Susana Torres

Activity of a hydroxybibenzyl bryophyte constituent against Leishmania spp. and Trypanosoma cruzi: in silico, in vitro and in vivo activity studies.

The synthesis and potent antiprotozoal activity of 14-hydroxylunularin, a natural hydroxybibenzyl bryophyte constituent is reported. 14-hydroxylunularin was highly active in vitro assays against culture and intracellular forms of Leishmania spp. and Trypanosoma. cruzi, in absence of cytotoxicity against mammalian cells. Preliminary structure-activity relationship studies showed that the reported bioactivity depends on hybridization at the carbon-carbon bridge, position and number of free hydroxy group on the aromatic rings. The reported results were also in agreement with the in silico prediction using Non-Stochastic Quadratic Fingerprints-based algorithms. The same compound also showed antiprotozoal activity in Leishmania spp. infected mice by oral and subcutaneous administration routes, with an optimal treatment of a daily subcutaneous administration of 10 mg/kg of body weight for 15 days. This study suggested that 14-hydroxylunularin may be chosen as a new candidate in the development of leishmanicidal therapy.

Zanthoxylum chiloperone leaves extract: First sustainable Chagas disease treatment

ETHNOPHARMACOLOGICAL RELEVANCE Zanthoxylum chiloperone var. angustifolium Engl. (Rutaceae) stem bark is used traditionally in Paraguay for its antiparasitic properties. Canthin-6-one is main compound isolated from Zanthoxylum chiloperone var angustifolium with broad spectrum antifungal, leishmanicidal and trypanocidal activities. AIM OF THE STUDY The qualitative and quantitative characterization and the isolation of main alkaloidal components of different organs of Zanthoxylum chiloperone are investigated by HPLC-UV-MS. The in vitro biological activity of each extract against trypomastigote and amastigote forms of Trypanosoma cruzi parasites were evaluated, then comparison the in vivo efficacy of the ethanolic leaves extract of Zanthoxylum chiloperone with reference drug, benznidazole, in acute Trypanosoma cruzi infected mice when administered by oral route. We have also evaluated the mutagenic and cytotoxic activity of the main component of Zanthoxylum chiloperone, i.e. canthin-6-one, by mouse bone marrow micronucleus test. MATERIALS AND METHODS The compositions of the ethanol extracts obtained after the maceration process were studied by HPLC-UV-MS methods. The quantitation analysis was performed by external standard method, using a calibration curve constructed utilizing solutions containing different concentrations of the reference samples. The anti-trypomastigote activity was evaluated by the lysis effect on mouse blood trypomastigotes (Y strain Trypanosoma cruzi). The anti-amastigote Trypanosoma cruzi activity was evaluated by a modified colorimetric method with chlorophenol red-β-d-galactopyranoside (CPRG). The cytotoxicity of extracts and compounds was performed on NCTC 929 cells. The in vivo efficacy of the ethanolic leaves extract of Zanthoxylum chiloperone and benznidazole, in acute Trypanosoma cruzi (two different strains) was evaluated in Trypanosoma cruzi infected mice; the drugs were administered by oral route. The mortality rates were recorded and parasitaemias in control and treated mice were determined once weekly for 70 days. The mutagenic and cytotoxic activity of the main component of Zanthoxylum chiloperone, canthin-6-one, by mouse bone marrow micronucleus test. RESULTS Canthin-6-one was the main compound of stem and root bark and 5-methoxy-canthin-6-one in leaves and fruits. The ethanolic leaves extract, canthin-6-one and benznidazole presented, approximately, the same level of in vitro activity against trypomastigote and amastigote forms of Trypanosoma cruzi. We have also evaluated the mutagenic and cytotoxic effects of canthin-6-one by micronucleus test in mice. This test showed any mutagenic and cytotoxic damages. The effects of oral or subcutaneous treatments at 10 mg/kg daily for 2 weeks with the ethanolic extract of leaves of Zanthoxylum chiloperone were examined in Balb/c mice infected acutely with Trypanosoma cruzi (CL or Y strain) and compared with benznidazole at 50 mg/kg for 2 weeks. In these experiments, 70 days after infection, parasitaemia and serological response were significantly reduced with the oral ethanolic extract treatment compared with reference drug. CONCLUSIONS This study have shown the efficacy of the leaves extract of Zanthoxylum chiloperone in reducing Trypanosoma cruzi parasitaemia in vivo assays and could be welcomed by scientific and rural communities of Paraguay because it could help them towards the use of local resources to treat an endemic infection, Chagas disease, affecting 20% of the population of this country.

Leishmanicidal and trypanocidal activity of extracts and secondary metabolites from basidiomycetes

Seventeen extracts and seven secondary metabolites isolated from basidiomycetes were tested in medium culture against promastigote forms of Leishmania spp. and bloodstream forms of Trypanosoma cruzi. Extracts from the culture filtrate or mycelium were generally inactive against the parasites except the Zucoagaricus genus mycelium extract which reduced by 47% the number of bloodstream forms. Striatin A, striatin B and podoscyphic acid exhibited in vitro activity at 10, 5 and 100 μg/mL, respectively. One compound showed activity against bloodstream forms of T. cruzi, the sesquiterpenoid naematolin, lysing the parasites by 79%. BALB/c mice infected with L. amazonensis were treated 3 weeks post‐infection with striatin A and striatin B by subcutaneous route for 15 days at 10 mg/kg daily. The reference drug, N‐methylglucamine antimonate, administered by subcutaneous injections at 28 mg Sbv/kg/day for 15 days reduced the parasite burden by 71.2% (p <0.05). Subcutaneous administration of straitin A at 10 mg/kg produced a weak decrease of the parasite burdens in the footpad by 17.6%. The treatment with striatin B had no effect and showed higher toxicity than striatin A.

Optimization of antitrypanosomatid agents: identification of nonmutagenic drug candidates with in vivo activity.

Chagas disease, caused by Trypanosoma cruzi parasite, was described thousands of years ago. Currently, it affects millions of people, mostly in Latin America, and there are not suitable drugs for treating it. As an attempt to find appropriate drugs to deal with this problem, we report here on the design, synthesis, and characterization of 82 new compounds. Trypanosomicidal behavior in vitro showed more than 20 outstanding derivatives with anti-Trypanosoma cruzi activity. Furthermore, we studied the nonspecific toxicity against mammalian cells determining their selectivity and also performed mutagenicity studies. Proof of concept, in vivo studies, was conducted with two of the most promising derivatives (77 and 80). They were identified as candidates because they have (i) very simple and cost-effective syntheses; (ii) activity against different stages and strains of the parasite showing excellent in vivo behavior during the acute phase of Chagas disease; and (iii) neither nonspecific toxicity nor mutagenic activity.

Studies on quinones. Part 34: The reaction of styrene with activated 1,4-benzoquinones: access to potential antiprotozoal pyranobenzoquinones ☆

Abstract Styrene reacts with highly electrophilic monosubstituted 1,4-benzoquinones to provide pyranobenzoquinone and phenanthrene derivatives, depending on the nature of the quinone substituent. The participation of transient Diels–Alder adduct intermediates in these reactions and their thermal-induced rearrangements are discussed. The in vitro antiparasital effects of the pyranobenzoquinones against Trypanosoma cruzi and Leishmania spp. are reported.

In Vivo Anti-Trypanosoma cruzi Activity of Hydro-Ethanolic Extract and Isolated Active Principles from Aristeguietia glutinosa and Mechanism of Action Studies

The currently available treatments for Chagas disease show limited therapeutic potential and are associated with serious side effects. Attempting to find alternative drugs isolated from Nature as agents against Trypanosoma cruzi has been our goal. Recently, we have demonstrated the in vitro anti-T. cruzi activities of two secondary metabolites isolated from the hydro-ethanolic extract of the aerial parts of Aristeguietia glutinosa (Lam.), (family Asteraceae). These active principles displayed poor hemolytic activity, low toxicity against murine macrophages, and absence of mutagenicity. Herein, proof of concept in vivo studies of the whole hydro-ethanolic extract of the aerial parts of Aristeguietia glutinosa and of the most active component isolated from the hydro-ethanolic extract, i.e., (+)-15-hydroxy-7-labden-17-al, was done in a murine acute model of Chagas disease. Both treatments caused a decrease in the animals’ parasitemia. Metabolomic mechanism of action studies were done by 1H-NMR, both on the extract and on the active compounds, examining the effects of the metabolites both on membrane sterol biosynthesis and mitochondrial dehydrogenases, whereby we found that one of the metabolites inhibited the activity of the parasite mitochondrial dehydrogenases and the other inhibited the biosynthesis of parasite membrane sterols. The results are interesting in the context of popular use of plants for the treatment of Chagas disease.

The effect of bisbenzylisoquinoline alkaloids on Trypanosoma cruzi infections in mice

Five bisbenzylisoquinoline (BBI) alkaloids, curine, cycleanine, isotet:andrine, limacine and pheanthine were tested for trypanocidal activity in C 3H He mice infected with Y or CL strain of Trypanosoma cruzi. The activity was compared with the baseline drug, benznidazole. Oral treatment was more effective with curine at 10 mg/kg or with cycleanine at 2 mg/kg daily for 10 days in mice infected with Y or CL strain. In these groups, the parasitemias were negative after 5-7 weeks after inoculation and mortality time 50 (MT(50)) was significantly higher than untreated mice. Benznidazole was effective in mice infected with CL strain but not in mice infected with Y strain. The other BBI showed a relative efficacy against both strains. The effect of BBI alkaloids could be due to a blocking of the Ca2+ channel for the regulation of T. cruzi infectivity to invade host cells or their selective immunosuppressive properties.

Potent and Selective Inhibitors of Trypanosoma cruzi Triosephosphate Isomerase with Concomitant Inhibition of Cruzipain: Inhibition of Parasite Growth through Multitarget Activity

Triosephosphate isomerase (TIM) is an essential Trypanosoma cruzi enzyme and one of the few validated drug targets for Chagas disease. The known inhibitors of this enzyme behave poorly or have low activity in the parasite. In this work, we used symmetrical diarylideneketones derived from structures with trypanosomicidal activity. We obtained an enzymatic inhibitor with an IC50 value of 86 nm without inhibition effects on the mammalian enzyme. These molecules also affected cruzipain, another essential proteolytic enzyme of the parasite. This dual activity is important to avoid resistance problems. The compounds were studied in vitro against the epimastigote form of the parasite, and nonspecific toxicity to mammalian cells was also evaluated. As a proof of concept, three of the best derivatives were also assayed in vivo. Some of these derivatives showed higher in vitro trypanosomicidal activity than the reference drugs and were effective in protecting infected mice. In addition, these molecules could be obtained by a simple and economic green synthetic route, which is an important feature in the research and development of future drugs for neglected diseases.

Finding of leishmanicidal activity of 14-hydroxylunularin in mice experimentally infected with Leishmania infantum

In this study, we report the in vivo efficacy of 14-hydroxylunularin evaluated in BALB/c mice experimentally infected with promastigotes of Leishmania infantum (syn L. chagasi), the major causative agent of visceral leishmaniasis in Latin America. Seven days post-infection, treatment with 14-hydroxylunularin started and it was administered by oral and subcutaneous routes in doses of 10 and 25 mg/kg of weight for ten days using Glucantime® as reference drug. In the liver, the evaluated compound showed parasite reduction above 90% by both administration routes being the oral route the most effective at both doses. Significant decreased numbers of parasites were also observed when the treated group was compared with the control group (p≤0.05). The subcutaneous route presented a remarkable difference with at least 80% parasite suppression in liver and spleen at 10 mg/kg dose and 90% in liver at 25 mg/kg. The leishmanicidal activity of 14-hydroxylunularin against L. infantum revealed by this study is another evidence in favor of this compound as a potential candidate for the development of a new oral treatment for leishmaniasis.

SYNTHESIS OF 4-ALKYLAMINO-6-CHLOROQUINOLINES AS POTENTIAL TRYPANOCIDAL AGENTS

A simple synthesis of 4-alkylamino-6-chloroquinolines 5-9 from 5,8-dimethoxy-6-chloro-4(lH)quinolone J, is described. Thermolysis of arylaminomethylene Meldrums acid derivative 2 is the key step on the preparation of compound 3. These compounds were tested in vitro against trypomastigote forms of Trypanosoma cruzi. Some derivatives were found to have a significant activity.