Avaliação da toxicidade e eficácia de nanoemulsões contendo Anfotericina B para o tratamento da Leishmaniose Visceral

Abstract

Amphotericin B (AmB), a second choice drug for the treatment of visceral leishmaniasis (VL), has a broad-spectrum of antifungal and antiparasitic activity. However, its conventional formulation (C-AmB), in which the AmB is associated to sodium deoxycholate, has high toxicity. Several adverse reactions have been linked to this formulation, like nephrotoxicity and anemia, and they are the major limitations for the treatment. Lipid formulations, such as nanoemulsions (NE), became an interesting alternative to overcome these drawbacks. Therefore, this study aimed to optimize, to characterize, to evaluate the in vitro toxic effects and to evaluate in vivo toxicity and activity of the AmB-loaded NE (NE-AmB) in BALB/c mice experimentally infected with Leishmania (Leishmania) infantum chagasi. The NE was characterized by the size, the polydispersity index (PI), and the drug encapsulation efficiency (EE). The hemolytic activity in vitro induced by AmB formulations was evaluated by their contact with red blood cells. The activity studies in a murine model of VL, C-AmB and NE-AmB (1 or 2 mg/kg/day) were administered intravenously on 5 alternate days. The in vivo toxicity studies were conducted in healthy mice, using AmB (1 mg/kg) on 3 alternate days. Biochemical analysis, blood urea nitrogen (BUN) and creatinine, and histopathology were carried out. Metabolomic analysis of the plasma on 3 different platforms coupled to mass spectrometry (MS) were also performed. After gradual increases in pressure in High Pressure Homogenizer (HAP), the NE had the droplet diameters (~ 140 nm) and PI values < 0.2, lower than those obtained previously and the EE close to 100%. The in vitro hemolytic activity of C-AmB was much higher than that observed for the NE-AmB. NE-AmB and C-AMB, both at 1 mg/kg, have reduced significantly the parasite burden in liver and spleen compared to the control, but differences between the two formulations were only observed when the NE-AmB was administered at 2 mg/kg without any sign of acute toxicity, unlike the C-AmB which proved to be lethal at this dose. BUN values, creatinine and histopathological analysis revealed no changes in kidney function. In metabolomics analysis, it was observed that the most affected metabolic pathways were the polyunsaturated fatty acids and arachidonic acid in the group treated with C-AmB, suggesting that this formulation may be associated with inflammatory processes, oxidative stress and renal ischemia. These data showed that the NE-AmB was able to reduce the toxic effects in vitro and in vivo and to show efficacy in vivo, revealing, therefore, to be a promising alternative for the treatment of VL