from thiol groups, resulted in a 70% recovery of caspase activity, which is consistent with S-nitrosation as a major mechanism for caspase inactivation. These data suggest that caspase S-nitrosation represents a mechanism for protection of colonic mucosal epithelial cells from death factor-induced death.

NCX-1000, a NO-releasing derivative of ursodeoxycholic acid, selectively delivers NO to the liver and protects against development of portal hypertension.

Portal hypertension resulting from increased intrahepatic resistance is a common complication of chronic liver diseases and a leading cause of death in patients with liver cirrhosis, a scarring process of the liver that includes components of both increased fibrogenesis and wound contraction. A reduced production of nitric oxide (NO) resulting from an impaired enzymatic function of endothelial NO synthase and an increased contraction of hepatic stellate cells (HSCs) have been demonstrated to contribute to high intrahepatic resistance in the cirrhotic liver. 2-(Acetyloxy) benzoic acid 3-(nitrooxymethyl) phenyl ester (NCX-1000) is a chemical entity obtained by adding an NO-releasing moiety to ursodeoxycholic acid (UDCA), a compound that is selectively metabolized by hepatocytes. In this study we have examined the effect of NCX-1000 and UDCA on liver fibrosis and portal hypertension induced by i.p. injection of carbon tetrachloride in rats. Our results demonstrated that although both treatments reduced liver collagen deposition, NCX-1000, but not UDCA, prevented ascite formation and reduced intrahepatic resistance in carbon tetrachloride-treated rats as measured by assessing portal perfusion pressure. In contrast to UDCA, NCX-1000 inhibited HSC contraction and exerted a relaxing effect similar to the NO donor S-nitroso-N-acetylpenicillamine. HSCs were able to metabolize NCX-1000 and release nitrite/nitrate in cell supernatants. In aggregate these data indicate that NCX-1000, releasing NO into the liver microcirculation, may provide a novel therapy for the treatment of patients with portal hypertension.

NCX4016 (NO-aspirin) inhibits thromboxane biosynthesis and tissue factor expression and activity in human monocytes.

BACKGROUND: NCX4016 (2 acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl ester, NicOx S.A., France) is an antithrombotic agent chemically related to acetylsalicylic acid (ASA). We hypothesised that NCX4016, being able to release nitric oxide (NO) and to inhibit cyclo-oxygenase, might inhibit the prothrombotic function in human monocytes. MATERIAL AND METHODS: The effects of NCX4016 and ASA on the release of thromboxane (TX) B2 and tissue factor expression and activity were compared using adherent human monocytes. The tested drugs were added before stimulation with 10 Kg/ml LPS and incubation lasted 6 hours. TXB2 concentration was measured by RIA in the supernatant of cultured cells. Immunoreactive tissue factor (TF) concentration was determined by enzyme-linked immunoassay and TF activity was assayed by measuring the peptidyl activity of the tissue factor/ factor VII complex. RESULTS: Both ASA and NCX4016 10-300 Kmol/L dose-dependently reduced TXB2 release. NCX4016 activity was comparable to that of equimolar ASA. Part of the activity of NCX4016 up to 100 Kmol/L was prevented by 10 Kml/L ODQ, inhibitor of cGMP generation. Immunoreactive TF was dose-dependently inhibited by 300 Kmol/L NCX4016, but not by ASA. Also tissue TF activity was reduced by 300 Kmol/L NCX4016, but not by ASA. CONCLUSIONS: The present results indicate that NCX4016 not only has anti-platelet effects but also inhibits prothrombotic activities in human monocytes, partly via NO-dependent mechanisms. NCX4016 may prove effective in the clinical setting of athero-thrombosis.

Antiproliferative effects of nitrosulindac on human colon adenocarcinoma cell lines.

Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the incidence of colon cancer, but their use is limited by toxicity in the gastrointestinal tract. The coupling of a nitric oxide-releasing moiety to NSAIDs strongly reduces these side effects. We demonstrated that the NO-releasing sulindac (nitrosulindac) has much more potent effects on colon adenocarcinoma cell lines compared to sulindac. Moreover, it could inhibit the growth of cells in soft agar experiments, demonstrating the antineoplastic activity at low concentration of nitrosulindac. However, this reduction in the growth of colon cancer cells seemed to be independent of the classical apoptosis pathway and could be explained by a cytostatic effect. Nitrosulindac caused a light perturbation of the cell cycle parameters not linked to a modification of the levels of p21 or the proliferating cell nuclear antigen. Moreover, neither sulindac, nor nitrosulindac, were able to inhibit the NF-kappa B pathway. These data suggested that nitrosulindac could be a better solution compared to other NSAIDs in the treatment of colon cancer.

Nitro-aspirin (NCX4016) reduces brain damage induced by focal cerebral ischemia in the rat.

The potential neuroprotective effects of the novel nitro-derivate of aspirin (NCX4016) on permanent focal cerebral ischemia in spontaneously hypertensive rats (SHRs) was investigated. Reference compounds were acetylsalicilic acid (ASA) and FK506 (tacrolimus). Ten minutes after surgery, SHRs were randomly divided into four groups of ten, pharmacologically treated and sacrificed 24 h after treatment. Brains were removed and processed to measure infarct volume, 70 kDa heat shock protein (hsp70), glial fibrillary acidic protein (GFAP) and vimentin (Vim) immunoreactivity (IR), and apoptosis using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay. NCX-4016 significantly reduced total infarct volume compared to ASA (-20%, P < 0.05), FK506 (-18%, P < 0.05) and vehicle treatment (-20%, P < 0.05). Experimental groups did not differ in hsp70-IR and GFAP-IR. Conversely, hyperplastic astrocytes, measured by Vim-IR, were significantly lower in NCX-4016 than in the vehicle group (-36%, P<0.01). TUNEL assay indicated a significantly lower degree of apoptosis in NCX-4016 group than vehicle in both the homolateral (-27%, P < 0.01) and contralateral hemisphere (-29%, P < 0.05). These findings indicate that NO release associated with aspirin confers neuroprotective effects against ischemic injury.

Role of the arginine-nitric oxide pathway in the regulation of vascular smooth muscle cell proliferation.

The objective of this study was to elucidate the mechanisms by which nitric oxide (NO) inhibits rat aortic smooth muscle cell (RASMC) proliferation. Two products of the arginine-NO pathway interfere with cell growth by distinct mechanisms. N(G)-hydroxyarginine and NO appear to interfere with cell proliferation by inhibiting arginase and ornithine decarboxylase (ODC), respectively. S-nitroso-N-acetylpenicillamine, (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate, and a nitroaspirin derivative (NCX 4016), each of which is a NO donor agent, inhibited RASMC growth at concentrations of 1-3 microM by cGMP-independent mechanisms. The cytostatic action of the NO donor agents as well as alpha-difluoromethylornithine (DFMO), a known ODC inhibitor, was prevented by addition of putrescine but not ornithine. These observations suggested that NO, like DFMO, may directly inhibit ODC. Experiments with purified, recombinant mammalian ODC revealed that NO inhibits ODC possibly by S-nitrosylation of the active site cysteine in ODC. DFMO, as well as the NO donor agents, interfered with cellular polyamine (putrescine, spermidine, spermine) production. Conversely, increasing the expression and catalytic activity of arginase I in RASMC either by transfection of cells with the arginase I gene or by induction of arginase I mRNA with IL-4 resulted in increased urea and polyamine production as well as cell proliferation. Finally, coculture of rat aortic endothelial cells, which had been pretreated with lipopolysaccharide plus a cytokine mixture to induce NO synthase and promote NO production, caused NO-dependent inhibition of target RASMC proliferation. This study confirms the inhibitory role of the arginine-NO pathway in vascular smooth muscle proliferation and indicates that one mechanism of action of NO is cGMP-independent and attributed to its capacity to inhibit ODC.