Challenging development of storable particles for oral delivery of a physiological nitric oxide donor.

Nitric oxide (NO) deficiency is often associated with several acute and chronic diseases. NO donors and especially S-nitrosothiols such as S-nitrosoglutathione (GSNO) have been identified as promising therapeutic agents. Although their permeability through the intestinal barrier have recently be proved, suitable drug delivery systems have to be designed for their oral administration. This is especially challenging due to the physico-chemical features of these drugs: high hydrophilicity and high lability. In this paper, three types of particles were prepared with an Eudragit® polymer: nanoparticles and microparticles obtained with a water-in-oil-in-water emulsion/evaporation process versus microparticles obtained with a solid-in-oil-in-water emulsion/evaporation process. They had a similar encapsulation efficiency (around 30%), and could be freeze-dried then be stored at least one month without modification of their critical attributes (size and GSNO content). However, microparticles had a slightly slower in vitro release of GSNO than nanoparticles, and were able to boost by a factor of two the drug intestinal permeability (Caco-2 model). Altogether, this study brings new data about GSNO intestinal permeability and three ready-to-use formulations suitable for further preclinical studies with oral administration.

Combined influence of nitric oxide and surface roughness in biofilm reduction across bacteria strains.

Effective use of medical device implants is often hindered by infection, which may cause the device to be rejected from the body and seriously endanger health. Such infections are often a result of biofilm formation or microbial colonies collecting on a surface. Therefore, a challenge in the medical field is to mitigate the impact of biofilm formation in order to save thousands of lives and millions of healthcare dollars annually. The proposed strategy is to target the attachment phase of the biofilm lifecycle to try to prevent the formation of antimicrobial resistant biofilms. Prevention of bacterial attachment may be induced through the introduction of nitric oxide (NO), a small biological signaling molecule known for its antibacterial properties. NO may be delivered via release from a donating molecule incorporated in the polymer composing the medical device. The NO donor S-nitrosoglutathione (GSNO) was utilized in this study because it is a relatively stable small molecule that naturally exists in the body, therefore negating possible adverse reactions when it is introduced to the body. Tygon®, a polymer commonly found in Food and Drug Administration approved medical devices such as catheters, was utilized as a platform for the inhibition of biofilms. To study the necessary amount of released NO needed to cause a reduction in attachment across varying strains, different concentrations of GSNO were applied. Two Gram-negative (Pseudomonas aeruginosa and Acinetobacter baumannii) and two Gram-positive species (Staphylococcus aureus and Methicillin Resistant Staphylococcus aureus), all strong biofilm formers listed as urgent threats by the Center for Disease Control, illustrated different responses to NO. Gram-positive species showed a decrease in viability over 80% with an average total NO release of 2.01 ± 2.11 × 10 - 4 µmols, while Gram-negative response was less, with viability decreasing to 38% (P. aeruginosa) and 71% (A. baumannii) with 1.25 ± 1.63 × 10-4 µmols NO. Further studies utilizing glutathione surface roughness controls highlight that increasing the surface roughness of the polymer platform produces no statistically significant difference in viability compared to the Tygon-only negative control in all strains except P. aeruginosa. Developing a quantitative understanding of how NO release and platform surface roughness impact biofilm attachment across Gram strains is key to reducing the incidence and impact of medical device associated infections.

Assessment of ocular surface toxicity after topical instillation of nitric oxide donors.

PURPOSE: To evaluate the ocular surface toxicity of two nitric oxide donors in ex vivo and in vivo animal models: S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylcysteine (SNAC) in a hydroxypropyl methylcellulose (HPMC) matrix at final concentrations 1.0 and 10.0 mM. METHODS: Ex vivo GSNO and SNAC toxicities were clinically and histologically analyzed using freshly excised pig eyeballs. In vivo experiments were performed with 20 albino rabbits which were randomized into 4 groups (5 animals each): Groups 1 and 2 received instillations of 150 µL of aqueous HPMC solution containing GSNO 1.0 and 10.0 mM, respectively, in one of the eyes; Groups 3 and 4 received instillations of 150 µL of aqueous HPMC solution-containing SNAC 1.0 and 10.0 mM, respectively, in one of the eyes. The contralateral eyes in each group received aqueous HPMC as a control. All animals underwent clinical evaluation on a slit lamp and the eyes were scored according to a modified Draize eye test and were histologically analyzed. RESULTS: Pig eyeballs showed no signs of perforation, erosion, corneal opacity or other gross damage. These findings were confirmed by histological analysis. There was no difference between control and treated rabbit eyes according to the Draize eye test score in all groups (p>0.05). All formulations showed a mean score under 1 and were classified as "non-irritating". There was no evidence of tissue toxicity in the histological analysis in all animals. CONCLUSION: Aqueous HPMC solutions containing GSNO and SNAC at concentrations up to 10.0 mM do not induce ocular irritation.

Effect of combined oxidative and nitrosative stresses on Staphylococcus aureus transcriptome.

Staphylococcus aureus is a pathogen responsible for severe community- and nosocomially acquired infections. To fight pathogen intrusion, the innate immune system uses a plethora of weapons, with the generation of oxidative and nitrosative stresses among the most efficient. In this work, the S. aureus genome-wide transcriptional responses to oxidative stress generated by hydrogen peroxide, to nitrosative stress imposed by S-nitrosoglutathione (GSNO), and to the combination of the two were investigated using microarray analysis. The results showed that these stresses have a significant impact on the transcriptome of S. aureus. Hydrogen peroxide modified mainly the mRNA abundance of genes involved in oxidative detoxification and DNA metabolism, which together represent 14 % of the total number of upregulated genes. GSNO caused significant alteration of the expression of gene products with regulatory function. However, the simultaneous addition of GSNO and hydrogen peroxide was found to cause the more significant transcriptomic alteration, affecting ∼10 % of the total transcriptome. In particular, exposure of S. aureus to GSNO plus hydrogen peroxide modified the transcription of genes associated with cell envelope and iron metabolism, including induction of ftnA and dps genes that encode iron-storage and oxidative-protecting proteins. Further studies revealed that when exposed to combined GSNO-hydrogen peroxide stresses, S. aureus has decreased viability, which is enhanced in the presence of iron, and low siderophore activity. Altogether, this study revealed, for the first time, how the combined oxidative and nitrosative stresses inflicted during phagocytosis interfere at the transcriptional level with the S. aureus cellular metabolism.

Assessment of ocular surface toxicity after topical instillation of nitric oxide donors / Avaliação da toxicidade na superfície ocular após instilação tópica de doadores de óxido nítrico

PURPOSE: To evaluate the ocular surface toxicity of two nitric oxide donors in ex vivo and in vivo animal models: S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylcysteine (SNAC) in a hydroxypropyl methylcellulose (HPMC) matrix at final concentrations 1.0 and 10.0 mM. METHODS: Ex vivo GSNO and SNAC toxicities were clinically and histologically analyzed using freshly excised pig eyeballs. In vivo experiments were performed with 20 albino rabbits which were randomized into 4 groups (5 animals each): Groups 1 and 2 received instillations of 150 µL of aqueous HPMC solution containing GSNO 1.0 and 10.0 mM, respectively, in one of the eyes; Groups 3 and 4 received instillations of 150 µL of aqueous HPMC solution-containing SNAC 1.0 and 10.0 mM, respectively, in one of the eyes. The contralateral eyes in each group received aqueous HPMC as a control. All animals underwent clinical evaluation on a slit lamp and the eyes were scored according to a modified Draize eye test and were histologically analyzed. RESULTS: Pig eyeballs showed no signs of perforation, erosion, corneal opacity or other gross damage. These findings were confirmed by histological analysis. There was no difference between control and treated rabbit eyes according to the Draize eye test score in all groups (p>0.05). All formulations showed a mean score under 1 and were classified as "non-irritating". There was no evidence of tissue toxicity in the histological analysis in all animals. CONCLUSION: Aqueous HPMC solutions containing GSNO and SNAC at concentrations up to 10.0 mM do not induce ocular irritation.

OBJETIVO: Avaliar a toxidade na superfície ocular de dois compostos doadores de óxido nítrico em modelos ex vivo e in vivo: S-nitrosoglutationa (GSNO) e S-nitroso-N-acetilcisteína (SNAC), em uma matriz de hidroxipropil metilcelulose (HPMC) nas concentrações finais de 1,0 and 10,0 mM. MÉTODOS: As toxicidades de GSNO e SNAC foram avaliadas clinicamente e histologicamente em modelo ex vivo usando globos oculares porcinos recém excisados. Experimentos in vivo foram realizados com 20 coelhos albinos que foram randomizados em 4 grupos (5 animais em cada): Os grupos 1 e 2 receberam instilações de 150 µL de solução aquosa de HPMC contendo GSNO 1,0 e 10,0 mM, respectivamente, em um dos olhos; Os grupos 3 e 4 receberam instilações de 150 µL de solução aquosa de HPMC contendo SNAC 1,0 and 10,0 mM, respectivamente, em um dos olhos. Os olhos contralaterias em cada grupo receberam solução aquosa de HPMC como controle. Todos os animais foram clinicamente avaliados em lâmpada de fenda e os olhos foram pontuados de acordo com o teste de Draize modificado e analisados histologicamente. RESULTADOS: Os globos oculares porcinos não apresentaram sinais de perfuração, erosão, opacidade da córnea ou outros danos graves. Esses resultados foram confirmados pela análise histológica. Não houve diferença entre os olhos dos coelhos tratados e controles de acordo com a pontuação do teste de Draize em todos os grupos (p>0,05). Todas as formulações apresentaram um escore médio menor do que 1 e foram classificadas como "não-irritantes". Não houve evidência de toxicidade tecidual nas análises histológicas em todos os animais. CONCLUSÃO: Soluções aquosas de HPMC contendo GSNO e SNAC em concentrações até 10,0 mM não induzem irritação ocular.

S-nitrosoglutathione-induced toxicity in Drosophila melanogaster: Delayed pupation and induced mild oxidative/nitrosative stress in eclosed flies.

The toxicity of the nitric oxide donor S-nitrosoglutathione (GSNO) was tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with GSNO at concentrations of 1.0, 1.5 or 4.0mM. Food supplementation with GSNO caused a developmental delay in the flies. Biochemical analyses of oxidative stress markers and activities of antioxidant and associated enzymes were carried out on 2-day-old flies that emerged from control larvae and larvae fed on food supplemented with GSNO. Larval exposure to GSNO resulted in lower activities of aconitase in both sexes and also lower activities of catalase and isocitrate dehydrogenase in adult males relative to the control cohort. Larval treatment with GSNO resulted in higher carbonyl protein content and higher activities of glucose-6-phosphate dehydrogenase in males and higher activities of superoxide dismutase and glutathione-S-transferase in both sexes. Among the parameters tested, aconitase activity and developmental end points may be useful early indicators of toxicity caused by GSNO.

Formation peculiarities of radiation-induced aberrations of chromosomes in human cells under the modifying influence of chemical agents (comparative aspects).

OBJECTIVE: The study objective was to determine and provide a comparative analysis of frequency and spectrum of the induced aberrations of chromosomes in culture of the human peripheral blood lymphocytes under the combined impact of radiation, co-mutagen, and chemical mutagen. METHODS: Culture of human peripheral blood lymphocytes and cytogenetic methods have been used. RESULTS: A co-mutagenic effect of the drug verapamil was established under the testing γ-irradiation of human peripheral blood lymphocytes in the dose range of 0.3-2.0 Gy at the expense of increased frequency of chromosomal aberrations (dicentrics). The combined effect of γ-irradiation and S-Nitrosoglutathione is directed on the induction and storage of chemical markers of exposure - the chromatid-type aberrations. CONCLUSION: A co-mutagenic effect of verapamil under the low-dose γ-irradiation as a 2-fold increase of the chromosome-type aberrations (radiation markers) incidence was revealed at a chromosomal level in human peripheral blood lymphocytes. Phenomenon of synergism of low-dose γ-irradiation and mutagen S-Nitrosoglutathione as a ~3-fold increased frequency of chromatid-type aberrations (chemical markers) was detected compared to the sole radiation effect.

Exogenous nitric oxide potentiate DNA damage and alter DNA repair in cells exposed to ionising radiation.

UNLABELLED: The aim of this study was to investigate impact of exogenous nitric oxide (NO) on generation of different types of DNA damages, their transformation, and specificity of DNA repair in cells treated with ionizing radiation (IR). METHODS: levels of single-strand and double-strand breaks assessed in peripheral blood lymphocytes (PBL) isolated from healthy humans and treated in vitro with NO donor -- S-nitrosoglutathione (GSNO) and IR. The rate of DNA repair estimated after 30 and 60 min of PBL treatment. The visualization and measuring the number of prompt and delayed DNA damages, including strand breaks, apurinic and thermolabile sites performed with single-cell gel electrophoresis. RESULTS: IR caused dose-dependent generation of single strand breaks (SSBs), double strand breaks (DSBs), and heat-labile sites (HLS) in cell DNA. However, particularly destructive was combined treatment IR with GSNO as NO donor that leads to a significant increase of DNA damage and a dose-dependent inhibition of the DNA repair rate. Obtained data proofs the ability of NO to inhibit fast and slow stages of SSBs, DSBs, and HLS repair resulting in significant growth of genotoxic effect. DNA breaks generation from HLS is able to affect DSBs yields especially in cells with altered DNA repair. The process of DNA repair of delayed DSBs formed from HLS was quite different from removal of DNA damages occurring immediately after treatment and was characterized by IR dose dependent inhibition of DNA repair. CONCLUSION: High level of DNA strand breaks, that are generated after the combined treatment with NO and IR, are accumulated for quite a long time after exposure due to altered DNA repair, indicating the development of genetic instability and increase of carcinogenic risk for organism exposed to combination of harmful environmental factors.

Preclinical 28-day inhalation toxicity assessment of s-nitrosoglutathione in beagle dogs and Wistar rats.

To support clinical development of S-nitrosoglutathione (GSNO) as a therapeutic agent, 28-day toxicology studies in rats and dogs were conducted. Rats (21-25/sex) and dogs (3-5/sex) were exposed for 4 hours or 1 hour, respectively, to inhaled GSNO (0, 3, 9.3, 19, and 28 mg/kg per d in rats and 0, 4.6, 9.0, and 16.2 mg/kg per d in dogs) or vehicle daily via a nebulizer. Animals were monitored throughout the 28-day dosing period and during a postexposure recovery period. Complete necropsy and tissue examinations were performed. Experimental end points included clinical pathology, toxicokinetics, and immunotoxicology. No biologically significant adverse findings were noted in either species, and the no observed adverse effect levels (NOAELs) under these conditions were the highest achieved doses (28 and 16.2 mg/kg per d in rats and dogs, respectively). These data demonstrate that GSNO is well tolerated in rodents and dogs and predict a favorable toxicity profile in humans, thus supporting future clinical development of GSNO or closely related compounds.

Identification of S-nitrosylation of proteins of Helicobacter pylori in response to nitric oxide stress.

Innate and adaptive immune responses are activated in humans when Helicobacter pylori invades the gastric mucosa. Nitric oxide (NO) and reactive nitrogen species are important immune effectors, which can exert their functions through oxidation and S-nitrosylation of proteins. S-nitrosoglutathione and sodium nitroprus-side were used as NO donors and H. pylori cells were incubated with these compounds to analyze the inhibitory effect of NO. The suppressing effect of NO on H. pylori has been shown in vitro. Furthermore, the proteins modified by S-nitrosylation in H. pylori were identified through the biotin switch method in association with matrix-assisted laser desorption ionization/time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). Five S-nitrosylated proteins identified were a chaperone and heat-shock protein (GroEL), alkyl hydroperoxide reductase (TsaA), urease alpha subunit (UreA), HP0721, and HP0129. Importantly, S-nitrosylation of TsaA and UreA were confirmed using purified recombinant proteins. Considering the importance of these enzymes in antioxidant defenses, adherence, and colonization, NO may exert its antibacterial actions by targeting enzymes through S-nitrosylation. Identification of protein S-nitrosylation may contribute to an understanding of the antibacterial actions of NO. Our findings provide an insight into potential targets for the development of novel therapeutic agents against H. pylori infection.

Dinitrosyl iron complexes with thiol-containing ligands and apoptosis: studies with HeLa cell cultures.

No pro-apoptotic effect of dinitrosyl iron complexes (DNIC) with glutathione, cysteine or thiosulfate was established after incubation of HeLa cells in Eagle's medium. However, DNIC with thiosulfate manifested pro-apoptotic activity during incubation of HeLa cells in Versene's solution supplemented with ethylene diamine tetraacetate (EDTA) known to induce the decomposition of these DNIC. The water-soluble о-phenanthroline derivative bathophenanthroline disulfonate (BPDS) had a similar effect on DNIC with glutathione during incubation of HeLa cells in Eagle's medium. It was assumed that EDTA- or BPDS-induced pro-apoptotic effect of DNIC with thiosulfate or glutathione is coupled with the ability of decomposing DNIC to initiate S-nitrosylation of proteins localized on the surface of HeLa cells. Presumably, the pro-apoptotic effect of S-nitrosoglutathione (GS-NO) on HeLa cells preincubated in Eagle's medium is mediated by the same mechanism, although the pro-apoptotic effect based on the ability of GS-NO to initiate the release of significant amounts of NO and its oxidation to cytotoxic peroxynitrite in a reaction with superoxide should not be ruled out either. No apoptotic activity was found in the presence of bivalent iron and glutathione favoring the conversion of GS-NO into DNIC with glutathione. It is suggested that interaction of HeLa cells with intact DNIC with glutathione or thiosulfate results in the formation of DNIC bound to cell surface proteins.

The induction of reactive oxygen species and loss of mitochondrial Omi/HtrA2 is associated with S-nitrosoglutathione-induced apoptosis in human endothelial cells.

The pathophysiological relevance of S-nitrosoglutathione (GSNO)-induced endothelial cell injury remains unclear. The main objective of this study was to elucidate the molecular mechanisms of GSNO-induced oxidative stress in endothelial cells. Morphological evaluation through DAPI staining and propidium iodide (PI) flow cytometry was used to detect apoptosis. In cultured EA.hy926 endothelial cells, exposure to GSNO led to a time- and dose-dependent apoptotic cascade. When intracellular reactive oxygen species (ROS) production was measured in GSNO-treated cells with the fluorescent probes 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate, we observed elevated ROS levels and a concomitant loss in mitochondrial membrane potential, indicating that GSNO-induced death signaling is mediated through a ROS-mitochondrial pathway. Importantly, we found that peroxynitrite formation and Omi/HtrA2 release from mitochondria were involved in this phenomenon, whereas changes of death-receptor dependent signaling were not detected in the same context. The inhibition of NADPH oxidase activation and Omi/HtrA2 by a pharmacological approach provided significant protection against caspase-3 activation and GSNO-induced cell death, confirming that GSNO triggers the death cascade in endothelial cells in a mitochondria-dependent manner. Taken together, our results indicate that ROS overproduction and loss of mitochondrial Omi/HtrA2 play a pivotal role in reactive nitrogen species-induced cell death, and the modulation of these pathways can be of significant therapeutic benefit.

Role of an inducible single-domain hemoglobin in mediating resistance to nitric oxide and nitrosative stress in Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni expresses two hemoglobins, each of which exhibits a heme pocket and structural signatures in common with vertebrate and plant globins. One of these, designated Cgb, is homologous to Vgb from Vitreoscilla stercoraria and does not possess the reductase domain seen in the flavohemoglobins. A Cgb-deficient mutant of C. jejuni was hypersensitive to nitrosating agents (S-nitrosoglutathione [GSNO] or sodium nitroprusside) and a nitric oxide-releasing compound (spermine NONOate). The sensitivity of the Cgb-deficient mutant to methyl viologen, hydrogen peroxide, and organic peroxides, however, was the same as for the wild type. Consistent with the protective role of Cgb against NO-related stress, cgb expression was minimal in standard laboratory media but strongly and specifically induced after exposure to nitrosative stress. In contrast, the expression of Cgb was independent of aeration and the presence of superoxide. In the absence of preinduction by exposure to nitrosative stress, no difference was seen in the degree of respiratory inhibition by NO or the half-life of the NO signal when cells of the wild type and the cgb mutant were compared. However, cells expressing GSNO-upregulated levels of Cgb exhibited robust NO consumption and respiration that was relatively NO insensitive compared to the respiration of the cgb mutant. Based on similar studies in Campylobacter coli, we also propose an identical role for Cgb in this closely related species. We conclude that, unlike the archetypal single-domain globin Vgb, Cgb forms a specific and inducible defense against NO and nitrosating agents.

The enhancement of the hyperglycemic effect of S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine by vitamin C in an animal model.

BACKGROUND: S-nitrosoglutathione (GSNO) and S-nitroso-N-acetlypenicillamine (SNAP) are two of the most common sources of nitric oxide (NO) in the biomedical field. Vitamin C has been known to accelerate the decomposition of GSNO and SNAP increasing the release and availability of NO which is cytotoxic at non-physiological concentrations. The study investigates any potential detrimental effect of vitamin C and GSNO, vitamin C and SNAP on glucose metabolism in normotensive and normoglycemic dogs. RESULTS: The results showed that administration of vitamin C (50 mg/kg) and GSNO (35 mg/kg & 50 mg/kg), or vitamin C (50 mg/kg) and SNAP (10 mg/kg) to overnight fasted dogs resulted in significant elevation of the blood glucose levels, attaining maximum level at the 2.0 or 2.5 h time point postprandially. The elevated blood glucose levels were due to significant reduction in plasma insulin levels in the dogs treated with vitamin C and GSNO, or vitamin C and SNAP (P < 0.05). The decreased insulin response was associated with significant elevation of nitric oxide produced from GSNO and SNAP co-administered with vitamin C, as assessed by plasma nitrate/nitrite levels. CONCLUSIONS: The results indicate that enhanced NO release by vitamin C affects postprandial blood glucose and plasma insulin levels and the reduced glucose tolerance is mainly due to impaired insulin release. The clinical relevance of the findings of this study suggest that hypertensive diabetic patients treated with GSNO or SNAP, who are on vitamin C supplements may be more predisposed to further decrease in their glycemic control.

Effect of hydrogen peroxide on nitric oxide (NO)-induced mutagenicity in Salmonella typhimurium.

Nitric oxide (NO) has been reported to impart, alone or in combination with reactive oxygen species (ROS), the cytotoxicity and putative genotoxicity associated with the immunological response. The present study examined the change in the mutagenic activity profile of the NO-donor spermine NONOate (SperNO) as a result of introduction of hydrogen peroxide (H(2)O(2)) to the Ames assay. The aim was to determine whether the assay could detect H(2)O(2)-induced co- or anti-mutagenic effects on NO-induced mutagenesis, and the Salmonella typhimurium base-pair substitution tester strain TA1535 provided an appropriate tool. While TA1535 was shown by the authors and others to be strongly sensitive to NO-induced mutagenesis, it has also been shown to be insensitive to H(2)O(2)-induced mutagenicity [1,2]. When H(2)O(2) (0.25-4.0 micromol/pl) was added directly to cells treated with SperNO (0.01-1.0 micromol/pl), co-mutagenicity was not detected, but a drop in reversion count and detectable toxicity was observed, especially at doses > 0.1 micromol/pl. When glucose/glucose oxidase (GOX) or reduced glutathione (GSH) were used as H(2)O(2)-generation systems the results varied. Reversion induced by SperNO (1 micromol/pl) was moderately enhanced by GOX (10-20 mUnits/pl), but the increase albeit reproducible did not reach a doubling (co-mutagenicity). GOX (40 micromol/pl) induced a reduction in reversion count, but no visible toxicity. On the other hand, GSH (20- 80 micromol/pl) gave a strong co-mutagenic effect. Co-mutagenicity was highest (> 5x) at 80 micromol/pl GSH and 0.1 micromol/pl SperNO. Based on these findings, it could be concluded that a) H(2)O(2), when steadily generated in the cell, has a modulatory effect on NO-mutagenicity, and such a conclusion is not inconsistent with the wide range of responses reported for the two chemicals, and/or b) the observed co-mutagenic effects of GSH may not be attributable solely to H(2)O(2) generation.