Leishmaniasis, caused by protozoan parasites of the Leishmania genus, is one of the most prevalent neglected tropical diseases. It is endemic in 98 countries, causing considerable morbidity and mortality. Pentavalent antimonials are the first line of treatment for leishmaniasis except in India. In resistant cases, miltefosine, amphotericin B and pentamidine are used. These treatments are unsatisfactory due to toxicity, limited efficacy, high cost and difficult administration. Thus, there is an urgent need to develop drugs that are efficacious, safe, and more accessible to patients. Trypanosomatids, including Leishmania spp. and Trypanosoma cruzi, have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. Inhibition of ergosterol biosynthesis is increasingly recognized as a promising target for the development of new chemotherapeutic agents. The aim of this work was to investigate the antiproliferative, physiological and ultrastructural effects against Leishmania amazonensis of itraconazole (ITZ) and posaconazole (POSA), two azole antifungal agents that inhibit sterol C14α-demethylase (CYP51). Antiproliferative studies demonstrated potent activity of POSA and ITZ: for promastigotes, the IC50 values were 2.74 µM and 0.44 µM for POSA and ITZ, respectively, and for intracellular amastigotes, the corresponding values were 1.63 µM and 0.08 µM, for both stages after 72 h of treatment. Physiological studies revealed that both inhibitors induced a collapse of the mitochondrial membrane potential (ΔΨm), which was consistent with ultrastructural alterations in the mitochondrion. Intense mitochondrial swelling, disorganization and rupture of mitochondrial membranes were observed by transmission electron microscopy. In addition, accumulation of lipid bodies, appearance of autophagosome-like structures and alterations in the kinetoplast were also observed. In conclusion, our results indicate that ITZ and POSA are potent inhibitors of L. amazonensis and suggest that these drugs could represent novel therapies for the treatment of leishmaniasis, either alone or in combination with other agents.

Amiodarone (AMIO), the most frequently antiarrhythmic drug used for the symptomatic treatment of chronic Chagas' disease patients with cardiac compromise, has recently been shown to have also specific activity against fungi, Trypanosoma cruzi and Leishmania. In this work, we characterized the effects of AMIO on proliferation, mitochondrial physiology, and ultrastructure of Leishmania amazonensis promastigotes and intracellular amastigotes. The IC50 values were 4.21 and 0.46 μM against promastigotes and intracellular amastigotes, respectively, indicating high selectivity for the clinically relevant stage. We also found that treatment with AMIO leads to a collapse of the mitochondrial membrane potential (ΔΨm) and to an increase in the production of reactive oxygen species, in a dose-dependent manner. Fluorescence microscopy of cells labeled with JC-1, a marker for mitochondrial energization, and transmission electron microscopy confirmed severe alterations of the mitochondrion, including intense swelling and modification of its membranes. Other ultrastructural alterations included (1) presence of numerous lipid-storage bodies, (2) presence of large autophagosomes containing part of the cytoplasm and membrane profiles, sometimes in close association with the mitochondrion and endoplasmic reticulum, and (3) alterations in the chromatin condensation and plasma membrane integrity. Taken together, our results indicate that AMIO is a potent inhibitor of L. amazonensis growth, acting through irreversible alterations in the mitochondrial structure and function, which lead to cell death by necrosis, apoptosis and/or autophagy.