Nitric oxide (NO) plays a critical role in skeletal muscle function, including control of blood flow and muscle repair. In muscular dystrophies, synthesis of NO in the skeletal muscle is known to be defective therefore contributing to damage progression. In the present study, we evaluated the effects of naproxcinod, an NO-donating anti-inflammatory compound, in two models of muscular dystrophy, the alpha-sarcoglycan (alpha-SG) null mice, a model for limb-girdle muscle dystrophy, and the mdx mouse model for Duchenne muscle dystrophy (DMD). Naproxcinod (10 and 30 mg/kg/day) was orally administered for 7 months to mdx mice and for 4 months to alpha-SG null mice starting at 4 weeks of age. Muscle function was assessed by treadmill test at 4 months (both mdx and alpha-SG null mice) and 7 months of treatment (mdx mice). Serum creatine kinase (CK) was measured as index of skeletal muscle damage. Inflammatory infiltrates, as well as muscle regeneration were studied in diaphragm and tibialis anterior muscles. In mdx mice 30 mg/kg/day of naproxcinod significantly improved muscle function in terms of resistance to exercise with a recovery score of 12%; significantly reduced skeletal muscle inflammation and serum CK activity; and increased muscle regeneration. Likewise in alpha-SG null mice, 4 months of naproxcinod treatment led to a significant improvement of resistance to fatigue (recovery score of 59%), and to reduction of muscle inflammation and damage. Furthermore, both diaphragm and tibialis anterior muscles appeared fully regenerated, thus supporting the marked beneficial effects observed for muscle function. The results demonstrate that naproxcinod, through NO donation together with anti-inflammatory activity, produces significant and persistent therapeutic effects improving muscle function, reducing muscle inflammation and maintaining regeneration capacity of the muscle in two models of muscular dystrophies. Nitric oxide (NO) plays a critical role in skeletal muscle function, including control of blood flow and muscle repair. In muscular dystrophies, synthesis of NO in the skeletal muscle is known to be defective therefore contributing to damage progression. In the present study, we evaluated the effects of naproxcinod, an NO-donating anti-inflammatory compound, in two models of muscular dystrophy, the alpha-sarcoglycan (alpha-SG) null mice, a model for limb-girdle muscle dystrophy, and the mdx mouse model for Duchenne muscle dystrophy (DMD). Naproxcinod (10 and 30 mg/kg/day) was orally administered for 7 months to mdx mice and for 4 months to alpha-SG null mice starting at 4 weeks of age. Muscle function was assessed by treadmill test at 4 months (both mdx and alpha-SG null mice) and 7 months of treatment (mdx mice). Serum creatine kinase (CK) was measured as index of skeletal muscle damage. Inflammatory infiltrates, as well as muscle regeneration were studied in diaphragm and tibialis anterior muscles. In mdx mice 30 mg/kg/day of naproxcinod significantly improved muscle function in terms of resistance to exercise with a recovery score of 12%; significantly reduced skeletal muscle inflammation and serum CK activity; and increased muscle regeneration. Likewise in alpha-SG null mice, 4 months of naproxcinod treatment led to a significant improvement of resistance to fatigue (recovery score of 59%), and to reduction of muscle inflammation and damage. Furthermore, both diaphragm and tibialis anterior muscles appeared fully regenerated, thus supporting the marked beneficial effects observed for muscle function. The results demonstrate that naproxcinod, through NO donation together with anti-inflammatory activity, produces significant and persistent therapeutic effects improving muscle function, reducing muscle inflammation and maintaining regeneration capacity of the muscle in two models of muscular dystrophies.

There is evidence that nitric oxide (NO) plays a critical role in skeletal muscle. In Duchenne muscular dystrophy (DMD) patients and the mdx mouse model of DMD, dystrophin deficiency causes a decrease and mislocalization of muscle-specific neuronal nitric oxide synthase (nNOSμ), leading to a variety of functional impairments such as muscle ischemia and compromised myogenesis. Previous studies have shown that NO donation associated with anti-inflammatory action showed beneficial effects in dystrophic mouse models. In this study, we have investigated the effects of naproxcinod, an NO donating naproxen, on skeletal and cardiac muscle function in mdx mice. 4-week-old mdx mice were orally treated for 9 months with three different doses of naproxcinod (10, 21 and 41 mg/kg/day) compared with 0.9 mg/kg of prednisolone. Functional and behavioral parameters using a grip strength meter and open field digiscan were measured at 3, 6, and 9 months of treatment using SOPs developed by TREAT-NMD. Additionally, in vitro EDL force contraction, optical imaging of inflammation, echocardiography and blood pressure were evaluated at the 9 months prior to sacrifice. Naproxcinod treatment at 10 and 21 mg/kg resulted in significant improvements in hindlimb grip strength as well as approximately a 25–30% decrease in inflammation in fore and hind limbs measured by in vivo optical imaging in mdx mice. Naproxcinod induced significant improvements in heart function as evidenced by ameliorated fraction shortening and ejection fraction measured using echocardiography along with improvements in systolic blood pressure. Moreover, the long term detrimental effects of prednisolone typically observed in mdx skeletal and heart function were not observed at the effective doses of naproxcinod. In conclusion, naproxcinod seems to have significant potential as a safe therapeutic option for the treatment of muscular dystrophies.

AIMS The aim of the present study was to assess whether the addition of a nitric oxide (NO)-donating moiety to atorvastatin enhances anti-inflammatory and anti-atherogenic effects in an animal model of endothelial dysfunction, systemic peroxidation and inflammation, and accelerated atherosclerosis. METHODS AND RESULTS Low-density lipoprotein receptor (LDLR)(-/-) mice kept on a high-fat diet (HFD) for 16 weeks underwent photochemical injury to the femoral artery with the local production of oxygen radicals. HFD markedly enhanced cholesterol, inflammatory biomarkers in plasma and in the femoral arterial wall, and atherosclerotic lesions in the aortic arch; inflammation and atherosclerosis were further increased by photochemically generated oxygen radicals. Treatment with the NO-donating atorvastatin NCX 6560 (11.7 mg/kg) was significantly more effective than atorvastatin (10 mg/kg) in reducing the following parameters: lipid-rich lesions in the aortic arch (surface covered: atorvastatin = 24 ± 5%; NCX 6560 = 14.7 ± 3.9%; P< 0.05); the production of radical oxygen species in the aorta (dichlorofluorescein fluorescence intensity per milligram of protein: atorvastatin = 2419 ± 136.7; NCX 6560 = 1766 ± 161.2; P< 0.05); femoral artery intima/media thickness (atorvastatin = 1.2 ± 0.11; NCX 6560 = 0.3 ± 0.14; P< 0.05); circulating interleukin-6 (atorvastatin = 34.3 ± 6.8 pg/mL; NCX 6560 = 17.7 ± 14.4 pg/mL; P< 0.05); and matrix metalloproteinase 2 in the arterial wall (atorvastatin = 55.2 ± 1.9 ng/µg of proteins; NCX 6560 = 45.8 ± 2.6 ng/µg of proteins; P < 0.05). CONCLUSION In conditions of severe endothelial dysfunction, systemic peroxidation and inflammation, and accelerated atherosclerosis, atorvastatin, even at high doses, displays suboptimal anti-atherogenic and anti-inflammatory effects, while the addition of a NO-donating property confers enhanced anti-atherogenic and anti-inflammatory effects.

BACKGROUND AND PURPOSE Statins, a major component of the prevention of cardiovascular disease, aid progenitor cell functions in vivo and in vitro. Statins bearing a NO‐releasing moiety were developed for their enhanced anti‐inflammatory/anti‐thrombotic properties. Here, we investigated if the NO‐donating atorvastatin (NCX 547) improved the functions of circulating angiogenic cells (CACs).EXPERIMENTAL APPROACH Circulating angiogenic cells (CACs) were prepared from peripheral blood monocytes of healthy volunteers and type‐2 diabetic patients and were cultured in low (LG) or high glucose (HG) conditions, in presence of atorvastatin or NCX 547 (both at 0.1 µM) or vehicle. Functional assays (outgrowth, proliferation, viability, senescence and apoptosis) were performed in presence of the endothelial NOS inhibitor L‐NIO, the NO scavenger c‐PTIO or vehicle.KEY RESULTS Culturing in HG conditions lowered NO in CACs, inhibited outgrowth, proliferation, viability and migration, and induced cell senescence and apoptosis. NCX 547 fully restored NO levels and functions of HG‐cultured CACs, while atorvastatin prevented only apoptosis in CACs. The activity of Akt, a pro‐survival kinase, was increased by atorvastatin in LG‐cultured but not in HG‐cultured CACs, whereas NCX 547 increased Akt activity in both conditions. L‐NIO partially blunted and c‐PTIO prevented NCX 547‐induced improvements in CAC functions. Finally, NCX 547 improved outgrowth and migration of CACs prepared from patients with type 2 diabetes.CONCLUSIONS AND IMPLICATIONS NCX 547 was more effective than atorvastatin in preserving functions of CACs. This property adds to the spectrum of favourable actions that would make NO‐releasing statins more effective agents for treating cardiovascular disease.

Background and purpose: Mature endothelial cells and their progenitors are dysfunctional in diabetes, resulting in deficient neovascularisation following arterial occlusion. This study aimed to evaluate the therapeutic activity of a nitric oxide (NO) releasing statin in the setting of experimental diabetes and peripheral ischaemia.Experimental approach: The effects of NCX 6550, an NO-releasing pravastatin derivative, on angiogenesis in ischaemic limbs was studied in normoglycaemic mice or mice made diabetic by treatment with streptozotocin (STZ). Control mice received an equimolar dosage of the parent statin compound, pravastatin. The therapeutic action of NCX 6550 was also tested in mice lacking the gene for endothelial nitric oxide synthase (eNOS).Key Results: In normoglycaemic or STZ-diabetic CD1 mice, only NCX 6550 stimulated skeletal muscle revascularisation. In addition, NCX 6550 induced greater improvement in limb reperfusion and salvage, than pravastatin. The number of circulating endothelial progenitor cells was decreased in STZ-diabetic mice, this defect being prevented by NCX 6550 and, to a lesser extent by pravastatin. In vitro, high glucose concentrations reduced the migratory capacity of endothelial progenitor EPCs, which was partly reversed by preincubation with pravastatin and completely reversed by NCX 6550. The postischaemic recovery of eNOS knockout mice was severely impaired as a consequence of depressed angiogenesis and this recovery was improved by treatment with NCX 6550, but not with pravastatin.Conclusions and implications: These findings indicate that incorporation of a bioactive NO moiety improves the therapeutic profile of statins for the treatment of peripheral vascular disease.

We have previously reported that NCX 2057, a new chemical entity bearing a nitric oxide (NO)-releasing moiety linked to the natural antioxidant ferulic acid, shows marked anti-inflammatory properties in a model of chronic brain inflammation. We have now studied the effects of NCX 2057 and its metabolic products, ferulic acid and NCX 2059, on inducible nitric oxide synthase (iNOS) expression and function in lipopolysaccharide/interferon-gamma (LPS/IFNgamma)-stimulated RAW 264.7 macrophages. NCX 2057 inhibited iNOS mRNA and protein expression (IC(50)=6.2+/-1.0 microM) without altering iNOS protein degradation rate. NCX 2057 also decreased the levels of LPS/IFNgamma-induced nitrite accumulation (IC(50)=4.3+/-0.7 microM) in RAW 264.7 cells. Conversely, NCX 2059, which does not possess NO-donating properties, was only weakly effective (IC(50) >100 microM) and ferulic acid was inactive. To understand further the mechanisms underlying anti-inflammatory properties we studied the effects of NCX 2057 on selected transcription factors. Unlike ferulic acid, NCX 2057 inhibited LPS-induced translocation/activation of the nuclear factor, NF-kappaB, while other transcription factors, such as, Sp1, NF-IL2A and STAT-1 were not affected. The present data support the concept that NO adds important anti-inflammatory properties to ferulic acid. Thus, NCX 2057 represents a new prototype drug for the treatment of disorders associated with chronic inflammation and oxidative stress.

Glucocorticoids, given at high-doses, improve recovery of function after spinal cord injury (SCI) in animals. However, side effects combined with a limited efficacy in clinical trials have restricted their usefulness for treatment of SCI patients. Recent studies have shown that incorporation of the nitric oxide releasing moiety into the glucocorticoid structure enhances anti-inflammatory properties and reduces side effects. One compound, a derivative of prednisolone (PRE), (NCX 1015, prednisolone 21 [(4′nitrooxymethyl)benzoate]), has interesting pharmacological properties. Therefore, we investigated its effects on apoptosis and recovery of function in rats after SCI. Rats received subcutaneously vehicle, NCX 1015 or PRE (37 μmol/kg, each) 3.5 h after a standardized thoracic lesion. The treatment was continued once a day for 3 days and the effect of both steroids on apoptosis was examined by immunohistochemistry 24 h after the last injection. NCX 1015 but not PRE reduced TUNEL and activated caspase 3 in both white and ventral gray matter as well as tumor necrosis factor immunoreactivity in ventral horn motorneurons, suggesting that NCX 1015 reduces SCI-induced apoptosis. The effect of NCX 1015 on motor function was then examined by a standard locomotion rating scale (BBB) starting at 1 day after injury and continuing up to 14 days. NCX 1015 improved significantly locomotor activity by 4 days after injury, whereas PRE had an effect equivalent to that of vehicle, thus providing a correlation between the antiapoptotic effect of NCX1015 and its ability to improve recovery of function. The data suggest that NCX 1015 might be a novel experimental therapeutic compound for recovery of function in SCI patients.

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is constitutively expressed in primary cultures of rat microglia, the main population of brain resident macrophages, and its ligand-dependent activation leads to the repression of several microglial functions. A few non-steroidal anti-inflammatory drugs (NSAIDs), e.g. indomethacin and ibuprofen, show PPAR-gamma agonistic properties. It has been proposed that PPAR-gamma activation contributes to the potential benefits of the long-term use of certain NSAIDs in delaying the progression of Alzheimer's disease (AD). Previous data have shown that the NSAID HCT1026 [2-fluoro-alpha-methyl(1,1'-biphenyl)4-acetic acid-4-(nitrooxy)butyl ester], a derivative of flurbiprofen which releases nitric oxide (NO), reduces the number of reactive microglial cells in a variety of models. This evidence together with the chemical analogy with ibuprofen led us to investigate whether flurbiprofen and HCT1026 interact with PPAR-gamma and interfere with microglial activation. We found that a low concentration (1 muM) of HCT1026, but not flurbiprofen, activated PPAR-gamma in primary cultures of rat microglia, with kinetics similar to those of the synthetic agonist ciglitazone. The PPAR-gamma antagonist GW9662 (2-chloro-5-nitrobenzanilide) prevented the activation of PPAR-gamma by HCT1026. Interestingly, unlike other NSAIDs that activate PPAR-gamma at concentrations higher than those required for cyclooxygenase inhibition, HCT1026 activated PPAR-gamma and inhibited prostaglandin E-2 synthesis at the same low concentration (1 muM). The results suggest that HCT1026 may exert additional anti-inflammatory actions through PPAR-gamma activation, allowing a more effective control of microglial activation and brain inflammation.

Summary.  Background: NO‐releasing statins are new chemical entities, combining HMG‐CoA reductase inhibition and slow NO release, that possess stronger anti‐inflammatory and antiproliferative activities than the native statins. Objective: We evaluated the antithrombotic effects of nitropravastatin (NCX‐6550) by assessing its activity on platelet activation and tissue factor (TF) expression by mononuclear cells in vitro and in vivo. Methods and results: In vitro, NCX‐6550 inhibited (1) U46619‐ and collagen‐induced platelet aggregation in buffer and plasma; (2) collagen‐induced P‐selectin expression in whole blood and (3) platelet adhesion to collagen‐coated coverslips under high shear stress. These effects were displayed at concentrations of NCX‐6550 ranging from 25 to 100 μm, and were totally reverted by the guanylylcyclase inhibitor ODQ (10 μm). Equimolar concentrations of pravastatin had no influence on these parameters of platelet function. LPS‐ and PMA‐induced TF expression by blood mononuclear cells was also inhibited by NCX‐6550 (IC50 13 μm), but not by pravastatin, as assessed by functional and immunological assays and by real‐time PCR. In a mouse model of platelet pulmonary thromboembolism, induced by the i.v. injection of collagen plus epinephrine, pretreatment with NCX‐6550 (24–48 mg kg−1) significantly reduced platelet consumption, lung vessel occlusion and mortality. Moreover, nitropravastatin markedly inhibited the generation of procoagulant activity by spleen mononuclear cells and peritoneal macrophages in mice treated with LPS. In these in vivo models too, pravastatin failed to affect platelet activation and monocyte/macrophage procoagulant activity. Conclusions: Our results show that nitropravastatin exerts strong antithrombotic effects in vitro and in vivo, and may represent an interesting antiatherothrombotic agent for testing in acute coronary syndromes.

The nitric oxide-releasing derivative of flurbiprofen, NCX 2216, has a safer gastrointestinal profile than the parent drug flurbiprofen and a strong anti-amyloidogenic activity. Here, we show that in primary microglial cultures, in addition to the expected inhibition of prostaglandin synthesis, NCX 2216 specifically activated the peroxisome proliferator-activated receptor-γ (PPAR-γ), a ligand-dependent transcription factor controlling several important microglial functions. Prolonged treatment (16 h) of microglial cultures with NCX 2216 induced PPAR-γ nitration and prevented further activation of the receptor by specific agonists. At functional levels, NCX 2216 treatment of LPS-activated microglial cultures resulted in the transient reduction of TNF-α and NO production and in the protracted inhibition of IL-1β and PGE2 synthesis. The dynamic regulation of the functional state of activated microglia by NCX 2216 helps explaining recent findings in Alzheimer's disease animal models and may offer new therapeutic opportunities for treating neurodegenerative diseases.

Currently, there is an intense debate on the potential use of nonsteroidal anti-inflammatory drugs (NSAIDs) in Alzheimer's disease (AD). NSAIDs are among the most widely prescribed drugs for the treatment of pain, fever, and inflammation. Their effects are largely attributed to the inhibition of the enzymatic activity of cyclooxygenase (COX)-1 and -2. The apparent activity of this class of drugs stems from one critical pathological process underlying AD and other neurodegenerative disorders, i.e., the presence of chronic neuroinflammation. In fact, prolonged use of NSAIDs is associated with reduced risk of AD. Besides COX inhibition, additional mechanisms could contribute to the potential activity of NSAIDs in AD. For example, several studies show that only a few selected NSAIDs also affect beta-amyloid (Abeta) deposition and metabolism. Among the Abeta-effective NSAIDs, flurbiprofen raised particular interest because of its multiple actions on key AD hallmarks. Studies in cell lines and animal models have shown that flurbiprofen racemate, its R-enantiomer and its nitric oxide (NO)-releasing derivatives, HCT 1026 and NCX 2216, are effective on AD amyloid pathology. Moreover, HCT 1026 and NCX 2216 differentially influence the cellular component of neuroinflammation (i.e., microglia activation) in some experimental settings, i.e., HCT 1026 inhibits the activation of microglia, while NCX 2216 can either enhance or inhibit microglial activation, depending upon the experimental conditions. It is still unclear which effects on microglia will prove most beneficial. Ultimately, clinical studies in AD patients will provide the best information as to whether selected NSAIDs will improve this devastating disease.