RESUMEN
Zika still poses a threat to global health owing to its association with serious neurological conditions and the absence of a vaccine and treatment. Sofosbuvir, an anti-hepatitis C drug, has shown anti-Zika effects in animal and cell models. Thus, this study aimed to develop and validate novel LC-MS/MS methods for the quantification of sofosbuvir and its major metabolite (GS-331007) in human plasma and cerebrospinal (CSF) and seminal fluid (SF), and apply the methods to a pilot clinical trial. The samples were prepared by liquid-liquid extraction and separated using isocratic mode on Gemini C18 columns. Analytical detection was performed using a triple quadrupole mass spectrometer equipped with an electrospray ionization source. The validated ranges for sofosbuvir were 0.5-2,000 ng/mL (plasma) and 0.5-100 ng/mL (CSF and SF), while for the metabolite they were 2.0-2,000 ng/mL (plasma), 5.0-200 ng/mL (CSF) and 10-1,500 ng/mL (SF). The intra-day and inter-day accuracies (90.8-113.8%) and precisions (1.4-14.8%) were within the acceptance range. The developed methods fulfilled all validation parameters concerning selectivity, matrix effect, carryover, linearity, dilution integrity, precision, accuracy and stability, confirming the suitability of the method for the analysis of clinical samples.
Asunto(s)
Infección por el Virus Zika , Virus Zika , Animales , Humanos , Cromatografía Liquida/métodos , Límite de Detección , Plasma , Reproducibilidad de los Resultados , Sofosbuvir , Espectrometría de Masas en Tándem/métodosRESUMEN
Prior investigations show that signaling activation through pattern recognition receptors can directly impact a number of inflammatory lung diseases. While toll-like receptor (TLR) 7 agonists have raised interest for their ability to inhibit allergen-induced pathological changes in experimental asthma conditions, the putative benefit of this treatment is limited by adverse effects. Our aim was to evaluate the therapeutic potential of two PEGylated purine-like compounds, TMX-302 and TMX-306, characterized by TLR7 partial agonistic activity; therefore, the compounds are expected to induce lower local and systemic adverse reactions. In vitro approaches and translation to murine models of obstructive and restrictive lung diseases were explored. In vitro studies with human PBMCs showed that both TMX-302 and TMX-306 marginally affects cytokine production as compared with equivalent concentrations of the TLR7 full agonist, TMX-202. The PEGylated compounds did not induce monocyte-derived DC maturation or B cell proliferation, differently from what observed after stimulation with TMX-202. Impact of PEGylated ligands on lung function and inflammatory changes was studied in animal models of acute lung injury, asthma, and silicosis following Lipopolysaccharide (LPS), allergen (ovalbumin), and silica inhalation, respectively. Subcutaneous injection of TMX-302 prevented LPS- and allergen-induced airway hyper-reactivity (AHR), leukocyte infiltration, and production of pro-inflammatory cytokines in the lung. However, intranasal instillation of TMX-302 led to neutrophil infiltration and failed to prevent allergen-induced AHR, despite inhibiting leukocyte counts in the BAL. Aerosolized TMX-306 given prophylactically, but not therapeutically, inhibited pivotal asthma features. Interventional treatment with intranasal instillation of TMX-306 significantly reduced the pulmonary fibrogranulomatous response and the number of silica particles in lung interstitial space in silicotic mice. These findings highlight the potential of TMX-306, emphasizing its value in drug development for lung diseases, and particularly silicosis.
RESUMEN
Galactosamine (GalN) is a hepatotoxic agent, which under determined situations provokes metabolic and energetic depletion as well as alterations in permeability, leading to cellular death. At the same time, it is known that fructose-1,6-bisphosphate (FBP) helps maintain cell energy levels and protects the cell against this lesive agent. We submitted two groups of male Wistar rats to the harmful intraperitoneal doses of GalN (400 mg/kg), one of which simultaneously received FBP (2 g/kg). Techniques were used in the analysis of the cellular components, adenosine triphosphate (ATP) and hepatic calcium and a close relationship between the types of cellular death unchained by these agents was established. The liver of the rats treated with GalN showed energy depletion and concomitant increase calcium in the hepatic tissue, which provoked higher levels of necrosis leading to reduce cellular viability. On the other hand, the group which had received GalN+FBP maintained calcium levels close to the control values and the energy rate did not decrease as much as in the GalN only group, but recovered the control values, within a period of 48 h. At the same time, the degree of apoptosis was greater than in the GalN group. This fact suggests that the FBP maintains cellular levels of ATP, thus protecting the cell from the toxic action of GalN, reducing the percentage of dead cells and causing an alteration in the pattern of the cell death, whereby there is an increase in the rate of apoptosis and a decrease in that of necrosis.
RESUMEN
Cadmium is an important toxic environmental heavy metal. Several studies have demonstrated that a major site of cadmium toxicity in humans and in other animals is the proximal tubule of the kidney. A well established model for nefrotoxicity is the use of in vitro technique with proximal tubule epithelial cell lines, as LLC-PK1. Herein, we have the intention to study the possible protective effect of high diluted CdCl2 solutions. In a blinding way, LLC-PK1 cells were pre-treated with high diluted cadmium chloride in the potencies 10 cH, 15 cH and 20cH. After 4 days, these cells have received CdCl2 in a pre-determined toxic concentration. The cell viability was assessed by MTT assay. We have identified a protective effect of two CdCl2 high diluted solutions, 10 cH and 20 cH, when cells were intoxicated by sublethal CdCl2 concentration. The results indicate that probably the high dilutions have an expressive action on cells in sublethal intoxication.
O Cádmio é um contaminante ambiental relevante. Muitos estudos demonstram que o sítio de toxicidade em humanos e outros animais é o túbulo proximal do rim. Um modelo bem estabelecido para nefrotoxicidade é o uso de técnicas in vitro com linhagens de células epiteliais do túbulo proximal, conhecidas por LLC-PK1. Assim, nossa proposta foi a de estudar os eventuais efeitos protetores de uma alta diluição de CdCl2. Em um ensaio cego, células LLC_PK1 foram pré-tratadas com altas diluições de cloreto de cádmio nas diluições 10 cH, 15 cH e 20 cH. Após 4 dias, estas células receberam CdCl2 em uma concentração tóxica, previamente deteminada. A viabilidade cellular foi estudada por ensaios MTT. Observamos um efeito protetor para duas altas diluições de CdCl2, 10 cH e 20 cH, quando as células foram intoxicadas por concentrações subletais de CdCl2. Estes resultados indicam a possibilidade de que altas diluições tenham ação expressiva em células, em intoxicações subletais.
El Cádmio es un metal pesado com relevante acción tóxica en el medio ambiente. Varios estudios han demostrado que un sitio importante de la toxicidad del cadmio en los humanos y en otros animales es el túbulo proximal del riñón. Un modelo bien establecido de nefrotoxicidad es el uso de la técnica in vitro con células epiteliales del túbulo proximal, como las LLC-PK1. Estudiamos el posible efecto protector de soluciones altamente diluidas de CdCl2. Com uma metodologia em ciego, las células LLC-PK1 fueron pre-tratados con cloruro de cadmio altamente diluídos en las potencias 10 cH, 15 cH y 20 cH. Después de 4 días, estas células han recibido CdCl2 en una concentración tóxica predeterminado. La viabilidad celular se evaluó por el ensayo MTT. Hemos identificado un efecto protector de dos soluciones de altamente diluída de CdCl2, 10 cH y 20 cH, cuando las células se intoxicaron por concentración CdCl2 subletales. Los resultados indican que probablemente las altas diluciones tienen una acción expresiva en las células, en la intoxicación subletal.