Juliana Q. Reimão

Antiparasitic, antineuroinflammatory, and cytotoxic polyketides from the marine sponge Plakortis angulospiculatus collected in Brazil.

Investigation of the bioactive crude extract from the sponge Plakortis angulospiculatus from Brazil led to the isolation of plakortenone ( 1) as a new polyketide, along with five known polyketides ( 2- 6) previously isolated from other Plakortis sponges. The known polyketides were tested in antileishmanial, antitrypanosomal, antineuroinflammatory, and cytotoxicity assays. The results show that plakortide P ( 3) is a potent antiparasitic compound, against both Leishmania chagasi and Trypanosona cruzi, and exhibited antineuroinflammatory activity. The known polyketides 2- 6 were tested for cytotoxicity against four human cancer cell lines, but displayed only moderate cytotoxic activity.

Isolation of an antileishmanial and antitrypanosomal flavanone from the leaves of Baccharis retusa DC. (Asteraceae).

In the course of selection of new bioactive compounds from Brazilian flora, the crude MeOH extract from the leaves of Baccharis retusa DC. (Asteraceae) showed potential against Leishmania sp. and Trypanosoma cruzi. Chromatographic fractionation of the dichloromethane phase from MeOH extract yielded great amounts of the bioactive derivative, which was characterized as 5,6,7-trihydroxy-4′-methoxyflavanone. The structure of this compound was established on the basis of spectroscopic data analysis, mainly nuclear magnetic resonance and mass spectrometry.

Effectiveness of liposomal buparvaquone in an experimental hamster model of Leishmania (L.) infantum chagasi

The objective of this study was to develop a novel liposomal formulation, containing phosphatidylserine (PS), of buparvaquone (BPQ) and to evaluate its in vivo effectiveness in Leishmania (L.) infantum chagasi-infected hamsters. The activity of BPQ was evaluated against both the promastigote forms of different Leishmania species and the intracellular amastigotes of L. (L.) infantum chagasi. Buparvaquone was entrapped in PS-liposomes (BPQ-PS-LP), and the drug was quantified by ultra-high-performance liquid chromatography. The treatment was quantified by detecting the RNA of the living amastigotes in the spleen and the liver by real-time PCR. In vitro assays with L. (L.) infantum chagasi intracellular amastigotes were performed in peritoneal macrophages for the evaluation of the 50% inhibitory concentration (IC(50)). BPQ-PS-LP at 0.33 mg/kg/day for eight consecutive days reduced the number of amastigotes by 89.4% (P<0.05) in the spleen and by 67.2% (P>0.05) in the liver, compared to 84.3% (P<0.05) and 99.7% (P<0.05), respectively, following Glucantime® treatment at 50 mg/kg/day. Free BPQ at 20 mg/kg/day failed to treat the hamsters when compared to the untreated group. BPQ was significantly (P<0.05) selective against L. (L.) infantum chagasi intracellular amastigotes, with an IC(50) value of 1.5 μM; no in vitro mammalian cytotoxicity could be detected. Other cutaneous species were also susceptible to BPQ, with IC(50) values in the range 1-4 μM. BPQ-PS-LP caused a significant reduction in the parasite burden at a 60-fold lower dose than did the free BPQ. These results show the potential of PS-liposome formulations for the successful targeted delivery of BPQ in visceral leishmaniasis.

Parasite burden in Leishmania (Leishmania) amazonensis-infected mice: validation of luciferase as a quantitative tool.

Given the lack of effective and safe alternatives to the drugs already in use, considerable efforts are being applied to the search of new therapeutic options to treat leishmaniasis. A necessary step in the discovery of antileishmanial drugs is the validation of drug candidates in mouse models. The standard methods to quantify the parasite burden in animal models, mainly culture-based, are time consuming and expensive. In recent years, in vivo imaging systems have been proposed as a tool to overcome these problems, allowing parasite detection in living organisms. Here we compared different treatment efficacy evaluation approaches. Recombinant Leishmania (L.) amazonensis lines expressing the luciferase gene (La-LUC) were obtained and characterized for biological properties as compared with the wild type (WT) parental line. Bioluminescence generated by La-LUC was shown to correlate with the number of promastigotes in vitro. La-LUC promastigotes and intracellular amastigotes were equally sensitive to amphotericin B (AmB) as the WT parasites. The clinical pattern of lesion development upon infection with the transgenic lines was similar to lesions observed after infection with the WT strain. The half maximal effective dose (ED50) of AmB was determined in La-LUC infected mice through quantification of bioluminescence in vivo and ex vivo, by limiting dilution and using clinical parameters. There was agreement in the ED50 determined by all methods. Quantification of bioluminescence in vivo and/or ex vivo was elected as the best tool for determining parasite burden to assess drug efficacy in infected mice. Furthermore, the detailed analysis of AmB effectiveness in this model generated useful data to be used in drug combination experiments.

In vitro and experimental therapeutic studies of the calcium channel blocker bepridil: detection of viable Leishmania (L.) chagasi by real-time PCR.

The need for novel and efficacious drugs against neglected parasitic diseases, such as Leishmaniasis and American Trypanosomiasis, is certainly apparent. In this work, we evaluated the in vitro potential of the calcium channel blocker bepridil against Leishmania spp. and Trypanosoma cruzi parasites and exploited an experimental assay using a hamster model with Leishmania (L.) chagasi, with a real-time PCR method for therapeutic evaluation. Bepridil was in vitro effective against promastigotes and intracellular amastigotes of L. (L.) chagasi, with 50% inhibitory concentration (IC(50)) values of 3.81 and 21.55 μM, respectively. Leishmania (L.) amazonensis, L. (L.) major and L. (V.) braziliensis promastigotes and T. cruzi trypomastigotes were also susceptible to bepridil, with in vitro selectivity toward parasites and IC(50) values in the range of 3 to 7 μM. The mammalian cytotoxicity using LLC-MK2 cells resulted in an IC(50) value of 62.67 μM. However, bepridil showed lack of activity at 12 mg/kg in the experimental hamster model infected with L. (L.) chagasi parasites. However, the real-time PCR was a promising tool for the accurate and fast quantification of RNA of living parasites in the liver and spleen of infected hamsters after treatment, eliminating time-consuming light microscopy evaluations.

Therapeutic evaluation of free and liposome-loaded furazolidone in experimental visceral leishmaniasis

Drug delivery systems are promising pharmaceutical formulations used to improve the therapeutic index of drugs. In this study, we developed a liposomal formulation of furazolidone that targets Leishmania (Leishmania) chagasi amastigotes in a hamster model. Using laser scanning confocal microscopy, it was demonstrated that the liposomal drug co-localised with L. (L.) chagasi amastigotes within macrophages. Liposomal furazolidone administered intraperitoneally at 0.5mg/kg for 12 consecutive days reduced spleen (74%) and liver (32%) parasite burden at a 100-fold lower dose than the free drug. Free furazolidone (50mg/kg) also effectively reduced spleen (82.5%) and liver (85%) parasites; its in vitro activity against promastigotes and intracellular amastigotes demonstrated a high degree of parasite selectivity. Thus, furazolidone, both in the free and liposome-loaded formulation, is an effective inhibitor of L. (L.) chagasi, representing a possible cost-effective drug candidate for the treatment of visceral leishmaniasis.

Antitrypanosomal activity of a diterpene and lignans isolated from Aristolochia cymbifera

Bioguided fractionation of extract from the leaves of Aristolochia cymbifera led to the isolation of the furofuran lignans fargesin, epieudesmin, and sesamin; the dibenzylbutyrolactone lignans hinokinin and kusunokinin; and an ENT-labdane diterpene named copalic acid. Our data demonstrated that copalic acid and kusunokinin were the most active compounds against trypomastigotes of Trypanosoma cruzi. Additionally, copalic acid demonstrated the highest parasite selectivity as a result of low toxicity to mammalian cells, despite a considerable hemolytic activity at higher concentrations. Among the isolated compounds, kusunokinin could be considered the most promising candidate, as it displayed significant activity against intracellular amastigotes (IC(50) = 17 µM) and trypomastigotes (IC(50) = 51 µM) without hemolytic activity. Fargesin, hinokinin, epieudesmin, and sesamin were also effective against trypomastigotes, but these compounds were highly toxic to mammalian cells and no parasite selectivity could be identified. The need for novel drugs for American trypanosomiasis is evident, and these secondary metabolites from A. cymbifera represent a useful tool for drug design.

Chemical constituents of the volatile oil from leaves of Annona coriacea and in vitro antiprotozoal activity

The essential oil of the leaves from Annona coriacea Mart., Annonaceae, was extracted by hydrodistillation in a Clevenger apparatus and analyzed by GC/MS and GC/FID. The oil yield was 0.05% m/m. Sixty compounds were identified, in a complex mixture of sesquiterpenes (76.7%), monoterpenes (20.0%) and other constituents (3.3%). Bicyclogermacrene was its major compound (39.8%) followed by other sesquiterpenes. Most of the monoterpenes were in low concentration (<1%). Only β-pinene and pseudolimonene presented the highest level of 1.6%. The volatile oil presented anti-leishmanial and trypanocidal activity against promastigotes of four species of Leishmania and trypomastigotes of Trypanosoma cruzi, showing to be more active against Leishmania (L.) chagasi (IC50 39.93 µ g/mL) (95% CI 28.00-56.95 µ g/mL).

Combination Therapy with Tamoxifen and Amphotericin B in Experimental Cutaneous Leishmaniasis

ABSTRACT Leishmaniasis chemotherapy remains very challenging. The high cost of active drugs, along with the severity of their side effects and the increasing failure rate of the current therapeutic schemes, calls for the discovery of new active drugs and schemes of treatment. The use of combination therapy has gained much attention in recent years as a possible strategy for overcoming the various shortcomings in the present arsenal. We recently described the effectiveness of tamoxifen in murine models of leishmaniasis, and here, we investigated the interactions between tamoxifen and amphotericin B, one of the most potent drugs used in leishmaniasis treatment. The in vitro interactions were indifferent for the association of tamoxifen and amphotericin B. The association was also assayed in vivo in Leishmania amazonensis-infected BALB/c mice and was found to yield at least additive effects at low doses of both drugs.

Generation of luciferase-expressing Leishmania infantum chagasi and assessment of miltefosine efficacy in infected hamsters through bioimaging.

Background The only oral drug available for the treatment of leishmaniasis is miltefosine, described and approved for visceral leishmaniasis in India. Miltefosine is under evaluation for the treatment of cutaneous leishmaniasis in the Americas although its efficacy for the treatment of human visceral leishmaniasis caused by Leishmania infantum chagasi has not been described. Drug efficacy for visceral leishmaniasis is ideally tested in hamsters, an experimental model that mimics human disease. Luciferase has been validated as a quantitative tool for the determination of parasite burden in experimental leishmaniasis. However, there are no reports of luciferase detection in the model of progressive visceral leishmaniasis in hamsters. Therefore, the aims of this study were to generate recombinant Leishmania infantum chagasi expressing the luciferase gene (Lc-LUC), characterize the biological properties of this transgenic line as compared with the wild-type parasites and evaluate miltefosine effectiveness in Lc-LUC infected hamsters. Methodology/Principal Findings A transgenic line containing a luciferase encoding gene integrated into the ribosomal DNA locus was obtained and shown to produce bioluminescence which correlated with the number of parasites. Lc-LUC growth curves and susceptibility to pentavalent antimony and miltefosine in vitro were indistinguishable from the wild-type parasites. The effectiveness of pentavalent antimony was evaluated in Lc-LUC infected hamsters through bioimaging and determination of Leishman Donovan Units. Both methods showed concordant results. Miltefosine was effective in the treatment of Lc-LUC-infected hamsters, as demonstrated by the reduction in parasite burden in a dose-dependent manner and by prolongation of animal survival. Conclusions/Significance Luciferase expressing parasites are a reliable alternative for parasite burden quantification in hamsters with advantages such as the possibility of estimating parasite load before drug treatment and therefore allowing distribution of animals in groups with equivalent mean parasite burden. Miltefosine was effective in vivo in an L. infantum chagasi experimental model of infection.