Avalia??o da atividade leishmanicida do antim?nio trivalente livre ou em formula??o lipossomal convencional, associado ou n?o ao ?cido asc?rbico, em camundongos BALB/c infectados com Leishmania infantum.

Programa de P?s-Gradua??o em Ci?ncias Farmac?uticas. CIPHARMA, Escola de Farm?cia, Universidade Federal de Ouro Preto.O presente estudo avaliou a utiliza??o de lipossomas convencionais como ve?culos vetores do antim?nio trivalente, na forma de t?rtaro em?tico, bem como sua associa??o ao ?cido asc?rbico, para o tratamento da leishmaniose visceral no modelo murino. Lipossomas constitu?dos por distearoilfosfatidilcolina/colesterol (5:4) foram obtidos pela metodologia de congelamento/descongelamento. A caracteriza??o da formula??o mostrou tamanho aproximado de 222,5 nm, ?ndice de polidispers?o de 0,214, efici?ncia de encapsula??o de 15%, concentra??o final de 4 mg SbIII/mL e cin?tica de libera??o bif?sica avaliada in vitro. Lipossomas vazios, igualmente obtidos e hidratados com tamp?o fosfato serviram como controle. Camundongos BALB/c com idade entre 6 e 8 semanas foram inoculados com 1 x 107 promastigotas de Leishmania (Leishmania) infantum (cepa C43) por via endovenosa. Ap?s seis semanas de infec??o, os animais foram divididos em sete grupos (n=8) e tratados por via intraperitoneal, em dose ?nica com: (1) tamp?o fosfato, (2) antim?nio trivalente (SbIII) - 9 mgSb/Kg, (3) lipossomas vazios, (4) lipossomas de SbIII (9 mgSb/Kg), (5) ?cido asc?rbico (AA) - 300 mg/kg; (6) associa??o do AA (300 mg/kg) ao SbIII (9 mgSb/kg) e (7) associa??o de AA (300 mg/kg) aos lipossomas de SbIII (9 mgSb/kg). Dez dias ap?s o tratamento, os animais foram eutanasiados e avaliou-se: a carga parasit?ria no f?gado, ba?o e medula ?ssea pelo m?todo de dilui??o limitante; o padr?o de c?lulas do ba?o por citometria de fluxo; a fun??o renal, hep?tica e card?aca por an?lises bioqu?micas e o padr?o histopatol?gico do f?gado, rins e cora??o. Observou-se redu??o significativa na carga parasit?ria ap?s tratamento com antim?nio trivalente lipossomal no f?gado, ba?o e medula ?ssea de 47%, 33% e 47%, respectivamente, indicando a capacidade do sistema de vetoriza??o de direcionar o medicamento para esses compartimentos. A administra??o concomitante do AA e SbIII livre ou em formula??o lipossomal n?o interferiu na atividade leishmanicida do SbIII. N?o foi observada diminui??o na carga parasit?ria ap?s tratamento com antim?nio trivalente livre em nenhum dos ?rg?os avaliados. Os resultados mostraram nenhuma altera??o no padr?o fenot?pico de c?lulas do ba?o por citometria de fluxo ou nas fun??es hep?tica, card?aca e renal por an?lises bioqu?micas. As an?lises histopatol?gicas mostraram que a coadministra??o dos lipossomas de SbIII e AA foi capaz de preservar os tecidos hep?tico e renal quando comparados os diferentes grupos experimentais. Os resultados desse trabalho permitiram concluir que a referida prepara??o lipossomal representa uma alternativa terap?utica capaz de reduzir a carga parasit?ria nos diferentes ?rg?os alvo, com potencial para a elimina??o do parasito na medula ?ssea. A coadministra??o do AA aos lipossomas de SbIII acarretou redu??o na toxicidade do SbIII. Em conjunto, nossos dados apontam os benef?cios do encapsulamento do SbIII em lipossomas unilamelares demonstrados pela redu??o das altera??es histopatol?gicas para f?gado e rins, particularmente em combina??o com AA. Acreditamos que nosso trabalho ser? ?til para a proposi??o de novos regimes terap?uticos para o tratamento da leishmaniose visceral, garantindo a ades?o do paciente ? terapia devido ? redu??o dos efeitos colaterais.The present study evaluated the utilization of conventional liposomes as carriers of trivalent antimony (SbIII), and its association with ascorbic acid, for the treatment of murine visceral leishmaniasis. Liposomes composed of diestearoylphosphatidylcholine and cholesterol (5:4) were obtained according to the freeze / thaw technique and hydrated in a solution of tartar emetic. Empty liposomes, hydrated in phosphate buffer, served as control vehicles. Characterization of the liposomal formulation revealed size, polydispersity index and encapsulation efficiency of 222.5 nm, 0.214 and 15%, respectively, for a formulation containing 4 mg SbIII/mL. It also displayed biphasic release kinetics in vitro. BALB/c mice, aged 6 to 8 weeks, were intravenously inoculated with 1 x 107 Leishmania (Leishmania) infantum promastigotes (C43 strain). Six weeks post infection, the animals were divided into seven groups (n=8) and treated intraperitoneally, as a single dose, with either (1) phosphate buffer, (2) SbIII at 9 mg Sb/kg, (3) empty liposomes, (4) SbIII-entrapped liposomes at 9 mg Sb/kg, (5) ascorbic acid (AA) at 300 mg/kg, (6) association of AA (300 mg/kg) with SbIII at 9 mg Sb/kg or (7) association of AA (300 mg/kg) with SbIII-entrapped liposomes at 9 mg Sb/kg. Ten days after treatment, animals were euthanized and the parasite load associated to the liver, spleen and bone marrow evaluated through the limiting dilution technique. Immunophenotyping of spleen cells was performed using flow cytometry. Enzyme activities in the serum were used to monitor hepatic, kidney and cardiac functions. Histopathological examinations of the liver, kidney and heart were also conducted. After treatment with SbIII-entrapped liposomes it was observed 47%, 33% and 47%, reductions in parasite load in the liver, spleen and bone marrow, respectively, indicating the ability of such delivery system to target the active principle to these compartments. Parallel administration of ascorbic acid with either free SbIII or SbIII-entrapped liposomes did not interfere with SbIII?s leishmanicide activity. No reduction in parasite load was observed after treatment with free SbIII for any of the investigated organs. Our results did not reveal any significant alteration in the profile of spleen cells. In addition, no impairment in the hepatic, renal and cardiac functions were observed. Histopathological analyses demonstrated that the co-administration of ascorbic acid with SbIII-entrapped liposomes was able to preserve the integrity of the hepatic and kidney tissues, in comparison to those from other experimental groups. The obtained results allowed us to conclude that our proposed SbIII liposomal preparation represents a therapeutic alternative able to reduce parasite burden with the potential to eliminate parasitism at the bone marrow. Co-administration of AA and SbIII-entrapped liposomes promoted reduced toxicity of SbIII. Collectively, our data extended the proposed benefits of unilamellar liposomes carrying SbIII by demonstrating their reduced toxicity, particularly in combination with ascorbic acid to the liver and kidney tissues. We believe our investigation can be useful for the proposal of novel therapeutic regimens to successfully treat visceral leishmaniasis aiming to guarantee patient compliance whilst minimizing potential side effects