Daniela Miglietta

Nitric oxide-donating carbonic anhydrase inhibitors for the treatment of open-angle glaucoma.

Novel bi-functional compounds with a nitric oxide (NO)-releasing moiety bound to a dorzolamide scaffold were investigated. Several compounds were synthesized and their activity as selective carbonic anhydrase inhibitors (CAI) evaluated in vitro on recombinant hCA type I, II and IV enzyme isoforms where they showed different degrees of potency and selectivity to hCA II. A high resolution X-ray crystal structure for the CA II adduct with 8 confirmed the high affinity of this class of compounds for the enzyme. Compounds 4, 6, and 8 showed highly potent and efficacious NO-mediated properties as assessed by their vascular relaxant effect on methoxamine-precontracted rabbit aortic rings. Finally, compounds 4 and 6 exerted potent intraocular pressure (IOP) lowering effects in vivo in normotensive rabbits thereby anticipating their potential for the treatment of hypertensive glaucoma.

Nitric oxide enhances the anti-inflammatory and anti-atherogenic activity of atorvastatin in a mouse model of accelerated atherosclerosis.

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.

Long-term treatment with naproxcinod significantly improves skeletal and cardiac disease phenotype in the mdx mouse model of dystrophy

In Duchenne muscular dystrophy (DMD) patients and the mouse model of DMD, mdx, dystrophin deficiency causes a decrease and mislocalization of muscle-specific neuronal nitric oxide synthase (nNOSμ), leading to functional impairments. Previous studies have shown that nitric oxide (NO) donation associated with anti-inflammatory action has beneficial effects in dystrophic mouse models. In this study, we have systematically investigated the effects of naproxcinod, an NO-donating naproxen derivative, on the skeletal and cardiac disease phenotype in mdx mice. Four-week-old mdx and C57BL/10 mice were treated with four different concentrations (0, 10, 21 and 41 mg/kg) of naproxcinod and 0.9 mg/kg of prednisolone in their food for 9 months. All mice were subjected to twice-weekly treadmill sessions, and functional and behavioral parameters were measured at 3, 6 and 9 months of treatment. In addition, we evaluated in vitro force contraction, optical imaging of inflammation, echocardiography and blood pressure (BP) at the 9-month endpoint prior to sacrifice. We found that naproxcinod treatment at 21 mg/kg resulted in significant improvement in hindlimb grip strength and a 30% decrease in inflammation in the fore- and hindlimbs of mdx mice. Furthermore, we found significant improvement in heart function, as evidenced by improved fraction shortening, ejection fraction and systolic BP. In addition, the long-term detrimental effects of prednisolone typically seen in mdx skeletal and heart function were not observed at the effective dose of naproxcinod. In conclusion, our results indicate that naproxcinod has significant potential as a safe therapeutic option for the treatment of muscular dystrophies.

Enhanced Oxygen Saturation in Optic Nerve Head of Non-Human Primate Eyes Following the Intravitreal Injection of NCX 434, an Innovative Nitric Oxide-Donating Glucocorticoid

PURPOSE Hypoxia of the retina and optic nerve head (ONH) is believed to be pivotal in the development of ocular vascular disorders, including diabetic macular edema (DME). Glucocorticoids are among the most effective agents for the treatment of back of the eye diseases. However, this class of compounds is highly liable to increase intraocular pressure (IOP) and does not improve ocular perfusion or tissue oxygenation. Nitric oxide (NO) has vasodilating properties and lowers IOP in experimental models and humans, suggesting that its properties might complement those of glucocorticoids. NCX 434 is an NO-donating triamcinolone acetonide (TA) that is less likely to increase IOP while targeting both the vascular and inflammatory components of DME. METHODS NCX 434 was studied in vitro with respect to its NO-releasing properties in isolated methoxamine-precontracted rabbit aortic rings and glucocorticoid-like activity in recombinant human glucocorticoid receptors. IOP and oxygen saturation in the ONH and overlaying arteries and veins were studied in the anesthetized cynomolgus monkey. Measurements were taken using, respectively, an applanation tonometer and a hyperspectral imaging system before and 7, 14, 21, 31 and 41 days after the intravitreal injection of NCX 434 (5.8 mg/eye) or TA equimolar doses (4.0 mg/eye). RESULTS NCX 434 inhibited (3)H-dexamethasone-specific binding (IC(50)=34±5 nM) on human glucocorticoid receptors and elicited NO-dependent aortic ring relaxation (EC(50) of 0.5±0.1 μM, E(max) 98.9%). In monkey eyes, NCX 434 enhanced, whereas TA did not, oxygen saturation in various ONH areas (*P<0.05 vs. basal), decreased it in veins, and did not affect it in the overlaying arteries. Neither NCX 434 nor TA altered IOP significantly at all time points. However, at 31 days post-treatment TA appeared to start increasing IOP (Δ(IOP)=+3.31±0.51 mmHg, 30.8%, over baseline, NS). CONCLUSIONS NCX 434 enhances ocular tissue oxygenation. This feature appears to depend on its NO-donating properties; thus, the compound deserves to be further investigated for the treatment of DME and other ocular disorders with impaired ocular perfusion.

NITRIC OXIDE PREVENTS ATORVASTATIN-INDUCED SKELETAL MUSCLE DYSFUNCTION AND ALTERATIONS IN MICE

Myopathy is the most common side effect of statins. Because nitric oxide (NO) has a key role in regulating skeletal muscle function, we studied whether the NO‐donating atorvastatin NCX 6560 could show a better profile on skeletal muscle function and structure compared with atorvastatin.

P.11.2 Naproxcinod, a nitric oxide-donating anti-inflammatory compound, is effective in two mouse models of muscle dystrophy

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.