Efeito de derivados de triazóis e seus sais em espécies de Leishmania associadas a manifestação visceral e estudos de modo de ação sobre o parasito

Abstract

Visceral leishmaniasis is a progressive disease and potentially fatal if untreated. However, current treatment has serious adverse effects, long-term treatment, and variability in effectiveness. The objective of this work was to analyze the activity of 34 1,2,3-triazole derivatives and their salts against Leishmania species associated with visceral manifestation of the disease, as well as toxicity in mammalian cells, mode of action studies and effect on murine model. The screening of the compounds was performed against the species of L. (L.) amazonensis, classically associated with cutaneous leishmaniasis, but also reported in cases of visceral leishmaniasis. Posteriorly, the most promising compound was evaluated in different strains of L. (L.) infantum, classically associated with visceral manifestation. The antipromastigote activity of Leishmania spp. and macrophages was evaluated by the MTT technique and the antimastigote activity by fluorimetry in the RFP transfected strain or parasite count after Giemsa staining. For the mode of action studies, specific markers were used and analyzed by fluorometry, flow cytometry, fluorescence microscopy or transmission electron microscopy (TEM). The triazolium salts showed significant activity in the promastigote and amastigote forms of L. (L.) amazonensis, and the most active compounds had 10 carbon atoms in the triazole side chain. These also had the smallest toxic effects to macrophages. Among the evaluated compounds, the triazole salt, 25c, was chosen to continue the tests. This compound showed significant activity in human and dog L. (L.) infantum strains, with IC50 values below 10 µM and selectivity index between 6-9 for the parasite. In addition, it did not exhibit human red blood cell toxicity up to the maximum concentration tested (18.75- 300 µM). Mode of action studies have indicated mitochondria as the main target of compound 25c at 4.55 µM in promastigote forms of L. (L.) infantum, associated with increased production of mitochondrial and total ROS, mitochondrial hyperpolarization, and lipid bodies accumulation. Phosphatidylserine exposure and DNA fragmentation in promastigotes treated with compound 25c did not occur, excluding the occurrence of apoptosis cell death. In the same way, occurrence of necrosis was rule out, since both the permeability and integrity of the plasma membrane were not affected by compound 25c. Studies of autophagy were performed and autophagic vacuoles were detected after treatment with compound 25c.The compound 25c at 7.92 µM decreased of levels ROS and did not interfere in the NO production and induced decrease of IL-10 interleukin levels in infected macrophages. In silico studies have demonstrated pharmacokinetic parameters and good oral bioavailability of compound 25c, stimulating the in vivo test by this route of administration. The compound at 20 mg/Kg decreased parasitic load in the spleen and liver of L. (L.) infantum infection in BALB/c mice. Toxicity studies in mice showed that compound 25c at 50 mg/Kg did not induce hepatic, renal toxicity, on the contrary, it reestablished the physiological conditions of infected mice. These results demonstrate the in vitro and in vivo potential of the triazole salt and encourage further studies on its action in vivo and in different animal models.