Preparo, caracterização e eficácia in vitro de formulações lipossomais de circulação prolongada contendo Miltefosina e Antimonato de Meglumina para o tratamento da Leishmaniose Visceral

Abstract

Visceral leishmaniasis (VL) is a disease that constitutes a serious public health problem. The therapy with the available drugs demands long treatment time and causes many side effects. New pharmacological approaches in the LV treatment have been suggested, such as those based on leishmanicidal drugs with liposomal formulations of prolonged circulation (stealth or PEGylated) seeking to increase therapeutic efficacy and reducing side effects. Therefore, the aim of this study was to prepare, characterize and evaluate, in vitro, the efficacy of PEGylated liposomal formulations containing miltefosine (HePC-PEG) and meglumine antimoniate (MA) (HePC-PEG/AM) for the LV treatment. PEGylated liposomes HePC-PEG and HePC-PEG/AM were prepared by the incorporation of the DSPE-PEG 2000 polymer in the vesicle membranes, using the DRV method. Effectiveness of the PEGylated formulations was compared with the conventional MA encapsulated-liposome (LAM), "empty" liposomes (Lemp), MA and “free” miltefosine. The characterization of the PEGylated formulations (hydrodynamic diameter, polydispersity index (PI) and zeta potential (Z)) were determined by photon correlation spectroscopy, the Sb encapsulation rate by graphite furnace atomic absorption spectrometry, and the presence of miltefosine in the formulations was determined by thin layer chromatography (TLC). The cytotoxicity concentration (CC50) and inhibitory concentration (CI50) assays of the formulations were evaluated in murine macrophages by MTT colorimetric method and in promastigote forms of Leishmania infantum by resazurin reduction colorimetric method, respectively. Also, the effectiveness of the formulations against intracellular amastigotes 24h, 48h and 72h after treatment was evaluated. The hydrodynamic diameter of the HePC-PEG and HePC-PEG/AM formulations were lower than 200nm, and PI˂0,3 e z˂-30mV. TLC analysis suggested the presence of miltefosine in the formulations. CC50 of HepC-PEG was 92,49 µg/mL and for HePC-PEG/MA was 103,09µg/mL, both higher rates than miltefosine in its free form (41,59µg/mL), suggesting less cell toxicity of the drug in liposomal formulation. The CI50 values were 33,19 µg/mL for HepC-PEG and 33,05 µg/mL for HePC-PEG/MA, whereas the free miltefosine IC50 was 49,59 µg/mL, suggesting that the leishmanicidal activity of miltefosine in PEGylated formulations remained. In the tests with intracellular amastigotes, the PEGylated formulations significantly reduced macrophage infection rates for MA, LAM and Lemp in 72h after the treatment, and at all times evaluated when compared to uninfected control. In conclusion, HePC-PEG and HePC-PEG/MA formulations presented reduced vesicle sizes, electrical stability, monodispersity and significant reduction in Leishmania infantum infection rates, in vitro.