Estudo pré-clínico dos fármacos letrozol e pamidronato como alternativas terapêuticas para tratamento da leishmaniose visceral humana

Abstract

Visceral leishmaniasis (VL) is an infectious disease caused in Brazil by the species Leishmania (Leishmania) infantum and, currently, there are no vaccines and/or prophylactic therapies for VL in humans, and the pharmacological approaches currently used, originating from the process drug repositioning. In this context, the objective of the present study was to carry out a preclinical study of the anticancer drugs letrozole (LET) and pamidronate (PAM), aiming to identify new therapeutic options for the treatment of human VL. The antileishmania activity of the drugs in vitro was evaluated by means of the antipromastigote and antiamastigote activities of L. infantum, with evaluation of cell cytotoxicity and production of nitric oxide (NO), for 72 hours. In the ex vivo context, the antileishmania and immunomodulatory activities of the drugs were evaluated using leukocytes from human peripheral blood, which were incubated with L. infantum, and with LET and PAM, for 6 hours. Antileishmania activity was evaluated in classic and non-classic monocytes and activated and non-activated neutrophils. Also, phenotypic functional aspects were evaluated in monocyte and neutrophil subpopulations, as well as CD8- and CD8+ and B lymphocytes. The antileishmania potential of LET and PAM in vivo was evaluated by quantifying the parasitic load of the spleen and liver, in Balb/c mice infected with L. infantum, and immunomodulatory activity was assessed by quantifying serum cytokines. The analysis of the results in vitro showed that both drugs showed antipromastigote activity, observing the formation of vacuoles in the cytoplasm of the promastigotes, as well as changes in the morphology of the kinetoplast and mitochondria. It was observed that LET and PAM reduced both the number of infected macrophages and the intracellular amastigotes, in a concentration-dependent manner. NO production was not observed, and in the evaluated conditions, LET and PAM, presented cell viability above 70%. By the ex vivo model used, using human leukocytes, LET and PAM reduced the internalized forms of L. infantum in the subpopulation of classic monocytes. The phenotypic characterization of monocyte and neutrophil subpopulations showed that the treatment promoted an increase in the expression of HLA-DR in monocytes, and the expression of Toll-like receptors (TLR) 2 was high and that of TLR-4, in all treatments, in the subpopulation of classic monocytes. Nor was NO production observed by this methodology. LET and PAM promoted an increase in IL-12, and a reduction in IL-10 and TGF-β, by monocytes and neutrophils. An increase in IFN- by CD8- and CD8+ T lymphocytes, and TNF by B-lymphocytes was demonstrated, as well as a reduction in IL-10 by the evaluated lymphocyte subpopulations. And through transmission electron microscopy analyzes, it was possible to suggest that LET and PAM promote damage to intracellular amastigotes. The in vivo results showed that LET at 2.0 mg/kg and PAM at 10.0 mg/kg promoted a reduction in the parasitic burden of the liver. In view of the results obtained, we demonstrated that LET and PAM may be eligible for evaluation as potential therapeutic alternatives and/or as prototypes for the treatment of human visceral leishmaniasis.