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A new platinum(II) complex with the amino acid L-tryptophan (trp), named Pt-trp, was synthesized and characterized. Elemental, thermogravimetric and ESI-QTOF mass spectrometric analyses led to the composition [Pt(C11H11N2O2)2]⋅6H2O. Infrared spectroscopic data indicate the coordination of trp to Pt(II) through the oxygen of the carboxylate group and also through the nitrogen atom of the amino group. The (13)C CP/MAS NMR spectroscopic data confirm coordination through the oxygen atom of the carboxylate group, while the (15)N CP/MAS NMR data confirm coordination of the nitrogen of the NH2 group to the metal. Density functional theory (DFT) studies were applied to evaluate the cis and trans coordination modes of trp to platinum(II). The trans isomer was shown to be energetically more stable than the cis one. The Pt-trp complex was evaluated as a cytotoxic agent against SK-Mel 103 (human melanoma) and Panc-1 (human pancreatic carcinoma) cell lines. The complex was shown to be cytotoxic over the considered cells.

We investigated the ability of S-nitroso-N-acetylcyseine (SNAC) to prevent structural and functional myocardial alterations in hypercholesterolemic mice. C57BL6 wild-type (WT) and LDL-R-/- male mice (S) were fed a standard diet for 15 days. LDL-R-/- mice (S) showed an 11% increase in blood pressure, 62% decrease in left atrial contractility, and lower CD40L and eNOS expression relative to WT. LDL-R-/- mice fed an atherogenic diet for 15 days (Chol) showed significant increased left ventricular mass compared to S, which was characterized by: (1) 1.25-fold increase in the LV weight/body weight ratio and cardiomyocyte diameter; (2) enhanced expression of the NOS isoforms, CD40L, and collagen amount; and (3) no alteration in the atrial contractile performance. Administration of SNAC to Chol mice (Chol + SNAC) (0.51 micromol/kg/day for 15 day, IP) prevented increased left ventricular mass, collagen deposit, NOS isoforms, and CD40L overexpression, but it had no effect on the increased blood pressure or atrial basal hypocontractility. Deletion of the LDL receptor gene in mice resulted in hypertension and a marked left atrial contractile deficit, which may be related to eNOS underexpression. Our data show that SNAC treatment has an antiinflammatory action that might contribute to prevention of structural and functional myocardial alterations in atherosclerotic mice independently of changes in blood pressure.

S-nitrosothiols have many biological activities and may act as nitric oxide (NO) carriers and donors, prolonging NO half-life in vivo. In spite of their great potential as therapeutic agents, most S-nitrosothiols are too unstable to isolate. We have shown that the S-nitroso adduct of N-acetylcysteine (SNAC) can be synthesized directly in aqueous and polyethylene glycol (PEG) 400 matrix by using a reactive gaseous (NO/O2) mixture. Spectral monitoring of the S-N bond cleavage showed that SNAC, synthesized by this method, is relatively stable in nonbuffered aqueous solution at 25 degrees C in the dark and that its stability is greatly increased in PEG matrix, resulting in a 28-fold decrease in its initial rate of thermal decomposition. Irradiation with UV light (lambda = 333 nm) accelerated the rate of decomposition of SNAC to NO in both matrices, indicating that SNAC may find use for the photogeneration of NO. The quantum yield for SNAC decomposition decreased from 0.65 +/- 0.15 in aqueous solution to 0.047 +/- 0.005 in PEG 400 matrix. This increased stability in PEG matrix was assigned to a cage effect promoted by the PEG microenvironment that increases the rate of geminated radical pair recombination in the homolytic S-N bond cleavage process. This effect allowed for the storage of SNAC in PEG at -20 degrees C in the dark for more than 10 weeks with negligible decomposition. Such stabilization may represent a viable option for the synthesis, storage and handling of S-nitrosothiol solutions for biomedical applications.

S-Nitrosothiols (RSNOs) are considered to play important roles in storing, transporting, and releasing nitric oxide (nitrogen monoxide, NO) in vivo. Although tertiary RSNOs are known to be intrinsically more stable than primary RSNOs, the correlation between the structure of primary RSNOs and the kinetics of thermal NO release in solution has not been established yet. We have characterized the kinetics of thermal NO release from three primary RSNOs: S-nitrosocysteine (CySNO), S-nitroso-N-acetylcysteine (SNAC), and S-nitrosoglutatione (GSNO) in aqueous solutions. It was found that the rates of NO release are strongly affected by the initial concentration of the solutions. Increasing the concentration of CySNO and SNAC from 1.0 × 10-1 to 61.0 mmol L-1 led to 5.7- and 14.6-fold increases in their initial rates of decomposition, respectively, whereas GSNO was much less affected (a 2-fold increase). However, a smaller increase in concentration (0.1 to 1.0 mM) led to a 4.6-fold decrease, on average, in the rat...

S-Nitrosothiols (RSNOs) are potent vasodilators found naturally in vivo. A variety of synthetic RSNOs have been considered as potential nitric oxide (NO) donors for biomedical applications. We have characterized the hypotensive effect of the RSNO S-nitroso-N-acetylcysteine (SNAC) in normotensive and hypertensive conscious rats. SNAC reduced the medium arterial pressure in a dose-response manner in both normotensive and hypertensive animals. At the same doses (EC(50) of SNAC), SNAC showed a vasodilator effect in normotensive rats more potent and more prolonged than that of sodium nitroprusside (SNP). The hypotensive effect of SNAC was also more potent in methylene blue-treated rats, where the cGMP-dependent pathway had been blockaded. These data indicate that SNAC acts by both cGMP-dependent and cGMP-independent pathways. It was also shown that the thiol N-acetylcysteine (NAC) potentiates the action of SNP in hypertensive rats, pointing to the mediation of thiols in the vasodilator action of SNP in this condition. Such mediation may involve the formation of a more potent thiol complex with the nitroprusside anion or the transfer of NO to NAC, generating SNAC as a primary vasoactive species. The kinetic monitoring of the decomposition reactions of SNAC and SNP showed that both compounds are quite stable under the infusion conditions used. Therefore, their vasodilator action cannot be assigned to their breakdown with release of free NO in solution. As the two compounds are unlikely to cross the plasmalemma of smooth muscle cells, their actions are probably associated with the mediation of endogenous thiols in transnitrosation reactions.

Photolysis of the pentacyanonitrosylferrate (II) ion in sodium nitroprusside (SNP) solutions involves a competition between photosubstitution and photo-oxidation reactions, where the nitrosyl and cyanide ligands can be released as free nitric oxide (NO) or NO+ and free CN- or CN radical. We have irradiated aqueous SNP solutions at several narrow wavelength ranges in the UV/Vis region (314–576 nm), with the aim of investigating the photolability of the CN and NO ligands. Kinetics of photolysis were used to characterize the photosensitivity of SNP solutions in the range 314–576 nm. Spectral changes in the UV/Vis and IR regions upon irradiation and assays for the detection of free CN- and NO, provided additional evidences for the absence of photoprocesses leading to the release of CN- with λirr > 480 nm. In this condition, the main photoproducts were found to be the [FeIII(CN)5(H2O)]2- ion and NO, formed in a intramolecular photo-oxidative process. Irradiation with unfiltered UV/Vis light and with λirr < 480 nm, lead to the release of both CN- and NO and to a mixture of ferrocyanide and ferricyanide products. These results confirm that the exposure of SNP solutions to UV/Vis light below λirr = 480 nm can lead to cyanide poisoning and further support that above this wavelength limit, a selective NO release from SNP can be achieved, with possible implications for its biochemical action.

Synthesis, characterization, DFT studies and biological assays of new gold(I) and gold(III) complexes of benzimidazole are reported. Molecular and structural characterizations of the compounds were based on elemental (C, H and N) and thermal (TG–DTA) analyses, and FT-IR and UV–Visible spectroscopic measurements. The structures of complexes were proposed based DFT calculations. The benzimidazole compounds (Lig1 and Lig2) and the gold complexes were tested against three Leishmania species related to cutaneous manifestations of leishmaniasis. The free benzimidazole compounds showed no leishmanicidal activity. On the other hand, the gold(I and III) complexes have shown to possess significant activity against Leishmania in both stages of parasite, and the gold(III) complex with Lig2 exhibited expressive leishmanicidal activity with IC50 values below 5.7 μM. Also, the gold complexes showed high leishmania selectivity. The gold(I) complex with Lig1, for example, is almost 50 times more toxic for the parasite than for macrophages. Besides the leishmanicidal activity, all complexes exhibited toxic effect against SK-Mel 103 and Balb/c 3T3, cancer cells.

Skin is the organ most exposed to environmental sunlight. Many in vitro and in vivo studies on skin have now unambiguously gathered evidence of the ultraviolet (UV) radiation involvement in the development of human skin pathologies such as sunburn, aging, autoimmunity, immunosuppression and cancer. Thus, the toxic effects of UV from natural sunlight and therapeutic artificial lamps are a major concern for human health. The mechanisms by which UV radiation promotes skin damage have been under intense investigation for decades, and much progress has been made in identifying the molecular alterations associated with changes in cellular functions. Furthermore, these studies are providing potential targets for molecular therapies. The aim of this chapter is to summarize the general knowledge available in the field of UV-induced skin damage, with an emphasis on the discussion of molecular markers of cellular senescence and inflammation. Besides the biological consequences of photodamage, this chapter also deals with technologies available for the detection of phototoxicity and characterization of the molecular action of nanostructures, and shows how helpful such approaches can be with a view to improving the photoprotection provided by skin products.

Drug-eluting stents (DES) were developed to combat the problem of in-stent restenosis, and evaluating the biological activity from DES systems is critical for its safety and efficacy. To test the cytotoxicity of nitric oxide (NO) donor-containing polymers for their potential use in DES applications, S-nitrosoglutathione (GSNO) or in combination with poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) in an aqueous polymeric solution (PVA/PVP/GSNO) was investigated using Balb/c 3T3 and Rabbit arterial smooth muscle (RASM) cells. The sensitivity of 3T3 cells to the cytotoxicity effects induced by GSNO was higher than that of RASM cells, while RASM cells were more susceptible to alterations in membrane permeability. Cell growth assays showed that GSNO and PVA/PVP/GSNO induced antiproliferative effects in RASM cells. Moreover, the presence of polymers can reduce the cytotoxicity and enhance the antiproliferative effects of GSNO. Dose-dependent inhibition of platelet aggregation was similar for both PVA/PVP/GSNO (EC50 of 3.4 ± 2.3 µM) and GSNO (EC50 of 2.8 ± 1.1 µM) solutions. Platelet adhesion assays showed that the inhibition caused by GSNO (EC50 of 5.0 mM) was dependent on the presence of plasma. These results demonstrate that the methodology adopted here is suitable to establish safety margins and evaluate the antithrombotic potential and antiproliferative effects of NO-eluting biomaterials and polymeric solutions for the new cardiovascular devices, and also to emphasize the importance of using more specific cell lines in these evaluations. Copyright © 2011 John Wiley & Sons, Ltd.

Background The pathophysiology of the NO/NO synthase system and dysfunctional changes in the endothelium in the early phases of the atherogenic process are incompletely understood. In this study, we investigated the effects of the nitrosothiol NO donor S -nitroso- N -acetylcysteine (SNAC) in the early prevention of plaque development in the hypercholesterolemic LDLr−/− mice as well as the changes in endothelium-dependent relaxation and NO synthase expression. Methods and results LDLr−/− mice were fed a 1.25% cholesterol-enriched diet for 15 days. Plasma cholesterol/triglyceride levels increased and this increase was accompanied by the development of aortic root lesions. Aortic vasorelaxation to acetylcholine was increased, although endothelium-independent relaxation in response to sodium nitroprusside did not change, which suggest stimulated NO release enhanced. This dysfunction was associated with enhanced aortic superoxide production and with increased levels of constitutive NOS isoform expression, particularly neuronal NOS. SNAC ( S -nitroso- N -acetylcysteine) administration (0.51 μmol/kg/day i.p. for 15 days) decreased the extent of the plaque by 55% in hypercholesterolemic mice, but had no effects on vasomotor changes. It did, however, lead to a decrease in constitutive NOS expression. The SNAC induced only minor changes in plasma lipid profile. Conclusion The present study has shown that, in early stages of plaque development in LDLr−/− mice, specific changes in NO/NO synthase system develop, that are characterized by increased endothelium-dependent vasorelaxation and increased constitutive NOS expression. Since the development of plaque and the indicator of endothelial cell dysfunction were prevented by SNAC, such treatment may constitute a novel strategy for the halting of progression of early plaque.

The local delivery of nitric oxide (nitrogen monoxide, NO) by thermal or photochemical means to target cells or organs has a great potential in several biomedical applications, especially if the NO donors are incorporated into non-toxic viscous matrices. In this work, we have shown that the NO donors S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylcysteine (SNAC) can be incorporated into F127 hydrogels, from where NO can be released thermally or photochemically (with lambda(irr)>480nm). High sensitivity differential scanning calorimetry (HSDSC) and a new spectrophotometric method, were used to characterize the micellization and the reversal thermal gelation processes of the F127 hydrogels containing NO donors, and to modulate the gelation temperatures to the range 29-32 degrees C. Spectral monitoring of the S-NO bond cleavage showed that the initial rates of thermal and photochemical NO release (ranging from 2 to 45 micromoll(-1)min(-1)) are decreased in the hydrogel matrices, relative to those obtained in aqueous solutions. This stabilization effect was assigned to a cage recombination mechanism and offers an additional advantage for the storage and handling of S-nitrosothiols. These results indicate that F127 hydrogels might be used for the thermal and photochemical delivery of NO from S-nitrosothiols to target areas in biomedical applications.

Orientador: Marcelo Ganzarolli de Oliveira Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica Doutorado

Orientador: Marcelo Ganzarolli de Oliveira Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica Mestrado

Melanoma is one of the most aggressive skin cancers due to its high capacity to metastasize. Treatment of metastatic melanomas is challenging for clinicians, as most therapeutic agents have failed to demonstrate improved survival. Thus, new candidates with antimetastatic activity are much needed. Riboavin (RF) is a component of the vitamin B complex and a potent photosensitizer. Previously, our group showed that the RF photoproducts (iRF) have potential as an antitumoral agent. Hence, we investigated the capacity of iRF on modulating melanoma B16F10 cells aggressiveness in vitro and in vivo. iRF decreases B16F10 cells survival by inhibiting mTOR as well as Src kinase. Moreover, melanoma cell migration was disrupted after treatment with iRF, mainly by inhibition of metalloproteinase (MMP) activity and expression, and by increasing TIMP expression. Interestingly, we observed that the Hedgehog (HH) pathway was inhibited by iRF. Two mediators of HH signaling, GLI1 and PTCH, were downregulated, while SUFU expression (an inhibitor of this cascade) was enhanced. Furthermore, inhibition of HH pathway signaling by cyclopamine and Gant 61 potentiated the antiproliferative action of RF. Accordingly, when a HH ligand was applied, the effect of iRF was almost completely abrogated. Our findings indicate that Hedgehog pathway is involved on the modulation of melanoma cell aggressiveness by iRF. Moreover, iRF treatment decreased pulmonary tumor formation in a murine experimental metastasis model. Research to clarify the molecular action of flavins, in vivo, is currently in progress. Taken together, the present data provides evidence that riboflavin photoproducts may provide potential candidates for improving the efficiency of melanoma treatment.