Parasitological and immunological evaluation of a quinoline derivative salt incorporated into a polymeric micelle formulation against Leishmania infantum infection.

Leishmaniasis is a parasitic disease caused by Leishmania protozoa, which presents a large spectrum of clinical manifestations. In the present study, a quinoline derivative salt named N-(2-((7-chloroquinolin-4-yl)amino)ethyl)-N-(prop-2-yn-1-yl)prop-2-yn-1-aminium chloride or QDS3 was in vitro and in vivo tested against L. infantum by means of its incorporation in Poloxamer 407-based polymeric micelles (QDS3/M). The in vitro antileishmanial activity of QDS3 and QDS3/M was investigated in L. infantum promastigotes, axenic amastigotes and infected macrophages. BALB/c mice were infected with L. infantum, and parasitological parameters were evaluated 1 and 15 days post-treatment by determining the parasite load by a limiting dilution assay, besides a quantitative PCR (qPCR) method. Immunological response was assessed based on production of cellular cytokines, as well as by quantification of nitrite levels and specific antibodies. In vitro results showed that QDS3 free or in micelles presented effective antileishmanial action against both parasite stages, being more effective in amastigotes. In vivo data showed that treatment using QDS3 or QDS3/M reduced the parasite load in the livers, spleens, draining lymph nodes (dLN) and bone marrows of the treated animals, 1 and 15 days after treatment, when compared to values found in the control groups. Additionally, treated mice developed a polarized Th1-type immune response, with higher levels of IL-12, IFN-γ, GM-CSF and nitrite, besides high production of specific IgG2a antibodies, when compared to the controls. Parasitological and immunological data obtained using the micellar composition were better than the others. In conclusion, QDS3, mainly when applied in a delivery adjuvant system, could be considered for future studies as therapeutic candidate against VL.

Nitrate and nitrite-based therapy to attenuate cardiovascular remodelling in arterial hypertension.

Hypertension is a highly prevalent disease marked by vascular and cardiac maladaptive remodelling induced mainly by renin-angiotensin system activation followed by oxidative stress. Here, we briefly describe these damages and review the current evidence supporting a potential role for nitrate and nitrite as antihypertensive molecules that act via nitric oxide (NO) formation-dependent and NO formation-independent mechanisms and how nitrate/nitrite inhibits cardiovascular remodelling in hypertension. The renin-angiotensin system activation and oxidative stress converge to activate proteases involved in cardiovascular remodelling in hypertension. Besides these proteases, several investigations have demonstrated that reduced endogenous NO bioavailability is a central pathological event in hypertension. In this regard, nitrate/nitrite, long considered inert products of NO, is now known as physiological molecules able to reduce blood pressure in hypertensive patients and in different experimental models of hypertension. These effects are associated with the formation of NO and other NO-related molecules, which could induce S-nitrosylation of target proteins. However, it remains unclear whether S-nitrosylation is an essential mechanism for the anti-remodelling effects of nitrate/nitrite in hypertension. Moreover, nitrate/nitrite produces antioxidant effects associated with the inhibition of signalling pathways involved in cardiovascular remodelling. Together, these findings may help to establish nitrate and nitrite as effective therapies in hypertension-induced cardiovascular remodelling.

Nitrite-stimulated Gastric Formation of S-nitrosothiols As An Antihypertensive Therapeutic Strategy.

Hypertension is usually associated with deficient nitric oxide (NO) bioavailability, and therefore stimulating NO activity is an important antihypertensive strategy. Recently, many studies have shown that both nitrite and nitrate anions are not simple products of NO metabolism and indeed may be reduced back to NO. While enzymes with nitrite-reductase activity capable of generating NO from nitrite may contribute to antihypertensive effects of nitrite, another mechanism involving the generation of NO-related species in the stomach from nitrite has been validated. Under the acidic conditions of the stomach, nitrite generates NO-related species that form S-nitrosothiols. Conversely, drugs that increase gastric pH may impair the gastric formation of S-nitrosothiols, which may mediate antihypertensive effects of oral nitrite or nitrate. Therefore, it is now becoming clear that promoting gastric formation of S-nitrosothiols may result in effective antihypertensive responses, and this mechanism opens a window of opportunity in the therapy of hypertension. In this review, we discuss the recent studies supporting the gastric generation of S-nitrosothiols as a potential antihypertensive mechanism of oral nitrite. We also highlight some drugs that increase S-nitrosothiols bioavailability, which may also improve the responses to nitrite/nitrate therapy. This new approach may result in increased nitrosation of critical pharmacological receptors and enzymes involved in the pathogenesis of hypertension, which tend to respond less to their activators resulting in lower blood pressure.

The nitroxyl donor Angeli's salt ameliorates Staphylococcus aureus-induced septic arthritis in mice.

Septic arthritis is a severe and rapidly debilitating disease associated with severe joint pain, inflammation and oxidative stress. Nitroxyl (HNO) has become a nitrogen oxide of significant interest due to its pharmacological endpoints that are potentially favorable for treating varied diseases. However, whether HNO also serves as a treatment to septic arthritis is currently unknown. The aim of this study was to investigate the effect of the HNO donor, Angeli's salt (AS), in the outcome of chronic Staphylococcus aureus (S. aureus)-induced septic arthritis in mice. Daily treatment with AS inhibited mechanical hyperalgesia and inflammation (edema, leukocyte migration, cytokines release and NF-κB activation, and oxidative stress) resulting in reduced disease severity (clinical course, histopathological changes, proteoglycan levels in the joints, and osteoclastogenesis). In addition, AS decreased the number of S. aureus colony forming unities in synovial tissue, enhanced the bactericidal effect of macrophages and inhibited the worsening of systemic inflammatory response (leukocyte counts in the lung and systemic proinflammatory cytokine concentration). Our results suggest for the first time the therapeutic potential of AS in a model of septic arthritis by mechanisms involving microbicidal effects, anti-inflammatory actions and reduction of disease severity.

The nitroxyl donor, Angeli's salt, inhibits inflammatory hyperalgesia in rats.

Nitric oxide modulates pain development. However, there is no evidence on the effect of nitroxyl (HNO/NO⁻) in nociception. Therefore, we addressed whether nitroxyl inhibits inflammatory hyperalgesia and its mechanism using the nitroxyl donor Angeli's salt (AS; Na2N2O3). Mechanical hyperalgesia was evaluated using a modified Randall and Selitto method in rats, cytokine production by ELISA and nitroxyl was determined by confocal microscopy in DAF (a cell permeable reagent that is converted into a fluorescent molecule by nitrogen oxides)-treated dorsal root ganglia neurons in culture. Local pre-treatment with AS (17-450 µg/paw, 30 min) inhibited the carrageenin-induced mechanical hyperalgesia in a dose- and time-dependent manner with maximum inhibition of 97%. AS also inhibited carrageenin-induced cytokine production. AS inhibited the hyperalgesia induced by other inflammatory stimuli including lipopolysaccharide, tumor necrosis factor-α, interleukin-1ß and prostaglandin E2. Furthermore, the analgesic effect of AS was prevented by treatment with ODQ (a soluble guanylate cyclase inhibitor), KT5823 (a protein kinase G [PKG] inhibitor) or glybenclamide (an ATP-sensitive K⁺ channel blocker), but not with naloxone (an opioid receptor antagonist). AS induced concentration-dependent increase in fluorescence intensity of DAF-treated neurons in a l-cysteine (nitroxyl scavenger) sensitive manner. l-cysteine did not affect the NO⁺ donor S-Nitroso-N-acetyl-DL- penicillamine (SNAP)-induced anti-hyperalgesia or fluorescence of DAF-treated neurons. This is the first study to demonstrate that nitroxyl inhibits inflammatory hyperalgesia by reducing cytokine production and activating the cGMP/PKG/ATP-sensitive K⁺ channel signaling pathway in vivo.

Circulating cell-free DNA levels in plasma increase with severity in experimental acute pulmonary thromboembolism.

BACKGROUND: The diagnosis of acute pulmonary thromboembolism (APT) and its severity is challenging. No previous study has examined whether there is a linear relation between plasma DNA concentrations and the severity of APT. We examined this hypothesis in anesthetized dogs. We also examined the changes in plasma DNA concentrations in microspheres lung embolization and whether the therapy of APT with nitrite could modify APT-induced changes in plasma DNA concentrations. In vitro DNA release from blood clots was also studied. METHODS: APT was induced with autologous blood clots (saline, 1, 3, or 5 ml/kg) injected into the right atrium. A group of dogs received 300 microm microspheres into the inferior vena cava to produce similar pulmonary hypertension. Another group of dogs received 6.75 micromol/kg nitrite after APT with blood clots of 5 ml/kg. Hemodynamic evaluations were carried out for 120 min. DNA was extracted from plasma samples using QIAamp DNA Blood Mini Kit and quantified using Quant-iT PicoGreen dsDNA detection kit at baseline and 120 min after APT. RESULTS: APT produced dose-dependent increases in plasma DNA concentrations, which correlated positively with pulmonary vascular resistance (P=0.002, r=0.897) and with mean pulmonary arterial pressure (P=0.006, r=0.856). Conversely, lung embolization with microspheres produced no significant changes in plasma DNA concentrations. While nitrite attenuated APT-induced pulmonary hypertension, it produced no changes in plasma DNA concentrations. Blood clots released dose-dependent amounts of DNA in vitro. CONCLUSIONS: Cell-free DNA concentrations increase in proportion to the severity of APT, probably as a result of increasing amounts of thrombi obstructing the pulmonary vessels.