Impaired calcium influx despite hyper-reactivity in contralateral carotid following balloon injury: eNOS involvement

Balloon catheter injury results in hyper-reactivity to phenylephrine in contralateral carotids. Decreased nitric oxide (NO) modulation and/or increased intracellular calcium concentration triggers vascular smooth muscle contraction. Therefore, this study explores the participation of NO signaling pathway and calcium mobilization on hyper-reactivity to phenylephrine in contralateral carotids. Concentration-response curves for calcium (CaCl(2)) and phenylephrine were obtained in control and contralateral carotids four days after balloon injury, in the presence and absence of the inhibitors (L-NAME, L-NNA, 1400W, 7-NI, Oxyhemoglobin, ODQ or Tiron). Confocal microscopy using Fluo-3AM or DHE was performed to detect the intracellular levels of calcium and reactive oxygen species, respectively. The modulation of NO on phenylephrine-induced contraction was absent in the contralateral carotid. Phenylephrine-induced intracellular calcium mobilization was not altered in contralateral carotids. However, extracellular calcium mobilization by phenylephrine was reduced in the contralateral carotid compared to control arteries, and this result was confirmed by confocal microscopy. L-NAME increased phenylephrine-induced extracellular calcium mobilization in the contralateral carotid to the control levels. Results obtained with L-NNA, 1400W, 7-NI, OxyHb, ODQ or Tiron showed that this response was mediated by products from endothelial NOS (eNOS) different from NO and without soluble guanylate cyclase activation, but it involved superoxide anions. Furthermore. Tiron or L-NNA reduced the levels of reactive oxygen species in contralateral carotids. Data suggest that balloon catheter injury promoted eNOS uncoupling in contralateral carotids, which generates superoxide rather than NO, and reduces phenylephrine-induced extracellular calcium mobilization, despite the hyper-reactivity to phenylephrine in contralateral carotids. (C) 2010 Elsevier B.V. All rights reserved.

Assignment of the absolute configuration at the sulfur atom of thioridazine metabolites by the analysis of their chiroptical properties: The case of thioridazine 2-sulfoxide

Thioridazine (THD) is a commonly prescribed phenotiazine neuroleptic drug, which is extensively biotransformed in the organism producing as main metabolites sulfoxides and a sulfone by sulfur oxidation Significant differences have been observed in the activity of the THD enantiomers as well as for its main metabolites, and enantioselectivity phenomena have been proved in the metabolic pathway. Here the assignment of the absolute configuration at the sulfur atom of enantiomeric THD-2-sulfoxide (THD-2-SO) has been carried out by circular dichroism (CD) spectroscopy The stereoisomers were separated by HPLC on Chiralpak AS column, recording the CD spectra for the two collected enantiomeric fractions The theoretical electronic CD spectrum has been obtained by the TDDFT/B3LYP/6-31G*. as Boltzmann averaging of the contributions calculated for the most stable conformations of the drug The comparison of the simulated and experimental spectra allowed the absolute configuration at the sulfur atom of the four THD-2-SO stereoisomers to be assigned The developed method should be useful for a reliable correlation between stereochemistry and activity and/or toxicity

Probing the electronic delocalization in a cyclic pyrazine ruthenium cluster hexamer

[Ru(3)O(CH(3)COO)(6)(pz)(CO)](6) is a cyclic hexamer species encompassing six triangular ruthenium cluster centers bridged by pyrazine ligands. The electronic communication among the cluster units strongly depends on their oxidation states, and has been successfully probed by means of cyclic voltammetry and UV-vis spectroelectrochemistry. (C) 2010 Elsevier B.V. All rights reserved.

Nitric oxide photorelease from hydrogels and from skin containing a nitro-ruthenium complex

Nitric oxide (NO) is a gaseous molecule that has specific functions dictated by its localization and its kinetics of release. As NO-donors have a range of potential uses in the skin, much attention has been paid to the development of topical NO delivery systems. The aim of this work was to study the release rate and the skin penetration of the NO-donor cis[Ru(NO(2))(bpy)(2)(4-pic)](+) from different gel formulations and their potential as topical NO delivery systems under light stimuli. Among the formulations developed, the anionic gel retarded the nitro-ruthenium complex diffusion and also obstructed NO release after light irradiation. On the other hand, NO release before light irradiation was observed when the complex was dispersed in the cationic chitosan gel, possibly due to oxi-redox reactions between the amino groups of the polymer and the drug molecule. Finally, the non-ionic gel released the NO after light irradiation to the same extent as a drug aqueous solution at the same pH. The drug dispersed in this gel also penetrated into the stratum corneum skin layer, and the nitro-ruthenium complex present in the skin was able to release the NO after light stimuli, suggesting the potential use of this formulation as a topical NO delivery system. (C) 2010 Elsevier B.V. All rights reserved.

Determination of beta-artemether and its main metabolite dihydroartemisinin in plasma employing liquid-phase microextraction prior to liquid chromatographic-tandem mass spectrometric analysis

A method for the determination of artemether (ART) and its main metabolite dihydroartemisinin (DHA) in plasma employing liquid-phase microextraction (LPME) for sample preparation prior to liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed. The analytes were extracted from 1 nil, of plasma utilizing a two-phase LPME procedure with artemisinin as internal standard. Using the optimized LPME conditions, mean absolute recovery rates of 25 and 32% for DHA and ART, respectively, were achieved using toluene-n-octanol (1:1, viv) as organic phase with an extraction time of 30 min. After extraction, the analytes were resolved within 5 min using a mobile phase consisting of methanol-ammonium acetate (10 mmol L(-1) pH 5.0, 80:20. v/v) on a laboratory-made column based on poly(methyltetradecylsiloxane) attached to a zirconized-silica support. MS-MS detection was employed using an electrospray interface in the positive ion mode. The method developed was linear over the range of 5-1000 ng mL(-1) for both analytes. Precision and accuracy were within acceptable levels of confidence (<15%). The assay was applied to the determination of these analytes in plasma from rats treated with ART. The two-phase LPME procedure is affordable and the solvent consumption was very low compared to the traditional methods of sample preparation. (C) 2010 Elsevier B.V. All rights reserved.

Enantioselective analysis of praziquantel and trans-4-hydroxypraziquantel in human plasma by chiral LC-MS/MS: Application to pharmacokinetics

A simple enantioselective method for the determination of praziquantel (PZQ) and trans-4-hydroxypraziquantel (4-OHPZQ) in human plasma was developed and validated by high-performance liquid chromatography/mass spectrometry. The plasma samples were prepared by liquid-liquid extraction using a mixture of methyl-tert-butylether/dichloromethane (2:1, v/v) as extraction solvent. The direct resolution of PZQ and 4-OHPZQ enantiomers was performed on a Chiralpak AD column using hexane-isopropanol (75:25, v/v) as the mobile phase. Diazepam was used as internal standard. The method described here is simple and reproducible. The quantitation limit of 1.25 ng/ml for each PZQ enantiomer and of 12.5 ng/ml for each 4-OHPZQ enantiomer permits the use of the method in studies investigating the kinetic disposition of a single dose of 1.5g racemic PZQ. Enantioselectivity in the kinetic disposition of PZQ and 4-OHPZQ was observed in the clinical study. with the demonstration of a higher proportion of the (+)-(S)-PZQ and (-)-(R)-4-OHPZQ enantiomers in plasma. (C) 2009 Elsevier B.V. All rights reserved.

Distinct conformational properties determined by implicit and explicit representation of protein-solvent interactions. An analytical and computer simulation study

In the protein folding problem, solvent-mediated forces are commonly represented by intra-chain pairwise contact energy. Although this approximation has proven to be useful in several circumstances, it is limited in some other aspects of the problem. Here we show that it is possible to achieve two models to represent the chain-solvent system. one of them with implicit and other with explicit solvent, such that both reproduce the same thermodynamic results. Firstly, lattice models treated by analytical methods, were used to show that the implicit and explicitly representation of solvent effects can be energetically equivalent only if local solvent properties are time and spatially invariant. Following, applying the same reasoning Used for the lattice models, two inter-consistent Monte Carlo off-lattice models for implicit and explicit solvent are constructed, being that now in the latter the solvent properties are allowed to fluctuate. Then, it is shown that the chain configurational evolution as well as the globule equilibrium conformation are significantly distinct for implicit and explicit solvent systems. Actually, strongly contrasting with the implicit solvent version, the explicit solvent model predicts: (i) a malleable globule, in agreement with the estimated large protein-volume fluctuations; (ii) thermal conformational stability, resembling the conformational hear resistance of globular proteins, in which radii of gyration are practically insensitive to thermal effects over a relatively wide range of temperatures; and (iii) smaller radii of gyration at higher temperatures, indicating that the chain conformational entropy in the unfolded state is significantly smaller than that estimated from random coil configurations. Finally, we comment on the meaning of these results with respect to the understanding of the folding process. (C) 2009 Elsevier B.V. All rights reserved.

Mechanisms underlying the vasorelaxant action of the pimarane ent-8(14),15-pimaradien-3 beta-ol in the isolated rat aorta

Pimarane-type diterpenes were described to exert antispasmodic and relaxant activities. Based on this observation we hypothesized that the diterpene ent-8(14),15-pimaradien-3 beta-ol (PA-3 beta-ol) induced vascular relaxation. With this purpose, the present work investigates the mechanisms involved in the vasorelaxant effect of the pimarane-type diterpene PA-3 beta-ol. Vascular reactivity experiments, using standard muscle bath procedures, were performed in isolated aortic rings from male Wistar rats. Cytosolic calcium concentration ([Ca(2+)]c) was measured by confocal microscopy using the fluorescent probe Fluo-3AM. PA-3 beta-ol (10, 50 and 100 mu mol/l) inhibited phenylephrine and KCl-induced contraction in either endothelium-intact or denuded rat aortic rings. PA-3 beta-ol also reduced CaCl(2)-induced contraction in Ca(2+)-free solution containing KCl (30 mmol/l) or phenylephrine (0.1 mu mol/l). PA-3 beta-ol (1-300 mu mol/l) concentration dependently relaxed phenylephrine-pre-contracted rings with intact or denuded endothelium. The diterpene also relaxed KCl-pre-contracted rings with intact or denuded endothelium. Moreover, Ca(2+) mobilization study showed that PA-3 beta-ol (100 mu mol/l) and verapamil (1 mu mol/l) inhibited the increase in Ca(2+)-concentration in smooth muscle and endothelial cells induced by phenylephrine (10 mu mol/l) or KCl (60 mmol/l). Pre-incubation of intact or denuded aortic rings with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 mu mol/l) and 1H-[1,2,4] Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ 1 mu mol/l) produced a rightward displacement of the PA-3 beta-ol concentration-response curves. On the other hand, 7-nitroindazole (100 mu mol/l), 1400 W (1 mu mol/l), indomethacin (10 mu mol/l) and tetraethylammonium (1 mmol/l) did not affect PA-3 beta-ol-induced relaxation. Collectively, our results provide evidence that the effects elicited by PA-3 beta-ol involve extracellular Ca(2+) influx blockade. Its effects are also partly mediated by the activation of NO-cGMP pathway. (C) 2009 Elsevier B.V. All rights reserved.

HPLC separation, NMR and QTOF/MS/MS structure elucidation of a prominent nitric oxide donor agent based on an isomeric composition of a nitrosyl ruthenium complex

A new and promising nitrosyl ruthenium complex, [Ru(NO)(bdqi-COOH)(terpy)](PF(6))(3), bdqi-COOH is 3,4-diiminebenzoic acid and terpy is 2,2`-terpyridine, has been synthesized as a NO donor agent. The procedure used for [Ru(NO)(bdqi-COOH)(terpy)](PF(6))(3) synthesis has, apparently, yielded the formation of two isomers in which the ligand bdqi-COOH appears to be coordinated in its reduced form (bdcat-COOH), which could have differences in their pharmacological properties. Therefore, it was intended to separate the two possible isomers by high-performance liquid chromatography (HPLC) and to characterize them by high resolution mass spectrometry (QTOF MS) and by magnetic nuclear resonance spectroscopy (NMR). The results obtained by MS showed that the ESI-MS mass spectra of both HPLC column fractions, e.g. peak 1 and peak 2, are essentially equal, showing that both isomers display nearly identical gas-phase behavior with clusters of isotopologue ions centered at m/z 573, m/z 543 and m/z 513. Regarding the NMR analysis, the results showed that the positional isomerism is located in the bdqi-COOH ligand. From the observed results it can be concluded that the synthesis procedure that has been used results in the formation of two [Ru(terpy)(bdqi-COOH)NO](PF(6))(3) isomers. (c) 2009 Elsevier B.V. All rights reserved.

Simultaneous determination of multibenzodiazepines by HPLC/UV: Investigation of liquid-liquid and solid-phase extractions in human plasma

A method for simultaneous determination of seven benzodiazepines (BZPs) (flunitrazepam, clonazepam, oxazepam, lorazepam, chlordiazepoxide, nordiazepam and diazepam using N-desalkylflurazepam as internal standard) in human plasma using liquid-liquid and solid-phase extractions followed by high-performance liquid chromatography (HPLC) is described. The analytes were separated employing a LC-18 DB column (250 mm x 4.6 mm, 5 mu m) at 35 degrees C under isocratic conditions using 5 mM KH(2)PO(4) buffer solution pH 6.0: methanol: diethyl ether (55:40:5, v/v/v) as mobile phase at a flow rate of 0.8 mL min(-1). UV detection was carried out at 245 nm. Employing LLE, the best conditions were achieved with double extraction of 0.5 mL, plasma using ethyl acetate and Na(2)HPO(4) pH 9.5 for pH adjusting. Employing SPE, the best conditions were achieved with 0.5 mL plasma plus 3 mL 0.1 M borate buffer pH 9.5, which were then passed through a C18 cartridge previously conditioned, washed for 3 times with these solvents: 3 mL 0.1 M borate buffer pH 9.5,4 mL Milli-Q water and 1 mL acetonitrile 5%, finally the BZPs elution was carried with diethyl ether: n-hexane: methanol (50:30:20). In both methods the solvent was evaporated at 40 degrees C under nitrogen flow. The validation parameters obtained in LLE were linearity range of 50-1200 ng mL(-1) plasma (r >= 0.9927), limits of quantification of 50 ng mL(-1) plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15%, and recovery above 65% for all BZPs. In SPE, the parameter obtained were linearity range of 30-1200 ng mL(-1) plasma (r >= 0.9900), limits of quantification of 30 ng mL(-1) plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15% and recovery above 55% for all BZPs. These extracting procedures followed by HPLC analysis showed their suitable applicability in order to examine one or more BZPs in human plasma. Moreover, it could be suggested that these procedures might be employed in various analytical applications, in special for toxicological/forensic analysis. (c) 2008 Elsevier B.V. All rights reserved.

3`3-Ditrifluoromethyldiphenyl diselenide: A new organoselenium compound with interesting antigenotoxic and antimutagenic activities

The trace element selenium (Se), once known only for its potential toxicity, is now a well-established essential micronutrient for mammals. The organoselenium compound diphenyl diselenide (DPDS) has shown interesting antioxidant and neuroprotective activities. On the other hand, this compound has also presented pro-oxidant and mutagenic effects. The compound 3`3-ditrifluoromethyldiphenyl diselenide (DFDD), a structural analog of diphenyl diselenide, has proven antipsychotic activity in mice. Nevertheless, as opposed to DPDS, little is known on the biological and toxicological properties of DFDD. In the present study, we report the genotoxic effects of the organoselenium compound DFDD on Salmonella typhimurium, Saccharomyces cerevisiae and Chinese hamster lung fibroblasts (V79 cells). DFDD protective effects against hydrogen peroxide (H(2)O(2))-induced DNA damage in vitro are demonstrated. DFDD did not cause mutagenic effects on S. typhimurium or S. cerevisiae strains; however, it induced DNA damage in V79 cells at doses higher than 25 mu M, as detected by comet assay. DFDD protected S. typhimurium and S. cerevisiae against H(2)O(2)-induced mutagenicity, and, at doses lower than 12.5 mu M, prevented H(2)O(2)-induced genotoxicity in V79 cells. The in vitro assays demonstrated that DFDD mimics catalase activity better than DPDS, but neither presents Superoxide dismutase action. The products of the reactions of DFDD or DPDS with H(2)O(2) were different. as determined by electrospray mass spectrometry analysis (ESI-MS). These results suggest that DFDD is not mutagenic for bacteria or yeast; however, it may induce weak genotoxic effects on mammalian cells. In addition, DFDD has a protective effect against H(2)O(2)-induced damage probably by mimicking catalase activity, and the distinct products of the reaction DFDD with H(2)O(2) probably have a fundamental role in the protective effects of DFDD. (C) 2009 Elsevier B.V. All rights reserved.

Ca(2+) binding to c-state of adenine nucleotide translocase (ANT)-surrounding cardiolipins enhances (ANT)-Cys(56) relative mobility: A computational-based mitochondrial permeability transition study

The oxidation of critical cysteines/related thiols of adenine nucleotide translocase (ANT) is believed to be an important event of the Ca(2+)-induced mitochondrial permeability transition (MPT), a process mediated by a cyclosporine A/ADP-sensitive permeability transition pores (PTP) opening. We addressed the ANT-Cys(56) relative mobility status resulting from the interaction of ANT/surrounding cardiolipins with Ca(2+) and/or ADP by means of computational chemistry analysis (Molecular Interaction Fields and Molecular Dynamics studies), supported by classic mitochondrial swelling assays. The following events were predicted: (i) Ca(2+) interacts preferentially with the ANT surrounding cardiolipins bound to the H4 helix of translocase, (ii) weakens the cardiolipins/ANT interactions and (iii) destabilizes the initial ANT-Cys(56) residue increasing its relative mobility. The binding of ADP that stabilizes the conformation ""m"" of ANT and/or cardiolipin, respectively to H5 and H4 helices, could stabilize their contacts with the short helix h56 that includes Cys(56), accounting for reducing its relative mobility. The results suggest that Ca(2+) binding to adenine nucleotide translocase (ANT)-surrounding cardiolipins in c-state of the translocase enhances (ANT)-Cys(56) relative mobility and that this may constitute a potential critical step of Ca(2+)-induced PTP opening. (C) 2009 Elsevier B.V. All rights reserved.

In situ gelling hexagonal phases for sustained release of an anti-addiction drug

In this study, fluid precursor formulations for subcutaneous injection and in situ formation of hexagonal phase gels upon water absorption were developed as a strategy to sustain the release of naltrexone, a drug used for treatment of drug addiction. Precursor formulations were obtained by combining BRIJ 97 with propylene glycol (PG, 5-70%, w/w). To study the phase behavior of these formulations, water was added at 10-90% (w/w), and the resulting systems were characterized by polarized light microscopy. Two precursor formulations containing BRIJ:PG at 95:5 (w/w, referred to as BRIJ-95) and at 80:20 (w/w, referred to as BRIJ-80) were chosen. Naltrexone was dissolved at 1% or suspended at 5% (w/w). Precursor formulations were transformed into hexagonal phases when water content exceeded 20%. Water uptake followed second-order kinetics, and after 2-4 h all precursor formulations were transformed into hexagonal phases. Drug release was prolonged by the precursor formulations (compared to a drug solution in PBS), and followed pseudo-first order kinetics regardless of naltrexone concentration. The release from BRIJ-80 was significantly higher than that from BRIJ-95 after 48 h. The relative safety of the precursor formulations was assessed in cultured fibroblasts. Even though BRIJ-95 was more cytotoxic than BRIJ-80, both precursor formulations were significantly less cytotoxic than sodium lauryl sulfate (considered moderate-to-severe irritant) at the same concentration (up to 50 mu g/mL). These results suggest the potential of BRIJ-based precursor formulations for sustained naltrexone release. (C) 2011 Elsevier By. All rights reserved.

Quercetin in w/o microemulsion: In vitro and in vivo skin penetration and efficacy against UVB-induced skin damages evaluated in vivo

The present study evaluated the potential of a w/o microemulsion as a topical carrier system for delivery of the antioxidant quercetin. Topical and transdermal delivery of quercetin were evaluated in vitro Using porcine car skin mounted on a Franz diffusion cell and in vivo on hairless-skin mice. Skin irritation by topical application of the microemulsion containing quercetin, and the protective effect of the formulation on UVB-induced decrease of endogenous reduced glutathione levels and increase of cutaneous proteinase secretion/activity were also investigated. The w/o microemulsion increased the penetration of quercetin into the stratum corneum and epidermis plus dermis at 3, 6. 9 and 12 h post-application in vitro and in vivo at 6 h post-application. No transdermal delivery of quercetin Occurred. By evaluating established endpoints of skin irritation (erythema formation, epidermis thickening and infiltration of inflammatory cells), the Study demonstrated that the daily application of the w/o microemulsion for up to 2 days did not cause skin irritation. W/o microemulsion containing quercetin significantly prevented the UVB irradiation-induced GSH depletion and secretion/activity of metalloproteinases. (C) 2008 Elsevier B.V. All rights reserved.

In vitro studies of cutaneous retention of magnetic nanoemulsion loaded with zinc phthalocyanine for synergic use in skin cancer treatment

In this study was developed a new nano drug delivery system (NDDS) based on association of biodegradable surfactants with biocompatible magnetic fluid of maguemita citrate derivative. This formulation consists in a magnetic emulsion with nanostructured colloidal particles. Preliminary in vitro experiments showed that the formulation presents a great potential for synergic application in the topical release of photosensitizer drug (PS) and excellent target tissue properties in the photodynamic therapy (PDT) combined with hyperthermia (HPT) protocols. The physical chemistry characterization and in vitro assays were carried out by Zn(II) Phtalocyanine (ZnPc) photosensitizer incorporated into NDDS in the absence and the presence of magnetic fluid, showed good results and high biocompatibility. In vitro experiments were accomplished by tape-stripping protocols for quanti. cation of drug association with different skin tissue layers. This technique is a classical method for analyses of drug release in stratum corneum and epidermis+ dermis skin layers. The NDDS formulations were applied directly in pig skin (tissue model) fixed in the cell`s Franz device with receptor medium container with a PBS/EtOH 20% solution (10mM, pH 7.4) at 37 degrees C. After 12 h of topical administration stratum corneum was removed from fifty tapes and the ZnPc retained was evaluated by solvent extraction in dimetil-sulphoxide under ultrasonic bath. These results indicated that magnetic nanoemulsion (MNE) increase the drug release on the deeper skin layers when compared with classical formulation in the absence of magnetic particles. This could be related with the increase of biocompatibility of NDDS due to the great affinity for the polar extracelullar matrix in the skin and also for the increase in the drug partition inside of corneocites wall. (C) 2008 Elsevier B.V. All rights reserved.