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Introduction: The human umbilical artery (HUA), rat-isolated right atrium, and rat-isolated vas deferens present a basal release of 6-nitrodopamine (6-ND). The basal release of 6-ND from these tissues was significantly decreased (but not abolished) when the tissues were pre-incubated with Nω-nitro-L-arginine methyl ester (L-NAME).Methods: In this study, the effect of the pharmacological modulation of the redox environment on the basal release of 6-ND was investigated. The basal release of 6-ND was measured using Liquid chromatography with tandem mass spectrometry (LC-MS/MS).Results and Discussion: Pre-incubation (30 min) of the tissues with GKT137831 (1 μM) caused a significant increase in the basal release of 6-ND from all tissues. In the HUA, pre-incubation with diphenyleneiodonium (DPI) (100 μM) also caused significant increases in the basal release of 6-ND. Preincubation of the HUA with hydrogen peroxide (H2O2) (100 μM) increased 6-ND basal release, whereas pre-incubation with catalase (1,000 U/mL) significantly decreased it. Pre-incubation of the HUA with superoxide dismutase (SOD) (250 U/mL; 30 min) also significantly increased the basal release of 6-ND. Preincubation of the HUA with either allopurinol (100 μM) or uric acid (1 mM) had no effect on the basal release of 6-ND. Pre-treatment of the HUA with L-NAME (100 μM) prevented the increase in the basal release of 6-ND induced by GKT137831, diphenyleneiodonium, and H2O2. The results obtained indicate a major role of endogenous H2O2 and peroxidases as modulators of 6- ND biosynthesis/release and a lack of peroxynitrite contribution.

Introduction: Hydrogen sulfide (H2S) is emerging as an important potential therapeutic option for respiratory inflammatory diseases. In this study, we investigated the effectiveness of a novel corticosteroid derivative, that is chemically linked to an H2S donor, in managing asthma features.Methods: The effects of prednisone (PS), H2S donor (4-hydroxybenzamide; TBZ), and their combination (PS-TBZ) have been evaluated in vitro and in vivo. The in vitro experiments were conducted using lipopolysaccharidestimulated J774 macrophages, while the in vivo experiments utilizing an experimental asthma model.Results: In the in vitro study we found that PS-TBZ exhibited an increased effect compared to the individual parent compounds in modulating the production of inflammatory mediators. TBZ also significantly reduced bronchial contractility and enhanced bronchial relaxation. In the in vivo experiments, where we administered PS, TBZ, or PS-TBZ to ovalbumin-sensitized BALB/c mice, we confirmed that PS-TBZ had a significantly better action in controlling airway hyperreactivity as compared to TBZ or PS alone. Moreover, PS-TBZ was more effective in restoring salbutamol-induced relaxation. The immunohistochemistry analysis demonstrated a significant reduction in the production of α-SMA and procollagen III, indicating the efficacy of PS-TBZ in controlling airway remodeling. Moreover, PS-TBZ also promoted epithelial repair, recovery of the bronchial and parenchyma structure and inhibited mucin production.Discussion: In conclusion, PS-TBZ offers an important opportunity to optimize the beneficial impact of corticosteroids on asthma features.

Neurounina-1 [chemical name: 7-nitro-5-phenyl-1-(pyrrolidin-1-ylmethyl)-1H-benzo[e][1,4]diazepin-2(3H)-one] is a new compound provided with relevant neuroprotective effect during stroke and in neonatal hypoxia by increasing the Na+/Ca2+ exchanger (NCX) isoforms NCX1 and NCX2 activity. This study shows for the first time, the development and validation of a sensitive and selective method for analysis of neurounina-1 in beagle dog plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The sample preparation consisted of extraction of the analyte and the internal standard (IS) (ropivacaine) from plasma (50 μL) by liquid-liquid extraction using acetonitrile (100 μL). The selected reaction monitoring mode of the positive ion was performed and the precursor to the product ion transitions of m/z 365 > 83 and m/z 275 > 126 were used to measure the derivative of neurounina-1 and ropivacaine. The chromatographic separation was achieved using a Phenomenex C18 Luna (150 mm × 4.6 mm × 5 μm) analytical column with an isocratic mobile phase composed of methanol/acetonitrile/water (50/40/10, v/v/v) + 0.1% formic acid + 1 M ammonium formate. The method was linear over a concentration range of 1–500 ng/mL. The method was applied to evaluate the pharmacokinetics of neurounina-1 after a single intravenous administration of three different doses (0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg) to beagle dogs (n = 5). The mean AUC0-tlast values were 26.10, 115.81, and 257.28 ng∗h/mL following intravenous administration of 0.1, 0.3, and 1 mg/kg, respectively. Linear pharmacokinetics was observed up to 1.0 mg/kg. The neurounina-1 was rapidly eliminated, with mean CL values of 46.24, 47.57, and 69.15 L/h, Vd of 130.31, 154.15, and 210.79 L and t1/2 of 2.14, 2.54, and 2.04 h after intravenous administration of 0.1, 0.3, and 1 mg/kg, respectively. This new analytical method allows the rapid determination of the neurounina-1, a new developed compound, able to exert a remarkable neuroprotective effect in the low nanomolar range.