Experimental chemotherapy for Chagas disease: 15 years of research contributions from in vivo and in vitro studies.

Chagas disease, which is caused by the intracellular parasite Trypanosoma cruzi, is a neglected illness with 12-14 million reported cases in endemic geographic regions of Latin America. While the disease still represents an important public health problem in these affected areas, the available therapy, which was introduced more than four decades ago, is far from ideal due to its substantial toxicity, its limited effects on different parasite stocks, and its poor activity during the chronic phase of the disease. For the past 15 years, our group, in collaboration with research groups focused on medicinal chemistry, has been working on experimental chemotherapies for Chagas disease, investigating the biological activity, toxicity, selectivity and cellular targets of different classes of compounds on T. cruzi. In this report, we present an overview of these in vitro and in vivo studies, focusing on the most promising classes of compounds with the aim of contributing to the current knowledge of the treatment of Chagas disease and aiding in the development of a new arsenal of candidates with anti-T. cruzi efficacy.

Experimental chemotherapy for Chagas disease: 15 years of research contributions from in vivo and in vitro studies

Chagas disease, which is caused by the intracellular parasite Trypanosoma cruzi, is a neglected illness with 12-14 million reported cases in endemic geographic regions of Latin America. While the disease still represents an important public health problem in these affected areas, the available therapy, which was introduced more than four decades ago, is far from ideal due to its substantial toxicity, its limited effects on different parasite stocks, and its poor activity during the chronic phase of the disease. For the past 15 years, our group, in collaboration with research groups focused on medicinal chemistry, has been working on experimental chemotherapies for Chagas disease, investigating the biological activity, toxicity, selectivity and cellular targets of different classes of compounds on T. cruzi. In this report, we present an overview of these in vitro and in vivo studies, focusing on the most promising classes of compounds with the aim of contributing to the current knowledge of the treatment of Chagas disease and aiding in the development of a new arsenal of candidates with anti-T. cruzi efficacy.

Different cell death pathways induced by drugs in Trypanosoma cruzi: an ultrastructural study.

Electron microscopy has proven to be a reliable and essential tool to determine morphological alterations and target organelles in the investigation of new drugs for Chagas disease. In this review, we focused on evaluating different agents that induce death of Trypanosoma cruzi, i.e. lysophospholipids analogues, naphthoquinones and derivatives, cytoskeletal inhibitors and natural products. Apoptosis-like presents as morphological characteristics DNA fragmentation, membrane blebbing and apoptotic body formation. Autophagy involves autophagosome formation, with the appearance of membranes surrounding organelles and cytosolic structures. Necrosis causes the loss of osmotic balance, an increase of cytoplasmic vacuolization and plasma membrane disruption. Mitochondrion appears as a central checkpoint in both apoptosis and necrosis. Our evidences of ultrastructural changes to T. cruzi treated with the different classes of compounds point to dramatic mitochondrial alterations and similar autophagic phenotypes. Lysophospholipid analogues interfere in the lipid biosynthesis in epimastigotes, altering the amount of both phospholipids and sterols, and consequently the physical properties of the membrane. Naphthoquinone derivatives led to a strong DNA fragmentation in trypomastigotes and to the release of cysteine proteases from reservosomes to cytosol in epimastigotes, starting a proteolytic process which results in parasite death. The susceptibility of reservosomes was also observed in parasites treated with propolis, suggesting impairment of lipid metabolism, compromising membrane fluidity and leading to lysis. The cytoskeletal agents blocked mitosis of epimastigotes, arresting cell cycle and impairing the parasite proliferation. The variety of drug stimuli converge to the same pathway of death suggests an intense cross-talking between the three types of PCD in the protozoa.

Anti-proliferative synergy of lysophospholipid analogues and ketoconazole against Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae): cellular and ultrastructural analysis.

OBJECTIVES: Investigation of the antiproliferative synergy of the lysophospholipid analogues (LPAs) edelfosine, ilmofosine and miltefosine with the ergosterol biosynthesis inhibitor ketoconazole against Trypanosoma cruzi. METHODS: The effect of LPAs, ketoconazole and their combination was evaluated against epimastigotes and intracellular amastigotes by the parameter IC50 leading to construction of isobolograms, for determination of a synergic effect. For epimastigotes, ultrastructural damage induced by these treatments was evaluated by transmission and scanning electron microscopy. RESULTS: Synergy was confirmed against both epimastigotes and amastigotes of the parasite. Edelfosine or ketoconazole alone induced morphological alterations in the plasma membrane and reservosomes of the parasites, while in combination, they also led to severe mitochondrial damage, formation of autophagic structures and multinucleation. Scanning electron microscopy confirmed the effect at the plasma membrane and also revealed alterations in the shape of the parasites. CONCLUSIONS: Our results describe the synergic anti-proliferative effect of LPAs and ketoconazole against epimastigotes and intracellular amastigotes and suggest that in epimastigotes, plasma membrane, reservosomes and mitochondria are targets of these drugs, possibly by interference with lipid metabolism.

Synthesis and antitrypanosomal profile of new functionalized 1,3,4-thiadiazole-2-arylhydrazone derivatives, designed as non-mutagenic megazol analogues.

In this work we reported the synthesis and the trypanocidal profile of new 1,3,4-thiadiazole-2-arylhydrazone derivatives of nitroimidazole series (4) or phenyl series (5), designed by exploring the molecular hybridization approach between megazol (2) and guanyl hydrazone derivative (3). The evaluation of the activity against bloodstream trypomastigote forms of Trypanosoma cruzi forms lead us to identify a new potent trypamomicide prototype, that is, brazilizone A (4k), which present an IC50/24 h=5.3 microM.

Effect of the lysophospholipid analogues edelfosine, ilmofosine and miltefosine against Leishmania amazonensis.

OBJECTIVES: Analysis of the effect of edelfosine, ilmofosine and miltefosine on Leishmania amazonensis and of potential targets of these lysophospholipid analogues. METHODS: Quantification and ultrastructural analysis of the effect of lysophospholipid analogues on promastigote forms and on infected peritoneal macrophages, and flow cytometry analysis of treated promastigotes labelled with propidium iodide and rhodamine 123 (Rh123). RESULTS: The lysophospholipid analogues presented potent antiproliferative activity with IC50/3 days of 1.9-3.4 microM for promastigotes and 4.2-9.0 microM for intracellular amastigotes. Treatment with these analogues in Schneider medium for 1 day led to a dose-dependent decrease in Rh123 fluorescence, an effect more accentuated in edelfosine-treated parasites, suggesting interference with the potential of the mitochondrial membrane. In both forms of L. amazonensis, edelfosine induced extensive mitochondrial damage, multinucleation and, in promastigotes, also led to plasma membrane alterations, formation of autophagic structures and membranous arrangements inside the flagellar pocket. CONCLUSIONS: The alkylglycerophosphocholines edelfosine and ilmofosine were more active than the alkylphosphocholine miltefosine against promastigotes and intracellular amastigotes of L. amazonensis, and ultrastructural and flow cytometry data indicate the mitochondrion as a target of edelfosine.

3-[4'-bromo-(1,1'-biphenyl)-4-yl]-N, N-dimethyl-3-(2-thienyl)-2-propen-1-amine: synthesis, cytotoxicity, and leishmanicidal, trypanocidal and antimycobacterial activities.

Current therapies for Chagas' disease, leishmaniasis and tuberculosis are unsatisfactory because of the failure rates, significant toxicity and/or drug resistance. In this study, the compound 3-[4'-bromo-(1,1'-biphenyl)-4-yl]-N,N-dimethyl-3-(2-thienyl)-2-propen-1-amine (IV) was synthesized and its trypanocidal, leishmanicidal and antimycobacterial activities were investigated. The cytotoxicity was determined on V79 cells with three endpoints: nucleic acid content, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide reduction and Neutral Red uptake. This compound was active against different species of mycobacteria and different life cycle stages of Trypanosoma cruzi. In experiments with trypomastigotes performed at 4 degrees C in the presence of blood, the activity was 8.8-fold more active than the standard drug, Crystal Violet. Higher activity was achieved against Leishmania amazonensis, with an ED(50)/24 h of 3.0 +/- 0.3 micro mol/L. The effect against trypanosomatids, which suggests high activity of compound IV against promastigotes of L. amazonensis and amastigotes of T. cruzi, stimulated further studies in vitro with amastigotes interiorized in macrophages and with in vivo models. Our results indicate that mammalian V79 cells are less susceptible to the action of compound IV than promastigotes of L. amazonensis (8.0-13.3-fold) and axenic amastigotes of T. cruzi (3.5-5.9-fold).