Eficácia in vitro de lipossomas furtivos contendo antimoniato de meglumina e miltefosina para o tratamento da leishmaniose tegumentar americana

Abstract

American tegumentary leishmaniasis (ATL) is a serious health problem worldwide distributed with case notified in all Brazilian regions. ATL is a zoonosis that involves primarily wild reservoirs and vectors which turns difficult the control of the disease. Despite of its limitations treatment still remains as main control measure for all clinical forms of leishmaniasis, including ATL. Even though the use of pentavalent antimonials (Sb5+) presents several limitations, these compounds are still the first-line drugs for the treatment of ATL, however, they can cause toxicity leading to important side effects. Because of this, a novel pharmacological approach based on leishmanicidal drugs in stealth liposome formulations have been suggested in order to increase the therapeutic efficacy and reduce side effects of these conventional drugs. The aim of this work was to prepare, characterize and evaluate, in vitro, the therapeutic efficacy of meglumine antimoniate encapsulated in stealth liposomes containing miltefosine for the treatment of cutaneous leishmaniasis. Stealth liposome containing miltefosine (HePC-PEG) was prepared using the "dehydration and rehydration vesicles" method (DRV) in water using miltefosine (HePC), cholesterol, dipalmitoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol 2000 and diethylphosphate. The stealth liposome containing miltefosine and meglumine antimoniate (HePC-PEG/AM) was obtained by rehydration of HePC-PEG vesicles with an aqueous solution of meglumine antimoniate. The vesicle characterization (hydrodynamic diameter, polydispersity index (PI) and zeta potential (Z)) of the formulations was determined by photon correlation spectroscopy and the Sb encapsulation rate by graphite furnace atomic absorption spectroscopy. The cytotoxicity (CC50) and leishmanicidal activity of the formulations (IC50) were performed in murine macrophages, using the MTT colorimetric method, and in promastigote forms of Leishmania amazonensis, using the colorimetric method of resazurin reduction, respectively. The efficacy of the formulation against intracellular amastigotes at 24h, 48h, and 72h after treatment was also evaluated. The HePC-PEG/AM formulation presented a hydrodynamic diameter of 200.85nm, PI= 0.074 and z= -66mV. HePC-PEG/AM presented CC50=103.21μg/mL, a higher rate than the CC50 obtained by the non-encapsulated miltefosine (41.59μg / mL) suggesting lower cellular toxicity of the drug in liposomal formulation. The IC50 value for HePC-PEG/AM was 28.34μg/mL whereas the non-encapsulated miltefosine the IC50 was 49.59 μg/mL. In the intracellular amastigote assays, the stealth formulation significantly reduced macrophage infection rates when compared to AM and miltefosine, 72h after treatment and to non treated control at all times evaluated. In conclusion, the HePC-PEG/AM formulation showed small hydrodynamic vesicle size, electrical stability and monodispersion, as well as providing significant reduction in the rates of macrophage infection by Leishmania amazonensis, in vitro, which was greater than the rates obtained by the conventional drugs suggesting synergy/addictive effect between these drugs when encapsulated in stealth liposomes.