2007 - 07

Eur J Pharm Sci. 2007 Jul;31(3-4):202-10. Epub 2007 Apr 1.

Binary polymeric blends to microencapsulate nitroflurbiprofen: physicochemical and in silico studies.

Cilurzo F, Selmin F, Vistoli G, Minghetti P, Montanari L. Istituto di Chimica Farmaceutica e Tossicologica P. Pratesi, Universita degli Studi di Milano, Milan, Italy.

Nitroflurbiprofen, NFP, a practically insoluble liquid drug, was microencapsulated in hydrophilic micromatrices made of poly(N-vinylpyrrolidone) (PVP), or polyaminomethacrylate (PAMA), or binary blends of polymers thereof. The PAMA/PVP miscibility was assessed both in the solid state (DSC and ATF-FTIR spectroscopy) and in solution by viscometric measurements. The in vitro NFP release test was carried out in over saturation condition to discriminate the increase of NFP apparent solubility (supersaturation degree, SD). Drug/polymer/polymer/water interactions were studied in silico by molecular dynamic (MD) simulations. PAMA and PVP resulted miscible only in aqueous solution. The release of NFP from microparticles occurred according to a non-monotonic pattern due to the formation of instable supersaturated systems and the drug separation in the dissolution medium. After 5 min, the SD was at least 3. The use of PVP/PAMA micromatrices reduced the instability of the supersaturated solutions. MD simulations evidenced that water molecules play a key role in the PAMA/PVP compatibilization process and in stabilization of NFP supersaturated systems by means of H-bond. The docking analyses here find a novel and successful application to predict the different ability of a drug to interact with polymeric blends in solution.

2007 - 04

Br J Pharmacol. 2007 Apr;150(7):873-82. Epub 2007 Mar 12.

Nitropravastatin stimulates reparative neovascularisation and improves recovery from limb Ischaemia in type-1 diabetic mice.

Emanueli C, Monopoli A, Kraenkel N, Meloni M, Gadau S, Campesi I, Ongini E, Madeddu P. Bristol Heart Institute, University of Bristol, Bristol, UK.

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. Invitro, 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.

Eur J Pharmacol. 2007 Apr 30;561(1-3):220-5. Epub 2007 Feb 1.

Effect of aspirin, paracetamol and their nitric oxide donating derivatives on exudate cytokine and PGE2 production in zymosan-induced air pouch inflammation in rats.

Mamuk S, Melli M. Ankara University, School of Medicine, Department of Pharmacology and Clinical Pharmacology, Morfoloji Binasi, Sihhiye 06100, Ankara, Turkey.

Effects of different doses of aspirin, compared to equimolar doses of nitric oxide (NO)-donating aspirin (NCX 4016), and of a single dose of paracetamol, compared to an equimolar dose of NO-donating paracetamol (NCX 701) were investigated in acute zymosan-induced air pouch inflammation in rats. Treatments were administered by orogastric route, and interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and prostaglandin E(2) (PGE(2)) levels in the exudates were analysed 4 h after zymosan injection by enzyme immunoassay (EIA). Aspirin, at 10, 30 and 100 mg/kg doses, increased IL-1beta levels in exudates, however, only the highest dose lead to a significant increase when compared to control, whereas a significant increase in TNF-alpha level was observed at all doses tested. NCX 4016, at equimolar doses for aspirin, i.e., 18.6, 55.8 and 186 mg/kg, respectively, did not cause any changes in exudate IL-1beta or TNF-alpha levels. These effects were significantly different, when aspirin was compared with the corresponding NCX 4016 group. Nevertheless, the ability of aspirin and NCX 4016 to inhibit PGE(2) synthesis in the exudate where comparable. Although paracetamol significantly increased exudate TNF-alpha level compared to the control group and NCX 701 group, neither paracetamol, nor NCX701 treatments changed the levels of exudate IL-1beta significantly. As expected, paracetamol and NCX 701 showed poor PGE(2) inhibition. At high doses, aspirin and NCX 4016 decreased the number of polymorphonuclear leukocytes in the exudate. However, this inhibition was not significantly different from the control group. Paracetamol and NO-paracetamol did not cause any change in the number of polymorphonuclear leukocytes in exudate. These results indicated that aspirin and NCX 4016 possessed different effects on cytokine production or release, despite the fact that both drugs inhibited the synthesis of PGE(2) in a similar way. Unlike paracetamol, which increased exudate TNF-alpha level, NCX 701 had no effect on TNF-alpha level in the exudates.

J Thromb Thrombolysis. 2007 Apr;23(2):129-33. Epub 2007 Jan 13.

Tissue factor and nitric oxide: a controversial relationship!

Dusse LM, Cooper AJ, Lwaleed BA. University of Southampton, Southampton, UK.

Tissue factor (TF) is the primary physiological initiator of blood coagulation. TF has a high-affinity for factor (F) VII resulting in the formation of (TF:FVII:FVIIa) bimolecular complex which, in the presence of Ca(2+), increases the enzymatic activity of FVIIa towards its natural substrates, FIX and FX, generating their active forms FIXa and FXa, respectively. This eventually leads to thrombin generation and a fibrin clot formation. Up-regulation of TF in injured blood vessels and atherosclerotic plaque can lead to undesirable vascular thrombosis. Nitric oxide (NO) is a free radical synthesized from L-arginine and molecular oxygen by nitric oxide synthases (NOS). NO participates in diverse physiological and pathophysiological process as an intra or extracellular messenger. A relationship between TF and NO has been proposed. Thus, models of TF regulation by NO has been studied in different cells and experimental animal models, but the results have been conflicting. The premise that NO donors can prevent TF expression in vivo has provided the foundation for a broad field of pharmacotherapeutics in vascular medicine. A new class of drugs combining a statin (inhibitors of coenzyme A reductase) with an NO-donating moiety has been described. The resulting drug, nitrostatin, has been suggested to increase the antithrombotic effects of native statin. However, it is questionable if NO release from these drugs had any significant role on TF inhibition. In summary, care must be taken in drawing conclusions about the relationship between NO and TF. Interpretation of NO studies must take several factors into consideration, including NO bioavailability, its half-life and inactivation, as well as the cell type and experimental model used.

2007 - 02

Gastroenterology. 2007 Feb;132(2):709-19. Epub 2006 Dec 20.

Nitroflurbiprofen, a nitric oxide-releasing cyclooxygenase inhibitor, improves cirrhotic portal hypertension in rats.

Laleman W, Van Landeghem L, Van der Elst I, Zeegers M, Fevery J, Nevens F. Department of Hepatology, University Hospital Gasthuisberg, Leuven, Belgium.

BACKGROUND & AIMS: We studied whether administration of nitroflurbiprofen (HCT-1026), a cyclooxygenase inhibitor with nitric oxide (NO)-donating properties, modulates the increased intrahepatic vascular tone in portal hypertensive cirrhotic rats. METHODS: In vivo hemodynamic measurements (n = 8/condition) and evaluation of the increased intrahepatic resistance by in situ perfusion (n = 5/condition) were performed in rats with thioacetamide-induced cirrhosis that received either nitroflurbiprofen (45 mg/kg), flurbiprofen (30 mg/kg, equimolar concentration to nitroflurbiprofen), or vehicle by intraperitoneal injection 24 hours and 1 hour prior to the measurements. Additionally, we evaluated the effect of acute administration of both drugs (250 micromol/L) on the intrahepatic vascular tone in the in situ perfused cirrhotic rat liver (endothelial dysfunction and hyperresponsiveness to methoxamine) and on hepatic stellate cell contraction in vitro. Typical systemic adverse effects of nonsteroidal anti-inflammatory drugs, such as gastrointestinal ulceration, renal insufficiency, and hepatotoxicity, were actively explored. RESULTS: In vivo, nitroflurbiprofen and flurbiprofen equally decreased portal pressure (8 +/- 0.8 and 8.4 +/- 0.1 mm Hg, respectively, vs 11.8 +/- 0.6 mm Hg) and reduced the total intrahepatic vascular resistance. Systemic hypotension was not aggravated in the different treatment groups (P = .291). In the perfused cirrhotic liver, both drugs improved endothelial dysfunction and hyperresponsiveness. This was associated with a decreased hepatic thromboxane A(2)-production and an increased intrahepatic nitrate/nitrite level. In vitro, nitroflurbiprofen, more than flurbiprofen, decreased hepatic stellate cells contraction. Flurbiprofen-treated rats showed severe gastrointestinal ulcerations (bleeding in 3/8 rats) and nefrotoxicity, which was not observed in nitroflurbiprofen-treated cirrhotic rats. CONCLUSIONS: Treatment with nitroflurbiprofen, an NO-releasing cyclooxygenase inhibitor, improves portal hypertension without major adverse effects in thioacetamide-induced cirrhotic rats by attenuating intrahepatic vascular resistance, endothelial dysfunction, and hepatic hyperreactivity to vasoconstrictors.

Neuroscience. 2007 Feb 9;144(3):950-60. Epub 2006 Nov 28.

Amyloid-beta vaccination, but not nitro-nonsteroidal anti-inflammatory drug treatment, increases vascular amyloid and microhemorrhage while both reduce parenchymal amyloid.

Wilcock DM, Jantzen PT, Li Q, Morgan D, Gordon MN. Department of Molecular Pharmacology and Physiology, School of Basic Biomedical Science, College of Medicine, University of South Florida, 12901 Bruce B. Downs Boulevard, MDC Box 9, Tampa, FL 33612, USA.

Vaccination with Abeta(1-42) and treatment with NCX-2216, a novel nitric oxide releasing flurbiprofen derivative, have each been shown separately to reduce amyloid deposition in transgenic mice and have been suggested as potential therapies for Alzheimer's disease. In the current study we treated doubly transgenic amyloid precursor protein and presenilin-1 (APP+PS1) mice with Abeta(1-42) vaccination, NCX-2216 or both drugs simultaneously for 9 months. We found that all treatments reduced amyloid deposition, both compact and diffuse, to the same extent while only vaccinated animals, with or without nonsteroidal anti-inflammatory drug (NSAID) treatment, showed increased microglial activation associated with the remaining amyloid deposits. We also found that active Abeta vaccination resulted in significantly increased cerebral amyloid angiopathy and associated microhemorrhages, while NCX-2216 did not, in spite of similar reductions in parenchymal amyloid. Co-administration of NCX-2216 did not attenuate this effect of the vaccine. This is the first report showing that active immunization can result in increased vascular amyloid and microhemorrhage, as has been observed with passive immunization. Co-administration of an NSAID agent with Abeta vaccination does not substantially modify the effects of Abeta immunotherapy. The difference between these treatments with respect to vascular amyloid development may reflect the clearance-promoting actions of the vaccine as opposed to the production-modifying effects proposed for flurbiprofen.

Can J Physiol Pharmacol. 2007 Feb;85(2):233-42.

Combining ursodeoxycholic acid or its NO-releasing derivative NCX-1000 with lipophilic antioxidants better protects mouse hepatocytes against amiodarone toxicity.

Ouazzani-Chahdi A, Elimadi A, Chabli A, Spanard J, Colin P, Haddad PS. Department of Pharmacology, Université de Montréal, Montréal, Canada.

Nonalcoholic steatohepatitis (NASH) is a common and potentially severe form of liver disease. This study aimed to determine the effect of ursodeoxycholic acid and its NO-releasing derivative NCX-1000 alone or in combination with antioxidants on cultured mouse hepatocytes treated with amiodarone to mimic certain aspects of hepatocyte injury found in NASH. Isolated mouse hepatocytes were incubated with ursodeoxycholic acid or NCX-1000 (0-100 micromol/L) combined or not combined with the hydrophilic antioxidants butylated hydroxytoluene and ascorbic acid (0-100 micromol/L) or with the lipophilic antioxidant alpha-tocopherol (0-100 micromol/L) 15 min before adding amiodarone (50 micromol/L) to the culture medium. Twenty hours later, necrosis, apoptosis, superoxide anion production, and malondialdehyde levels were assessed in cultured cells. Amiodarone led to a dose-dependent decrease in cell viability with an LD50 of 50 micromol/L and increased production of superoxide anion and lipid peroxidation. NCX-1000 showed a better protective potential than ursodeoxycholic acid against the toxic effects of amiodarone. The hydrophilic antioxidants had no effect on the toxicity of amiodarone, whereas alpha-tocopherol at a concentration >100 micromol/L almost completely suppressed it. Ursodeoxycholic acid and NCX-1000 protection was additive only when they were combined with alpha-tocopherol, not with butylated hydroxytoluene or ascorbic acid. In addition, all the antioxidants tested reduced the superoxide anion detected, but only alpha-tocopherol prevented lipid peroxidation induced by amiodarone. The combination of lipophilic antioxidants with ursodeoxycholic acid or NCX-1000 enhances their protective potential and could represent an interesting therapeutic approach to explore for the treatment of NASH.

2007 - 02

J Pharmacol Exp Ther. 2007 Jan;320(1):419-26. Epub 2006 Sep 27.

The nitric oxide-donating pravastatin derivative, NCX 6550 [(1S-[1alpha(betaS*, deltaS*), 2alpha, 6alpha, 8beta-(R*), 8a alpha]]-1,2,6,7,8,8a-Hexahydro-beta, delta, 6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphtalene-heptanoic acid 4-(nitrooxy)butyl ester)], reduces splenocyte adhesion and reactive oxygen species generation in normal and atherosclerotic mice.

Dever G, Spickett CM, Kennedy S, Rush C, Tennant G, Monopoli A, Wainwright CL. Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom.

Statins possess anti-inflammatory effects that may contribute to their ability to slow atherogenesis, whereas nitric oxide (NO) also influences inflammatory cell adhesion. This study aimed to determine whether a novel NO-donating pravastatin derivative, NCX 6550 [(1S-[1alpha(betaS*,deltaS*),2alpha,6alpha,8beta-(R*),8a alpha]]-1,2,6,7,8,8a-hexahydro-beta,delta,6-trihydroxy-2-methyl-8-(2-methyl-1-oxo butoxy)-1-naphthalene-heptanoic acid 4-(nitrooxy)butyl ester)], has greater anti-inflammatory properties compared with pravastatin in normal and atherosclerotic apolipoprotein E receptor knockout (ApoE-/-) mice. C57BL/6 and ApoE-/- mice were administered pravastatin (40 mg/kg), NCX 6550 (48.5 mg/kg), or vehicle orally for 5 days. Ex vivo studies assessed splenocyte adhesion to arterial segments and splenocyte reactive oxygen species (ROS) generation. NCX 6550 significantly reduced splenocyte adhesion to artery segments in both C57BL/6 (8.8 +/- 1.9% versus 16.6 +/- 6.7% adhesion; P < 0.05) and ApoE-/- mice (9.3 +/- 2.9% versus 23.4 +/- 4.6% adhesion; P < 0.05) concomitant with an inhibition of endothelial intercellular adhesion molecule-1 expression. NCX 6550 also significantly reduced phorbol 12-myristate 13-acetate-induced ROS production that was enhanced in isolated ApoE-/- splenocytes. Conversely, pravastatin had no significant effects on adhesion in normal or ApoE-/- mice but reduced the enhanced ROS production from ApoE-/- splenocytes. In separate groups of ApoE-/- mice, NCX 6550 significantly enhanced endothelium-dependent relaxation to carbachol in aortic segments precon-tracted with phenylephrine (-logEC(50), 6.37 +/- 0.37) compared with both vehicle-treated (-logEC50, 5.81 +/- 0.15; P < 0.001) and pravastatin-treated (-logEC50, 5.57 +/- 0.45; P < 0.05) mice. NCX 6550 also significantly reduced plasma monocyte chemoattractant protein-1 levels (648.8 pg/ml) compared with both vehicle (1191.1 pg/ml; P < 0.001) and pravastatin (847 +/- 71.0 pg/ml; P < 0.05) treatment. These data show that NCX 6550 exerts superior anti-inflammatory actions compared with pravastatin, possibly through NO-related mechanisms.

Proc Natl Acad Sci U S A. 2007 Jan 2;104(1):264-9. Epub 2006 Dec 20.

Nitric oxide release combined with nonsteroidal antiinflammatory activity prevents muscular dystrophy pathology and enhances stem cell therapy.

Brunelli S, Sciorati C, D'Antona G, Innocenzi A, Covarello D, Galvez BG, Perrotta C, Monopoli A, Sanvito F, Bottinelli R, Ongini E, Cossu G, Clementi E. Department of Experimental Medicine, University of Milano-Bicocca, 20052 Monza, Italy.

Duchenne muscular dystrophy is a relatively common disease that affects skeletal muscle, leading to progressive paralysis and death. There is currently no resolutive therapy. We have developed a treatment in which we combined the effects of nitric oxide with nonsteroidal antiinflammatory activity by using HCT 1026, a nitric oxide-releasing derivative of flurbiprofen. Here, we report the results of long-term (1-year) oral treatment with HCT 1026 of two murine models for limb girdle and Duchenne muscular dystrophies (alpha-sarcoglycan-null and mdx mice). In both models, HCT 1026 significantly ameliorated the morphological, biochemical, and functional phenotype in the absence of secondary effects, efficiently slowing down disease progression. HCT 1026 acted by reducing inflammation, preventing muscle damage, and preserving the number and function of satellite cells. HCT 1026 was significantly more effective than the corticosteroid prednisolone, which was analyzed in parallel. As an additional beneficial effect, HCT 1026 enhanced the therapeutic efficacy of arterially delivered donor stem cells, by increasing 4-fold their ability to migrate and reconstitute muscle fibers. The therapeutic strategy we propose is not selective for a subset of mutations; it provides ground for immediate clinical experimentation with HCT 1026 alone, which is approved for use in humans; and it sets the stage for combined therapies with donor or autologous, genetically corrected stem cells.

2006 - 11

Br J Pharmacol. 2006 Nov;149(5):516-22. Epub 2006 Aug 29.

Effect of a nitric oxide releasing derivative of paracetamol in a rat model of endotoxaemia.

Marshall M, Keeble J, Moore PK. King's College London, Cardiovascular Division, UK.

BACKGROUND AND PURPOSE: Nitroparacetamol is a nitric oxide-releasing paracetamol with novel anti-inflammatory properties compared to the parent compound. This study has investigated the anti-inflammatory activity of nitroparacetamol in a model of endotoxaemia in rats to probe the mechanisms underlying this effect. EXPERIMENTAL APPROACH: Nitroparacetamol (92 mg kg(-1)), paracetamol (50 mg kg(-1)) or vehicle were administered to male, Wistar rats 15 min prior to or 3 h after lipopolysaccharide (0.5 mg kg(-1), serotype 0127:B8). Mean arterial pressure and heart rate were measured for 5 h and plasma and organs were then obtained to determine organ dysfunction, inducible nitric oxide synthase and cyclooxygenase-2 expression (lung, liver and kidney tissue) and plasma nitrate/nitrite. In separate experiments, nitroparacetamol, paracetamol or vehicle was administered 1 h before acetylcholine (0.1 microg kg(-1)) or sodium nitroprusside (0.25 microg kg(-1)) to determine if nitroparacetamol desensitizes responses to exogenous/endogenous nitric oxide. KEY RESULTS: Nitroparacetamol prevented but did not reverse the lipopolysaccharide-induced hypotension. There was no effect on heart rate or plasma markers of organ dysfunction. Nitroparacetamol prevented the increased plasma nitrate/nitrite and expression of COX-2 and iNOS, whereas paracetamol exerted partial inhibition of COX-2 in lung alone. Nitroparacetamol also reduced responses to acetylcholine and sodium nitroprusside. CONCLUSIONS AND IMPLICATIONS: NO is the active component of nitroparacetamol in this model of endotoxaemia. Pro-inflammatory processes targeted by nitroparacetamol have been shown to include iNOS/COX-2 induction and possibly vascular soluble guanylyl cyclase. Precise mechanisms underlying the NO effect are unclear but inhibition of cytokine formation may be important.

Inflamm Res. 2006 Nov;55(11):498-503.

Flurbiprofen and its nitric oxide-releasing derivative protect against septic shock in rats.

Anuar F, Whiteman M, Bhatia M, Moore PK. Department of Pharmacology, National University of Singapore, Block MD2, 18 Medical Drive, Singapore, 117597, Singapore.

OBJECTIVE: Flurbiprofen and nitroflurbiprofen were evaluated in a caecal ligation puncture (CLP) model of septic shock in the rat. METHODS AND RESULTS: CLP (12 h) reduced blood pressure (72.5 +/- 1.0 mm Hg c. f. 101.0 +/- 3.6 mm Hg, P < 0.05), and increased plasma NOx (153.0 +/- 11.5 muM c. f. 36.2 +/- 3.2 microM, P < 0.05), IL-1beta (534.0 +/- 93.1 pg/mL c. f.; 9.6 +/- 9.6 pg/mL, P < 0.05), TNF-alpha (88.0 +/- 13.6 pg/mL, P < 0.05), inflammatory damage in lung and liver,and mortality. Both flurbiprofen (21 mg/kg, p. o.) and nitroflurbiprofen (30 mg/kg, p. o.) prevented the fall in blood pressure (e. g. 80.4 +/- 2.1 mm Hg and 79.8 +/- 1.2 mm Hg respectively, 12 h, P < 0.05), reduced organ damage and prolonged survival. Nitroflurbiprofen (but not flurbiprofen) increased plasma NOx and reduced plasma TNF-alpha concentration at all time points (except 1 h). Neither drug affected plasma IL-1beta-levels. CONCLUSIONS: These results suggest a protective effect of flurbiprofen and nitroflurbiprofen in septic shock.

2006 - 10

Drug News Perspect. 2006 Oct;19(8):485-9.

Chronicles in drug discovery.

Moral MA, Khurdayan VK, Bozzo J. Prous Science Medical Information Department, Barcelona, Spain.

Chronicles in Drug Discovery features special interest reports on advances in drug discovery and development. This month we focus on the progress of the ongoing search for safe and effective chemopreventive agents. Chemoprevention is a strategy to decrease the risk of developing cancer by using agents that prevent or abrogate carcinogenic processes. Bowman- Birk inhibitor concentrate, budesonide, NCX-4016 and statins are all undergoing investigation in the clinical setting as potential chemopreventive agents for head and neck, lung, colon and breast cancers, respectively.

2006 - 09

Neuropharmacology. 2006 Sep;51(4):858-65. Epub 2006 Jul 25.

Enhancement of the analgesic activity of paracetamol and nitroparacetamol by the oral administration of all-trans retinoic acid.

Alfonso Romero-Sandoval E, Molina C, Herrero JF. Departamento de Fisiologia, Facultad de Medicina, Campus Universitario, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain.

All-trans retinoic acid (ATRA), the active metabolite of vitamin A, is involved in the inflammatory reaction and modulates the expression of cyclooxygenase (COX) enzymes and nitric oxide (NO) activity. Since COX enzymes are the substrate of action of COX inhibitors, we studied the analgesic activity of paracetamol (PAR) and its NO-derivative nitroparacetamol (NOP) in the presence and absence of oral ATRA. Nociceptive responses were studied using the recording of single motor units technique in alpha-chloralose anesthetized normal and monoarthritic male Wistar rats. Intravenous PAR was not effective in normal rats. However, after pre-treatment with ATRA, PAR reduced dose-dependently the responses to noxious mechanical stimulation (ID50: 60+/-7 micromol/kg; 9.1 mg/kg), but not wind-up. The analgesic activity of NOP was enhanced after pre-treatment with ATRA either on responses to noxious mechanical stimulation (ID50s: 147+/-2 vs. 46+/-2 micromol/kg) or wind-up (maximal effect of 46+/-1% with 480 micromol/kg vs. 33+/-3% of control with 240 micromol/kg). The administration of ATRA did not modify the effect of PAR and NOP in monoarthritic rats. We conclude that pre-treatment with oral ATRA enhances the analgesic activity of PAR and NOP in acute pain, probably due to a positive modulation of their activity on spinal cord COX enzymes.

2006 - 05

J Pharmacol Exp Ther. 2006 May;317(2):752-61. Epub 2006 Jan 19.

Erratum in: J Pharmacol Exp Ther. 2007 Jan;320(1):497.

In vitro metabolism of (nitrooxy)butyl ester nitric oxide-releasing compounds: comparison with glyceryl trinitrate.

Govoni M, Casagrande S, Maucci R, Chiroli V, Tocchetti P. Department of Drug Metabolism and Pharmacokinetics, NicOx Research Institute, Via Ariosto 21, 20091 Bresso, Milan, Italy.

We investigated the in vitro metabolism of two (nitrooxy)butyl ester nitric oxide (NO) donor derivatives of flurbiprofen and ferulic acid, [1,1'-biphenyl]-4-acetic acid-2-fluoro-alpha-methyl-4-(nitrooxy)butyl ester (HCT 1026) and 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid 4-(nitrooxy)butyl ester (NCX 2057), respectively, in rat blood plasma and liver subcellular fractions compared with (nitrooxy)butyl alcohol (NOBA) and glyceryl trinitrate (GTN). HCT 1026 and NCX 2057 undergo rapid ubiquitous carboxyl ester hydrolysis to their respective parent compounds and NOBA. The nitrate moiety of this latter is subsequently metabolized to inorganic nitrogen oxides (NOx), predominantly in liver cytosol by glutathione S-transferase (GST) and to a lesser extent in liver mitochondria. If, however, in liver cytosol, the carboxyl ester hydrolysis is prevented by an esterase inhibitor, the metabolism at the nitrate moiety level does not occur. In blood plasma, HCT 1026 and NCX 2057 are not metabolized to NOx, whereas a slow but sustained NO generation in deoxygenated whole blood as detected by electron paramagnetic resonance indicates the involvement of erythrocytes in the bioactivation of these compounds. Differently from NOBA, GTN is also metabolized in blood plasma and more quickly metabolized by different GST isoforms in liver cytosol. The cytosolic GST-mediated denitration of these organic nitrates in liver limits their interaction with other intracellular compartments to possible generation of NO and/or their subsequent availability and bioactivation in the systemic circulation and extrahepatic tissues. We show the possibility of modulating the activity of hepatic cytosolic enzymes involved in the metabolism of (nitrooxy)butyl ester compounds, thus increasing the therapeutic potential of this class of compounds.

2006 - 04

Inflamm Allergy Drug Targets. 2006 Apr;5(2):115-9.

Nitric oxide and inflammation.

Cirino G, Distrutti E, Wallace JL. Department of Experimental Pharmacology, Universita di Napoli-Federico II, Napoli, Italy.

There are several pre-clinical studies on the involvement of NO in inflammation. From this large amount of information it is clear that virtually every cell and many immunological parameters are modulated by NO. Thus, the final outcome is that NO cannot be rigidly classified as an anti-inflammatory or pro-inflammatory molecule. This peculiar aspect of the pathophysiology of NO has hampered the development of new drugs based on the concepts developed. Recent therapeutic approach are targeted to increase endogenous NO by activating the gene and some promising early data are available. At the present stage one of the most promising approach in the inflammation field is represented by a new class of NO-releasing compounds namely NO-NSAIDs that have recently enrolled in phase 2 clinical studies.

Neurobiol Dis. 2006 Apr;22(1):25-32. Epub 2005 Nov 22.

Dynamic regulation of microglial functions by the non-steroidal anti-inflammatory drug NCX 2216: implications for chronic treatments of neurodegenerative diseases.

Bernardo A, Gasparini L, Ongini E, Minghetti L. Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome, Italy.

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-gamma (PPAR-gamma), a ligand-dependent transcription factor controlling several important microglial functions. Prolonged treatment (16 h) of microglial cultures with NCX 2216 induced PPAR-gamma 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-alpha and NO production and in the protracted inhibition of IL-1beta 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.

Br J Pharmacol. 2006 Apr;147(8):966-74.

Nitric oxide-releasing flurbiprofen reduces formation of proinflammatory hydrogen sulfide in lipopolysaccharide-treated rat.

Anuar F, Whiteman M, Siau JL, Kwong SE, Bhatia M, Moore PK. Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD2, 18 Medical Drive, Singapore 117597, Singapore.

The biosynthesis of both nitric oxide (NO) and hydrogen sulfide (H2S) is increased in lipopolysaccharide (LPS)-injected mice and rats but their interaction in these models is not known. In this study we examined the effect of the NO donor, nitroflurbiprofen (and the parent molecule flurbiprofen) on NO and H2S metabolism in tissues from LPS-pretreated rats. Administration of LPS (10 mg kg(-1), i.p.; 6 h) resulted in an increase (P<0.05) in plasma TNF-alpha, IL-1beta and nitrate/nitrite (NO(x)) concentrations, liver H2S synthesis (from added cysteine), CSE mRNA, inducible nitric oxide synthase (iNOS), myeloperoxidase (MPO) activity (marker for neutrophil infiltration) and nuclear factor-kappa B (NF-kappaB) activation. Nitroflurbiprofen (3-30 mg kg(-1), i.p.) administration resulted in a dose-dependent inhibition of the LPS-mediated increase in plasma TNF-alpha, IL-1beta and NO(x) concentration, liver H2S synthesis (55.00+/-0.95 nmole mg protein(-1), c.f. 62.38+/-0.47 nmole mg protein(-1), n = 5, P<0.05), CSE mRNA, iNOS, MPO activity and NF-kappaB activation. Flurbiprofen (21 mg kg(-1), i.p.) was without effect. These results show for the first time that nitroflurbiprofen downregulates the biosynthesis of proinflammatory H2S and suggest that such an effect may contribute to the augmented anti-inflammatory activity of this compound. These data also highlight the existence of 'crosstalk' between NO and H2S in this model of endotoxic shock.

2006 - 02

Am J Physiol Heart Circ Physiol. 2006 Feb;290(2):H517-24. Epub 2005 Sep 19.

Cardioprotective effects of nitroparacetamol and paracetamol in acute phase of myocardial infarction in experimental rats.

Zhu YZ, Chong CL, Chuah SC, Huang SH, Nai HS, Tong HT, Whiteman M, Moore PK. Dept. of Pharmacology, National Univ. of Singapore, Singapore 117597.

We aimed to determine whether nitroparacetamol (NO-paracetamol) and paracetamol exhibit cardioprotective effects. Myocardial infarction (MI) was induced in rats, and drug treatment was started 1 wk before surgery. Mortality rate and infarct size at 2 days after MI were compared. Treatment groups included vehicle (saline), paracetamol (5 mg x kg(-1) x day(-1)) and NO-paracetamol (15 mg x kg(-1) x day(-1)). Mortality rates for vehicle (n = 80), paracetamol (n = 79), and NO-paracetamol (n = 76) groups were 37.5%, 21.5%, and 26.3%, respectively. Infarct size for the vehicle group was 44.8% (+/-6.1%) of the left ventricle (LV). For the paracetamol and NO-paracetamol groups, infarct size was 31.3% (+/-5.6%) and 30.7% (+/-8.1%) of the LV, respectively. Both paracetamol- and NO-paracetamol-treated groups showed increased activities of catalase and SOD compared with the vehicle group. They could attenuate endothelial, inducible, and neuronal nitric oxide synthase and cyclooxygenase-1 and -2 gene expression after MI. The observation indicates the potential clinical significance of the cardioprotective effects of these drugs.

2005 - 11

J Thromb Haemost. 2005 Nov;3(11):2554-62.

A novel nitric oxide-releasing statin derivative exerts an antiplatelet/antithrombotic activity and inhibits tissue factor expression.

Rossiello MR, Momi S, Caracchini R, Giannini S, Guglielmini G, Monopoli A, Ongini E, Semeraro N, Colucci M, Gresele P. Department of Biomedical Sciences, Section of General Pathology, University of Bari, Bari, Italy.

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 mum, and were totally reverted by the guanylylcyclase inhibitor ODQ (10 microm). 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 microm), 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.

2005 - 09

Br J Pharmacol. 2005 Sep;146(1):109-17.

Sildenafil citrate and sildenafil nitrate (NCX 911) are potent inhibitors of superoxide formation and gp91phox expression in porcine pulmonary artery endothelial cells.

Muzaffar S, Shukla N, Srivastava A, Angelini GD, Jeremy JY. Department of Cardiac Surgery, Bristol Heart Institute, Bristol Royal Infirmary, University of Bristol, UK.

Acute respiratory distress syndrome (ARDS) is associated with increased superoxide (O(2)(*-)) formation in the pulmonary vasculature and negation of the bioavailability of nitric oxide (NO). Since NO inhibits NADPH oxidase expression through a cyclic GMP-mediated mechanism, sildenafil, a type V phosphodiesterase inhibitor, may be therapeutically effective in ARDS through an augmentation of NO-mediated inhibition of NADPH oxidase. Therefore, the effect of sildenafil citrate and NO-donating sildenafil (NCX 911) on O(2)(*-) formation and gp91(phox) (active catalytic subunit of NADPH oxidase) expression was investigated in cultured porcine pulmonary artery endothelial cells (PAECs). PAECs were incubated with 10 nM TXA(2) analogue, 9,11-dideoxy-9alpha,11alpha-methanoepoxy-prostaglandin F(2alpha) (U46619) (+/-sildenafil or NCX 911), for 16 h and O(2)(*-) formation measured spectrophometrically and gp91(phox) using Western blotting. The role of the NO-cGMP axis was studied using morpholinosydnonimine hydrochloride (SIN-1), the diethylamine/NO complex (DETA-NONOate), the guanylyl cyclase inhibitor, 1H-{1,2,4}oxadiazolo{4,3-a}quinoxalin-1-one (ODQ), and the protein kinase G inhibitor, 8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-cGMPS). NO release was studied using a fluorescence assay and O(2)(*-)-NO interactions by measuring nitrites. After a 16-h incubation with 10 nM U46619, both NCX 911 and sildenafil elicited a concentration-dependent inhibition of O(2)(*-) formation and gp91(phox) expression, NCX 911 being more potent (IC(50); 0.26 nM) than sildenafil citrate (IC(50); 1.85 nM). These inhibitory effects were reversed by 1 microM ODQ and 10 microM Rp-8-Br-cGMPS. NCX 911 stimulated the formation of cGMP in PAECs and generated NO in a cell-free system to a greater degree than sildenafil citrate. The inhibitory effect of sildenafil was augmented by 1 muM SIN-1 and blocked partially by the eNOS inhibitor 10 microM N(5)-(1-iminoethyl)-ornithine (L-NIO). Acutely, sildenafil and NCX 911 also inhibited O(2)(*-) formation, again blocked by 1 microM ODQ. NCX 911 reacted with O(2)(*-) generated by xanthine oxidase, an effect that was inhibited by superoxide dismutase (500 U ml(-1)). Since O(2)(*-) formation plays contributory role in ARDS, both sildenafil citrate and NCX 911 may be indicated for treating ARDS through suppression of NADPH oxidase expression and therefore of O(2)(*-) formation and preservation of NO bioavailability.

2005 - 08

Shock. 2005 Aug;24(2):182-7.

Flurbiprofen and HCT1026 protect mice against acute pancreatitis-associated lung injury.

Huang J, Moochhala SM, Moore PK, Bhatia M. Department of Pharmacology and Cardiovascular Biology Research Group, National University of Singapore, Singapore.

Impaired lung function in severe acute pancreatitis is the primary cause of morbidity and mortality in this condition. Flurbiprofen is a powerful nonsteroidal anti-inflammatory drug (NSAID). However, administration of this drug is associated with severe gastrointestinal side effects. The NO-releasing derivative of flubiprofen (nitroflurbiprofen, HCT1026) has recently been developed by the addition of a nitroxybutyl moiety to the flurbiprofen structure. This modification does not interfere with the anti-inflammatory activity of the drug but markedly reduces its ability to induce gastric injury. The effects of treatment with flurbiprofen and HCT1026 on the severity of pancreatitis and the associated lung injury were investigated in a mouse model. Acute pancreatitis was induced in mice by hourly intraperitoneal injections of cerulein. Flurbiprofen and HCT1026 were administered either 30 min before or 1 h after starting cerulein injections, and the severity of acute pancreatitis and associated lung injury were assessed. The severity of acute pancreatitis was determined by hyperamylasemia, neutrophil sequestration in the pancreas (pancreatic MPO activity), and pancreatic acinar cell injury/necrosis on histological examination of pancreas sections. The severity of acute pancreatitis-associated lung injury was assessed by neutrophil sequestration in the lungs (lung MPO activity) and by histological examination of lung sections. HCT1026 and flurbiprofen, given prophylactically as well as therapeutically, significantly reduced lung inflammation without having any significant effect on pancreatic injury. These results suggest the usefulness of flurbiprofen as well as HCT1026 as potential treatments for pancreatitis-associated lung injury.

2005 - 07

Eur J Pharmacol. 2005 Jul 11;517(3):224-31.

Effect of sildenafil citrate and a nitric oxide donating sildenafil derivative, NCX 911, on cavernosal relaxation and superoxide formation in hypercholesterolaemic rabbits.

Shukla N, Jones R, Persad R, Angelini GD, Jeremy JY. Bristol Heart Institute, Department of Cardiac Surgery, Bristol Royal Infirmary, Bristol BS2 8HW, UK.

Hypercholesterolaemia promotes erectile dysfunction through increased superoxide formation and negation of nitric oxide (NO) bioactivity in cavernosal tissue. The source of superoxide has not been clearly defined, however. Sildenafil (Viagra), the standard therapy for erectile dysfunction, may also be rendered more effective by the presence of an NO donor. One drug that intrinsically fulfils this criterion is sildenafil nitrate (NCX 911), an NO donating derivative of sildenafil. The objective of this study, therefore, was to determine the source of superoxide and its effect on erectile function in corpus cavernosum from hypercholesterolaemic rabbits and to determine whether NCX 911 confers an improvement over sildenafil citrate in this model. Hypercholesterolaemia elicited an increase in superoxide formation by rabbit cavernosal tissue and a reduction of carbachol-stimulated relaxation both of which were reversed by diphenylene iodonium chloride and apocynin (NADPH oxidase inhibitors). In response to sodium nitroprusside, hypercholesterolaemia also caused an attenuation of cavernosal relaxation which was not reversed with NADPH oxidase inhibitors. Both sildenafil citrate and NCX 911 significantly reversed impaired carbachol-stimulated relaxation and inhibited superoxide formation by cavernosal tissue from hypercholesterolaemic rabbits, NCX 911 being more potent. NCX 911 also augmented cavernosal cGMP levels, an effect blocked by the guanylyl cyclase inhibitor, 1H-{1,2,4}oxadiazolo {4,3-a}quinoxalin-1-one (ODQ). These data demonstrate that hypercholesterolaemia promotes erectile dysfunction through an augmentation of superoxide derived from NADPH oxidase in cavernosal tissue. It also indicates that NO donating sildenafil may be therapeutically more beneficial than conventional sildenafil in treating erectile dysfunction with an oxidative stress-related aetiology.

2005 - 05

Life Sci. 2005 May 20;77(1):85-95.

Enhancement of fentanyl antinociception by subeffective doses of nitroparacetamol (NCX-701) in acute nociception and in carrageenan-induced monoarthritis.

Gaitan G, Ahuir FJ, Herrero JF. Departamento de Fisiologia, Campus Universitario, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain.

We have reported that subanalgesic doses of new generation non-steroidal anti-inflammatory drugs (NSAIDs) enhance the antinociceptive activity of the mu-opiate fentanyl, and the duration of its effect, in acute nociception. Since this therapy is intended for situations of hyperalgesia, we have compared the antinociceptive activity of fentanyl in the absence and in the presence of subeffective doses of NCX-701 (nitroparacetamol) in normal animals and in animals with carrageenan-induced monoarthritis. Subanalgesic dose of NCX-701 did not modify any of the nociceptive responses on its own but reduced the ID50 of fentanyl more than two-fold in both the normal and sensitized states. When administered alone, full recovery from fentanyl was always observed within 15 to 20 minutes, however, full recovery was not observed in the presence of NCX-701. Naloxone was unable to reverse the effect, suggesting a possible reduction of other opiate-mediated secondary effects. We therefore studied the possibility that combining administration of fentanyl and nitroparacetamol (NCX-701) would reduce the development of acute tolerance to fentanyl in behavioral experiments. Acute tolerance to fentanyl in behavioral nociceptive reflexes was developed within 72 h after the constant infusion of the drug, whereas in animals treated with small doses of NCX-701 tolerance was not observed. In summary, our results, both in normal animals and in animals with hyperalgesia, show that fentanyl antinociception can be strongly potentiated with subanalgesic doses of the NSAID NCX-701 and that the development of acute tolerance to fentanyl in normal animals is prevented by this combination of drugs.

2005 - 04

Brain Res Brain Res Rev. 2005 Apr;48(2):400-8. Epub 2005 Jan 28.

Activity of flurbiprofen and chemically related anti-inflammatory drugs in models of Alzheimer's disease.

Gasparini L, Ongini E, Wilcock D, Morgan D. Nicox Research Institute, Bresso, Milan, Italy.

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.

Brain Res Brain Res Rev. 2005 Apr;48(2):370-8.

Transgenic AD model mice, effects of potential anti-AD treatments on inflammation and pathology.

van Groen T, Kadish I. Department of Neuroscience and Neurology, University of Kuopio, Canthia Building, Finland.

The extracellular deposition of amyloid (A) peptides in plaques, and neurofibrillary tangles are the two characteristic pathological features of Alzheimer's disease (AD). Plaques are surrounded by activated astrocytes and microglia, to study the relation between amyloid neuropathology and inflammation, we examined the changes in amyloid pathology in the hippocampus following three different treatments aimed at reducing the amyloid burden. (1) To investigate the effects of long-term cholinergic deafferentation, we lesioned the fimbria-fornix pathway in our AD-model mice at 7 months of age, and 11 months post-lesion the mice were sacrificed for histopathological analysis. The fimbria-fornix transection resulted in a substantial depletion of cholinergic markers in the hippocampus, but the lesion did not result in an alteration in hippocampal A deposition and inflammation (i.e., numbers or staining density of astrocytes and microglia). (2) To investigate the effects of estrogen, we ovariectomized mice and treated them with estrogen (sham-lesion, zero dose, low dose, and high dose) and studied the pathology at different postsurgery intervals. Estrogen depletion (i.e., ovariectomy) or estrogen replacement did not affect A deposition or inflammation at any time point. (3) In the final studies, we treated mice with flurbiprofen and an NO-donating derivative of flurbiprofen (HCT 1026) for several months (from 6 till 14 months of age), and studied the A pathology and inflammation in the brain. Sham treatment, flurbiprofen, and the low-dose HCT 1026 did not affect pathology; however, a higher dose of HCT 1026 reduced both A load and amount of microglial activation surrounding plaques.

2005 - 02

Pharmacol Biochem Behav. 2005 Feb;80(2):327-32. Epub 2004 Dec 23.

Subanalgesic doses of dexketoprofen and HCT-2037 (nitrodexketoprofen) enhance fentanyl antinociception in monoarthritic rats.

Gaitan G, Herrero JF. Departamento de Fisiologia, Facultad de Medicina, Campus Universitario, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain.

Subanalgesic doses of the non-steroidal antiinflammatory drugs (NSAID) dexketoprofen trometamol and nitroparacetamol (NCX-701) enhance mu-opiate fentanyl effect in acute nociception. It is not known if a similar combination of drugs is effective in situations of spinal cord sensitization. The aim of this study was to assess if the enhancement of fentanyl antinociception can be observed in carrageenan-induced monoarthritis, when combined with dexketoprofen (DKT) or nitrodexketoprofen (HCT-2037). Withdrawal reflexes were recorded as single motor units in male Wistar rats anesthetized with alpha-chloralose. Fentanyl was studied alone and in the presence of 0.4, 0.8 micromol/kg of DKT or 0.3 micromol/kg of HCT-2037. In responses to noxious mechanical stimulation, the ID50 of fentanyl was enhanced twofold by 0.8 micromol/kg DKT and more than fourfold by HCT-2037 and no significant recovery was observed 45 min later. DKT 0.4 micromol/kg was, however, very little effective. The opioid antagonist naloxone did not reverse the effect. Enhancement of fentanyl effect on wind-up was only observed with HCT-2037 but not with DKT. We conclude that the combined administration of subanalgesic doses of dexketoprofen derivatives, specially its nitroderivative, and the mu-opiate fentanyl is an effective antinociceptive therapy in situations of articular inflammation involving a naloxone-independent mechanism of action.

J Neurochem. 2005 Feb;92(4):895-903.

Nuclear receptor peroxisome proliferator-activated receptor-gamma is activated in rat microglial cells by the anti-inflammatory drug HCT1026, a derivative of flurbiprofen.

Bernardo A, Ajmone-Cat MA, Gasparini L, Ongini E, Minghetti L. Department of Cell Biology and Neuroscience, Istituto Superiore di Sanita, Rome, Italy.

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 microm) 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 E2 synthesis at the same low concentration (1 microm). 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.

2005 - 01

J Sex Med. 2005 Jan;2(1):53-7.

A nitric oxide-releasing PDE5 inhibitor relaxes human corpus cavernosum in the absence of endogenous nitric oxide.

Kalsi JS, Ralph DJ, Thomas P, Bellringer J, Minhas S, Kell PD, Cellek S. The St. Peter's Andrology Centre and Wolfson Institute for Biomedical Research, University College London, UK.

INTRODUCTION: In conditions with severe deficiency of endogenous nitric oxide (NO), such as long-term diabetes and cavernosal nerve injury, phosphodiesterase type 5 (PDE5) inhibitors have reduced efficacy in the treatment of erectile dysfunction. NO-releasing PDE5 inhibitors could be an alternative therapeutic approach in such cases. AIM: We therefore aimed to compare sildenafil and NO-releasing sildenafil (NCX-911) in relaxing human corpus cavernosum in the absence or presence of endogenous NO. METHODS: The two compounds were compared in reducing the phenylephrine-induced tone of human corpus cavernosum in the presence or absence of an inhibitor of NO synthase (L-NAME; 500 microM) or an inhibitor of soluble guanylate cyclase (ODQ, 10 microM). RESULTS: NCX-911 was as potent as sildenafil in control conditions (EC(50) = 733.1 +/- 94.4 nM and 800.7 +/- 155.8 nM, respectively). The potency of NCX-911 was not altered but that of sildenafil decreased significantly in the presence of L-NAME (EC(50) = 980.4 +/- 106.7 nM and 2446.7 +/- 256.8 nM, respectively; P < 0.001 for sildenafil vs. control). Both compounds below 1 microM failed to induce relaxation in the presence of ODQ (EC(50) = 6,578 +/- 1150 nM and 6,488 +/- 938 nM for NCX-911 and sildenafil, respectively). CONCLUSION: These results show that the potency of NCX-911 was maintained unlike sildenafil in the absence of endogenous NO confirming the potential use of NO-releasing PDE5 inhibitors in NO-deficient conditions.

Eur J Pharmacol. 2005 Jan 31;508(1-3):7-13. Epub 2004 Dec 28.

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

Zacharowski P, Breese E, Wood E, Del Soldato P, Warner T, Mitchell J. Cardiac, Vascular and Inflammation Research, The William Harvey Research Institute, Bart's and The London, Queen Mary School of Medicine and Dentistry, London, UK.

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.

2004 - 12

Int J Impot Res. 2004 Dec;16(6):479-85.

A comparative study of sildenafil, NCX-911 and BAY41-2272 on the anococcygeus muscle of diabetic rats.

Kalsi JS, Ralph DJ, Madge DJ, Kell PD, Cellek S. Wolfson Institute for Biomedical Research, University College London, Gower Street, Cruciform Building, London, UK.

We compared the effects of a nitric oxide (NO)-releasing sildenafil (NCX-911), NO-independent soluble guanylate cyclase activator (BAY41-2272) and sildenafil on the anococcygeus muscle from streptozotocin-induced 16-weeks diabetic rats. NCX-911, BAY41-2272 and sildenafil reduced the phenylephrine-induced tone in the control group (EC50=1088.8+/-165.0, 151.6+/-9.3 and 827.1+/-167.3 nM, respectively). The potencies of NCX-911 and BAY41-2272 were not altered, but that of sildenafil was significantly reduced in the diabetic group. EC50 values for NCX-911, BAY41-2272 and sildenafil in the diabetic group were 1765.9+/-303.5, 209.7+/-27.3 and 2842.2+/-640.3 nM, respectively (P<0.05 for sildenafil). Nitrergic relaxation responses were significantly decreased in the diabetic group. The remaining nitrergic relaxation responses were potentiated by BAY41-2272 but not by sildenafil or NCX-911. These results confirm that endogenous NO derived from nitrergic nerves is significantly decreased in diabetes, and suggest that NO-releasing PDE5 inhibitors and NO-independent soluble guanylate cyclase activators could be more useful than PDE5 inhibitors in the treatment of ED in long-term diabetes.

2004 - 10

Clin Pharmacol Ther. 2004 Oct;76(4):350-8.

The effects and metabolic fate of nitroflurbiprofen in healthy volunteers.

Zacharowski P, Zacharowski K, Donnellan C, Johnston A, Vojnovic I, Forte P, Del Soldato P, Benjamin N, O'Byrne S. William Harvey Research Institute, Barts and The London, Queen Mary's School of Medicine and Dentistry.

OBJECTIVE: Nitric oxide-donating nonsteroidal anti-inflammatory drugs (NO-NSAIDs) are a new class of cyclooxygenase (COX) inhibitors. To investigate whether these drugs actually release nitric oxide (NO), we labeled the nitroxy group of nitroflurbiprofen with nitrogen 15 to determine the metabolic fate of this compound in humans. METHOD: Six healthy volunteers who fasted were given an oral dose of 15 N-nitroflurbiprofen (100 mg). Samples of blood, urine, and gastric headspace gas were taken over a 24-hour period to determine the levels of nitroflurbiprofen, flurbiprofen, total nitrate/nitrite, 15 N-nitrate/nitrite, COX activity, and gastric NO. In a crossover study (1 week apart), a further 6 healthy volunteers who fasted were given an oral dose of nitroflurbiprofen (100 mg) or flurbiprofen (65 mg) and levels of gastric NO were determined. RESULTS: Nitroflurbiprofen was undetectable in the systemic circulation. Levels of 15 N-nitrate/nitrite (5.2% +/- 1.5% enrichment) and flurbiprofen (2.4 +/- 0.7 microg/mL) peaked at 4 hours in the plasma and gradually decreased thereafter. In unstimulated blood, the plasma levels of thromboxane B 2 (COX-1 activity) were 2 to 3 ng/mL, and after calcium ionophore stimulation, large amounts of thromboxane B 2 were produced (112 +/- 31 ng/mL). Prostaglandin E 2 was undetectable in unstimulated blood. After lipopolysaccharide stimulation, the plasma levels of prostaglandin E 2 increased to 15 +/- 4 ng/mL. The metabolite flurbiprofen inhibited plasma COX-1 activity for the duration of the study period (maximum inhibition at 4 hours), whereas COX-2 activity recovered after 6 hours. In the crossover study, levels of gastric NO were higher in subjects given nitroflurbiprofen, when compared with those given flurbiprofen. (The area under the curve for gastric NO was 435 +/- 107 ppm . h versus 305 +/- 94 ppm . h [95% confidence interval of the difference, 89-172 ppm . h; P < .001]). CONCLUSION: Nitroflurbiprofen was undetectable in the systemic circulation, suggesting metabolism to 15 N-nitrate/nitrite and flurbiprofen in the presystemic circulation. Levels of gastric NO were significantly higher after ingestion of nitroflurbiprofen than flurbiprofen.

2004 - 09

Int J Immunopathol Pharmacol. 2004 Sep-Dec;17(3):317-30.

Comparison between flurbiprofen and its nitric oxide-releasing derivatives HCT-1026 and NCX-2216 on Abeta(1-42)-induced brain inflammation and neuronal damage in the rat.

Prosperi C, Scali C, Barba M, Bellucci A, Giovannini MG, Pepeu G, Casamenti F. Department of Pharmacology, University of Florence, Florence, Italy.

Brain inflammation is an underlying factor in the pathogenesis of Alzheimers disease (AD). We investigated, in vivo, whether differences exist in the anti-inflammatory and neuroprotective actions of flurbiprofen and its two nitric oxide-donor derivatives, HCT-1026 and NCX-2216, and the ability of these two derivatives to release nitric oxide in the brain. In adult rats injected into the nucleus basalis with preaggregated Abeta(1-42) we investigated glia reaction, the induction of inducible nitric oxide synthase (iNOS), the activation of p38 mitogen-activated protein kinase (p38MAPK) pathway and the number of choline acetyltransferase (ChAT)-positive neurons and, in naive rats we investigated, by microdialysis, cortical extracellular levels of nitrite. Injection of Abeta(1-42) induced iNOS and activation of p38MAPK 7 days after injection and an intense microglia and astrocyte reaction along with a marked reduction in the number ChAT-positive neurons, persisting up to at least 21 days. Flurbiprofen, HCT-1026 and NCX-2216 (15 mg/kg) significantly attenuated the Abeta(1-42)-induced glia reaction, iNOS induction and p38MAPK activation 7 days after treatment and astrocytes reaction 21 days after treatment. On an equimolar basis, HCT-1026 resulted the most active agent in reducing the Abeta(1-42)-induced microglia reaction. The cholinergic cell loss was also significantly reduced by 21 days of HCT-1026 treatment. No differences in body weight were found between the animals treated for 21 days with 15 mg/kg of either HCT-1026 or NCX-2216 and the controls. Oral administration of HCT-1026 (15 mg/kg) or NCX-2216 (100 mg/kg) to naive rats was followed by significant and long lasting increases in cortical nitrite levels. These findings indicate that the addition of a nitric oxide donor potentiates the anti-inflammatory activity of flurbiprofen in a model of brain inflammation.

Bone. 2004 Sep;35(3):636-43.

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

Idris AI, Del Soldato P, Ralston SH, van't Hof RJ. Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen AB25 2ZD, UK.

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.

Br J Pharmacol. 2004 Sep;143(1):33-42.

Comment in:
Br J Pharmacol. 2004 Sep;143(1):1-2.

Liver delivery of NO by NCX-1000 protects against acute liver failure and mitochondrial dysfunction induced by APAP in mice.

Fiorucci S, Antonelli E, Distrutti E, Mencarelli A, Farneti S, Del Soldato P, Morelli A. Dipartimento di Medicina Clinica e Sperimentale, Clinica di Gastroenterologia ed Epatologia, Universita degli Studi di Perugia, Italy.

1. NCX-1000, (3alpha, 5beta, 7beta)-3,7-dihydroxycholan-24oic acid[2-methoxy-4-[3-[4-(nitroxy)butoxy]-3-oxo-1-propenyl]phenyl ester, is a nitric oxide (NO)-derivative of ursodeoxyxholic acid (UDCA) that selectively release NO in the liver. 2. Here, we demonstrated that administering mice with 40 micromol kg(-1) NCX-1000, but not UDCA, improves liver histopathology and reduces mortality caused by 330 micromol kg(-1) APAP from 60 to 25% (P<0.01). Administration of NCX-1000, in a therapeutic manner, that is, 2 h after acetaminophen (APAP) intoxication reduced mortality, improved liver histopathology and prevented liver IFN-gamma, TNF-alpha, Fas/Fas ligand and inducible nitric oxide synthase (iNOS) mRNA accumulation caused by APAP. 3. In vitro exposure of primary cultures of mouse hepatocytes to APAP, 6.6 mm, resulted in apoptosis followed by necrosis. Loss of cell viability correlates with early mitochondrial membrane potential (Deltapsi(m)) hyperpolarization followed by depolarization and cytochrome c translocation from mitochondria to cytosol. APAP-induced apoptosis associated with procaspase-3 and -9 cleavage, appearance of truncated Bid and activation of poly(ADP-ribose) polymerase (PARP). 4. Treating primary culture of hepatocytes with 5 microm cyclosporine and 10 microm trifluoperazine for eight resulted in significant reduction of apoptosis induced by APAP suggesting that loss of Deltapsim was mechanistically involved in apoptosis induced by APAP in vitro. 5. NCX-1000, but not UDCA, concentration-dependently (ED(50)=16 microm) protected against Deltapsi(m) depolarization and reduced transition from apoptosis to necrosis caused by 6.6 mm APAP. 6. Treating primary cultures of hepatocytes with the NO-donor DETA-NO, 100 microm, reduced apoptosis induced by APAP and prevented caspase activation. 7. In conclusion, NCX-1000 is effective in protecting against APAP-induced hepatotoxicity when administered in a therapeutic manner. This protection may involve the inhibition of apoptosis and the maintenance of mitochondrial integrity.

Br J Pharmacol. 2004 Sep;143(1):1-2.

Comment on:
Br J Pharmacol. 2004 Sep;143(1):33-42.

Acetaminophen hepatotoxicity: NO to the rescue.

Wallace JL. Department of Pharmacology & Therapeutics, Mucosal Inflammation Research Group, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada.

Severe liver injury as a result of overdose or chronic use of acetaminophen (paracetamol) remains a significant clinical problem, accounting for as much as 40% of cases of acute liver failure. The mechanisms underlying the liver injury caused by acetaminophen have become much better understood in recent years. In this issue, Fiorucci et al. report that delivery of nitric oxide (NO) in small amounts to the liver, via a novel derivative of the bile acid ursodeoxycholic acid, results in significant protection of the liver from acetaminophen-induced damage. NO appears to produce these beneficial actions through several mechanisms, including the suppression of synthesis of several proinflammatory cytokines. There is also substantial evidence that a NO-releasing derivative of acetaminophen offers several advantages over acetaminophen itself, including enhanced analgesic potency and reduced liver toxicity.

2004 - 07

Cardiovasc Drug Rev. 2004 Summer;22(2):135-46.

Treatment of portal hypertension with NCX-1000, a liver-specific NO donor. A review of its current status.

Fiorucci S, Antonelli E, Tocchetti P, Morelli A. Dipartimento di Medicina Clinica e Sperimentale, Clinica di Gastroenterologia ed Epatologia, Universita degli Studi di Perugia, Via E dal Pozzo, 06122, Perugia, Italy.

Portal hypertension, a life threatening complication of liver cirrhosis, results from increased intrahepatic resistance and increased portal blood inflow through a hyperdynamic splanchnic system. The increased intrahepatic vascular tone is the result of an enhanced activity of endogenous vasoconstrictors and a deficiency of nitric oxide (NO) release by sinusoidal endothelial cells. These pathophysiological events provide the rational basis for using NO-based therapies for the treatment of portal hypertension. Clinical studies have demonstrated that nitrate therapy results in a significant reduction of portal pressure as assessed by hepatic venous portal gradient but causes vasodilation in both systemic arterial and venous vascular beds, aggravating the progression of the vasodilatory syndrome of cirrhotic patients. For this reason, the ideal drug for the treatment of portal hypertension should act by decreasing intrahepatic vascular resistance, without worsening the splanchnic/systemic vasodilatation. NCX-1000 is the prototype of a family of NO-releasing derivatives of ursodeoxycholic acid (UDCA). These compounds are releasing selectively, from parenchymal and non-parenchymal hepatic cells, biologically active NO into the liver microcirculation with no detectable effect on systemic circulation. Preclinical studies have shown that long- and short-term administration of NCX-1000 to rodents with chronic liver injury protects against the development of portal hypertension and reduces the intrahepatic hyperreactivity to alpha1-adrenoceptor agonists. The finding of increased liver nitrite/nitrate content in NCX-1000-treated animals together with an increase in cGMP levels in their liver homogenates suggests that this nitro-compound behaves as a liver-selective NO donor. In contrast to conventional NO-donors such as isosorbide mono- and di-nitrate, which are also used for primary and secondary prevention of gastrointestinal bleeding, NCX-1000 has no effect on mean arterial pressure in either normal or cirrhotic animals indicating the absence of adverse systemic effect. In summary, these data suggest that NCX-1000 may provide a novel therapy for the treatment of patients with portal hypertension.

J Pharmacol Exp Ther. 2004 Jul;310(1):367-75. Epub 2004 Apr 14.

A nitric oxide-releasing salbutamol elicits potent relaxant and anti-inflammatory activities.

Lagente V, Naline E, Guenon I, Corbel M, Boichot E, Burgaud JL, Del Soldato P, Advenier C. Laboratoire de Pharmacodynamie et de Pharmacologie Moléculaire, Institut National de la Santé et de la Recherche Médicale, Université de Rennes 1, Rennes, France.

Beta2-adrenoceptor agonists are widely used in the treatment of pulmonary diseases. We have investigated the relaxant and anti-inflammatory activities of NCX-950 (alpha'-[[(1,1-dimethylethy)amino]methyl]-4-hydroxy-1,3-benzenedimethanol nitrate) (a nitric oxide-releasing salbutamol) in human isolated bronchi and on lipopolysaccharide (LPS)-induced acute airway inflammation in mice. NCX-950 (10(-8)-10(-5) M) elicited a relaxation of human isolated bronchi moderately higher than salbutamol, which was reduced by a beta-adrenergic blocking drug, propranolol, but not by an inhibitor of guanylate cyclase, ODQ (1H-[1,2,4]oxadiazolo[4,3-] quinolaxin-1-one). The treatment of mice with NCX-950 (1, 10, and 100 microM aerosol) markedly inhibited the neutrophil influx induced by LPS aerosol in bronchoalveolar lavage (BAL) fluid, whereas salbutamol at equimolar doses elicited a moderate inhibition. Pretreatment of mice with NCX-950 (100 microM) also significantly reduced tumor necrosis factor-alpha, interleukin-6 (IL-6), transforming growth factor-beta, and matrix metalloproteinase-9 release in BAL fluid, whereas salbutamol was ineffective. Propranolol, but not ODQ, suppressed the inhibitory activity of NCX-950 on neutrophil influx and IL-6 release in BAL fluids. A nitric oxide-releasing sildenafil NCX-911 [(5-[2-ethoxy-5-(4-methylpiperidinylsulfonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihy dro-7H-pyrazolo[4,3-d]pyrimidin-7-one nitrate)], but not sildenafil (100 microM) also reduced the neutrophil influx following LPS exposure in mice. This study reported that NCX-950 elicits potent relaxant and anti-inflammatory activities compared with salbutamol, and these effects may be mainly due to the activation of the beta2-adrenoceptor rather than the cGMP pathway.

Curr Opin Investig Drugs. 2004 Jul;5(7):755-9.

NCX-701. NicOx.

Joshi GP. University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9068, USA.

NicOx is developing NCX-701, a nitric oxide-releasing derivative of paracetamol (acetaminophen), for the potential treatment of inflammation and pain. The compound is currently undergoing phase III clinical trials.

J Pharmacol Exp Ther. 2004 Jul;310(1):367-75. Epub 2004 Apr 14.

A nitric oxide-releasing salbutamol elicits potent relaxant and anti-inflammatory activities.

Lagente V, Naline E, Guenon I, Corbel M, Boichot E, Burgaud JL, Del Soldato P, Advenier C. Laboratoire de Pharmacodynamie et de Pharmacologie Moléculaire, Institut National de la Santé et de la Recherche Médicale, Université de Rennes 1, Rennes, France.

Beta2-adrenoceptor agonists are widely used in the treatment of pulmonary diseases. We have investigated the relaxant and anti-inflammatory activities of NCX-950 (alpha'-[[(1,1-dimethylethy)amino]methyl]-4-hydroxy-1,3-benzenedimethanol nitrate) (a nitric oxide-releasing salbutamol) in human isolated bronchi and on lipopolysaccharide (LPS)-induced acute airway inflammation in mice. NCX-950 (10(-8)-10(-5) M) elicited a relaxation of human isolated bronchi moderately higher than salbutamol, which was reduced by a beta-adrenergic blocking drug, propranolol, but not by an inhibitor of guanylate cyclase, ODQ (1H-[1,2,4]oxadiazolo[4,3-] quinolaxin-1-one). The treatment of mice with NCX-950 (1, 10, and 100 microM aerosol) markedly inhibited the neutrophil influx induced by LPS aerosol in bronchoalveolar lavage (BAL) fluid, whereas salbutamol at equimolar doses elicited a moderate inhibition. Pretreatment of mice with NCX-950 (100 microM) also significantly reduced tumor necrosis factor-alpha, interleukin-6 (IL-6), transforming growth factor-beta, and matrix metalloproteinase-9 release in BAL fluid, whereas salbutamol was ineffective. Propranolol, but not ODQ, suppressed the inhibitory activity of NCX-950 on neutrophil influx and IL-6 release in BAL fluids. A nitric oxide-releasing sildenafil NCX-911 [(5-[2-ethoxy-5-(4-methylpiperidinylsulfonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one nitrate)], but not sildenafil (100 microM) also reduced the neutrophil influx following LPS exposure in mice. This study reported that NCX-950 elicits potent relaxant and anti-inflammatory activities compared with salbutamol, and these effects may be mainly due to the activation of the beta2-adrenoceptor rather than the cGMP pathway.

2004 - 06

Proc Natl Acad Sci U S A. 2004 Jun 1;101(22):8497-502.

Nitric oxide (NO)-releasing statin derivatives, a class of drugs showing enhanced antiproliferative and antiinflammatory properties.

Ongini E, Impagnatiello F, Bonazzi A, Guzzetta M, Govoni M, Monopoli A, Del Soldato P, Ignarro LJ. Nicox Research Institute, Via Ariosto 21, 20091 Bresso, Milan, Italy.

Inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, namely statins, exert pleiotropic actions beyond lipid-lowering effects. Their pharmacological activity on atherosclerotic plaque stability and vascular inflammation appears to be mediated, at least in part, by nitric oxide (NO). With the aim of enhancing the nonlipid-lowering properties of selected statins, we introduced a NO-releasing moiety into the structure of pravastatin (NCX 6550) and fluvastatin (NCX 6553). NO release was evaluated as nitrosylhemoglobin adduct formation by using EPR spectroscopy in rat blood. Both compounds produced a linear time-dependent increase in nitrosylhemoglobin formation, which is consistent with slow NO release kinetics. In PC12 cells, unlike their native statins, both compounds stimulated cGMP formation (NCX 6550, EC(50) = 2.3 +/- 0.2 microM; NCX 6553, EC(50) = 2.7 +/- 0.2 microM). Moreover, NCX 6550 potently inhibited cell proliferation in rat aortic smooth muscle cells (IC(50) = 2.2 +/- 0.3 microM) with a mechanism that involved both the polyamine and HMG-CoA reductase signalling pathways. Hence, mevalonate or putrescine partially reverted the effects of NCX 6550 and their combination was fully effective. In RAW 264.7 murine macrophage cells stimulated with lipopolysaccharide (1 microg/ml), NCX 6550, but not pravastatin, significantly decreased inducible NO synthase and cyclooxygenase-2 protein expression as well as nitrite accumulation. All together, the data show that the previously undescribed NO-releasing statins retain HMG-CoA reductase inhibitory activity and release bioactive NO slowly. Among the additional properties, compared with native statins, the NO-releasing statins show enhanced antiinflammatory effects. Thus, NO-releasing statins represent an interesting class of drugs having potential in the therapy of disorders associated with endothelial dysfunction and vascular inflammation.

2004 - 05

J Pharmacol Exp Ther. 2004 May;309(2):626-33. Epub 2004 Jan 30.

Gastric tolerability and prolonged prostaglandin inhibition in the brain with a nitric oxide-releasing flurbiprofen derivative, NCX-2216 [3-[4-(2-fluoro-alpha-methyl-[1,1'-biphenyl]-4-acetyloxy)-3 methoxyphenyl]-2-prop enoic acid 4-nitrooxy butyl ester].

Wallace JL, Muscara MN, de Nucci G, Zamuner S, Cirino G, del Soldato P, Ongini E. Department of Pharmacology and Therapeutics, University of Calgary, Alberta,

[3-[4-(2-fluoro-alpha-methyl-[1,1'-biphenyl]-4-acetyloxy)-3-methoxyphenyl]-2-propenoic acid 4-nitrooxy butyl ester] is an NO-releasing flurbiprofen derivative that also contains a ferulic acid (antioxidant) moiety. NCX-2216 has been shown to be effective in reducing beta-amyloid deposition in a transgenic mouse model of Alzheimer's disease. The tolerability of this compound in the stomach and its ability to suppress prostaglandin synthesis in the brain are not known. The purpose of this study was to assess the contribution of nitric oxide (NO) and ferulic acid to the pharmacological properties of NCX-2216 versus flurbiprofen;thus, we compared their gastric tolerability and suppression of prostaglandin synthesis, peripherally and centrally. Oral flurbiprofen produced extensive gastric damage and suppressed gastric prostaglandin synthesis. In contrast, while suppressing prostaglandin production, equimolar doses of NCX-2216 did not cause detectable gastric injury. The NO-releasing moiety of NCX-2216 (but not the ferulic acid moiety) was crucial for the gastric safety of this compound. NCX-2216 substantially inhibited prostanoid synthesis despite not being detectable in plasma and despite producing only low amounts of flurbiprofen in plasma and in the brain. Inhibition of brain prostaglandin synthesis by NCX-2216 (22 mg/kg) persisted for a much longer period of time (up to 48 h) than was seen with flurbiprofen (inf. or=12 h). These results demonstrate that a single administration of NCX-2216 can produce prolonged suppression of brain prostaglandin synthesis without causing gastric injury. It is likely that an active metabolite of NCX-2216 contributes to the suppression of cyclooxygenase activity. NCX-2216 may represent an attractive alternative to conventional nonsteroidal anti-inflammatory drugs for long-term treatment of a variety of inflammatory disorders, especially those occurring in the central nervous system.

Curr Opin Investig Drugs. 2004 May;5(5):551-6.

Nitroflurbiprofen (NicOx).

Scatena R. Istituto di Biochimica e Biochimica Clinica, Faculty of Medicine, Universita' Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00168 Rome, Italy.

Nitroflurbiprofen is a nitrosylated flurbiprofen analog under development by NicOx for the potential treatment of urinary incontinence, Alzheimer's disease (AD) and the prevention and treatment of accelerated bone resorption associated with disorders such as osteoporosis, inflammatory joint disease and Paget's disease. In addition, a topical formulation of nitroflurbiprofen is under development for the potential treatment of dermatological disorders, including contact urticaria. By 1999, nitroflurbiprofen was in phase IIa trials for urinary incontinence, Paget's disease and osteoporosis, and phase II trials of the topical formulation were underway in contact urticaria by March 2002. Phase I trials for AD had commenced by May 2003, and in September 2003, NicOx was intending to conduct further phase II trials for micturition disorders in 2003.

J Neuroimmunol. 2004 May;150(1-2):10-9.

A nitric oxide releasing derivative of flurbiprofen inhibits experimental autoimmune encephalomyelitis.

Furlan R, Kurne A, Bergami A, Brambilla E, Maucci R, Gasparini L, Butti E, Comi G, Ongini E, Martino G. Neuroimmunology Unit, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.

Nitric oxide (NO)-releasing non-steroidal anti-inflammatory drugs (NSAIDs) have been reported to have a safer profile and additional anti-inflammatory and immuno-modulatory properties compared to parent compounds. Preventive treatment of experimental autoimmune encephalomyelitis (EAE)-induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55-with the NO-releasing derivative of flurbiprofen HCT1026 delayed disease onset and significantly decreased disease severity. HCT1026 treatment was associated to (i) decreased mRNA levels of pro-inflammatory cytokines, caspase-1, and iNOS in blood cells; (ii) decreased ability of encephalitogenic T cells to proliferate; (iii) reduced number of central nervous system (CNS)-infiltrating T cells; (iv) decreased axonal loss and demyelination; (v) increased CD4(+) CD69(-) CD25(+) regulatory T cells in the spleen.

Am J Physiol Renal Physiol. 2004 May;286(5):F945-54. Epub 2003 Dec 16.

Renal expression of COX-2, ANG II, and AT1 receptor in remnant kidney: strong renoprotection by therapy with losartan and a nonsteroidal anti-inflammatory.

Gonzalves AR, Fujihara CK, Mattar AL, Malheiros DM, Noronha Ide L, de Nucci G, Zatz R. Renal Division, Department of Clinical Medicine, Faculty of Medicine, University of Sao Paulo, Brazil.

Chronic renal injury can be mediated by angiotensin II (ANG II) and prostanoids through hemodynamic and inflammatory mechanisms and attenuated by individual suppression of these mediators. In rats with (5/6) renal ablation (Nx), we investigated 1) the intrarenal distribution of COX-2, ANG II, and the AT(1) receptor (AT(1)R); 2) the renoprotective and antiinflammatory effects of an association between the AT(1)R blocker, losartan (Los), and the gastric sparing anti-inflammatory nitroflurbiprofen (NOF). Adult male Munich-Wistar rats underwent Nx or sham operation (S), remaining untreated for 30 days, after which renal structure was examined in 12 Nx rats (Nx(pre)). The remaining rats were followed during an additional 90 days, distributed among 4 treatment groups: Nx(V) (vehicle), Nx(Los) (Los), Nx(NOF) (NOF), and Nx(Los/NOF) (Los/NOF). Nx(pre) rats exhibited marked albuminuria, hypertension, glomerulosclerosis, interstitial expansion, and macrophage infiltration, accompanied by abnormal glomerular, vascular, and interstitial COX-2 expression. ANG II appeared in interstitial cells, in contrast to S, in which ANG II was virtually confined to afferent arterioles. Intrarenal AT(1)R distribution shifted from mostly tubular in S to predominantly interstitial in Nx(pre). All these changes were aggravated at 120 days and attenuated by Los and NOF monotherapies. Los/NOF treatment arrested renal structural injury and ANG II expression and reversed hypertension, albuminuria, and renal inflammation. In conclusion, abnormal expression of COX-2, ANG II, and AT(1)R may be key to development of renal injury in Nx. Concomitant COX-2 inhibition and AT(1)R blockade arrested renal injury and may represent a useful strategy in the treatment of chronic nephropathies.

2004 - 04

Int J Impot Res. 2004 Apr;16(2):195-200.

NCX-911, a novel nitric oxide-releasing PDE5 inhibitor relaxes rabbit corpus cavernosum in the absence of endogenous nitric oxide.

Kalsi JS, Kell PD, Cellek S, Ralph DJ. Institute of Urology and Nephrology, University College London, London, UK.

Phosphodiesterase type 5 (PDE5) inhibitors have reduced efficacy in treating erectile dysfunction (ED) in conditions where there is a lack of endogenous nitric oxide (NO). Therefore, NO-releasing PDE5 inhibitors have been developed. Here we report the effect of such a compound, NCX-911, on the tone and nitrergic relaxations of rabbit corpus cavernosum in the absence or presence of a NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME; 500 microM). NCX-911 was found to be as potent as sildenafil at inducing relaxation of rabbit cavernosum (EC(50) values 997.8+/-195.7 and 1000.5+/-140.8 nM, respectively). The potency of NCX-911 was not altered, but that of sildenafil decreased five-fold in the presence of L-NAME (EC(50) values 1281.2+/-268.3 and 4959.1+/-882.1, nM respectively, P<0.001 for sildenafil). Both compounds potentiated nitrergic relaxations with similar potencies. These results suggest that NO-releasing PDE5 inhibitors could potentially be more useful than PDE5 inhibitors in the treatment of ED in conditions where there is a lack of endogenous NO.

J Bone Joint Surg Br. 2004 Apr;86(3):444-9.

The influence on human osteoblasts in vitro of non-steroidal anti-inflammatory drugs which act on different cyclooxygenase enzymes.

Evans CE, Butcher C. Medical School, University of Manchester, Manchester, England.

There is increasing evidence that non-steroidal anti-inflammatory drugs (NSAIDs) can adversely affect bone repair. We have, therefore, studied the in vitro effects of NSAIDs, which differentially inhibit cyclooxygenases (COX), the prostaglandin/thromboxane synthesising enzymes, on human osteoblasts. Indomethacin and the new nitric oxide (NO)-donating NSAIDs block the activity of both COX-1 and COX-2. Indomethacin and 5,5-dimethyl-3-(3 fluorophenyl)-4-(4 methylsulphonal) phenyl-2 (5H)-furanone (DFU) reduced osteoblast numbers in a dose-dependant manner and increased collagen synthesis and alkaline phosphatase activity. The reduction in osteoblast numbers was not caused by loss of adhesion and was reversible. Neither NSAID influenced DNA synthesis. There was no difference between the effects of indomethacin and DFU. NO-NSAIDs did not affect cell numbers. These results suggest that care should be taken when administering NSAIDs to patients with existing skeletal problems and that NO-NSAIDs may be safer.

2004 - 01

Eur J Pharmacol. 2004 Jan 12;483(2-3):317-22.

Effect of nitric oxide releasing paracetamol and flurbiprofen on cytokine production in human blood.

Marshall M, Moore PK. Department of Pharmacology, King's College, University of London, Guys' Site, Hodgkin Building, London SE1 9RT, UK.

Exposure of anti-coagulated human blood to Escherichia coli lipopolysaccharide (50 ng/ml) resulted in the time-dependent (maximum at 5 h) biosynthesis of interleukin-1beta and tumour necrosis factor-alpha (TNF-alpha). Preincubation with nitroparacetamol or nitroflurbiprofen (but not paracetamol or flurbiprofen) caused dose-related inhibition of the formation of interleukin 1 beta (IC(50)s, 44.5 and 362 microM, n=12) and tumour necrosis factor-alpha (IC(50)s, 9.0 and 0.0009 microM, n=12). The inhibitory effect of nitroparacetamol was completely reversed by (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazol-1-yloxy-3-oxide potassium (carboxy-PTIO, 100 microM; NO scavenging agent). Neither the nuclear factor-kappaB transduction inhibitor, pyrrolidinedithiocarbamate (10-1000 microM) nor the nitric oxide donor, 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene (NOC-5, 10-1000 microM), affected cytokine formation in these experiments.

Pulm Pharmacol Ther. 2004;17(4):219-32.

Comparative effects of inhaled budesonide and the NO-donating budesonide derivative, NCX 1020, against leukocyte influx and airway hyperreactivity following lipopolysaccharide challenge.

Nevin BJ, Broadley KJ. Division of Pharmacology, Welsh School of Pharmacy, Cardiff University, Cathays Park, Cardiff CF10 3XF, UK.

Lipopolysaccharide (LPS) inhalation (30 microg ml(-1), 1 h) caused airway hypereactivity (AHR) to histamine (1 mM, 20 s) 1 h later in conscious guinea-pigs. Bronchoalveolar lavage fluid (BALF) levels of neutrophils, myeloperoxidase (MPO) and protein were elevated whereas nitric oxide (NO) metabolites were reduced 1 h after LPS compared with saline challenge. 24 h after LPS, there was no AHR, but BALF neutrophils, eosinophils, macrophages, MPO, protein and NO metabolites were all raised. Budesonide (0.7 mM) and a molar equivalent concentration of the NO-donating budesonide derivative, NCX 1020, were inhaled (15 min) at 24 h and 45 min before LPS. The only change produced by budesonide was to reduce eosinophil influx at 24 h after LPS, compared with vehicle treated animals. NCX 1020, however, blocked AHR and reduced neutrophils (1 and 24 h) and MPO (1 and 24 h), while NO levels were raised at 1 and reduced at 24 h after LPS. The combined inhalation before LPS of the NO donor, SNAP (1.4 mM), with budesonide (0.7 mM) blocked the AHR to histamine and significantly reduced neutrophils (1 and 24 h) and MPO (1 and 24 h), while NO levels were raised at 1 h after LPS. Thus, NO and a corticosteroid co-administered as NCX 1020 or budesonide with a NO donor, have an additive effect against LPS-induced inflammatory responses and may have value in the treatment of neutrophil-driven airways disease such as COPD.

2003 - 12

J Hepatol. 2003 Dec;39(6):940-6.

Comment in:
J Hepatol. 2003 Dec;39(6):1072-5.

A liver-specific nitric oxide donor improves the intra-hepatic vascular response to both portal blood flow increase and methoxamine in cirrhotic rats.

Loureiro-Silva MR, Cadelina GW, Iwakiri Y, Groszmann RJ. Hepatic Hemodynamic Laboratory, Digestive Diseases Section/111H, VA Medical Center, 950 Campbell Avenue, West Haven, CT 06516, USA.

BACKGROUND/AIMS: A decreased intra-hepatic nitric oxide (NO) production participates on the pathogenesis of portal hypertension in cirrhosis. We tested the hemodynamic effects of a liver-specific NO donor (NCX-1000) derived from ursodeoxycholic acid in portal hypertensive cirrhotic rats. METHODS: After a 14-day treatment with ursodeoxycholic acid or NCX-1000 by gavage, ascitic cirrhotic rats (CCl4-induced) were used in two studies: (1) in vivo mean arterial pressure (MAP), portal pressure (PP) and superior mesenteric artery (SMA) blood flow measurements before and during progressive blood volume expansion (blood infusion); and (2) in situ liver perfusion to obtain dose/response curves to methoxamine (alpha1-adrenergic agonist) and flow/pressure curves. RESULTS: Basal heart rate, MAP, and PP were similar in both groups. During blood infusion, similar MAP and SMA flow increases were observed in both groups; however, PP increase observed in control rats was blunted in NCX-1000 treated rats (P=0.015). In liver perfusions, flow/pressure curves were similar in both groups; however, NCX-1000-treated livers showed a lower response to methoxamine (P=0.016). cGMP concentration in NCX-1000-treated livers was higher (P=0.015) than in controls. CONCLUSIONS: Treatment with a liver-specific NO donor improves the portal system adaptability to portal blood flow increase and ameliorates the intra-hepatic response to methoxamine in cirrhotic rats.

J Hepatol. 2003 Dec;39(6):932-9.

Comment in:
J Hepatol. 2003 Dec;39(6):1072-5.

NCX-1000, a nitric oxide-releasing derivative of ursodeoxycholic acid, ameliorates portal hypertension and lowers norepinephrine-induced intrahepatic resistance in the isolated and perfused rat liver.

Fiorucci S, Antonelli E, Brancaleone V, Sanpaolo L, Orlandi S, Distrutti E, Acuto G, Clerici C, Baldoni M, Del Soldato P, Morelli A. Clinica di Gastroenterologia ed Epatologia, Dipartimento di Medicina Clinica e Sperimentale, Universita degli Studi di Perugia, Perugia, Italy.

BACKGROUND/AIMS: We studied whether acute administration of NCX-1000, a nitric oxide (NO)-releasing derivative of ursodeoxycholic acid (UDCA), to animals with established liver cirrhosis decreases intrahepatic resistance and modulates hepatic vascular hypereactivity to norepinephrine (NE). METHODS: Four-week bile duct ligated (BDL) cirrhotic and control, sham-operated, rats were treated orally with 28 mg/kg per day NCX-1000 or 15 mg/kg per day UDCA for 5 days. Isolated normal and cirrhotic livers were perfused with NE, from 10 nM to 30 microM, in a recirculating system. RESULTS: NCX-1000 administration to BDL cirrhotic rats decreased portal pressure (P<0.01) without affecting mean arterial pressure and heart rate. In the isolated perfused liver system, administration of NE resulted in a dose-dependent increase of intrahepatic resistance. Vasoconstriction caused by 30 microM NE was reduced by 60% in animals treated with NCX-1000 (P<0.001), while UDCA was uneffective. The same portal pressure lowering effect was documented in cirrhotic and sham operated rats. Administration of NCX-1000 to BDL and sham operated rats resulted in a similar increase of nitrite/nitrate and cGMP concentrations in the liver. CONCLUSIONS: By selectively delivering NO to the liver, NCX-1000 increases cGMP concentrations and effectively counteracts the effect of endogenous vasoconstrictors on the hepatic vascular tone.