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We recently reported the synthesis of NOSH-aspirin, a novel hybrid that releases both nitric oxide (NO) and hydrogen sulfide (H2S). In NOSH-aspirin, the two moieties that release NO and H2S are covalently linked at the 1, 2 positions of acetyl salicylic acid, i.e. ortho-NOSH-aspirin (o-NOSH-aspirin). In the present study, we compared the effects of the positional isomers of NOSH-ASA (o-NOSH-aspirin, m-NOSH-aspirin and p-NOSH-aspirin) to that of aspirin on growth of HT-29 and HCT 15 colon cancer cells, belonging to the same histological subtype, but with different expression of cyclooxygenase (COX) enzymes; HT-29 express both COX-1 and COX-2, whereas HCT 15 is COX-null. We also analyzed the effect of these compounds on proliferation and apoptosis in HT-29 cells. Since the parent compound aspirin, inhibits both COX-1 and COX-2, we also evaluated the effects of these compounds on COX-1 and COX-2 enzyme activities and also performed modeling of the interactions between the positional isomers of NOSH-aspirin and COX-1 and COX-2 enzymes. We observed that the three positional isomers of NOSH aspirin inhibited the growth of both colon cancer cell lines with IC50s in the nano-molar range. In particular in HT-29 cells the IC50s for growth inhibition were: o-NOSH-ASA, 0.04±0.011 µM; m-NOSH-ASA, 0.24±0.11 µM; p-NOSH-ASA, 0.46±0.17 µM; and in HCT 15 cells the IC50s for o-NOSH-ASA, m-NOSH-ASA, and p-NOSH-ASA were 0.062 ±0.006 µM, 0.092±0.004 µM, and 0.37±0.04 µM, respectively. The IC50 for aspirin in both cell lines was >5mM at 24h. The reduction of cell growth appeared to be mediated through inhibition of proliferation, and induction of apoptosis. All 3 positional isomers of NOSH-aspirin preferentially inhibited COX-1 over COX-2. These results suggest that the three positional isomers of NOSH-aspirin have the same biological actions, but that o-NOSH-ASA displayed the strongest anti-neoplastic potential., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Nitric oxide (NO) plays a relevant role during cell death regulation in tumor cells. The overexpression of nitric oxide synthase type III (NOS-3) induces oxidative and nitrosative stress, p53 and cell death receptor expression and apoptosis in hepatoblastoma cells. S-nitrosylation of cell death receptor modulates apoptosis. Sorafenib is the unique recommended molecular-targeted drug for the treatment of patients with advanced hepatocellular carcinoma. The present study was addressed to elucidate the potential role of NO during Sorafenib-induced cell death in HepG2 cells. We determined the intra- and extracellular NO concentration, cell death receptor expression and their S-nitrosylation modifications, and apoptotic signaling in Sorafenib-treated HepG2 cells. The effect of NO donors on above parameters has also been determined. Sorafenib induced apoptosis in HepG2 cells. However, low concentration of the drug (10nM) increased cell death receptor expression, as well as caspase-8 and -9 activation, but without activation of downstream apoptotic markers. In contrast, Sorafenib (10 µM) reduced upstream apoptotic parameters but increased caspase-3 activation and DNA fragmentation in HepG2 cells. The shift of cell death signaling pathway was associated with a reduction of S-nitrosylation of cell death receptors in Sorafenib-treated cells. The administration of NO donors increased S-nitrosylation of cell death receptors and overall induction of cell death markers in control and Sorafenib-treated cells. In conclusion, Sorafenib induced alteration of cell death receptor S-nitrosylation status which may have a relevant repercussion on cell death signaling in hepatoblastoma cells., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

The influence of (1H-1,2,3-triazol-1-yl)-1,2,5-oxadiazole derivatives: 4-amino-3-(5-methyl-4- ethoxycarbonyl-(1H-1,2,3-triazol-1-yl)-1,2,5-oxadiazole (TF4CH3) and 4,4'-bis(5-methel-4-ethoxycarbonyl-1H- 1,2,3-triazol-1-yl)-3,3'-azo-1,2,5-oxadiazole (2TF4CH3) on stimulation of human platelet soluble guanylate cyclase by YC-1, NO-donors (sodium nitroprusside, SNP, and spermine NONO) and on a synergistic increase of NO-dependent enzyme activation in the presence of YC-1 has been investigated. Both compounds increased guanylate cyclase activation by YC-1, potentiated guanylate cyclase stimulation by NO-donors and increased the synergistic effect of YC-1 on NO-dependent activation of soluble guanylate cyclase. The similarity in the properties of the examined TF4CH3 and 2TF4CH3 with that of YC-1 and the possible mechanism underlying the revealed properties of compounds used are discussed.

Recently, an increasing number of publications have demonstrated the importance of the small molecule nitric oxide (NO) in several physiological and pathophysiological processes. NO acts as a key modulator in cardiovascular, immunological, neurological, and respiratory systems, and deficiencies in the production of NO or its inactivation has been associated with several pathologic conditions, ranging from hypertension to sexual dysfunction. Although the clinical administration of NO is still a challenge owing to its transient chemical nature, the combination of NO and nanocarriers based on biocompatible polymeric scaffolds has emerged as an efficient approach to overcome the difficulties associated with the biomedical administration of NO. Indeed, significant progress has been achieved by designing NO-releasing polymeric nanomaterials able to promote the spatiotemporal generation of physiologically relevant amounts of NO in diverse pharmacological applications. In this review, we summarize the recent advances in the preparation of versatile NO-releasing nanocarriers based on polymeric nanoparticles, dendrimers and micelles. Despite the significant innovative progress achieved using nanomaterials to tailor NO release, certain drawbacks still need to be overcome to successfully translate these research innovations into clinical applications. In this regard, this review discusses the state of the art regarding the preparation of innovative NO-releasing polymeric nanomaterials, their impact in the biological field and the challenges that need to be overcome. We hope to inspire new research in this exciting area based on NO and nanotechnology., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Earlier it has been found that the hypotensive drug Oxacom containing binuclear dinitrosyl iron complexes (B-DNIC) with glutathione can effectively decrease, as a nitric monooxide (NO) donor, the mean arterial pressure (МАР) in rats upon intravenous bolus injection in the form of an aqueous solution (Chazov et al., 2012). The aim of this study was to investigate the hypotensive effects of Oxacom administered to experimental rats by intravenous, intramuscular, subcutaneous, intraperitoneal, intragastric, rectal routes.MAP and heart rate (HR) were measured with the help of arterial catheters equipped with tensometric sensors. Oxacom was administered to rats at the dose of 2.0 μmole of B-DNIC/kg. The concentration of paramagnetic mononuclear protein-bound DNIC (М-DNIC) formed in the blood and tissues of various internal organs of the rat was determined by the EPR method. Upon subcutaneous, intramuscular or intraperitoneal administration of Oxacom, the maximum amplitude of the МАР decrease varies from 30% to 70%, respectively, in comparison with the corresponding parameter for the intravenously injected Oxacom. Another difference is the lack of the fast phase in the initial stage of the МАР decrease and the longer persistence of protein-bound M-DNIC formed in the circulating blood after intramuscular, subcutaneous or intraperitoneal administration of Oxacom. Thus, the NO donor Oxacom exerts pronounced hypotensive effects on rats not only upon intravenous, but also upon intramuscular, subcutaneous or intraperitoneal administration., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Pseudomonas aeruginosa is an opportunistic pathogen causing a variety of life-threatening diseases such as cystic fibrosis and nosocomial infections in burn victims. The ability of P. aeruginosa to cause infection is attributed to the production of virulence factors such as pyocyanin and elastases. These virulence factors are under the control of quorum sensing (QS) a cell to cell communication process controlled by small diffusible signalling molecules based on N-acyl-homoserine lactones (AHLs) known as autoinducers. The inhibition of QS and thereby virulence factors is seen as a potential new anti-infective strategy. Additionally, the role of nitric oxide (NO) in downstream processes in bacteria such as biofilm dispersal, motility, virulence and antimicrobial defence systems is gaining attention and could be used to control bacterial. Herein we report the design and synthesis of hybrid compounds based on AHL signalling molecules and NO donors as anti-infective agents. A series of AHL-NO hybrids were synthesised and potent inhibitors of QS and virulence factors of P. aeruginosa were identified. This research has led to conversion of agonist AHLs to antagonist AHLs with dual properties of QS inhibition and NO release.

Composites of a collagen matrix and dinitrosyl iron complexes with glutathione (DNIC-GS) (in a dose of 4.0 μmoles per item) in the form of spongy sheets (DNIC-Col) were prepared and then topically applied in rat excisional full-thickness skin wound model. The effects of DNIC-Col were studied in comparison with spontaneously healing wounds (SpWH) and wounds treated with collagen sponges (Col) without DNIC-GS. The composites induced statistically and clinically significant acceleration of complete wound closure (21±1 day versus 23±1 day and 26±1 day for DNIC-Col, Col and SpWH, respectively). Histological examination of wound tissues on days 4, 14, 18 and 21 after surgery demonstrated that this improvement was supported by enhanced growth, maturation and fibrous transformation of granulation tissue and earlier epithelization of the injured area in rats treated with DNIC-Col composites benchmarked against Col and SpWH. It is suggested that the positive effect of the new pharmaceutical material on wound healing is based on the release of NO from decomposing DNIC. This effect is believed to be potentiated by the synergy of DNIC and collagen., (Copyright © 2015. Published by Elsevier B.V.)

Hybrid products in which the dihydroartemisinin scaffold is combined with NO-donor furoxan and NONOate moieties have been synthesized and studied as potential tools for the treatment of cerebral malaria (CM). The designed products were able to dilate rat aorta strips precontracted with phenylephrine with a NO-dependent mechanism. All hybrid compounds showed preserved antiplasmodial activity in vitro and in vivo against Plasmodium berghei ANKA, comparable to artesunate and artemether. Hybrid 10, selected for additional studies, was capable of increasing survival of mice with late-stage CM from 27.5% to 51.6% compared with artemether. Artemisinin-NO-donor hybrid compounds show promise as potential new drugs for treating cerebral malaria.

Photocontrollable NO donors enable precise spatiotemporal release of NO under physiological conditions. We designed and synthesized a novel dimethylnitrobenzene-type NO donor, Flu-DNB-DB, which contains a carbon-carbon double bond in place of the amide bond of previously reported Flu-DNB. Flu-DNB-DB releases NO in response to one-photon activation in the blue wavelength region, and shows a greatly increased two-photon cross-section (δu) at 720 nm (Flu-DNB: 0.12 GM, Flu-DNB-DB: 0.98 GM). We show that Flu-DNB-DB enables precisely controlled intracellular release of NO in response to 950 nm pulse laser irradiation for as little as 1s. This near-infrared-light-controllable NO source should be a valuable tool for studies on the biological roles of NO., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Telokin phosphorylation by cyclic GMP-dependent protein kinase facilitates smooth muscle relaxation. In this study we examined the relaxation of gastric fundus smooth muscles from basal tone, or pre-contracted with KCl or carbachol (CCh), and the phosphorylation of telokin S13, myosin light chain (MLC) S19, MYPT1 T853, T696, and CPI-17 T38 in response to 8-Bromo-cGMP, the NO donor sodium nitroprusside (SNP), or nitrergic neurotransmission. We compared MLC phosphorylation and the contraction and relaxation responses of gastric fundus smooth muscles from telokin-/- mice and their wild-type littermates to KCl or CCh, and 8-Bromo-cGMP, SNP, or nitrergic neurotransmission, respectively. We compared the relaxation responses and telokin phosphorylation of gastric fundus smooth muscles from wild-type mice and W/WV mice which lack ICC-IM, to 8-Bromo-cGMP, SNP, or nitrergic neurotransmission. We found that telokin S13 is basally phosphorylated and that 8-Bromo-cGMP and SNP increased basal telokin phosphorylation. In muscles pre-contracted with KCl or CCh, 8-Bromo-cGMP and SNP had no effect on CPI-17 or MYPT1 phosphorylation, but increased telokin phosphorylation and reduced MLC phosphorylation. In telokin-/- gastric fundus smooth muscles, basal tone and constitutive MLC S19 phosphorylation were increased. Pre-contracted telokin-/- gastric fundus smooth muscles have increased contractile responses to KCl, CCh, or cholinergic neurotransmission and reduced relaxation to 8-Bromo-cGMP, SNP, and nitrergic neurotransmission. However, basal telokin phosphorylation was not increased when muscles were stimulated with lower concentrations of SNP or when the muscles were stimulated by nitrergic neurotransmission. SNP, but not nitrergic neurotransmission, increased telokin Ser13 phosphorylation in both wild-type and W/WV gastric fundus smooth muscles. Our findings indicate that telokin may play a role in attenuating constitutive MLC phosphorylation and provide an additional mechanism to augment gastric fundus mechanical responses to inhibitory neurotransmission.

A highly sensitive amperometric gas-phase nitric oxide (NO) sensor based on a Pt working electrode chemically deposited on a Nafion film is described. The Pt electrode is chemically deposited on a Nafion 117 membrane by impregnating the film with Pt(NH3)4(2+) ions, which are then exposed to NaBH4 to precipitate conductive Pt metal. The sensor was characterized with a mass-flow controlled 1 ppm NO standard gas and has an electrochemical surface area of 34 ± 9 cm(2), low limits of detection (4.3 ± 1.1 ppb) and a fast response time (<5s) toward changes in gas phase NO levels. Good correlation was found for measurements with the new amperometric Pt-Nafion sensor vs. chemiluminescence results for detecting the rates of NO release from Carbosil2080A polymer films doped with S-nitroso-N-acetylpenicillamine (R = 0.999, m = 0.999, n = 6) and for electrochemical reduction of nitrite to NO (R = 0.999, m = 0.938, n = 3) mediated by a copper(II)-tri(2-pyridylmethyl)amine complex., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objectives: The aim of this work is to synthesize a novel stable and biodegradable nitric oxide (NO) donor polymer based on a chitosan backbone. This polymer needed to be linked to glutathione (GSH), which was nitrosated in a second step. This polymer has been developed as an NO delivery platform that could be further evaluated for an oral delivery in Crohn's disease., Methods: The new polymer (named S-nitrosoglutathione-oligosaccharide-chitosan or SNOC) was obtained using a two-step procedure involving the linkage of GSH to chitosan via an amidine reaction followed by a post-nitrosation with NaNO2. The GSH linkage was assessed using NMR, FTIR and an Ellman's test, whereas the final NO amount was determined by the Griess and Saville method., Results: Polymers with different numbers of NO groups were obtained (159.04 ± 64.16 µmol/g of polymer for SNOC G1 and 525.08 ± 151.35 µmol/g of polymer for SNOC G2) depending on the procedure used for production. When tested in an Ussing chamber, SNOC G2 had a sustained release of NO and nitrites for at least 6 h., Conclusion: We believe that this type of polymer is adapted for the development of various formulations, including microparticles.

Nitric oxide (NO) is a promising therapeutic agent with antibacterial and wound-healing properties. However, the gaseous state and short half-life of NO necessitate a formulation that can control its storage and release. In this study, we developed NO-releasing films (CS/NO film) composed of chitosan (CS) and S-nitrosoglutathione (GSNO) as a NO donor. Thermal analysis demonstrated molecular dispersion of GSNO in the films. In vitro release study revealed that NO release from CS/NO films followed Korsmeyer-Peppas model with Fickian diffusion kinetics. Moreover, the CS/NO film showed a stronger antibacterial activity against Pseudomonas aeruginosa (Gram-negative) and Staphylococcus aureus (Gram-positive) than the CS film. Further, the CS/NO film accelerated wound healing and epithelialization in a rat model of full-thickness wounds as compared to the CS film. Histopathological studies revealed that CS/NO films favorably enhanced the re-epithelialization and reconstruction of wounded skin. Therefore, our results suggest that CS/NO films could be a suitable formulation for treating full-thickness wounds., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Dinitrosyl iron(i) complexes (DNICs), intracellular NO donors, are important factors in nitric oxide-dependent regulation of cellular metabolism and signal transduction. It has been shown that NO diminishes the toxicity of iron ions and vice versa. To gain insight into the possible role of DNIC in this phenomenon, we examined the effect of GS-DNIC formation on the ability of iron ions to mediate DNA damage, by treatment of the pUC19 plasmid with physiologically relevant concentrations of GS-DNIC. It was shown that GS-DNIC formation protects against the genotoxic effect of iron ions alone and iron ions in the presence of a naturally abundant antioxidant, GSH. This sheds new light on the iron-related protective effect of NO under the circumstances of oxidative stress.

To evaluate the biological preference of synthetic small drugs towards DNA target, new metal based chemotherapeutic agents of Cu(II), Co(II), Ni(II) and Zn(II), 2,4-diiodo-6-((pyridin-2-ylmethylimino)methyl)phenol (L) Schiff base complexes (1, 2, 3 &4) having N,N,O donor system respectively were synthesized and thoroughly characterized. The IR results confirmed the tridentate binding of the ligand with metal centre during complexation and reflects the proposed structure. The density function theory calculations were also used to further investigate the electronic structure and properties of ligand and complexes. The preliminary investigation of herring Sperm (HS-DNA) interaction propensity of complexes 1-4 were carried out in Tris-HCl buffer at pH 7.1 to demonstrate their mode of interactions. The obtained results reveal that these complexes significantly interact with DNA on the grooves, further, this observed mode of interactions was also confirmed by molecular docking evaluations. The complexes 1-4 were also screened for antimicrobial evaluations which demonstrated that their significant activity against various human pathogens. The cleavage studies with pBR322 plasmid DNA revealed higher nuclease activity of 1 as compared to other complexes., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cell wall is the major component of root apoplast which is the main reservoir for iron in roots, while nitric oxide (NO) is involved in regulating the synthesis of cell wall. However, whether such regulation could influence the reutilization of iron stored in root apoplast remains unclear. In this study, we observed that iron deficiency elevated NO level in tomato (Solanum lycopersicum) roots. However, application of S-nitrosoglutathione, a NO donor, significantly enhanced iron retention in root apoplast of iron-deficient plants, accompanied with a decrease of iron level in xylem sap. Consequently, S-nitrosoglutathione treatment increased iron concentration in roots, but decreased it in shoots. The opposite was true for the NO scavenging treatment with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Interestingly, S-nitrosoglutathione treatment increased pectin methylesterase activity and decreased degree of pectin methylation in root cell wall of both iron-deficient and iron-sufficient plants, which led to an increased iron retention in pectin fraction, thus increasing the binding capacity of iron to the extracted cell wall. Altogether, these results suggested that iron-deficiency-induced elevation of NO increases iron immobilization in root apoplast by decreasing pectin methylation in cell wall.

On the basis of the strategy of creating multifunctional drugs, a novel series of phenylsulfonylfuroxan-based hydroxamates with histone deacetylase (HDAC) inhibitory and nitric oxide (NO) donating activities were designed, synthesized, and evaluated. The most potent NO donor-HDAC inhibitor (HDACI) hybrid, 5c, exhibited a much greater in vitro antiproliferative activity against the human erythroleukemia (HEL) cell line than that of the approved drug SAHA (Vorinostat), and its antiproliferative activity was diminished by the NO scavenger hemoglobin in a dose-dependent manner. Further mechanism studies revealed that 5c strongly induced cellular apoptosis and G1 phase arrest in HEL cells. Animal experiment identified 5c as an orally active agent with potent antitumor activity in a HEL cell xenograft model. Interestingly, although compound 5c was remarkably HDAC6-selective at the molecular level, it exhibited pan-HDAC inhibition in a western blot assay, which is likely due to class I HDACs inhibition caused by NO release at the cellular level.

The fact that drugs currently used in the treatment of Leishmania are highly toxic and associated with acquired resistance has promoted the search for new therapies for treating American tegumentary leishmaniasis (ATL). In this study, BALB/c mice were injected in the hind paw with Leishmania (Leishmania) amazonensis and subsequently treated with a combination of nitric oxide (NO) donor (cis-[Ru(bpy) 2imN(NO)](PF6)3) (Ru-NO), given by intraperitoneal injection, and oral Brazilian propolis for 30 days. Ru-NO reached the center of the lesion and increased the NO level in the injured hind paw without lesion exacerbation. Histological and immunological parameters of chronic inflammation showed that this combined treatment increased the efficacy of macrophages, determined by the decrease in the number of parasitized cells, leading to reduced expression of proinflammatory and tissue damage markers. In addition, these drugs in combination fostered wound healing, enhanced the number of fibroblasts, pro-healing cytokines and induced collagen synthesis at the lesion site. Overall, our findings suggest that the combination of the NO donor Ru-NO and Brazilian propolis alleviates experimental ATL lesions, highlighting a new therapeutic option that can be considered for further in vivo investigations as a candidate for the treatment of cutaneous leishmaniasis.

After vascular interventions, endothelial cells are typically injured or lacking, resulting in decreased NO synthesis to maintain vascular health. Moreover, inflammation as a result of the tissue injury and/or the presence of an implanted foreign polymer such as a vascular graft causes excessive generation of reactive oxygen species (ROS) (e.g., superoxide), which can react with NO. The combination of the above creates a general decline in NO bioavailability, as well as oxidative stress due to less available NO to scavenge ROS. Localized NO delivery is an attractive solution to alleviate these issues; however, NO donors typically exhibit unpredictable NO payload release when using nitrosothiols or the risk of nitrosamine formation for synthetic diazeniumdiolates. The objective of this study was therefore to synthesize an NO donor from a biological peptide that could revert to its native form upon NO release. To this effect, protamine sulfate (PS), an FDA-approved peptide with reported vasodilator and anticoagulant properties, was diazeniumdiolated to form PS/NO. PS/NO showed diazeniumdiolate-characteristic UV peaks and NO release in physiological solutions and was capable of scavenging radicals to decrease oxidative stress. Furthermore, PS/NO selectively inhibits the proliferation of smooth muscle cells and adventitial fibroblasts, thereby reversing reported mitogenic properties of PS. Endothelial cell growth, on the other hand, was promoted by PS/NO. Finally, PS retained its anticoagulant properties upon diazeniumdiolation at clinically relevant concentrations. In conclusion, we have synthesized an NO prodrug from a biological peptide, PS/NO, that selectively inhibits proliferation of smooth muscle cells and fibroblasts, retains anticoagulant properties, and reverts back to its native PS form upon NO payload release., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Two series of novel NO-releasing benzimidazole derivatives (8a-e, 9a-g) were designed and synthesized by coupling nitro ester and furoxan NO-donor moieties with benzimidazole biphenyl skeleton. The NO-releasing assay indicated that all the target compounds had different level of NO-releasing ability. Furthermore, the isolated organ assay (rat aortic strips) was used to evaluate the antagonism of Ang II-induced vasoconstriction ability. It was observed that the pA2 values of compounds 8e and 9e were better than that of lead compound 6. Moreover, the pharmacological investigation showed that the antagonism of Ang II-induced pressure response by oral administration of compound 8e was obviously superior to that of lead compound 6, and comparable to that of the positive control losartan. These results suggested that NO-releasing hybrids may provide a promising approach for the discovery of novel antihypertensive agents., (© 2014 John Wiley & Sons A/S.)

Background: Nitric oxide (NO) is a vital signalling molecule in a variety of tissues including the neuronal, vascular and reproductive system. However, its high diffusibility and inactivation make characterisation of nitrergic signalling difficult. The use of NO donors is essential to characterise downstream signalling pathways but knowledge of donor release capacities is lacking, thus making comparisons of donor responses difficult., New Method: This study characterises NO profiles of commonly used NO donors. Donors were stored under defined conditions and temporal release profiles detected to allow determination of released NO concentrations., Results: Using NO-sensitive microsensors we assessed release profiles of NO donors following different storage times and conditions. We found that donors such as NOC-5 and PAPA-NONOate decayed substantially within days, whereas SNP and GSNO showed greater stability releasing consistent levels of NO over days. In all donors tested, the amount of released NO differs between frozen and unfrozen stocks., Comparison With Existing Method(s): Fluorescent and amperometric approaches to measure NO concentrations yield a wide range of levels. However, due to a lack of characterisation of the release profiles, inconsistent effects on NO signalling have been widely documented. Our systematic assessment of release profiles of a range of NO donors therefore provides new essential data allowing for improved and defined investigations of nitrergic signalling., Conclusions: This is the first systematic comparison of temporal release profiles of different NO donors allowing researchers to compare conditions across different studies and the use of defined NO levels by choosing specific donors and concentrations., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Photochemical Nitric oxide releasing composite materials (Photo-NORMs) were prepared using biocompatible polymers and the photochemical nitric oxide donor complex (CrONO). We have demonstrated nitric oxide (NO) release from the solid composites for extended (>30 hours) and controlled (20-100 pmoles s(-1)) durations after visible light irradiation. Quantitation of the efficiency of NO release from the composites shows that polymer gas permeability most dramatically affects the overall efficiency (QY) of photochemical NO release, where polymers with higher gas permeability have a higher QY of nitric oxide release. Composites were also prepared with β-phase lanthanide-doped NaYF4 upconverting nanoparticles (UCNPs). Controlled Nitric oxide release was achieved via near infrared (NIR) irradiation. A prototype LED device shows proof-of-concept that such photoresponsive NO-releasing composites could be applied to implantable systems, where the amount of NO released is modulated by changing irradiation time and light intensity. This research provides the guidelines necessary to move towards device fabrication and testing in actual tissue to evaluate the photo-NORMS as a reliable option for nitric oxide release in vivo.

The antiglaucoma drugs dorzolamide (1) and brinzolamide (2) lower intraocular pressure (IOP) by inhibiting the carbonic anhydrase (CA) enzyme to reduce aqueous humor production. The introduction of a nitric oxide (NO) donor into the alkyl side chain of dorzolamide (1) and brinzolamide (2) has led to the discovery of NO-dorzolamide 3a and NO-brinzolamide 4a, which could lower IOP through two mechanisms: CA inhibition to decrease aqueous humor secretion (reduce inflow) and NO release to increase aqueous humor drainage (increase outflow). Compounds 3a and 4a have shown improved efficacy of lowering IOP in both rabbits and monkeys compared to brinzolamide (2).

Seventeen CDDO-amino acid-NO donor trihybrids (4a-q) were designed and synthesized. Biological evaluation indicated that the most active compound 4c produced high levels of NO and inhibited the proliferation of drug-sensitive (HCT-8, IC50 = 0.294 μM) and drug-resistant (HCT-8/5-FU, IC50 = 0.232 μM) colon cancer cells, which were attenuated by an NO scavenger or typical substrate of PepT1. Furthermore, 4c triggered HCT-8 and HCT-8/5-FU cell apoptosis more strongly than CDDO-Me, inhibited the HIF-1α, Stat3, AKT, and ERK signaling, and induced the nitration of P-gp, MRP1, and BCRP proteins in HCT-8/5-FU cells. Finally, 4c had 4.36-5.53-fold less inhibitory activity against nontumor colon epithelial-like cells (CCD841, IC50 = 1.282 μM) in vitro and inhibited the growth of implanted human drug-resistant colon cancers in mice more potently than CDDO-Me. Together, 4c is a novel trihybrid with potent antitumor activity and may be a promising candidate for the treatment of drug-resistant colon cancer.

Objective: Increasing evidence suggests that endothelial nitric oxide (NO) deficit and baroreflex dysfunction are associated with a variety of cardiovascular conditions, ranging from arterial hypertension to stroke and coronary heart disease, importantly appearing even in preclinical stages of the disease. To test the hypothesis that the arterial baroreflex has a modulatory effect on NO-dependent vasodilation, sodium nitroprusside (SNP), a spontaneous NO-donor, vasodilatory effect was studied in conjunction with sinocarotid baroreceptor magnetic stimulation and potential implementation in NO deficiency states., Methods: Mean femoral artery blood pressure (MAP), heart rate (HR) and ear lobe skin microcirculatory blood flow, measured by a microphotoelectric plethysmogram (MPPG), were simultaneously recorded in conscious rabbits before and after 40-min sinocarotid baroreceptor exposure to 350 mT static magnetic field (SMF), generated by Nd2-Fe4-B alloy (n=8) or sham magnets (n=8, controls). Arterial baroreflex sensitivity (BRS) was measured by changes in HR and MAP (ΔHR/ΔMAP) after intravenous bolus injections of SNP and phenylephrine., Results: The vasodilatory effect of SNP significantly increased after SMF sinocarotid baroreceptor exposure (MPPGbeforeSMF: 2.57 ± 0.81 V vs. MPPGafterSMF: 7.82 ± 1.61 V, p<0.0001) and positively correlated with significant increase in BRS (r=0.51, p=0.01)., Conclusions: Baroreflex-mediated increment in vessel sensitivity to NO is suggested to be a new mechanism in baroreflex physiology with potential implementation in cardiovascular conditions where NO deficit and autonomic dysfunction increase the risk of morbidity and mortality substantially., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A range of fac-[Mn(CO)3(L,L'-Bid)(H2O)](n) (L,L'-Bid = neutral or monoanionic bidentate ligands with varied L,L' donor atoms, N,N' and N,O, 1,10-phenanthroline, 2,2'-bipyridine, 2-picolinate, 2,4-quinolinate; n = 0, +1) has been synthesized and the methanol substitution has been investigated for the first time. The complexes were characterized by UV/vis, IR and NMR spectroscopy and X-ray crystallographic studies of the compounds fac-[Mn(CO)3(Bipy)(H2O)][CF3SO3] () and fac-[Mn(CO)3(Phen)(H2O)][CF3SO3] () are reported. A two order-of-magnitude of activation for the methanol substitution is induced as manifested by the second order rate constants with (N,N'-Bid) < (N,O-Bid). Forward and reverse rate and stability constants from slow and stopped-flow UV/vis measurements (k1, M(-1) s(-1); k-1, s(-1); K1, M(-1)) for pyridine as entering nucleophile are as follows: fac-[Mn(CO)3(Phen)(CH3OH)](+) (2.39 ± 5) × 10(-3), (1.5 ± 0.3) × 10(-5), 159 ± 32; fac-[Mn(CO)3(2,4-QuinH)(CH3OH)] (4.5 ± 0.2), (4 ± 1) × 10(-2), 113 ± 29. Activation parameters (ΔH, kJ mol(-1); ΔS, J K(-1) mol(-1)) from Eyring plots for entering nucleophiles as indicated are as follows: fac-[Mn(CO)3(Phen)(CH3OH)](+) (bromide ions) 66.7 ± 0.6, -27 ± 2; (pyridine) 80 ± 3, -25 ± 11; fac-[Mn(CO)3(Pico)(CH3OH)] (bromide ions) 68 ± 2, -24 ± 5. A dissociative interchange mechanism is proposed.

Here, we have evaluated the protective effect of the NO donor cis-[Ru(bpy)2(SO3)NO](PF6) (FOR0810) in experimental models of gastric damage induced by naproxen or ethanol in mice, and the involvement of soluble guanylate cyclase (sGC) and ATP-sensitive K(+) channels (KATP) in these events. Swiss mice were pre-treated with saline, ODQ (a soluble guanylate cyclase inhibitor; 10 mg kg(-1)) or glibenclamide (a KATP channels blocker; 10 mg kg(-1)). After either 30 min or 1 h, FOR0810 (3 mg kg(-1)) was administered. At the end of 30 min, the animals received naproxen (300 mg kg(-1)) by gavage. After 6 h, the animals were sacrificed and gastric damage, myeloperoxidase (MPO) activity, and TNF-α and IL-1β gastric concentrations were evaluated. In addition, the effects of FOR0810 on naproxen-induced mesenteric leukocyte adherence were determined by intravital microscopy. Other groups, were pre-treated with saline, ODQ or glibenclamide. After either 30 min or 1 h, FOR0810 was administered. At the end of 30 min, the animals received 50% ethanol by gavage. After 1 h, the animals were sacrificed, and gastric damage, gastric reduced glutathione (GSH) concentration and malondialdehyde (MDA) levels were determined. In naproxen-induced gastric damage, FOR0810 prevented gastric injury, decreased gastric MPO activity and leukocyte adherence, associated with a decrease in TNFα and IL-1β gastric concentrations. FOR0810 also prevented ethanol-induced gastric damage by increase in GSH levels and decrease in MDA levels. ODQ and glibenclamide completely reversed FOR0810's ability to prevent gastric damage by either naproxen or ethanol. We infer that FOR0810 prevented gastric damage through the activation of both sGC and KATP channels, which triggered a decrease in both free radical and cytokine production via the blocking of neutrophil adhesion and infiltration., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Three new NOS donor ligands have been prepared by addition ethanolic suspension of 2-hydrazino-2-oxo-N-phenyl-acetamide to phenyl isocyanate (H2PAPS), phenyl isothiocyanate (H2PAPT) and benzoyl isothiocyanate (H2PABT). The Ni(II) complexes prepared from the chloride salt and characterized by conventional techniques. The isolated complexes were assigned the formulaes, [Ni2(PAPS)(H2O)2](H2O)2, [Ni(H2PAPT)Cl2(H2O)](H2O)2 and [(Ni)2(HPABT)2Cl2(H2O)2], respectively. The IR spectra of complexes shows that H2PAPS behaves as a binegative pentadentate via both CO of hydrazide moiety in keto and enol form, enolized CO of cyanate moiety and the CN (azomethine) groups of enolization. H2PAPT behaves as neutral tridentate via both CO of hydrazide moiety and CN (azomethine) group due to SH formation and finally H2PABT behaves as mononegative tetradentate via CO and enolized CO of hydrazide moiety, CO of benzoyl moiety and C=S groups. The experimental IR spectra of ligands are compared with those obtained theoretically from DFT calculations. Also, the bond lengths, bond angles, HOMO (Highest Occupied Molecular Orbitals), LUMO (Lowest Unoccupied Molecular Orbital) and dipole moments have been calculated. The calculated HOMO-LUMO energy gap reveals that charge transfer occurs within the molecule. The theoretical values of binding energies indicate the higher stability of complexes than of ligands. Also, the kinetic and thermodynamic parameters for the different thermal degradation steps of the complexes were determined by Coats-Redfern and Horowitz-Metzger methods. The antibacterial activities were also tested against B. Subtilis and E. coli bacteria. The free ligands showed a higher antibacterial effect than their Ni(II) complexes. The antitumor activities of the Ligands and their Ni(II) complexes have been evaluated against liver (HePG2) and breast (MCF-7) cancer cells. All ligands were found to display cytotoxicity that are better than that of Fluorouracil (5-FU), while Ni(II) complexes show low activity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A new chelating agent, N'-(4-methoxybenzylidene)-2-oxo-2-(phenylamino)acetohydrazide (H2OMPH) and its complexes with Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Hg(II) and U(IV)O2(2+) ions have been prepared and characterized by conventional techniques. The spectral data indicated that the ligand coordinates as neutral bidentate with Cu(II), Mn(II), U(IV)O2(2+) and Hg(II), neutral tridentate with Ni(II), mononegative tridentate with Co(II) and binegative tetradentate with Zn(II) ions. On basis of magnetic and electronic spectral data an octahedral geometry for Mn(II), Co(II) and Ni(II) complexes and a square planar geometry for Cu(II) complex have been proposed and confirmed by applying geometry optimization and conformational analysis. The protonation constants of H2OMPH and the stepwise stability constants of its complexes are calculated at 298, 308 and 318 k as well as their thermodynamic parameters. Also, the Kinetic parameters (Ea, A, ΔH(*), ΔS(*) and ΔG(*)) were determined for each thermal degradation stage of some complexes using Coats-Redfern and Horowitz-Metzger methods. Moreover, the ligand and some complexes were screened for in vitro antibacterial activity against Staphylococcus epidermalies (St. epid); Streptococcus pyagenies (Strp. py.) as Gram +ve bacteria and Escherichia coli (E. coli); Klebsiella spp. (kleb. spp.) as Gram -ve bacteria using inhibition zone diameter., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Edaravone (5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one, EDV) is a free-radical scavenger reduces organ ischemic injury. Here we investigated whether the protective effects of EDV in renal ischemia/reperfusion (I/R) injury may be enhanced by an EDV derivative bearing a nitric oxide- (NO-) donor furoxan moiety (NO-EDV). Male Wistar rats were subjected to renal ischemia (45 minutes), followed by reperfusion (6 hours). Administration of either EDV (1.2-6-30 µmol/kg, i.v.) or NO-EDV (0.3-1.2-6 µmol/kg, i.v.) dose-dependently attenuated markers of renal dysfunction (serum urea and creatinine, creatinine clearance, urine flow, urinary N-acetyl-β-D-glucosaminidase, and neutrophil gelatinase-associated lipocalin/lipocalin-2). NO-EDV exerted protective effects in the dose-range 1.2-6 µmol/kg, while a higher dose (30 µmol/kg) was needed to obtain protection by EDV. Both EDV and NO-EDV modulated tissue markers of oxidative stress and lipid peroxidation. NO-EDV, but not EDV, activated endothelial NO synthase (NOS) and blunted I/R-induced upregulation of inducible NOS, secondary to modulation of Akt and NF-κB activation, respectively. Besides NO-EDV administration inhibited I/R-induced IL-1β, IL-18, IL-6, and TNF-α overproduction. Overall, these findings demonstrate that the NO-donor moiety contributes to the protection against early renal I/R injury and suggest that NO-donor EDV codrugs are worthy of additional study as innovative pharmacological tools.

Nitric oxide (NO) is known to have dichotomous effects on cancer biology, acting as a pro- or antineoplastic agent. Low concentrations of NO are reported to promote tumor growth, whereas high NO influx acts as a potent tumor repressor, leading to cytotoxicity and apoptosis. There is increasing interest in developing NO-releasing materials as potent tumoricidal agents in which high and localized concentrations of NO may be directly released in a sustained manner to the tumor site. Nanomaterials allied to NO donors have emerged as a promising strategy in cancer treatment. In this context, this review summarizes the roles of NO in cancer biology and highlights the therapeutic potential effects of NO-releasing nanomaterials based on polymeric nanoparticles, dendritic polymers, liposomes, silica nanoparticles, metallic nanoparticles and quantum dots in combating tumor cells.

While physiological levels of nitric oxide (NO) protect the endothelium and have vasodilatory effects, excessive NO has adverse effects on the cardiovascular system. Recently, new NO-releasing pharmacodynamic hybrids of angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) have been developed.We analyzed whether olmesartan with NO-donor side chains (Olm-NO) was superior to olmesartan (Olm) for the control of blood pressure (BP). Although there was no significant difference in binding affinity to AT1 wild-type (WT) receptor between Olm and Olm-NO in a cell-based binding assay, the suppressive effect of Olm-NO on Ang II-induced inositol phosphate (IP) production was significantly weaker than that of Olm in AT1 WT receptor-expressing cells. While Olm had a strong inverse agonistic effect on IP production, Olm-NO did not. Next, we divided 18 C57BL mice into 3 groups: Ang II (infusion using an osmotic mini-pump) as a control group, Ang II (n = 6) + Olm, and Ang II (n = 6) + Olm-NO groups (n = 6). Olm-NO did not block Ang II-induced high BP after 10 days, whereas Olm significantly decreased BP. In addition, Olm, but not Olm-NO, significantly reduced the ratio of heart weight to body weight (HW/BW) with downregulation of the mRNA levels of atrial natriuretic peptide.An ARB with a NO-donor may cancel BP-lowering effects probably due to excessive NO and a weak blocking effect by Olm-NO toward AT1 receptor activation.

Physiological S-nitrosothiols (RSNO), such as S-nitrosoglutathione (GSNO), can be used as nitric oxide (NO) donor for the treatment of vascular diseases. However, despite a half-life measured in hours, the stability of RSNO, limited by enzymatic and non-enzymatic degradations, is too low for clinical application. So, to provide a long-lasting effect and to deliver appropriate NO concentrations to target tissues, RSNO have to be protected. RSNO encapsulation is an interesting response to overcome degradation and provide protection. However, RSNO such as GSNO raise difficulties for encapsulation due to its hydrophilic nature and the instability of the S-NO bound during the formulation process. To our knowledge, the present study is the first description of the direct encapsulation of GSNO within polymeric nanoparticles (NP). The GSNO-loaded NP (GSNO-NP) formulated by a double emulsion process, presented a mean diameter of 289 ± 7 nm. They were positively charged (+40 mV) due to the methacrylic acid and ethylacrylate polymer (Eudragit® RL) used and encapsulated GSNO with a satisfactory efficiency (i.e. 54% or 40 mM GSNO loaded in the NP). In phosphate buffer (37 °C; pH 7.4), GSNO-NP released 100% of encapsulated GSNO within 3h and remained stable still 6h. However, in contact with smooth muscle cells, maximum protein nitrosation (a marker of NO bioavailability) was delayed from 1h for free GSNO to 18h for GSNO-NP. Therefore, protection and sustained release of NO were achieved by the association of a NO donor with a drug delivery system (such as polymeric NP), providing opportunities for vascular diseases treatment., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Localized nitric oxide (NO) release from polymeric materials holds much promise for the prevention of coagulation often associated with implantable and extracorporeal blood-contacting devices. Films of polyurethane (PU) containing incorporated polyethyleneimine were thus exposed to NO gas to form diazeniumdiolates (NONOates) in situ. Donor incorporation and NO gas exposure did not affect the mechanical properties of the films. The NO release capacity increased with increasing polydimethylsiloxane (PDMS) content in the soft segment of the PU: total capacity could be more than doubled (P<0.05) from 0.093 ± 0.028 to 0.225 ± 0.004 mmol/g when the PDMS content was increased from 0 to 100%. Release kinetics were best approximated using a modified Korsemeyer-Peppas power law (R2=0.95-0.99). Despite the resultant rapid initial decrease in NO release rates, values above that observed for quiescent endothelial cells (0.83 pmol·cm(-2)·s(-1)) were maintained for extended periods of 5-10 days, while rates above that of a stimulated endothelium (2.7-6.8 pmol·cm(-2)·s(-1)) were achieved for the first 24 hours. This method of NONOate formation may be advantageous, as potential premature NO release by exposure of diazeniumdiolated donors during incorporation, processing and storage, can be avoided by in situ diazoniumdiolation closer to the time of implantation.

At the cardiovascular level, nitric oxide (NO) controls smooth muscle functions, maintains vascular integrity, and exerts an antihypertensive effect. Metal-nonoates are a recently discovered class of NO donors, with NO release modulated through the complexation of the N-aminoethylpiperazine N-diazeniumdiolate ligand to metal ions, and thus representing a significant innovation with respect to the drugs traditionally used. In this study, we characterized the vascular protective effects of the most effective compound of this class, Ni(PipNONO)Cl, compared with the commercial N-diazeniumdiolate group derivate, diethylenetriamine/nitric oxide (DETA/NO). Ni(PipNONO)Cl induced a concentration-dependent relaxation of precontracted rat aortic rings. The ED50 was 0.67 µM, compared with 4.3 µM obtained with DETA/NO. When tested on cultured microvascular endothelial cells, Ni(PipNONO)Cl exerted a protective effect on the endothelium, promoting cell proliferation and survival in the picomolar range. The administration of Ni(PipNONO)Cl to vascular smooth muscle cells reduced the cell number, promoting their apoptosis at a high concentration (10 µM). Inhibition of smooth muscle cell migration, a hallmark of atherosclerosis, was accompanied by cytoskeletal rearrangement and loss of lamellipodia. When added to isolated platelets, Ni(PipNONO)Cl significantly reduced ADP-induced aggregation. Since atherosclerosis is accompanied by an inflammatory environment, cultured endothelial cells were exposed to interleukin (IL)-1β. In the presence of IL-1β, Ni(PipNONO)Cl inhibited cyclooxygenase-2 and inducible nitric oxide synthase upregulation, and reduced endothelial permeability and the platelet and monocyte adhesion markers CD31 and CD40 at the plasma membrane. Overall, these data indicate that Ni(PipNONO)Cl exerts vascular protective effects relevant for vascular dysfunction and prevention of atherosclerosis and thrombosis., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Although diazeniumdiolates (X[N(O)NO](-)) are extensively used in biochemical, physiological, and pharmacological studies due to their ability to release NO and/or its congeneric nitroxyl, the mechanisms of these processes remain obscure. In this work, we used a combination of spectroscopic, kinetic, and computational techniques to arrive at a quantitatively consistent molecular mechanism for decomposition of amino diazeniumdiolates (amino NONOates: R2N[N(O)NO](-), where R=N(C2H5)2 (1), N(C3H4NH2)2 (2), or N(C2H4NH2)2 (3)). Decomposition of these NONOates is triggered by protonation of their [NN(O)NO](-) group with the apparent pKa and decomposition rate constants of 4.6 and 1 s(-1) for 1; 3.5 and 0.083 s(-1) for 2; and 3.8 and 0.0033 s(-1) for 3. Although protonation occurs mainly on the O atoms of the functional group, only the minor R2N(H)N(O)NO tautomer (population ~10(-7), for 1) undergoes the NN heterolytic bond cleavage (kd~10(7) s(-1) for 1) leading to amine and NO. Decompositions of protonated amino NONOates are strongly temperature-dependent; activation enthalpies are 20.4 and 19.4 kcal/mol for 1 and 2, respectively, which includes contributions from both the tautomerization and bond cleavage. The bond cleavage rates exhibit exceptional sensitivity to the nature of R substituents which strongly modulate activation entropy. At pH<2, decompositions of all three NONOates that have been investigated are subject to additional acid catalysis that occurs through di-protonation of the [NN(O)NO](-) group., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Nitric oxide plays an important role in various biological processes including antinociception. The control of its local concentration is crucial for obtaining the desired effect and can be achieved with exogenous nitric oxide-carriers such as ruthenium complexes. Therefore, we evaluated the analgesic effect and mechanism of action of the ruthenium nitric oxide donor [Ru(HEDTA)NO] focusing on the role of cytokines, oxidative stress and activation of the cyclic guanosine monophosphate/protein kinase G/ATP-sensitive potassium channel signaling pathway. It was observed that [Ru(HEDTA)NO] inhibited in a dose-dependent (1-10 mg/kg) manner the acetic acid-induced writhing response. At the dose of 1 mg/kg, [Ru(HEDTA)NO] inhibited the phenyl-p-benzoquinone-induced writhing response, and formalin- and complete Freund's adjuvant-induced licking and flinching responses. Systemic and local treatments with [Ru(HEDTA)NO] also inhibited the carrageenin-induced mechanical hyperalgesia and increase of myeloperoxidase activity in paw skin samples. Mechanistically, [Ru(HEDTA)NO] inhibited carrageenin-induced production of the hyperalgesic cytokines tumor necrosis factor-α and interleukin-1β, and decrease of reduced glutathione levels. Furthermore, the inhibitory effect of [Ru(HEDTA)NO] in the carrageenin-induced hyperalgesia and myeloperoxidase activity was prevented by the treatment with ODQ (soluble guanylyl cyclase inhibitor), KT5823 (protein kinase G inhibitor) and glybenclamide (ATP-sensitive potassium channel inhibitor), indicating that [Ru(HEDTA)NO] inhibits inflammatory hyperalgesia by activating the cyclic guanosine monophosphate/protein kinase G/ATP-sensitive potassium channel signaling pathway, respectively. These results demonstrate that [Ru(HEDTA)NO] exerts its analgesic effect in inflammation by inhibiting pro-nociceptive cytokine production, oxidative imbalance and activation of the nitric oxide/cyclic guanosine monophosphate/protein kinase G/ATP-sensitive potassium channel signaling pathway in mice.

Nitric oxide (NO) is one of the most important vasodilator molecules produced by the endothelium. It has already been established that NO/cGMP signaling pathway deficiencies are involved in the pathophysiological mechanisms of many cardiovascular diseases. In this context, the development of NO-releasing drugs for therapeutic use appears to be an effective alternative to replace the deficient endogenous NO and mimic the role of this molecule in the body. Organic nitrates represent the oldest class of NO donors that have been clinically used. Considering that tolerance can occur when these drugs are applied chronically, the search for new compounds of this class with lower tolerance potential is increasing. Here, we briefly discuss the mechanisms involved in nitrate tolerance and highlight some achievements from our group in the development of new organic nitrates and their preclinical application in cardiovascular disorders.

A series of NO-donor praziquantel hybrid compounds was obtained by combining praziquantel (PZQ) and furoxan moieties in a single entity. NO-donor properties of the furoxan derivatives were evaluated by detecting nitrite after incubation of the products in 7.4 pH buffered solution in the presence of L-cysteine. Structurally-related furazans, devoid of NO release capacity, were also synthesized for control purposes. All products were studied for their ability to inhibit recombinant Schistosoma mansoni thioredoxin glutathione reductase (TGR). Mobility and death of adult Schistosoma mansoni worms cultured in the presence of the products were evaluated versus PZQ. Analysis of the results showed that some products were endowed with both PZQ and NO-dependent antiparasitic properties. Compounds 6, 7, 18, and 24 emerged as the most interesting balanced hybrids, worthy of additional study on PZQ-resistant parasites., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

We have recently developed tert-dodecane S-nitrosothiol (tDodSNO) as a photoactivated nitric oxide (NO) donor. We here compare the potency of tDodSNO to S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP), drugs which are also based upon the S-nitrosothiol functionality and have been extensively used for NO release studies. Photoactivation in vitro, at a clinically relevant light fluence rate (200W/m(2)), demonstrated that tDodSNO released much higher levels of NO than either GSNO or SNAP. When evaluated in an ex vivo aortic ring vasorelaxation assay, tDodSNO was also the only drug to exhibit a photodynamic response, with an 8 fold decrease in EC50 (8.1-1.0µM) upon irradiation. While both GSNO and SNAP induced NO dependent vasorelaxation at lower concentrations than tDodSNO (EC50׳s of 158 and 38nM respectively), this activity was due to their rapid metabolic decomposition, and could not be modulated by photoactivation. Additionally, tDodSNO׳s photodynamic response allowed vascular tone to be directly regulated by light intensity. Molecular modeling of drug properties suggested that these differences in activity could be attributed to a combination of an increase in tDodSNO׳s hydrophobicity, and substantial steric shielding of molecule׳s S-nitrosothiol group from solvent interactions. In conclusion, our study demonstrates that tDodSNO is currently the most effective known s-nitrosothiol for phototherapeutic applications., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The in vivo analytical performance of percutaneously implanted nitric oxide (NO)-releasing amperometric glucose biosensors was evaluated in swine for 10 d. Needle-type glucose biosensors were functionalized with NO-releasing polyurethane coatings designed to release similar total amounts of NO (3.1 μmol cm(-2)) for rapid (16.0 ± 4.4 h) or slower (>74.6 ± 16.6 h) durations and remain functional as outer glucose sensor membranes. Relative to controls, NO-releasing sensors were characterized with improved numerical accuracy on days 1 and 3. Furthermore, the clinical accuracy and sensitivity of rapid NO-releasing sensors were superior to control and slower NO-releasing sensors at both 1 and 3 d implantation. In contrast, the slower, extended, NO-releasing sensors were characterized by shorter sensor lag times (<4.2 min) in response to intravenous glucose tolerance tests versus burst NO-releasing and control sensors (>5.8 min) at 3, 7, and 10 d. Collectively, these results highlight the potential for NO release to enhance the analytical utility of in vivo glucose biosensors. Initial results also suggest that this analytical performance benefit is dependent on the NO-release duration.

It has been established that oximes cause endothelium-independent relaxation in blood vessels. In the present study, the cardiovascular effects of the new oxime 3-hydroxy-4-(hydroxyimino)-2-(3-methylbut-2-enylnaphtalen-1(4H)-one (Oxime S1) derived from lapachol were evaluated. In normotensive rats, administration of Oxime S1 (10, 15, 20 and 30 mg/Kg, i.v.) produced dose-dependent reduction in blood pressure. In isolated aorta and superior mesenteric artery rings, Oxime S1 induced endothelium-independent and concentration-dependent relaxations (10(-8) M to 10(-4) M). In addition, Oxime S1-induced vasorelaxations were attenuated by hydroxocobalamin or methylene blue in aorta and by PTIO or ODQ in mesenteric artery rings, suggesting a role for the nitric oxide (NO) pathway. Additionally, Oxime S1 (30 and 100 µM) significantly increased NO concentrations (13.9 ± 1.6 nM and 17.9 ± 4.1 nM, respectively) measured by nitric oxide microsensors. Furthermore, pre-contraction with KCl (80 mM) prevented Oxime S1-derived vasorelaxation in endothelium-denuded aortic rings. Of note, combined treatment with potassium channel inhibitors also reduced Oxime S1-mediated vasorelaxation suggesting a role for potassium channels, more precisely Kir, Kv and KATP channels. We observed the involvement of BKCa channels in Oxime S1-induced relaxation in mesenteric artery rings. In conclusion, these data suggest that the Oxime S1 induces hypotension and vasorelaxation via NO pathway by activating soluble guanylate cyclase (sGC) and K+ channels.

Nitric oxide (NO) donors are substances that can release NO. Vascular relaxation induction is among the several functions of NO, and the administration of NO donors is a pharmacological alternative to treat hypertension. This review will focus on the physicochemical description of ruthenium-derived NO donor complexes that release NO via reduction and light stimulation. In particular, we will discuss the complexes synthesized by our research group over the last ten years, and we will focus on the vasodilation and arterial pressure control elicited by these complexes. Soluble guanylyl cyclase (sGC) and potassium channels are the main targets of the NO species released from the inorganic compounds. We will consider the importance of the chemical structure of the ruthenium complexes and their vascular effects.

Here, we report the design, synthesis, and evaluation of arylboronate ester based diazeniumdiolates (BORO/NO), a class of nitric oxide (NO) donors activated by hydrogen peroxide (H2O2), a reactive oxygen species (ROS), to generate NO. We provide evidence for the NO donors' ability to permeate bacteria to produce NO when exposed to H2O2 supporting possible applications for BORO/NO to study molecular mechanisms of NO generation in response to elevated ROS.

Spatiotemporally controllable nitric oxide (NO)-releasers allow us to analyze the physiological effects of NO, a gaseous mediator that modulates many biological signaling networks, and are also candidate chemotherapeutic agents. We designed and synthesized a blue-light-controllable NO releaser, named NOBL-1, which bears an N-nitrosoaminophenol moiety for NO release tethered to a BODIPY dye moiety for harvesting blue light. Photoinduced electron transfer from N-nitrosoaniline to the antenna moiety upon irradiation with relatively noncytotoxic blue light (470-500 nm) should result in NO release with formation of a stable quinone moiety. NO release from NOBL-1 was confirmed by ESR spin trapping and fluorescence detection. Spatially controlled NO release in cells was observed with DAR-4M AM, a fluorogenic NO probe. We also demonstrated temporally controlled vasodilation of rat aorta ex vivo by blue-light-induced NO release from NOBL-1. This compound should be useful for precise examination of the functions of NO with excellent spatiotemporal control.

S-nitrosothiols (RSNO) are considered as potential drugs for delivering nitric oxide (NO) or related species in cardiovascular disorders associated with decrease in NO bioavailability. We have synthesized a new RSNO, i.e. S,S'-dinitrosobucillamine (BUC(NO)2), which combines in its structure two S-mononitrosothiols, S-nitroso-N-acetylpenicillamine (SNAP) and S-nitroso-N-acetylcysteine (NACNO). Synthesized BUC(NO)2 was structurally characterized using high-performance liquid chromatography/mass spectrometry (HPLC/MS), (1)H nuclear magnetic resonance ((1)H NMR), infrared (IR) and UV-visible spectroscopies, and thermal analysis; resulting data are consistent with the expected structure. The vasorelaxant effect of BUC(NO)2 was evaluated using isolated rat aortic rings and compared to SNAP, NACNO, and to an equimolar mixture of NACNO plus SNAP in order to mimic the number of NO contained in a BUC(NO)2 molecule. BUC(NO)2 (pD2=7.8±0.1) was more potent in vasorelaxation than NACNO (pD2=6.4±0.2), SNAP (pD2=6.7±0.1) and the mixture of SNAP plus NACNO (pD2=6.7±0.2). The release of NO from BUC(NO)2 was 6-fold that of the basal value and significantly higher than the release of NO from the SNAP plus NACNO mixture (4-fold increase versus basal value). Finally, the role of protein disulfide isomerase (PDI) in BUC(NO)2 metabolism was investigated. Vasorelaxant effect (pD2=6.8±0.2) and NO release decreased in the presence of a PDI inhibitor (both P<0.05 versus BUC(NO)2). In conclusion, BUC(NO)2 releases a larger amount of NO into the aorta, partially through PDI activation, and induces vasorelaxation at lower concentrations than other RSNO previously reported., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Fifteen novel hybrids containing diterpene skeleton and nitric oxide (NO) donor were prepared from isosteviol. All the compounds were tested on preliminary cytotoxicity, and the results showed that six target compounds (8c, 10b, 14a, 14c, 18c, and 18d) exhibited anti-proliferation activity on HepG2 cells, with 8c (IC50=4.24 μM) and 18d (IC50=2.75 μM) superior to the positive control CDDO-Me (2-cyano-3,12-dioxooleana-1,9(11)-dien-28-acid methyl ester, IC50=4.99 μM); eleven target compounds (8a-c, 9a-c, 10a-b, 14a, 14c, 18d) exhibited anti-proliferation activities on B16F10 cells at different levels, among them, seven compounds were more potent than comptothecin (IC50=2.78 μM) and CDDO-Me (IC50=5.85 μM), particularly, 10b (IC50=0.02 μM) presented the strongest effect, which was selected as a candidate for further study., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Ruthenium-derived complexes have emerged as new nitric oxide (NO) donors that may help circumvent the NO deficiency that impairs vasodilation. NO in vessels can be produced by the endothelial cells and/or released by NO donors. NO interacts with soluble guanylyl-cyclase to produce cGMP to activate the kinase-G pathway. As a result, conductance arteries, veins and resistance arteries dilate, whereas the cytosolic Ca(2+) levels in the smooth muscle cells decrease. NO also reacts with oxygen or the superoxide anion, to generate reactive oxygen species that modulate NO-induced vasodilation. In this article, we focus on NO production by NO synthase and discuss the vascular changes taking place during hypertension originating from endothelial dysfunction. We will describe how the NO released from ruthenium-derived complexes enhances the vascular effects arising from failed NO generation or lack of NO bioavailability. In addition, how ruthenium-derived NO donors induce the hypotensive effect by vasodilation is also discussed.

In vitro cytotoxicity study of the [Ru3O(CH3COO)6(4-pic)2(NO)]PF6 triruthenium nitrosyl cluster (compound 1, 4-pic=4-methylpyridine) against B16F10 melanoma cell line was evaluated in the presence and absence of visible light irradiation. The nitrosyl cluster 1 showed a significant tumoricidal activity when irradiated at λ=532 nm, reducing cell viability up to 90% at a concentration of 62.5 μM. However, cell death of 60% is also observed in the dark which can be assigned to the NO release mediated by a redox reaction of the cluster in cell medium. This possibility was confirmed by amperometric detection of NO after the addition of ascorbic acid to compound 1 in phosphate buffer. A control experiment was performed with the solvated cluster [Ru3O(CH3COO)6(4-pic)2(CH3OH)]PF6 (compound 2) and no significant lowering of cell viability was observed. These results suggest that the nitrosyl cluster acts as a pro-drug, delivering NO, which is the actual active species., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Reversible chemical modifications of protein cysteine residues by S-nitrosylation and S-oxidation are increasingly recognized as important regulatory mechanisms for many protein classes associated with cellular signaling and stress response. Both modifications may theoretically occur under cellular nitrosative or nitroxidative stress. Therefore, a proteomic isotope-coded approach to parallel, quantitative analysis of cysteome S-nitrosylation and S-oxidation was developed. Modifications of cysteine residues of (i) human glutathione-S-transferase P1-1 (GSTP1) and (ii) the schistosomiasis drug target thioredoxin glutathione reductase (TGR) were studied. Both S-nitrosylation (SNO) and S-oxidation to disulfide (SS) were observed for reactive cysteines, dependent on concentration of added S-nitrosocysteine (CysNO) and independent of oxygen. SNO and SS modifications of GSTP1 were quantified and compared for therapeutically relevant NO and HNO donors from different chemical classes, revealing oxidative modification for all donors. Observations on GSTP1 were extended to cell cultures, analyzed after lysis and in-gel digestion. Treatment of living neuronal cells with CysNO, to induce nitrosative stress, caused levels of S-nitrosylation and S-oxidation of GSTP1 comparable to those of cell-free studies. Cysteine modifications of PARK7/DJ-1, peroxiredoxin-2, and other proteins were identified, quantified, and compared to overall levels of protein S-nitrosylation. The new methodology has allowed identification and quantitation of specific cysteome modifications, demonstrating that nitroxidation to protein disulfides occurs concurrently with S-nitrosylation to protein-SNO in recombinant proteins and living cells under nitrosative stress.