A Nature-Inspired Design Yields a New Class of Steroids Against Trypanosomatids.

Chagas disease and Leishmaniasis are neglected endemic protozoan diseases recognized as public health problems by the World Health Organization. These diseases affect millions of people around the world however, efficient and low-cost treatments are not available. Different steroid molecules with antimicrobial and antiparasitic activity were isolated from diverse organisms (ticks, plants, fungi). These molecules have complex structures that make de novo synthesis extremely difficult. In this work, we designed new and simpler compounds with antiparasitic potential inspired in natural steroids and synthesized a series of nineteen steroidal arylideneketones and thiazolidenehydrazines. We explored their biological activity against Leishmania infantum, Leishmania amazonensis, and Trypanosoma cruzi in vitro and in vivo. We also assayed their genotoxicity and acute toxicity in vitro and in mice. The best compound, a steroidal thiosemicarbazone compound 8 (ID_1260) was active in vitro (IC50 200 nM) and in vivo (60% infection reduction at 50 mg/kg) in Leishmania and T. cruzi. It also has low toxicity in vitro and in vivo (LD50 >2000 mg/kg) and no genotoxic effects, being a promising compound for anti-trypanosomatid drug development.

Looking for combination of benznidazole and Trypanosoma cruzi-triosephosphate isomerase inhibitors for Chagas disease treatment.

BACKGROUND: The current chemotherapy for Chagas disease is based on monopharmacology with low efficacy and drug tolerance. Polypharmacology is one of the strategies to overcome these limitations. OBJECTIVES: Study the anti-Trypanosoma cruzi activity of associations of benznidazole (Bnz) with three new synthetic T. cruzi-triosephosphate isomerase inhibitors, 2, 3, and 4, in order to potentiate their actions. METHODS: The in vitro effect of the drug combinations were determined constructing the corresponding isobolograms. In vivo activities were assessed using an acute murine model of Chagas disease evaluating parasitaemias, mortalities and IgG anti-T. cruzi antibodies. FINDINGS: The effect of Bnz combined with each of these compounds, on the growth of epimastigotes, indicated an additive action or a synergic action, when combining it with 2 or 3, respectively, and an antagonic action when combining it with 4. In vivo studies, for the two chosen combinations, 2 or 3 plus one fifth equivalent of Bnz, showed that Bnz can also potentiate the in vivo therapeutic effects. For both combinations a decrease in the number of trypomastigote and lower levels of anti-T. cruzi IgG-antibodies were detected, as well clear protection against death. MAIN CONCLUSIONS: These results suggest the studied combinations could be used in the treatment of Chagas disease.

Identification of a new amide-containing thiazole as a drug candidate for treatment of Chagas' disease.

Although the parasitic infection Chagas' disease was described over 100 years ago, even now there are not suitable drugs. The available drugs nifurtimox and benznidazole have limited efficacies and tolerances, with proven mutagenic effects. Attempting to find appropriate drugs to deal with this problem, here we report on the development and pharmacological characterization of new amide-containing thiazoles. In the present study, we evaluated the in vitro and in vivo effects of new candidates against Trypanosoma cruzi, the etiological agent of Chagas' disease. The lead amide-containing thiazole derivative had potent in vitro activity, an absence of both in vitro mutagenic and in vivo clastogenic effects, and excellent in vitro selectivity and in vivo tolerance. The compound suppressed parasitemia in mice, modifying the anti-T. cruzi antibodies like the reference drug, benznidazole, and displayed the lowest mortality among the tested drugs. The present evidence suggests that this compound is a promising anti-T. cruzi agent surpassing the lead optimization stage in drug development and leading to a candidate for preclinical study.

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.

Preclinical Studies and Drug Combination of Low-Cost Molecules for Chagas Disease.

Chagas disease is caused by the protozoan Trypanosoma cruzi (T. cruzi). It remains the major parasitic disease in Latin America and is spreading worldwide, affecting over 10 million people. Hundreds of new compounds with trypanosomicidal action have been identified from different sources such as synthetic or natural molecules, but they have been deficient in several stages of drug development (toxicology, scaling-up, and pharmacokinetics). Previously, we described a series of compounds with simple structures, low cost, and environmentally friendly production with potent trypanosomicidal activity in vitro and in vivo. These molecules are from three different families: thiazolidenehydrazines, diarylideneketones, and steroids. From this collection, we explored their capacity to inhibit the triosephosphate isomerase and cruzipain of T. cruzi. Then, the mechanism of action was explored using NMR metabolomics and computational molecular dynamics. Moreover, the mechanism of death was studied by flow cytometry. Consequently, five compounds, 314, 793, 1018, 1019, and 1260, were pre-clinically studied and their pharmacologic profiles indicated low unspecific toxicity. Interestingly, synergetic effects of diarylideneketones 793 plus 1018 and 793 plus 1019 were evidenced in vitro and in vivo. In vivo, the combination of compounds 793 plus 1018 induced a reduction of more than 90% of the peak of parasitemia in the acute murine model of Chagas disease.

Preclinical Studies in Anti-Trypanosomatidae Drug Development.

The trypanosomatid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania are the causative agents of human African trypanosomiasis, Chagas Disease and Leishmaniasis, respectively. These infections primarily affect poor, rural communities in the developing world, and are responsible for trapping sufferers and their families in a disease/poverty cycle. The development of new chemotherapies is a priority given that existing drug treatments are problematic. In our search for novel anti-trypanosomatid agents, we assess the growth-inhibitory properties of >450 compounds from in-house and/or "Pathogen Box" (PBox) libraries against L. infantum, L. amazonensis, L.braziliensis, T. cruzi and T. brucei and evaluate the toxicities of the most promising agents towards murine macrophages. Screens using the in-house series identified 17 structures with activity against and selective toward Leishmania: Compounds displayed 50% inhibitory concentrations between 0.09 and 25 µM and had selectivity index values >10. For the PBox library, ~20% of chemicals exhibited anti-parasitic properties including five structures whose activity against L. infantum had not been reported before. These five compounds displayed no toxicity towards murine macrophages over the range tested with three being active in an in vivo murine model of the cutaneous disease, with 100% survival of infected animals. Additionally, the oral combination of three of them in the in vivo Chagas disease murine model demonstrated full control of the parasitemia. Interestingly, phenotyping revealed that the reference strain responds differently to the five PBox-derived chemicals relative to parasites isolated from a dog. Together, our data identified one drug candidate that displays activity against Leishmania and other Trypanosomatidae in vitro and in vivo, while exhibiting low toxicity to cultured mammalian cells and low in vivo acute toxicity.

In vitro susceptibility to benznidazole, nifurtimox and posaconazole of Trypanosoma cruzi isolates from Paraguay / [Sensibilidad in vitro a benznidazol, nifurtimox y posaconazol de cepas de Trypanosoma cruzi de Paraguay].

Introduction: Trypanosoma cruzi, the causative agent of Chagas disease, shows substantial phenotypic and genotypic heterogeneity, which can influence the epidemiological and clinical variations of the disease and the sensitivity to the drugs used in the treatment. Objective: To assess the in vitro susceptibility to benznidazole, nifurtimox, and posaconazole of 40 cloned strains of T. cruzi isolated in Paraguay belonging to different genotypes, hosts, and localities. Materials and methods: We incubated the parasites in their epimastigote stage in LIT culture medium with different concentrations of each drug in triplicate assays. The degree of susceptibility was estimated by the inhibitory concentrations of 50 and 90% (IC50 and IC90) to obtain the average values and the standard deviation for each strain and drug. We determined the statistical significance between groups by analysis of variances with the Wilcoxon/Kruskal-Wallis non-parametric test and values of p<0.05. Results: A wide range of drug response was observed. Two groups of parasites (A and B) were identified as having significant differences in susceptibility to benznidazole (p<0.0001), and three groups (A, B, C) to nifurtimox and posaconazole (p<0.0001). Conclusions: Overall, the isolates were more susceptible to nifurtimox than benznidazole and posaconazole. Such differences highlight the heterogeneity of T. cruzi populations circulating in Paraguay, an aspect to consider in the treatment and follow up of patients.

Introducción. Trypanosoma cruzi, agente causal de la enfermedad de Chagas, exhibe una sustancial heterogeneidad fenotípica y genotípica que puede influir en las variaciones epidemiológicas y clínicas de la enfermedad, así como en la sensibilidad a los fármacos utilizados en el tratamiento. Objetivo. Evaluar la sensibilidad in vitro al benznidazol, el nifurtimox y el posaconazol de 40 cepas clonadas de T. cruzi de Paraguay, con distintos genotipos, huéspedes y localidades de origen. Materiales y métodos. En su estado epimastigote, los parásitos se incubaron en medio de cultivo LIT (Liver Infusion Tryptose) con diferentes concentraciones de cada fármaco en ensayos por triplicado. El grado de sensibilidad se estimó a partir de las concentraciones inhibitorias del 50 y el 90% (IC50 e IC90) y se obtuvieron los valores promedio y la desviación estándar de cada cepa y fármaco. La significación estadística entre grupos se determinó mediante análisis de varianzas con el test no paramétrico de Wilcoxon/Kruskal-Wallis y valores de p<0,05. Resultados. Se observó un amplio rango de respuesta a los fármacos. Se identificaron dos grupos de parásitos (A y B) con diferencias significativas en la sensibilidad al benznidazol (p<0,0001), y tres grupos (A, B, C) en cuanto a la sensibilidad al nifurtimox y el posaconazol (p<0,0001). Conclusiones. En general, las cepas fueron más sensibles al nifurtimox que al benznidazol y el posaconazol. Estas diferencias evidencian la heterogeneidad de las poblaciones de T. cruzi que circulan en Paraguay, lo que debe considerarse en el tratamiento y el seguimiento de las personas afectadas.

Helietta apiculata: a tropical weapon against Chagas disease.

The present study pretends to evaluate the in vivo efficacy of the crude chloroform bark extract of Helietta apiculata, then the activity will be compared with the reference drug, benznidazole, in acute Trypanosoma cruzi infected mice when administered by oral route. The chloroformic extract of Helieta apiculata was administered by oral route at 5, 10 and 50 mg/kg daily for two weeks. This study has shown a moderate efficacy of the H. apiculata bark extract in reducing T. cruzi parasitaemia in 42 to 54% after a monitoring of 60 days post-infection and when compared with control groups. Concerning mice mortality, only two only two mice died, one from the control group and the other one from the group threated with 10 mg of the chlorofom extract of H. apiculata, suggesting the potential of H. apiculta extracts as a safe and inexpensive treatment of Chagas disease.

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.

In vitro antiprotozoal activity of (S)-cis-Verbenol against Leishmania spp. and Trypanosoma cruzi.

(S)-cis-Verbenol, a monoterpene frequently found as a component of essential oils, was assayed against Leishmania amazonensis, Leishmania infantum, Leishmania brasiliensis and against two strains of Trypanosoma cruzi. The cytotoxicity of the compound was also assayed against human fibroblast cells using a colorimetric method. Benznidazole was used as reference drug against T. cruzi and amphotericin B was used against Leishmania spp. The compound showed good activity against the trypanosomes, being more active against the CL Brenner strain, with an IC50 value of 8.3µg/mL. Against Leishmania, the IC50 values were between 2.1 and 3.8µg/mL. The compound showed no cytotoxicity against human fibroblasts at the concentrations assayed and was 100-500 times more toxic for the parasites than for the human cells, as indicated by the selectivity indexes. The results open interesting perspectives about the potential of (S)-cis-Verbenol and other individual components of essential oils for the treatment of these diseases.

Evaluación biológica in vitro del extracto etanólico de Kaa tái, Polygonum punctatum Elliot. (POLYGONACEAE)

En Paraguay, así como en las Américas, la leishmaniasis constituye una de las problemáticas de salud pública importante, debido a su complejidad tanto epidemiológica, clínica y biológica, afectando especialmente a los más pobres y en los países en vía de desarrollo. Polygonum punctatum Elliot. pertenece a la familia Polygonaceae, la medicina popular le atribuye varias propiedades, por medio de estudios anteriores se confirma que diferentes extractos de esta planta presentan actividad antibacteriana, antiinflamatoria y antifúngica. En este trabajo, se evaluó la actividad citotóxica del extracto etanólico de Polygonum punctatum de la familia Polygonaceae en concentraciones de 100, 50 y 25 µg/ml en células de macrófagos de ratones, en los cuales no se encontró actividad citotóxica. Además, se evaluó la actividad leishmanicida por medio de estudios experimentales in vitro a concentraciones de 100, 75, 50, 25, 20 y 10 µg/ml frente a tres cepas de parásitos: Leishmania infantum, L. amazonensis y L. braziliensis, en los cuales se observó una actividad de 58 y 56% de lisis de parásitos con el extracto de 100µg/ml frente a las cepas de L. braziliensis y L. infantum, respectivamente y 51% para L. amazonensis. Estos resultados son prometedores, y aportan una base para el desarrollo y búsqueda de nuevos tratamientos para la leishmaniasis, sin embargo, son necesarios estudios posteriores en cuanto a aislamiento e identificación del compuesto y evaluación en modelos animales in vivo.

In Paraguay, as well as in the Americas, leishmaniasis constitutes one of the important public health problems, due to its epidemiological, clinical, and biological complexity, especially affecting the poorest and developing countries. Polygonum punctatum Elliot belongs to the Polygonaceae family, and popular medicine attributes several properties to it. Previous studies confirmed that different extracts of this plant have antibacterial, anti-inflammatory and antifungal activity. In this work, the cytotoxic activity of the ethanolic extract of Polygonum punctatum was evaluated at concentrations of 100, 50 and 25 µg/ml in mouse macrophage cells, in which no cytotoxic activity was found. In addition, the leishmanicidal activity was evaluated through experimental in vitro studies at concentrations 100, 75, 50, 25, 20 and 10 µg/ml against three strains of parasites: Leishmania infantum, L. amazonensis and L. braziliensis. Activities of 58 and 56% of parasite lysis were observed with the 100 µg/ml extract against strains of L. braziliensis and L. infantum, respectively, and 51% for L. amazonensis. These results are promising and provide a basis for the development and search of a new treatment for leishmaniasis. However, further studies are necessary regarding the isolation and identification of the compound and its evaluation in in vivo animal models.

2H-benzimidazole 1,3-dioxide derivatives: a new family of water-soluble anti-trypanosomatid agents.

Three series of benzimidazole N-oxide derivatives were developed and were examined for their activity against trypanosomatid parasites (Trypanosoma cruzi and Leishmania spp.). 2H-benzimidazole 1,3-dioxides displayed remarkable in vitro activities against both parasites, with derivatives 28, 29, and 32 being the most potent (IC50 < 5 microM) against the epimastigote form of T. cruzi and 28, 33, and 35 the most potent against the promastigote form of Leishmania spp. Unspecific cytotoxicity was evaluated using murine macrophages, and derivative 33 was not toxic at a concentration 30 times that of its IC50 against T. cruzi that was completely toxic for Leishmania spp., implying that the series of 2H-benzimidazole 1,3-dioxides is selective toward both trypanosomatid parasites. Derivatives 33 and 35 were submitted to an in vivo assay using an acute model of Chagas' disease and a short-term treatment (30 mg/kg/day orally administrated as aqueous solution, during 10 days). While in the control (untreated) and Benznidazole (50 mg/kg/day) groups survival fraction was 60.0% and 87.5%, respectively, none of the animals treated with derivatives 33 and 35 died. From the preliminary structure-activity relationship studies reduction potential and electrophilicity were found relevant to anti-T. cruzi activity. Active compounds are better electrophiles and more easily reduced than inactive ones.

New approach towards the synthesis of selenosemicarbazones, useful compounds for Chagas' disease.

Herein, we describe a new approach towards the synthesis of selenosemicarbazones. The reaction involves an O-Se exchange of semicarbazones using Ishihara reagent. Eleven selenosemicarbazones were prepared using this methodology, with low to moderate yields. Among the prepared compounds the m-bromo phenyl methyl derivative 1b was selected to be evaluated in vivo, in a murine model of acute Chagas' disease. Compound 1b 10 mg/kg bw/day reduced 50% of parasitaemia profile compared with the control group, but was less effective than Benznidazole (50 mg/kg bw/day reduced 90%) and toxic. These studies are important to guide future Chagas drug design.

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.

Evaluación genotóxica de las moléculas con efectos tripanocida y leishmanicida cantin-6-ona y 5-metoxicantin-6-ona en células de médula ósea de ratones / Genotoxic evaluation of the molecules with trypanocidal and leishmanicidal effects, canthin-6-one and 5-methoxycanthin-6-one in mouse bone marrow cells

La tripanosomiasis americana y la leishmaniasis son problemas de salud pública relevantes en Iberoamérica. Las drogas utilizadas actualmente para el tratamiento de estas enfermedades poseen efectos colaterales tóxicos severos. Varios grupos de investigación están abocados a la búsqueda de productos naturales y sintéticos para encontrar nuevos agentes terapéuticos efectivos que no presenten reacciones colaterales adversas. En la evaluación de compuestos de la especie vegetal Zanthoxylum chiloperone (Rutaceae), se demostró que compuestos aislados del extracto presentaban actividad leishmanicida, tripanocida y antifúngica in vivo. Teniendo como antecedentes estos resultados, en el presente estudio se evaluaron los efectos genotóxico y citotóxico del cantín-6-ona y del 5-metoxicantin-6-ona, moléculas aisladas de la planta, en células de médula ósea de animales tratados. El estudio de los efectos genotóxicos se hizo a través del ensayo de modificaciones en la frecuencia de micronúcleos y el efecto citotóxico por modificaciones en la relación entre eritrocitos policromáticos y eritrocitos normocromáticos. Se realizaron 2 ensayos independientes y en cada ensayo los animales fueron divididos en tres grupos de tratamiento: GRUPO I: control negativo que recibió 200 uL de agua y 2.1% de DMSO, vía oral, GRUPO II: compuesto a ser evaluado (canthin-6-ona o 5-methoxicantin-6-ona) con 2.1% de DMSO, y GRUPO III: control positivo que recibió ciclofosfamida 50mg/kg/peso del animal, vía intraperitoneal. El análisis estadístico mostró que ambos compuestos no presentaron efectos genotóxicos ni citotóxicos. Estos resultados permiten proponer a estas moléculas como candidatas a ser sometidas a estudios más detallados como potenciales fármacos contra estas dos enfermedades

American trypanosomiasis and leishmaniasis are relevant public health problems in Latin America. The drugs currently used to treat these diseases have severe toxic side effects. Several research groups are dedicated to the search of natural and synthetic products to find new effective therapeutic agents that do not present adverse collateral reactions. In the evaluation of compounds of the plant species Zanthoxylum chiloperone (Rutaceae), it was shown that isolated compounds of the extract had leishmanicidal, trypanocidal and antifungal in vivo activities. Based on these results, the genotoxic and cytotoxic effects of canthin-6-one and 5-methoxycanthin-6-one, molecules isolated from the plant, on bone marrow cells of treated mice were evaluated in the present study. The study of genotoxic effects was made through the test of modifications in the frequency of micronuclei and the cytotoxic effects by modifications in the relationship between polychromatic erythrocytes and normochromic erythrocytes. Two independent assays were performed and in each assay the animals were divided into three treatment groups: GROUP I: negative control that received 200 µL of water and 2.1% of DMSO, orally, GROUP II: compound to be evaluated (canthin-6 -one or 5-methoxycanthin-6-one) with 2.1% DMSO, and GROUP III: positive control that received cyclophosphamide 50mg /kg animal weight, intraperitoneal. Statistical analysis showed that both compounds had neither genotoxic nor cytotoxic effects. These results allow these molecules to be proposed as candidates to be subjected to more detailed studies as potential drugs against these two diseases

Sensibilidad in vitro a benznidazol, nifurtimox y posaconazol de cepas de Trypanosoma cruzi de Paraguay / In vitro susceptibility to benznidazole, nifurtimox and posaconazole of Trypanosoma cruzi isolates from Paraguay

Resumen: Introducción. Trypanosoma cruzi, agente causal de la enfermedad de Chagas, exhibe una sustancial heterogeneidad fenotípica y genotípica que puede influir en las variaciones epidemiológicas y clínicas de la enfermedad, así como en la sensibilidad a los fármacos utilizados en el tratamiento. Objetivo. Evaluar la sensibilidad in vitro al benznidazol, el nifurtimox y el posaconazol de 40 cepas clonadas de T. cruzi de Paraguay, con distintos genotipos, huéspedes y localidades de origen. Materiales y métodos. En su estado epimastigote, los parásitos se incubaron en medio de cultivo LIT (Liver Infusion Tryptose) con diferentes concentraciones de cada fármaco en ensayos por triplicado. El grado de sensibilidad se estimó a partir de las concentraciones inhibitorias del 50 y el 90% (IC50 e IC90) y se obtuvieron los valores promedio y la desviación estándar de cada cepa y fármaco. La significación estadística entre grupos se determinó mediante análisis de varianzas con el test no paramétrico de Wilcoxon/Kruskal-Wallis y valores de p<0,05. Resultados. Se observó un amplio rango de respuesta a los fármacos. Se identificaron dos grupos de parásitos (A y B) con diferencias significativas en la sensibilidad al benznidazol (p<0,0001), y tres grupos (A, B, C) en cuanto a la sensibilidad al nifurtimox y el posaconazol (p<0,0001). Conclusiones. En general, las cepas fueron más sensibles al nifurtimox que al benznidazol y el posaconazol. Estas diferencias evidencian la heterogeneidad de las poblaciones de T. cruzi que circulan en Paraguay, lo que debe considerarse en el tratamiento y el seguimiento de las personas afectadas.

Abstract: Introduction: Trypanosoma cruzi, the causative agent of Chagas disease, shows substantial phenotypic and genotypic heterogeneity, which can influence the epidemiological and clinical variations of the disease and the sensitivity to the drugs used in the treatment. Objective: To assess the in vitro susceptibility to benznidazole, nifurtimox, and posaconazole of 40 cloned strains of T. cruzi isolated in Paraguay belonging to different genotypes, hosts, and localities. Materials and methods: We incubated the parasites in their epimastigote stage in LIT culture medium with different concentrations of each drug in triplicate assays. The degree of susceptibility was estimated by the inhibitory concentrations of 50 and 90% (IC50 and IC90) to obtain the average values and the standard deviation for each strain and drug. We determined the statistical significance between groups by analysis of variances with the Wilcoxon/Kruskal-Wallis non-parametric test and values of p<0.05. Results: A wide range of drug response was observed. Two groups of parasites (A and B) were identified as having significant differences in susceptibility to benznidazole (p<0.0001), and three groups (A, B, C) to nifurtimox and posaconazole (p<0.0001). Conclusions: Overall, the isolates were more susceptible to nifurtimox than benznidazole and posaconazole. Such differences highlight the heterogeneity of T. cruzi populations circulating in Paraguay, an aspect to consider in the treatment and follow up of patients.

Development of bis-thiazoles as inhibitors of triosephosphate isomerase from Trypanosoma cruzi. Identification of new non-mutagenic agents that are active in vivo.

The neglected disease American trypanosomiasis is one of the major health problems in Latin America. Triosephosphate isomerase from Trypanosoma cruzi (TcTIM), the etiologic agent of this disease, has been proposed as a druggable target. Some bis-benzothiazoles have been described as irreversible inhibitors of this enzyme. On the other hand, new bioactive furane-containing thiazoles have been described as excellent in vivo anti-T. cruzi agents. This encouraged us to design and develop new bis-thiazoles with potential use as drugs for American trypanosomiasis. The bis-thiazol 5, 3,3'-allyl-2,2'-bis[3-(2-furyl)-2-propenylidenehydrazono]-2,2',3,3'-tetrahydro-4,4'-bisthiazole, showed the best in vitro anti-T. cruzi profile with a higher selectivity index than the reference drugs Nifurtimox and Benznidazole against amastigote form of the parasite. This derivative displayed marginal activity against TcTIM however the bis-thiazol 14, 3-allyl-2-[3-(2-furyl)-2-propenylidenehydrazono]-3'-phenyl-2'-(3-phenyl-2-propenylidenehydrazono]-2,2',3,3'-tetrahydro-4,4'-bisthiazole, was an excellent inhibitor of the enzyme of the parasite. The absence of both in vitro mutagenic and in vivo toxicity effects, together with the activity of bis-thiazol 5in vivo, suggests that this compound is a promising anti-T. cruzi agent surpassing the "hit-to-lead" stage in the drug development process.

Bioactive alkyl phenols and embelin from Oxalis erythrorhiza.

The benzoquinone embelin and four alkyl phenols were isolated from an Argentinean collection of Oxalis erythrorhiza. 3-Heptadecyl-5-methoxy-phenol is reported for the first time. The structures were determined by spectroscopic methods. Embelin presented inhibitory effect on methicillin-resistant Staphylococcus aureus, Escherichia coli and the dermatophytic fungi Epidermophyton floccosum, Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes and Trichophyton rubrum with MICs ranging between 50 and 100 microg/ml. Furthermore, embelin was active against Trypanosoma cruzi trypomastigotes with 100% lysis at 100 microg/ml and cytotoxicity below the trypanocidal concentration. The new alkyl phenol 3-heptadecyl-5-methoxy-phenol was active towards Leishmania amazonensis and Leishmania donovani promastigotes with 100% lysis at 100 microg/ml. The cytotoxicity (IC50) of embelin and the new alkyl phenol on human lung fibroblasts were 739 and 366 microM, respectively. The plant is used to treat heart complains, a symptomatology related to Chagas' disease which is endemic in the San Juan Province, Argentine.

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.

Looking for combination of benznidazole and Trypanosoma cruzi-triosephosphate isomerase inhibitors for Chagas disease treatment

BACKGROUND The current chemotherapy for Chagas disease is based on monopharmacology with low efficacy and drug tolerance. Polypharmacology is one of the strategies to overcome these limitations. OBJECTIVES Study the anti-Trypanosoma cruzi activity of associations of benznidazole (Bnz) with three new synthetic T. cruzi-triosephosphate isomerase inhibitors, 2, 3, and 4, in order to potentiate their actions. METHODS The in vitro effect of the drug combinations were determined constructing the corresponding isobolograms. In vivo activities were assessed using an acute murine model of Chagas disease evaluating parasitaemias, mortalities and IgG anti-T. cruzi antibodies. FINDINGS The effect of Bnz combined with each of these compounds, on the growth of epimastigotes, indicated an additive action or a synergic action, when combining it with 2 or 3, respectively, and an antagonic action when combining it with 4. In vivo studies, for the two chosen combinations, 2 or 3 plus one fifth equivalent of Bnz, showed that Bnz can also potentiate the in vivo therapeutic effects. For both combinations a decrease in the number of trypomastigote and lower levels of anti-T. cruzi IgG-antibodies were detected, as well clear protection against death. MAIN CONCLUSIONS These results suggest the studied combinations could be used in the treatment of Chagas disease.