[show abstract][hide abstract] ABSTRACT: PURPOSE: Polymorphonuclear neutrophils, the first leukocytes to infiltrate the inflamed tissue, can make important contributions to vascular inflammatory processes driving the development of atherosclerosis. We herein investigated the effects of atorvastatin and NCX 6560 (a nitric oxide (NO)-donating atorvastatin derivative that has completed a successful phase 1b study) on neutrophilic inflammation in carotid arteries of normocholesterolemic rabbits subjected to perivascular collar placement. METHODS: Atorvastatin or NCX 6560 were administered orally (5 mg/kg/day or equimolar dose) to New Zealand White rabbits for 6 days, followed by collar implantation 1 h after the last dose. Twenty-four hours later carotids were harvested for neutrophil quantification by immunostaining. RESULTS: Treatment with NCX 6560 was associated with a lower neutrophil infiltration (-39.5 %), while atorvastatin did not affect neutrophil content. The result was independent of effects on plasma cholesterol or differences in atorvastatin bioavailability, which suggests an important role of NO-related mechanisms in mediating this effect. Consistent with these in vivo findings, in vitro studies showed that NCX 6560, as compared to atorvastatin, had greater inhibitory activity on processes involved in neutrophil recruitment, such as migration in response to IL-8 and IL-8 release by endothelial cells and by neutrophils themselves. Pretreatment with NCX 6560, but not with atorvastatin, reduced the ability of neutrophil supernatants to promote monocyte chemotaxis, a well-known pro-inflammatory activity of neutrophils. CONCLUSION: Experimental data suggest a potential role of NO-releasing statins in the control of the vascular inflammatory process mediated by polymorphonuclear neutrophils.
Cardiovascular Drugs and Therapy 02/2013; · 2.67 Impact Factor
[show abstract][hide abstract] ABSTRACT: INTRODUCTION: 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. METHODS: C57BL/6 mice received atorvastatin 40 mg/kg/day or an equivalent dose of NCX 6560 for 2 months. Muscle function assessed by treadmill test, serum creatine kinase (CK) activity, citrate synthase (CS) activity, and muscle histology were evaluated. RESULTS: Atorvastatin significantly (P < 0.001) reduced muscle endurance, increased serum CK by 6-fold, and induced muscle fiber atrophy. Conversely, NCX 6560 preserved muscle function, prevented CK increase and did not modify muscle structure. Interestingly, atorvastatin reduced CS activity, a marker for mitochondrial function, in gastrocnemius, diaphragm, and heart, whereas NCX 6560 prevented such decrease. CONCLUSIONS: These findings suggest that NO may prevent statin-induced myopathy. Muscle Nerve, 2012.
[show abstract][hide abstract] ABSTRACT: 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.
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).
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.
Cardiovascular research 02/2012; 94(3):428-38. · 5.80 Impact Factor
[show abstract][hide abstract] ABSTRACT: NCX 434 is a nitric oxide (NO)-donating triamcinolone acetonide (TA), shown to enhance optic nerve head (ONH) oxygen saturation in non-human primate eyes. Here, the effects of a single intravitreal (IVT) injection of TA were compared with those of NCX 434 on intraocular pressure (IOP), retinal function and retrobulbar haemodymamics in endothelin-1 (ET-1) induced ONH ischaemia/reperfusion in rabbits. Biochemical changes were also assessed in the aqueous humour and in retinal biopsies.
IOP and resistivity index of ophthalmic artery (RI-OA) were recorded using TonoPen and ecocolor Doppler, respectively. Retinal function was assessed using photopic electroretinography. Cytokine expression and oxidative stress markers were evaluated with immunoassay techniques.
At 4 weeks post IVT treatment, TA increased IOP and RI-OA while NCX 434 did not (IOP(Vehicle)=13.6±1.3, IOP(NCX 434)=16.9±2.2, IOP(TA)=20.9±1.9 mm Hg; p<0.05 vs vehicle; RI-OA(Vehicle)=0.44±0.03; RI-OA(NCX 434)=0.47±0.02; RI-OA(TA)=0.60±0.04). Both NCX 434 and TA reversed ET-1 induced decrease in electroretinography amplitude to similar extents. NCX 434 attenuated ET-1 induced oxidative stress markers and nitrotyrosine in retinal tissue, and interleukin-6 and tumour necrosis factor-α in aqueous humour more effectively than TA.
NCX 434 attenuates ET-1 induced ischaemia/reperfusion damage without increasing IOP, probably due to NO release. If data are confirmed in other species and models, this compound could represent an interesting new therapeutic option for retinal and ONH diseases, including diabetic retinopathy.
The British journal of ophthalmology 01/2012; 96(5):757-61. · 2.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: In patients with Duchenne muscular dystrophy (DMD) and the standard mdx mouse model of DMD, dystrophin deficiency causes loss of neuronal nitric oxide synthase (nNOSμ) from the sarcolemma, producing functional ischemia when the muscles are exercised. We asked if functional muscle ischemia would be eliminated and normal blood flow regulation restored by treatment with an exogenous nitric oxide (NO)-donating drug. Beginning at 8 weeks of age, mdx mice were fed a standard diet supplemented with 1% soybean oil alone or in combination with a low (15 mg/kg) or high (45 mg/kg) dose of HCT 1026, a NO-donating nonsteroidal anti-inflammatory agent which has previously been shown to slow disease progression in the mdx model. After 1 month of treatment, vasoconstrictor responses to intra-arterial norepinephrine (NE) were compared in resting and contracting hindlimbs. In untreated mdx mice, the usual effect of muscle contraction to attenuate NE-mediated vasoconstriction was impaired, resulting in functional ischemia: NE evoked similar decreases in femoral blood flow velocity and femoral vascular conductance (FVC) in the contracting compared to resting hindlimbs (ΔFVC contraction/ΔFVC rest = 0.88±0.03). NE-induced functional ischemia was unaffected by low dose HCT 1026 (ΔFVC ratio = 0.92±0.04; P>0.05 vs untreated), but was alleviated by the high dose of the drug (ΔFVC ratio = 0.22±0.03; P<0.05 vs untreated or low dose). The beneficial effect of high dose HCT 1026 was maintained with treatment up to 3 months. The effect of the NO-donating drug HCT 1026 to normalize blood flow regulation in contracting mdx mouse hindlimb muscles suggests a putative novel treatment for DMD. Further translational research is warranted.
PLoS ONE 01/2012; 7(11):e49350. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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).
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.
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.
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.
British Journal of Pharmacology 04/2011; 164(2b):570-83. · 5.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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).
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).
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.
Journal of ocular pharmacology and therapeutics: the official journal of the Association for Ocular Pharmacology and Therapeutics 03/2011; 27(2):115-21. · 1.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of the study was to investigate the ocular hypotensive activity of a nitric oxide (NO)-donating latanoprost, BOL-303259-X, following topical administration. The effect of BOL-303259-X (also known as NCX 116 and PF-3187207) on intraocular pressure (IOP) was investigated in monkeys with laser-induced ocular hypertension, dogs with naturally-occurring glaucoma and rabbits with saline-induced ocular hypertension. Latanoprost was used as reference drug. NO, downstream effector cGMP, and latanoprost acid were determined in ocular tissues following BOL-303259-X administration as an index of prostaglandin and NO-mediated activities. In primates, a maximum decrease in IOP of 31% and 35% relative to baseline was achieved with BOL-303259-X at doses of 0.036% (9 μg) and 0.12% (36 μg), respectively. In comparison, latanoprost elicited a greater response than vehicle only at 0.1% (30 μg) with a peak effect of 26%. In glaucomatous dogs, IOP decreased from baseline by 44% and 10% following BOL-303259-X (0.036%) and vehicle, respectively. Latanoprost (0.030%) lowered IOP by 27% and vehicle by 9%. Intravitreal injection of hypertonic saline in rabbits increased IOP transiently. Latanoprost did not modulate this response, whereas BOL-303259-X (0.036%) significantly blunted the hypertensive phase. Following BOL-303259-X treatment, latanoprost acid was significantly elevated in rabbit and primate cornea, iris/ciliary body and aqueous humor as was cGMP in aqueous humor. BOL-303259-X lowered IOP more effectively than latanoprost presumably as a consequence of a contribution by NO in addition to its prostaglandin activity. The compound is now in clinical development for the treatment of glaucoma and ocular hypertension.
Experimental Eye Research 03/2011; 93(3):250-5. · 3.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: The IOP lowering effects of NCX 139, a new chemical entity comprising latanoprost amide and a NO-donating moiety, were compared to those of the respective des-nitro analog in in vitro assays and in rabbit and dog models of ocular hypertension. The NO donor, molsidomine as well as the prostamide bimatoprost (Lumigan(®)) and the prostaglandin agonist, latanoprost (Xalatan(®)) were also investigated for comparison. NCX 139 but not its des-nitro analog resulted in NO-mediated vascular relaxant effect in pre-contracted rabbit aortic rings (EC(50)=0.70±0.06 μM; E(max)=80.6±2.9%). Like bimatoprost (IC(50)=3.07±1.3 μM) or latanoprost (IC(50)=0.48±0.15 μM), NCX 139 displaced (3)H-PGF2α binding on recombinant human prostaglandin-F (FP) receptors with an estimated potency of 0.77±0.13 μM. In transient ocular hypertensive rabbits, bimatoprost and latanoprost were not effective while molsidomine elicited a dose-dependent reduction of IOP confirming the responsiveness of rabbits to NO but not to FP receptor agonists. NCX 139 tested at a therapeutically relevant dose, significantly lowered IOP while the des-nitro analog was not effective (0.03% NCX 139, Δ(max)=-12.8±2.0 mmHg). In glaucomatous dogs, 0.03% NCX 139 decreased IOP to a greater extent compared to an equimolar dose of the respective des-nitro derivative (Δ(max)=-4.6±1.0 and -2.7±1.3 mmHg, respectively for NCX 139 and its des-nitro analog). Albeit with low potency, NCX 139 also resulted effective in normotensive dogs while it did not reduce IOP in normotensive rabbits. NCX 139, a compound targeting two different and important mechanisms, is endowed with ocular hypotensive effects more evident in hypertensive conditions which may be of interest in the search of more effective treatments for hypertensive glaucoma.
Experimental Eye Research 02/2011; 93(3):243-9. · 3.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Determine the cardio-protective properties of a nitric oxide-releasing pravastatin (Ncx-6550), in comparison to pravastatin.
A mouse model of myocardial infarct was used assessing tissue damage both at 2 and 24 hour post-reperfusion, administering compounds both prophylactically and therapeutically.
Ncx-6550 induced a significant dose-dependent (2.24-22.4 micromol/kg i.p.) cardioprotection in the two hour reperfusion protocol. In vehicle-treated mice, infarct size (expressed as fraction of area at risk; IS/AR) was 41.2 +/- 1%, and it was reduced to 22.2 +/- 0.9% and 32.6 +/- 0.9% following 22.4 and 6.72 micromol/kg Ncx-6550 (p < 0.05). 22.4 micromol/kg Ncx-6550 also increased cardiac levels of the enzyme heme oxygenase-1. Treatment of mice with pravastatin induced significant reduction of myocardial injury only at 22.4 micromol/kg (IS/AR value: 33.7 +/- 0.9%). In a 24 hour reperfusion protocol, Ncx-6550 and pravastatin were tested only at 22.4 micromol/kg i.p. being given either one hour prior to ischemia (prophylactic protocol) or one hour into reperfusion (therapeutic protocol). With either treatment scheme, Ncx-6550 produced higher cardioprotection compared to pravastatin, as reflected also by a reduction in the incidence of lethality as well as in circulating troponin I and interleukin-1beta levels.
These results indicate Ncx-6550 as a novel therapeutic agent with a potential for the treatment of myocardial infarct.
Microcirculation (New York, N.Y.: 1994) 08/2010; 17(6):417-26. · 2.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nitric oxide (NO) is involved in a variety of physiological processes including ocular aqueous humor dynamics by targeting mechanisms that are complementary to those of prostaglandins. Here, we have characterized a newly synthesized compound, NCX 125, comprising latanoprost acid and NO-donating moieties.
NCX 125 was synthesized and tested in vitro for its ability to release functionally active NO and then compared with core latanoprost for its intraocular pressure (IOP)-lowering effects in rabbit, dog, and nonhuman primate models of glaucoma.
NCX 125 elicited cGMP formation (EC(50) = 3.8 + or - 1.0 microM) in PC12 cells and exerted NO-dependent iNOS inhibition (IC(50) = 55 + or - 11 microM) in RAW 264.7 macrophages. NCX 125 lowered IOP to a greater extent compared with equimolar latanoprost in: (a) rabbit model of transient ocular hypertension (0.030% latanoprost, not effective; 0.039% NCX 125, Delta(max) = -10.6 + or - 2.3 mm Hg), (b) ocular hypertensive glaucomatous dogs (0.030% latanoprost, Delta(max)= -6.7 + or - 1.2 mm Hg; 0.039% NCX 125, Delta(max) = -9.1 + or - 3.1 mm Hg), and (c) laser-induced ocular hypertensive non-human primates (0.10% latanoprost, Delta(max) = -11.9 + or - 3.7 mm Hg, 0.13% NCX 125, Delta(max) = -16.7 + or - 2.2 mm Hg). In pharmacokinetic studies, NCX 125 and latanoprost resulted in similar latanoprost-free acid exposure in anterior segment ocular tissues.
NCX 125, a compound targeting 2 different mechanisms, is endowed with potent ocular hypotensive effects. This may lead to potential new perspectives in the treatment of patients at risk of glaucoma.
Journal of ocular pharmacology and therapeutics: the official journal of the Association for Ocular Pharmacology and Therapeutics 04/2010; 26(2):125-32. · 1.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: Adenosine, an ubiquitous neuromodulator, and its analogues have been shown to produce ‘depressant’ effects in animal models believed to be relevant to depressive disorders, while adenosine receptor antagonists have been found to reverse adenosine-mediated ‘depressant’ effect.We have designed studies to assess whether adenosine A2A receptor antagonists, or genetic inactivation of the receptor would be effective in established screening procedures, such as tail suspension and forced swim tests, which are predictive of clinical antidepressant activity.Adenosine A2A receptor knockout mice were found to be less sensitive to ‘depressant’ challenges than their wildtype littermates. Consistently, the adenosine A2A receptor blockers SCH 58261 (1 – 10 mg kg−1, i.p.) and KW 6002 (0.1 – 10 mg kg−1, p.o.) reduced the total immobility time in the tail suspension test.The efficacy of adenosine A2A receptor antagonists in reducing immobility time in the tail suspension test was confirmed and extended in two groups of mice. Specifically, SCH 58261 (1 – 10 mg kg−1) and ZM 241385 (15 – 60 mg kg−1) were effective in mice previously screened for having high immobility time, while SCH 58261 at 10 mg kg−1 reduced immobility of mice that were selectively bred for their spontaneous ‘helplessness’ in this assay.Additional experiments were carried out using the forced swim test. SCH 58261 at 10 mg kg−1 reduced the immobility time by 61%, while KW 6002 decreased the total immobility time at the doses of 1 and 10 mg kg−1 by 75 and 79%, respectively.Administration of the dopamine D2 receptor antagonist haloperidol (50 – 200 μg kg−1 i.p.) prevented the antidepressant-like effects elicited by SCH 58261 (10 mg kg−1 i.p.) in forced swim test whereas it left unaltered its stimulant motor effects.In conclusion, these data support the hypothesis that A2A receptor antagonists prolong escape-directed behaviour in two screening tests for antidepressants. Altogether the results support the hypothesis that blockade of the adenosine A2A receptor might be an interesting target for the development of effective antidepressant agents.British Journal of Pharmacology (2001) 134, 68–77; doi:10.1038/sj.bjp.0704240
British Journal of Pharmacology 02/2009; 134(1):68 - 77. · 5.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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).
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.
These findings indicate that incorporation of a bioactive NO moiety improves the therapeutic profile of statins for the treatment of peripheral vascular disease.
British Journal of Pharmacology 05/2007; 150(7):873-82. · 5.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: A novel series of 3-substituted-8-aryl-[1,2,4]-triazolo[5,1-i]purin-5-amine analogs related to Sch 58261 was synthesized in order to identify potent adenosine A(2A) receptor antagonists with improved selectivity over the A(1) receptor, physiochemical properties, and pharmacokinetic profiles as compared to those of Sch 58261. As a result of structural modifications, numerous analogs with excellent in vitro binding affinities and selectivities were identified. Moreover, compound 27 displayed both superior in vitro and highly promising in vivo profiles.
[show abstract][hide abstract] ABSTRACT: Antagonism of the adenosine A2A receptor offers great promise in the treatment of Parkinson's disease. Employing the known pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine A2A antagonist SCH 58261 as a starting point, we identified the potent and selective (vs. A1) antagonist 11h, orally active in the rat haloperidol-induced catalepsy model. We further optimized this lead to the methoxyethoxyethyl ether 12a (SCH 420814), which shows broad selectivity, good pharmacokinetic properties, and excellent in vivo activity.