Antileishmanial and antitrypanosomal activity of bufadienolides isolated from the toad Rhinella jimi parotoid macrogland secretion

Amphibian skin secretions are considered a rich source of biologically active compounds and are known to be rich in peptides, bufadienolides and alkaloids. Bufadienolides are cardioactive steroids from animals and plants that have also been reported to possess antimicrobial activities. Leishmaniasis and American Trypanosomiasis are parasitic diseases found in tropical and subtropical regions. The efforts toward the discovery of new treatments for these diseases have been largely neglected, despite the fact that the only available treatments are highly toxic drugs. In this work, we have isolated, through bioguided assays, the major antileishmanial compounds of the toad Rhinella jimi parotoid macrogland secretion. Mass spectrometry and (1)H and (13)C NMR spectroscopic analyses were able to demonstrate that the active molecules are telocinobufagin and hellebrigenin. Both steroids demonstrated activity against Leishmania (L.) chagasi promastigotes, but only hellebrigenin was active against Trypanosoma cruzi trypomastigotes. These steroids were active against the intracellular amastigotes of Leishmania, with no activation of nitric oxide production by macrophages. Neither cytotoxicity against mouse macrophages nor hemolytic activities were observed. The ultrastructural studies with promastigotes revealed the induction of mitochondrial damage and plasma membrane disturbances by telocinobufagin, resulting in cellular death. This novel biological effect of R. jimi steroids could be used as a template for the design of new therapeutics against Leishmaniasis and American Trypanosomiasis. (C) 2008 Elsevier Ltd. All rights reserved.FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao Paulo[05/00974-9]CNPq[303516/2005-4]CNPq[473614/20065

Mammalian cell invasion and intracellular trafficking by Trypanosoma cruzi infective forms

Trypanosoma cruzi, the etiological agent of Chagas’ disease, occurs as different strains or isolates that may be grouped in two major phylogenetic lineages: T. cruzi I, associated with the sylvatic cycle and T. cruzi II, linked to the human disease. In the mammalian host the parasite has to invade cells and many studies implicated the flagellated trypomastigotes in this process. Several parasite surface components and some of host cell receptors with which they interact have been identified. Our work focused on how amastigotes, usually found growing in the cytoplasm, can invade mammalian cells with infectivities comparable to that of trypomastigotes. We found differences in cellular responses induced by amastigotes and trypomastigotes regarding cytoskeletal components and actin-rich projections. Extracellularly generated amastigotes of T. cruzi I strains may display greater infectivity than metacyclic trypomastigotes towards cultured cell lines as well as target cells that have modified expression of different classes of cellular components. Cultured host cells harboring the bacterium Coxiella burnetii allowed us to gain new insights into the trafficking properties of the different infective forms of T. cruzi, disclosing unexpected requirements for the parasite to transit between the parasitophorous vacuole to its final destination in the host cell cytoplasm

Anti-leishmanial and anti-trypanosomal potential of polygodial isolated from stem barks of Drimys brasiliensis Miers (Winteraceae)

Parasitic protozoan diseases affect the poorest population in developing countries. Leishmaniasis and Chagas disease have been included among the most important threats for public health in Central and South American continent, with few therapeutic alternatives and highly toxic drugs. in the course of selection of novel drug candidates, we studied the anti-protozoal potential of Drimys brasiliensis. Thus, the crude hexane extract from stem bark as well as its main derivative, the sesquiterpene polygodial, were tested using in vitro assays. the crude hexane extract and polygodial showed activity against Leishmania spp. in the range between 22 and 62 mu g/mL, but polygodial demonstrated high parasite selectivity towards Trypanosoma cruzi trypomastigotes (2 mu g/mL), with a selectivity index of 19. Finally, polygodial showed a leishmanicidal effect, inducing intense ultrastructural damages in Leishmania in short-time incubation. the obtained results suggested that polygodial could be used as a tool for drug design studies against protozoan diseases and as a candidate molecule for further in vivo studies against T. cruzi.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Inst Ciencias Ambientais Quim & Farmaceut, BR-09972270 Diadema, SP, BrazilUniv Presbiteriana Mackenzie, Ctr Ciencias & Humanidades, BR-01302907 São Paulo, BrazilUniv Presbiteriana Mackenzie, Ctr Ciencias Biol & Saude, BR-01302907 São Paulo, BrazilInst Adolfo Lutz Registro, Dept Parasitol, Lab Toxinol Aplicada, BR-01246000 São Paulo, BrazilUniversidade Federal de São Paulo, Inst Ciencias Ambientais Quim & Farmaceut, BR-09972270 Diadema, SP, BrazilFAPESP: 06/57626-5CNPq: 473405/2008-3CNPq: 477422/2009-8Web of Scienc

Antileishmanial Activity of the Estrogen Receptor Modulator Raloxifene

<div><p>Background</p><p>The treatment of leishmaniasis relies mostly on parenteral drugs with potentially serious adverse effects. Additionally, parasite resistance in the treatment of leishmaniasis has been demonstrated for the majority of drugs available, making the search for more effective and less toxic drugs and treatment regimens a priority for the control of leishmaniasis. The aims of this study were to evaluate the antileishmanial activity of raloxifene <i>in vitro</i> and <i>in vivo</i> and to investigate its mechanism of action against <i>Leishmania amazonensis</i>.</p><p>Methodology/Principal Findings</p><p>Raloxifene was shown to possess antileishmanial activity <i>in vitro</i> against several species with EC₅₀ values ranging from 30.2 to 38.0 µM against promastigotes and from 8.8 to 16.2 µM against intracellular amastigotes. Raloxifene's mechanism of action was investigated through transmission electron microscopy and labeling with propidium iodide, DiSBAC₂(3), rhodamine 123 and monodansylcadaverine. Microscopic examinations showed that raloxifene treated parasites displayed autophagosomes and mitochondrial damage while the plasma membrane remained continuous. Nonetheless, plasma membrane potential was rapidly altered upon raloxifene treatment with initial hyperpolarization followed by depolarization. Loss of mitochondrial membrane potential was also verified. Treatment of <i>L. amazonensis</i> – infected BALB/c mice with raloxifene led to significant decrease in lesion size and parasite burden.</p><p>Conclusions/Significance</p><p>The results of this work extend the investigation of selective estrogen receptor modulators as potential candidates for leishmaniasis treatment. The antileishmanial activity of raloxifene was demonstrated <i>in vitro</i> and <i>in vivo</i>. Raloxifene produces functional disorder on the plasma membrane of <i>L. amazonensis</i> promastigotes and leads to functional and morphological disruption of mitochondria, which culminate in cell death.</p></div

Ultrastructural aspects in raloxifene treated <i>Leishmania</i> promastigotes and intracellular amastigotes.

<p>(A to F): Ultrathin sections of <i>L. amazonensis</i> promastigotes. Promastigotes untreated (A) or incubated with 60 µM raloxifene for 30 min (B and C), 2 h (D and E) or 14 h (F) in M199 observed under transmission electron microscopy. The structure indicated by the arrow in (B) is shown in higher magnification in the inset. (G to I): Ultrastructural morphology of intracellular amastigotes. BMDM were infected with <i>L. amazonensis</i> and cultured for 48 hours in the absence (G) or in the presence of 9 µM raloxifene (H and I). (I) shows the same field as in (H) under higher magnification. Raloxifene induced the formation of autophagosomes (arrows) and mitochondrial swelling (stars), with no disruption of the plasma membrane. Arrowheads indicate remnants of vacuolated cell bodies compatible with dead amastigotes. N: nucleus; M: mitochondrion.</p

Mitochondrial membrane potential in raloxifene treated parasites.

<p><i>L. amazonensis</i> promastigotes preincubated with raloxifene in Hank's balanced salt solution supplemented with glucose for 20 min were loaded with 0.3 µg/mL Rh123, and the fluorescence level was measured by flow cytometry. Parasites treated with 100 µM FCCP were used as a positive control. Untreated parasites (NT) and parasites incubated with the highest volume of drug diluent (DMSO 1.2%) were used as negative controls. (A) Representative fluorescence histograms with untreated parasites (gray), 11 µM raloxifene (blue), 120 µM raloxifene (red) or 100 µM FCCP (black). (B) Mean fluorescence intensity compared to the control in parasites treated with increasing concentrations of raloxifene. Bars represent the mean and standard deviation of triplicates in an experiment representative of three independent experiments.</p

Half maximal effective concentration (EC₅₀) of raloxifene against <i>Leishmania</i> spp.

a<p>Parasite form: P: promastigotes; LA: lesion derived amastigotes; IA: intracelullar amastigotes. Half maximal effective concentration against promastigotes and axenic lesion-derived amastigotes was determined through MTT or MTS as described <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002842#pntd.0002842-ZauliNascimento1" target="_blank">[20]</a>; activity against <i>L. amazonensis</i> and <i>L. infantum chagasi</i> intracellular amastigotes was determined through luciferase activity or microscopic counting, respectively, as described in Material and Methods.</p>b<p>The results are expressed as the mean and standard deviation (± SD) of three independent experiments, each one performed in triplicate.</p

Clinical follow up and determination of parasite burden in raloxifene-treated mice.

<p>BALB/c mice were infected with 1×10⁶ promastigotes at the proximal end of the tail. Treatment was initiated 3 weeks post-infection. (A) Mice infected with wild type <i>L. amazonensis</i> and treated with 40 mg/kg/day raloxifene for 10 doses in weekdays (n = 10/group). (B–E) Mice infected with LaLUC and treated with 100 mg/kg/day raloxifene in a total of 10 doses in alternate days (n = 5 per group). (A and B) Progression of lesion thickness (mean ± standard deviation) in untreated (circles) and raloxifene-treated mice (squares). The horizontal bar indicates the period of raloxifene administration. (C) Macroscopical aspect of the lesion in representative animals from the untreated and treated groups. (D) Lesion bioluminescence (mean ± standard deviation) recorded from untreated and raloxifene-treated mice 6 weeks post-infection. (E) Bioluminescence imaging from untreated and raloxifene-treated mice 6 weeks post-infection. Ph/sec/cm²/sr: photons per second per square centimeter per steradian. (*) <i>p</i><0.01 in comparison with untreated group. Results shown are from one experiment representative of two independent experiments.</p

Propidium iodide labeling after raloxifene treatment.

<p>Parasites were incubated at 25°C in M199 medium, left untreated or treated with 30 or 60 µM raloxifene and analysed upon addition of propidium iodide. Parasites treated with 25 µM digitonin were used as a positive control. Untreated parasites and parasites incubated with the highest volume of drug diluent (DMSO 0.6%) were used as negative controls. Fluorescence histograms are representative of three independent experiments with untreated parasites (gray), 30 µM raloxifene (blue), 60 µM raloxifene (red) or 25 µM digitonin (black) for 20 min (A) or 2 hours (B). (C) Values shown are the mean fluorescence intensity ± standard deviation of three independent experiments. (*) indicates significant difference relative to the untreated group (p<0.0001). NT: untreated; RAL: raloxifene.</p