Análise in vitro da ação combinada de compostos leishmanicidas

Leishmaniasis belong to the distinct group of neglected diseases, named so for their endemicity mostly in countries under development, therefore attracting low interest of pharma industries to research new medicines. The treatment of leishmaniasis uses pentavalent antimonials as the first choice: pentamidine (Pt), miltefosine (Mt), amphotericin B and its liposomal formulation. All of them cause adverse effects and resistant cases have been increasing. Thus, there is an urgent need to develop new alternative therapies that are safer, more effective, in short-term treatment, and accessible for all the patients. The present work, first, tested the phenotypic activity of 18 new compounds (ftalazinones and imidazoles inhibitors of phosphodiesterase (PDE), imidazoles inhibitors of ergosterol biosynthesis (EBI), and quinolines), comparing them with two reference medicines, Pt and Mt, in monotherapy activity assays with amastigotes obtained from footpad lesion of mice experimentally infected with Leishmania amazonensis, and also comparing with the in vitro activity on intracellular forms present in infected macrophages. Among the compounds, nine showed no activity, with EC50 > 50 μM. The most promising were the ftalazinones and imidazoles inhibitors of PDEs (NPD001, NPD008, NPD040, NPD029, NPD3115, NPD3124, NPD3125, NPD3126) and VNI (EBI), that presented moderate leishmanicidal activity (EC50 from 10.15 to 44 μM), being less effective than Mt and Pt, which EC50 values were 4 and 1.5 μM, respectively. The best compounds were also tested for toxicity alone and in combination on macrophage cultures. In the monotherapy toxicity assays, LC50 > pentamidine (LC50 = 15.2 μM), being, therefore, less toxic. However, when compared to Mt (LC50 = 157.6 μM), the ftalazinones and the imidazole NPD3126 were more cytotoxic. Selectivity index (SI) was also calculated, and all tested compounds had SI lower than the two medicines. The most potent compounds (NPD001, NPD008, NPD040, NPD3115 e NPD3126) were selected for combined therapy assays with Mt and Pt, and also with one another, being evaluated in ex vivo models. All combinations showed additivity or “no interaction”, according to the classification of Odds (2003). Regarding the toxicity assays of the tested combinations, there was loss of cell viability (> 56 %) only at the highest concentrations of the proportions between the compounds. Finally, to compare and validate the data obtained, in vitro assays were performed with intracellular amastigotes of L. amazonenses in macrophage culture. NPD008, NPD3126 and Pt exhibited higher leishmanicidal potency upon the intracellular parasites in comparison to the free amastigotes obtained from footpad lesion, with the following values of EC50: Pt: 0.35 μM and 1.5 μM; NPD008: 2.38 μM and 10.8 μM; and NPD3126: 5.0 μM and 10.15 μM, respectively. NPD008 presented lower EC50 (2.38 μM) than Mt (5.0 μM) and better SI (17) in comparison to the others tested compounds, therefore being suitable to follow to combination studies on intracellular amastigotes of L. amazonensis. Similarly, to the result upon free amastigotes, NPD008 + Pt resulted in additivity (xΣFICI = 1.39). However, in the non-toxic proportions of 80:20, 60:40 and 40:60 of NPD008 + Pt, the reduction of parasite load observed was higher than 85 %, reaching suppression indexes equal to 98 % or 100 % reduction of parasites inside the host cell in some proportions. The analysis with scanning electron microscopy revealed the incubation of the NPD008 + Pt with free amastigotes, there were alterations suggestive of inhibition of cell division processes, with a high number of duplicated non-dissociated amastigotes, resulting in extensive parasite agglomerates, as previously reported for Trypanosoma brucei incubated with PDE inhibitors. The present study shows the promising effect of PDE inhibitors on L. amazonensis amastigotes, particularly in combination schemes, that deserve to be further investigated in in vivo studies, aiming to the development of new drugs for therapy of leishmaniasis.