Formulação de anfotericina B em lipossomas de circulação prolongada para o tratamento da leishmaniose visceral

Abstract

Visceral leishmaniasis (VL) is a serious parasitic disease, caused by Leishmania (L.) infantum and L. (L.) donovani. It is estimated that around 300,000 new cases result in ~30,000 deaths/year worldwide. Treatment is the main control measure, but it has limitations, such as toxicity and high cost lines. The objective of this study was to prepare, characterize and evaluate the therapeutic efficacy, in vitro and in vivo, of an innovative prolonged circulation liposomal formulation containing AmB (LAmB-PEG), and of a conventional liposomal formulation (LAmB-Conv) for the treatment of LV. The liposomes were prepared using the "Vesicle dehydration and rehydration" method and the average hydrodynamic diameter, zeta potential (Z) and the vesicles polydispersion index (IP) were determined by photon correlation spectroscopy. The cytotoxicity assay (CC50) was performed on the RAW 264.7 cell line by the MTT colorimetric method. The values of the inhibitory concentration (IC50) were determined by the colorimetric assay of resazurin in the Leishmania (L.) infantum strains (MCAN/BR/2002/BH401). Then, the selectivity index (SI) was calculated, established by the CC50/IC50 ratio. BALB/c mice were treated according to the following protocols (n = 6): i- two doses of LAmB-PEG (5mg/kg/96h/IP); ii- two doses of LAmB-Conv (5mg/kg/96h/IP); iii- ten doses of AmB (5mg/kg/24h/IP); iv- 36 animals that received no treatment. After treatment, blood was collected and plasma aliquots were used to assess renal (serum urea and creatinine levels) and liver functions (levels of pyruvic transaminase, oxaloacetic transaminase, total serum proteins and albumin). BALB/c mice were infected with 107 L. infantum promastigotes (MCAN/BR/2002/BH401) and twenty-five days after infection, the animals were treated with two therapeutic protocols (n = 6): Protocol 1: i- two doses of LAmB-PEG (5mg / kg / 96h / IP); ii- two doses of LAmB-Conv (5mg / kg / 96h / IP); iii- ten doses of AmB (5mg / kg / 24h / IP); iv- ten doses of sterile saline solution (PBS) (24h). Protocol 2: i- two doses of LAmB-PEG (10mg / kg / 96h / IP); ii- two doses of LAmB-Conv (10mg / kg / 96h / IP); iii- ten doses of AmB (5mg / kg / 24h / IP); iv- ten doses of sterile saline solution (PBS) (24h). After treatment, the animals were sacrificed and the parasitic load on the liver and spleen was determined by qPCR. The formulations prepared in this work showed characteristics of stable, monodispersed nanosystems with an average hydrodynamic diameter of less than 200nm. LAmB-PEG showed CC50, equal to 328.55μg/mL and LAmB-Conv equal to 316.1μg/mL, both rates higher than free AmB (265.61μg/mL), suggesting less cellular toxicity of the drug in liposomal formulation. IC50 values were similar for LAmB-PEG (20.365 ng/mL), LAmB-Conv (25.215ng/mL) and for free AmB (38.305 ng/mL), suggesting that there was no reduction in AmB leishmanicidal activity in the formulations. Against intracellular amastigotes, LAmB-PEG significantly reduced infection rates of macrophages compared to control (untreated macrophages), and macrophages treated with LAmB-Conv and AmB-D. There was no change in renal function in the toxicity and therapeutic efficacy tests performed in a murine model. However, the enzymes TGO and albumin, used to assess liver function, were increased in the groups treated with LAmB-PEG, LAmB-Conv and AmB-D. The other parameters to assess liver function were within the reference levels for the formulations. The animals infected with L. infantum and treated with the formulation LAmB-PEG showed a significant reduction in the parasitic load on the liver and spleen in relation to the control group, in the evaluated therapeutic protocols (5mg/kg and 10mg/kg). At a dose of 10 mg/kg, in the spleen, the formulation LAmB-PEG showed a statistical difference with AmB, indicating an improvement in the efficacy of the drug when it was encapsulated in a liposomal vesicle. For the first time, proof of concept that the innovative formulations of LAmB-PEG and LAmB-Conv induce a significant reduction in infection rates of L. infantum, in vitro and in vivo, has been confirmed. These results reinforce the therapeutic potential of these formulations and open new perspectives for the treatment of VL.