Search

Aromatic diamidines (ADs) have been recognized as promising antiparasitic agents. Therefore, in the present work, the in vitro trypanocidal effect of 11 ADs upon the relevant clinical forms of Trypanosoma cruzi was evaluated, as well as determining their toxicity to mammalian cells and their subcellular localization.The trypanocidal effect upon trypomastigotes and amastigotes was evaluated by light microscopy through the determination of the IC(50) values. The cytotoxicity was determined by the MTT colorimetric assay against mouse cardiomyocytes. For the subcellular localization, transmission electron microscopy and fluorescence approaches were used. The fluorescence intensity within the kinetoplast DNA (kDNA) and nuclear DNA (nDNA) of treated parasites was determined using the Image J program.Compounds 2, 5 and 7 showed the lowest IC(50) values (micromolar range) against intracellular amastigotes and trypomastigotes. In the presence of blood, all the tested ADs exhibited a reduction of their activity. The compounds did not exhibit toxicity to cardiac cells and the highest selectivity index (SI) was achieved by compound 5 with an SI of137 for trypomastigotes and compound 7 with an SI of107 for intracellular parasites. The subcellular effects upon bloodstream forms treated with compounds 5 and 7 were mainly on kDNA, leading to its disorganization. The higher accumulation in the kDNA observed for all tested ADs was not directly related to their efficacy.Our results show the high activity of this new series of ADs against both trypomastigote and amastigote forms, with excellent SIs, especially compound 7, which merits further in vivo evaluation.

Received 14 January 2005; returned 15 May 2005; revised 24 May 2005; accepted 7 October 2005Objectives: Investigation of the mode of action of the naphthoimidazole N1, obtained from the reaction ofb-lapachonewithbenzaldehyde,whichamong45semi-syntheticderivativesofnaphthoquinonesisolatedfrom Tabebuia sp. was one of the most active compounds against Trypanosoma cruzi trypomastigotes.Methods: Quantification of the effect of N1 against the proliferative forms of T. cruzi, and investigation ofpotential targets in the parasite using electron microscopy and flow cytometry techniques.Results:N1presentedthefollowingorderofactivity:amastigotes>trypomastigotes>epimastigotes.Theeffectonintracellularformswas 25timeshigherthanonmacrophagesandheartmusclecells.N1-treatedparasites presented an abnormal chromatin condensation and mitochondrial damage. In epimastigotes,alterations of reservosomes were observed, and in trypomastigotes, a decrease in the electron density ofacidocalcisomeswasobserved.Inepimastigotes,thenaphthoimidazoleinhibitedtheactivityofsuccinatecytochrome c reductase. Labelling with rhodamine 123 or Acridine Orange was decreased in both formstreated with N1.Conclusions:Theresultssuggestthatepimastigotes,reservosomes,mitochondrion,andnucleuscontainN1 targets. In trypomastigotes, in which reservosomes are absent, the organelles affected by thecompound were also the mitochondrion and nucleus, as well as acidocalcisomes, in which the decreasein electron density could be due to the use of polyphosphate as an alternative energy supply.Keywords: T. cruzi, chemotherapy, naphthoquinones, parasites

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.

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 1.9‐3.4mM for promastigotes and 4.2‐9.0mM 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.