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.

Enfermedad de chagas congénito: presentación de caso clínico / Congenital chagas disease: case report

El agente etiológico de la enfermedad de Chagas es un protozoario parásito (Tripano-soma Cruzi) que causa una infección aguda y crónica, en humanos. La extensión del daño varía de acuerdo a las cepas parasita-rias y características individuales del hués-ped; puede causar incapacidad y muerte. Han sido descritas diferentes vías de infec-ción: vectorial, transfusional, accidental, digestiva y congénita. Esta última vía de la infección depende de dos indicadores bási-cos, la prevalencia de gestantes chagásicas e incidencia de la transmisión vertical. El diagnóstico presenta un desafío en aquellos lugares del país en donde, si bien la enfer-medad es considerada endémica, no se realiza actualmente el screening obligatorio en la embarazada. Es importante saber que el tratamiento antes del primer año de vida tiene una excelente respuesta y evita secue-las crónicas que pueden ser invalidantes en etapa adulta...(AU)

Doença de Chagas: do surgimento ao tratamento - revisão da literatura / Chagas Disease: from emergence to treatment - a literature review

Embora a doença de Chagas seja endêmica em certas regiões da América Latina, os fluxos migratórios recentes permitiram sua expansão para áreas onde antes era desconhecida. Mais de 8 milhões de pessoas estão infectadas pelo Trypanosoma cruzi, o que resulta em aproximadamente 10.000 mortes por ano. Esta revisão tem como objetivo fornecer uma compilação sobre os tópicos mais importantes da doença de Chagas em um único trabalho: a descoberta por Carlos Chagas (1909), sua ocorrência, epidemiologia, vetores, via de transmissão, patologia, sinais e sintomas, diagnóstico, e tratamentos, ainda limitado a duas drogas utilizadas há mais de 40 anos: nifurtimox e benzonidazol

Although Chagas disease is endemic in certain regions of Latin America, recent migratory flows have allowed it to expand into areas where it was previously unknown. More than 8 million people are infected with Trypanosoma cruzi, causing around 10,000 deaths a year. This review aims to provide a compilation on the most important topics about the Chagas disease in a single place: its discovery by Carlos Chagas (1909), its occurrence, epidemiology, vectors, transmission route, pathology, signs and symptoms, diagnosis, and current treatments, which is still limited to two drugs for more than 40 years: nifurtimox and benzonidazole

Therapeutic drug monitoring of benznidazole and nifurtimox: a systematic review and quality assessment of published clinical practice guidelines

Abstract The pharmacological management of adults with chronic-phase Chagas disease is challenging despite it being the recent focus of extensive research. One of the challenges in the current clinical practice guidelines (CPGs) landscape is the existence of non-evidence-based recommendations for the use of laboratory tests in treatment monitoring. This study aimed to systematically assess the quality and consistency of recommendations of CPGs on the pharmacological management of adults with chronic-phase Chagas disease. Systematic literature searches were conducted in MEDLINE, EMBASE, SciELO and Google to identify all published CPGs relevant to the pharmacological management of Chagas disease, between January 2010 and March 2016. Three independent reviewers assessed the quality of each CPG using the Appraisal of Guidelines Research and Evaluation (AGREE) II instrument. A total of five CPGs were included and the overall quality of the guidelines for therapeutic drug monitoring of Chagas disease was moderate-to-low. There was considerable variation in the quality of the CPGs across the AGREE II domains. The domains of scope/purpose, stakeholder involvement, and clarity of presentation were rated well, and the domains of applicability and editorial independence received poor ratings. This review showed that the methodological quality of CPGs for Chagas disease was generally inappropriate, and there was no explicit link between the best available evidence and current recommendations.

Dipyridamole potentiated the trypanocidal effect of nifurtimox and improved the cardiac function in NMRI mice with acute chagasic myocarditis

BACKGROUND As chronic Chagas disease does not have a definitive treatment, the development of alternative therapeutic protocols is a priority. Dipyridamole (DPY) is an alternative to counteract the pathophysiological phenomena involved in Chagas cardiomyopathy. OBJECTIVE To evaluate the therapeutic efficacy of DPY associated with nifurtimox (Nfx) in epimastigote axenic cultures and in mice with acute Chagas disease. METHODS NMRI adult male mice were divided into nine groups: three healthy and six Trypanosoma cruzi-infected groups. Mice received vehicle, Nfx or DPY, alone or combined. The doses assayed were Nfx 10 and 40 mg/kg and DPY 30 mg/kg. The treatment efficacy was evaluated by clinical, electrocardiographic, parasitological, biochemical and histopathological methods. FINDINGS In vitro, DPY and Nfx had a trypanocidal effect with IC50 values of 372 ± 52 and 21.53 ± 2.13 µM, respectively; DPY potentiated the Nfx effect. In vivo, Nfx (40 mg/kg) with or without DPY had a therapeutic effect, which was reflected in the 84-92% survival rate and elimination of parasitaemia and heart tissue amastigotes. Nfx (10 mg/kg) had a subtherapeutic effect with no survival and persistence of amastigotes, inflammation and fibrosis in heart tissue; adding DPY increased the survival rate to 85%, and all tested parameters were significantly improved. MAIN CONCLUSION DPY has a trypanocidal effect in vitro and enhances the Nfx therapeutic effect in an in vivo murine model.

Terapia de Resgate com Nifurtimox e Dipiridamol na Miocardite Chagásica Aguda Grave com Insuficiência Cardíaca em Camundongos NMRI / Rescue Therapy with Nifurtimox and Dipyridamole for Severe Acute Chagas Myocarditis with Congestive Heart Failure in NMRI Albino Mice

Fundamento: A doença de Chagas é um problema de saúde global, sendo necessário o desenvolvimento de novos protocolos terapêuticos. Nosso grupo demonstrou recentemente que o nifurtimox associado ao dipiridamol tem efeitos curativos em camundongos com doença de Chagas aguda. Neste estudo, avaliamos o efeito deste protocolo terapêutico em camundongos chagásicos com insuficiência cardíaca. Objetivo: Avaliar se o nifurtimox e o dipiridamol são úteis no tratamento de resgate em camundongos com miocardite chagásica aguda com insuficiência cardíaca. Métodos: Foram divididos em três grupos 42 camundongos com miocardite chagásica aguda e insuficiência cardíaca congestiva: Controle Chagas (n = 11); Nif-Dip, tratados com nifurtimox e dipiridamol (n = 14); e Nif-Dip-Insuficiência Cardíaca, tratados com nifurtimox e dipiridamol, associados com digoxina, furosemida e captopril (n = 17). As doses de nifurtimox e dipiridamol foram de 40 e 30mg/kg/dia, respectivamente, durante 6 semanas. Os camundongos foram submetidos a avaliações clínicas, eletrocardiográficas, hemoparasitológicas e histopatológicas. Resultados: Observou-se menor mortalidade no Grupo Nif-Dip (n = 4; 28,57%) em relação ao Controle Chagas (n = 6; 54,54%) e ao Nif-Dip-Insuficiência Cardíaca (n = 9; 52,9%). Clinicamente, os camundongos tratados com nifurtimox e dipiridamol aumentaram o peso corporal e melhoraram a insuficiência cardíaca, sem mostrar esplenomegalia. Nestes grupos, foram erradicadas as parasitemias e os parasitas teciduais; a fibrose, a miocitólise, o infiltrado de células inflamatórias e os mastócitos diminuíram. Os distúrbios de repolarização, os intervalos QRS e o QT prolongados, o aumento da amplitude da onda S e a dissociação atrioventricular foram revertidos pelo tratamento. Conclusão: O tratamento com nifurtimox e dipiridamol pode ser usado no resgate em camundongos com doença chagásica aguda grave, já que o nifurtimox teve atividade tripanocida, e o dipiridamole potenciou seu efeito. O dipiridamol seria útil na insuficiência cardíaca chagásica

Background: Chagas disease is a global health problem; therefore, the development of new therapeutic protocols is necessary. Our group recently demonstrated that nifurtimox associated with dipyridamole has curative effects in mice with acute Chagas disease. In this study, we assess the effect of this therapeutic protocol in chagasic mice with heart failure. Objective: To evaluate whether nifurtimox and dipyridamole are useful to rescue mice with severe acute chagasic myocarditis with heart failure. Methods: 42 mice with acute chagasic myocarditis and congestive heart failure were divided into three groups: control chagas (n = 11), Nif-Dip treated with nifurtimox and dipyridamole (n = 14) and Nif-Dip-heart failure treated with nifurtimox and dipyridamole associated with digoxin, furosemide, and captopril (n = 17). Nifurtimox and dipyridamole doses were 40 and 30 mg/kg/day, respectively, for 6 weeks. Mice underwent clinical, electrocardiographic, hemoparasitological and histopathological assessments. Results: Lower mortality in Nif-Dip (28.57%; n = 4) compared to control chagas (54.54%; n = 6) and Nif-Dip-heart failure (52.9%; n = 9) was observed. Clinically, nifurtimox and dipyridamole-treated mice increased body weight and improved heart failure without splenomegaly. In these groups, parasitemia and tissue parasites were eradicated; fibrosis, myocytolysis, inflammatory cell infiltrate and mast cells decreased. Repolarization disorders, prolonged QRS and QT intervals, increase of S wave amplitude and atrioventricular dissociation were reversed by the treatment. Conclusion: Nifurtimox with dipyridamole can rescue NMRI mice from severe acute chagas disease, as nifurtimox showed trypanocidal activity and dipyridamole potentiated its effect. Dipyridamole would be useful in chagasic heart failure

Herbal extracts differentially inhibit oxidative effects caused by the biotransformation of nifurtimox, nitrofurantoin and acetaminophen on rat liver microsomes / Extractos herbales inhiben diferencialmente los efectos oxidativos causados por la biotransformación de nifurtimox, nitrofurantoína y acetaminofeno en microsomas hepáticos de rata]

Inflammation is a cellular defensive mechanism associated to oxidative stress. The administration of nitrofurantoin, nifurtimox and acetaminophen generates oxidative stress by their biotransformation through CYP450 system. The main adverse effect described for the first two drugs is gastrointestinal inflammation and that of the last, hepatitis. Therefore, standardised dry extracts from Rosmarinus officinalis, Buddleja globosa Hope, Cynara scolymus L., Echinacea purpurea and Hedera helix were tested to evaluate their capacity to decrease drug-induced oxidative stress. For that, rat liver microsomes were incubated with drugs in the presence of NADPH (specific CYP450 system cofactor) to test oxidative damage on microsomal lipids, thiols, and GST activity. All drugs tested induced oxidation of microsomal lipids and thiols, and inhibition of GST activity. Herbal extracts prevented these phenomena in different extension. These results show that antioxidant phytodrugs previously evaluated could alleviate drugs adverse effects associated to oxidative stress.

Inflamación es un mecanismo de defensa el cual está asociado a estrés oxidativo. La administración de nitrofurantoína, nifurtimox y paracetamol genera estrés oxidativo al metabolizarse a través del sistema CYP450. El principal efecto adverso de los dos primeros fármacos es inflamación gastrointestinal y del tercero, hepatitis. Por lo tanto, utilizamos diversos extractos herbales para disminuir el estrés oxidativo inducido por estos fármacos. Para esto se incubaron microsomas hepáticos de rata con dichos fármacos en presencia de NADPH (cofactor específico del sistema CYP450) y se evaluó el daño oxidativo generado sobre los lípidos, los tioles y la actividad GST microsómica. Todos los fármacos indujeron oxidación de los lípidos y los tioles microsómicos e inhibieron la actividad GST. Los extractos herbales previnieron estos fenómenos oxidativos en diferente extensión. Estos resultados indican que fitofármacos antioxidantes previamente evaluados, podrían aliviar los efectos adversos asociados a estrés oxidativo de los fármacos.

Aspectos epidemiológicos e clínicos da Doença de Chagas aguda no Brasil e na américa Latina / Epidemiological and clinical aspects of acute Chagas Disease in Brazil and Letin America

surgindo em um cenário não habitual, relacionado com a forma de transmissão. Atualmente, a transmissão por via oral envolvendo alimentos contaminados é a principal via de infecção humana, com surtos em toda a América Latina. Entre os sintomas da fase aguda, destacam-se febre prolongada, quase sempre acompanhada de mal-estar, adinamia, cefaleia e perda do apetite, acompanhados de manifestações sistêmicas, tais como adenomegalias e hepatoesplenomegalias. As manifestações cardíacas variam desde quadros assintomáticos até graus variáveis de insuficiência cardíaca aguda ou choque cardiogênico e morte. O tratamento da fase aguda com antiparasitários deve sempre ser instituído, pois existem algumas evidencias de melhor evolução clínica em relatos de casos

Acute Chagas disease shows changes in the epidemiological situation, reappearing in an unusual scenario related to the mode of transmission. Currently, the oral transmission involving contaminated food is the main route of human infection with outbreaks throughout Latin America. Among the symptoms of the acute phase stand out prolonged fever, often accompanied by malaise, asthenia, headache and loss of appetite, accompanied by systemic manifestations such as lymphadenopathy and hepatosplenomegaly. Cardiac manifestations range from asymptomatic clinical statuses to varying degrees of acute heart failure or cardiogenic shock and death. Treatment of acute phase with antiparasitic agents should always be provided, as there is some evidence of better clinical outcome in case reports

Chagas congénito de segunda generación en Uruguay: Primer caso sintomático descrito en el país / Second generation congenital Chagas disease in Uruguay: First symptomatic case described in the country

El Chagas congénito se produce cuando la embarazada infectada trasmite el parásito al feto, situación que puede suceder en cualquier estadio de la enfermedad y momento del embarazo. Es el único mecanismo de trasmisión presente actualmente en Uruguay, con una incidencia cercana al 4%. La mayoría de los neonatos infectados nacen asintomáticos y un 10% al 40% presentan síntomas que son indistinguibles de otras infecciones de trasmisión vertical. La prematurez, el bajo peso, la hepatoesplenomegalia y las alteraciones hematológicas son los síntomas más frecuentes. El diagnóstico representa un desafío en aquellos lugares del país en donde, si bien la enfermedad era considerada endémica, no se realiza actualmente el screening obligatorio de la embarazada. Se describe un lactante procedente de Paysandú, de medio socioeconómico deficitario, que fue pretérmino severo y que presentó al nacer alteraciones hematológicas de las tres series y hepatoesplenomegalia, lo cual motivó múltiples estudios. Sin embargo, no se sospechó la enfermedad hasta los 5 meses de vida, cuando volvió a ingresar por otra patología. Se confirmó la infección a los 9 meses mediante tres técnicas serológicas diferentes. Se indicó tratamiento con nifurtimox por 2 meses y a los 26 y 36 meses de vida presentó serología negativa. Consideramos importante tener alto índice de sospecha de la enfermedad en neonatos con signos de infección congénita y sin serología materna que la descarte. Es importante saber que el tratamiento antes del primer año de vida tiene una excelente respuesta y evita secuelas crónicas que pueden ser invalidantes en etapa adulta.

Congenital Chagas disease occurs when an infected pregnant woman transmits the parasite to the fetus, a situation that can happen at any stage of disease and time of pregnancy. It is the only transmission mechanism currently present in Uruguay, with an incidence close to 4%. Most infected infants are born asymptomatic and 10 to 40% have symptoms that are indistinguishable from other infections’ vertical transmission. Prematurity, low birth weight, hepatosplenomegaly and hematological disorders are the most common symptoms. Diagnosis is a challenge in those areas where there is no current compulsory screening for pregnant women in spite of the disease being considered endemic. The study presents the case of an infant from Paysandú, coming from a low socio-economic environment, which was a severe preterm, and presented hematologic disorders of the three series at birth and hepatosplenomegaly, which caused many studies. However, there was no suspicion of the disease until the infant was five months old when re-entering by other pathology. Infection at nine months is confirmed by three different serological techniques. Nifurtimox therapy is indicated for two months and at twenty six and thirty six months of life the patient presents negative serology. A high level of suspicion is needed in order to diagnose the disease in infants with signs of congenital infection without maternal serology. It is important to know that treatment before the first year of life has excellent response and prevents chronic sequelae that can be disabling in adulthood.

Tripanosomiasis americana, una mirada desde eltratamiento / American trypanosomiasis, a new look at its treatment

La tripanosomiasis americana o enfermedad de Chagas es una infección parasitaria causada por el parásito flagelado Trypanosomacruzi, cuyo principal vector es un insecto de la clase hemíptera conocido como triatomino. La quimioterapia, adicional a un controloportuno de vectores y rigurosidad en el control de las transfusiones, son los elementos más importantes para el manejo de estaparasitosis. En la actualidad, el mercado farmacéutico solo ofrece nifurtimox y benznidazol como opciones terapéuticas, y a pesar deque se han obtenido resultados satisfactorios con el uso de estos medicamentos en fases agudas de la enfermedad, tripanosomiasiscongénita y accidentes de laboratorio, la efectividad en las fases crónicas es notoriamente menor. Además, ambos fármacos generanciertos efectos adversos que deben ser tenidos en cuenta por el personal de la salud, antes de iniciar el debido manejo de estaenfermedad. El objetivo del presente artículo es revisar el estado actual del tratamiento farmacológico de la tripanosomiasis americana,buscando resumir los nuevos avances terapéuticos y dar a conocer las limitaciones de los mismos debido a sus efectos adversos.

American trypanosomiasis or Chagas disease is a parasitic infection caused by the flagellate parasite Trypanosoma cruzi, whosemain vector is an insect of the Hemiptera class, called triatomine. Chemotherapy, in addition to an appropriate vector control and arigorous control of transfusions, are the most important strategies for the management of this parasitosis. Currently, the pharmaceuticalmarket only offers nifurtimox and benznidazole as treatment options, and although satisfactory results have been obtained with theuse of these drugs in either acute stages of the disease, congenital trypanosomiasis and laboratory accidents, its effectiveness in thechronic phases is significantly smaller. In addition, both drugs produce some side effects that must be taken into account by healthworkers, before starting the proper management of the disease. The aim of this article is to review the current state of the Americantrypanosomiasis treatment, in order to summarize the new advances in therapeutics and to introduce their limitations due to adverseeffects.

Genotoxicidad del nifurtimox en diferentes líneas celulares utilizando el ensayo cometa / Genotoxiciyt from Nifurtimox in comet assay

El nifurtimox es un 5-nitrofurano sintético utilizado en el tratamiento dela enfermedad de Chagas. El objetivo de este estudio fue determinar la toxicidad celular y el daño del ADN causado por el nifurtimox en células Vero, J774, NIH/3T3 y THP-1. Se utilizó la coloración vital con azul tripan y elmétodo colorimétrico MTT para determinar la toxicidad y el ensayo cometa alcalino para determinar el daño al ADN. Los cometas fueron contadosen un microscopio de fluorescencia y el porcentaje de daño total del ADN fue calculado y clasificado de 0 (sin daño) a 4 (daño severo). En el ensayo de toxicidad, las células J774 fueron las líneas celulares más sensibles y las células THP-1 las menos sensibles al nifurtimox con valores de CC50 34,04-138,58 μg/ml y CC90 130,58->300 μg/ml de nifurtimox, respectivamente.En el ensayo cometa, el porcentaje de daño total de ADN a 100 μg/ml de nifurtimox fue 79,75%, 85,30% y 10,25% en células NIH/3T3, J774 y THP-1 respectivamente. En las células Vero el daño del ADN fue del 80% en células tratadas y no tratadas. El nifurtimox presentó toxicidad y genotoxicidad con actividades que dependieron del tipo de célula y de la concentración del medicamento utilizada. Es importante tomar en cuenta estas diferencias al realizar conclusiones finales de resultados obtenidos utilizando estos ensayos, especialmente el ensayo cometa...

Antichagásicos potenciais: síntese e modelagem molecular de híbridos de hidrazonas e liberadores de óxido nítrico / Potential antichagasic agents: synthesis and molecular modeling of hydrazones and nitric oxide releasing hybrids

A doença de Chagas é uma parasitose extremamente negligenciada, cujo agente etiológico é o protozoário Trypanosoma cruzi. Atualmente, 21 países da América Latina são considerados regiões endêmicas, onde 75-90 milhões de pessoas estão expostas à infecção, 6-7 milhões estão infectadas e mais de 41 mil novos casos surgem por ano. Entretanto, apenas os fármacos nifurtimox e benznidazol estão disponíveis no mercado. Estes, além da baixa eficácia na fase crônica da parasitose, apresentam diversos efeitos adversos, sendo que no Brasil apenas o benznidazol é utilizado. Este fato mostra a importância de se ampliar o número de fármacos disponíveis e propor quimioterapia mais eficaz para o tratamento da doença de Chagas. Como forma de contribuir para essa busca, este trabalho objetiva a síntese de compostos híbridos bioisostéricos N-acilidrazônicos e sulfonilidrazônicos, contendo grupo liberador de óxido nítrico, com potencial de interação com cisteíno-proteases parasitárias, tais como a cruzaína. Nestes derivados, os grupos liberadores de óxido nítrico utilizados foram os grupos furoxano (contendo substituinte metílico e fenílico) e éster nitrato. Propôs-se a variação de anéis aromáticos substituídos e não-substituídos, com o intuito de avaliar a possível relação estrutura-atividade (REA) desses análogos. Até o momento, somente os compostos da série N-acilidrazônica tiveram avaliação biológica realizada. Os valores de IC50 dos compostos na forma amastigota do parasita variaram entre >100 a 2,88 µM, sendo este último valor comparável ao fármaco de referência. A atividade inibitória frente à cruzaína foi de 25,2 µM a 2,2 µM. Já a liberação de óxido nítrico foi avaliada pelo método indireto de detecção de nitrato e os valores variaram entre 52,0 µM e 4.232,0 µM. Estes são bem inferiores ao composto padrão, além de não se identificar correlação direta entre a atividade biológica e a liberação de NO. Na sequência, os dois compostos mais ativos (6 e 14) foram submetidos a estudos de permeabilidade e de citotoxicidade. O composto 6 foi considerado o de maior permeabilidade segundo o Sistema de Classificação Biofarmacêutica (SCB) e todos os compostos apresentaram a taxa de fluxo menor que 2, indicando a ausência de mecanismo de efluxo. Na avaliação do potencial citotóxico desses compostos em células humanas, o derivado 6 apresentou índice de seletividade superior ao do benznidazol. Em estudos de modelagem molecular usando análise exploratória de dados (HCA e PCA), propriedades estéricas/geométricas e eletrônicas foram consideradas as mais relevantes para a atividade biológica. Além disso, estudos de docking mostraram que a posição do grupo nitro no anel aromático é importante para a interação com a cruzaína. Ademais o composto 6 não provocou mudanças significativas no ciclo celular e na fragmentação de DNA em células humanas, mostrando-se como líder promissor para futuros estudos in vivo. Atividade tripanomicida, citotoxicidade, potencial de liberação de NO e estudos de permeabilidade dos 23 derivados sulfonilidrazônicos e ésteres nitrato estão sendo avaliados

Chagas disease is an extremely neglected parasitic disease whose etiologic agent is the protozoan Trypanosoma cruzi. Currently 21 Latin American countries are considered endemic regions, where 75-90 million people are exposed to infection, 6-7 million are infected and more than 41,000 new cases occur annually. However only nifurtimox and benznidazole are available on the market. These drugs, besides low efficacy in the chronic phase of the parasite have numerous adverse effects, and in Brazil only benznidazole is used. This fact shows the importance of increasing the number of drugs available and propose more effective chemotherapy for the Chagas disease treatment. As a contribution to the problem, this study aims the synthesis of biososteric compounds from N-acylhydrazone and sulfonylhydrazone, which have the potential to interact with parasitic cysteine protease, such as cruzain, containing nitric oxide releasing groups, which also has inhibitory activity in this enzyme class. In these derivatives nitric oxide releasing groups used were furoxan (containing methyl and phenyl substituent) and nitrate ester groups. The variation of aromatic rings substituted and unsubstituted was proposed in order to evaluate the possible structure-activity relationship (SAR) of these analogs. Only N-acylhydrazone series had its biological profile evaluated up to now. The IC50 values of the compounds against the amastigote form of the parasite ranged from >100 µM to 2.88 µM, the last value being comparable to that of reference drug. Cruzain inhibitory activity ranged from 25.2 µM to 2.2 µM. The nitric oxide releasing potential was evaluated using the indirect method of detection and nitrate values ranged between 52.0 µM and 4,232.0 µM. These results are below than those of the standard compound, and there is no direct correlation between the biological activity and nitric oxide releasing potential as well. Further, the two most active compounds (6 and 14) were submitted to permeability and cytotoxicity studies. Compound 6 showed the highest permeability value according to Biopharmaceutics Classification System (BCS), and both compounds showed flow rate lower than 2, indicating no efflux mechanism. In the cytotoxicity studies of these compounds in human cells, the derivative 6 showed selectivity index greater than benznidazole. In molecular modeling studies using exploratory data analysis (HCA and PCA) steric/geometric and electronic properties were considered the most relevant for biological activity. In addition, docking studies were performed and showed that the position of the nitro group on the aromatic ring is important for the interaction with cruzain. Compound 6 did not cause significant changes in cell cycle and DNA fragmentation in human cells, showing to be a promising lead compound for future in vivo studies. Trypanocidal activity, cytotoxicity assay, NO releasing potential and permeability studies of the 23 sulfonylhydrazones and nitrate ester derivatives are being evaluated

Planejamento de fármacos anti-T. cruzi: Síntese de compostos nitrofurânicos, avaliação da atividade biológica in vitro e estudos de relações estrutura-atividade / Design of anti-T. cruzi compounds: Synthesis of 5-nitrofuran derivatives, in vitro biological activity and study of structure-activity relationships

A doença de Chagas afeta cerca de 6 a 7 millhões de pessoas no mundo, principalmente América Latina. A busca de alternativas terapêuticas para esta enfermidade tem grande relevância para a sociedade, já que as opções atuais são limitadas, sendo disponível apenas o benznidazol (BZD) e nifurtimox. Os derivados nitroheterocíclicos são considerados compostos bioativos com número crescente de estudos na comunidade científica contra seu agente etiológico, o Trypanosoma cruzi. Neste sentido, o presente trabalho tem por objetivo a identificação de derivados do 5-nitrofurano com atividade frente a diferentes cepas do T. cruzi, assim como estudar possíveis modo de ação desta classe de compostos. Esta investigação envolve estudos computacionais com o propósito de construir modelos quantitativos de relações estrutura-atividade (QSAR multivariado) que possam auxiliar na previsão de novas estruturas com perfil farmacológico otimizado. No presente trabalho foram realizadas as etapas de planejamento, síntese e identificação de 36 compostos com resultados satisfatórios quanto à identificação estrutural, pureza e rendimento, que foi da ordem de 70%. A determinação da atividade anti-T. cruzi in vitro dos compostos obtidos foi realizada frente às cepas Silvio X10 cl1, Y, Bug 2149 cl10 e Colombiana na forma epimastigota do parasito. A maioria dos compostos analisados apresentou maior capacidade de inibição de crescimento do parasito, comparado ao BZD: Silvio X10 cl1 - IC50 = 29,16 ±2,90 µM, Y - IC50 = 40,40 ±3,37µM, Bug 2149 cl10 - IC50 = 30,63 ±3,21 µM, Colombiana - IC50 = 47,91 ±4,96 µM. O composto mais ativo (BSF-35) apresentou os seguintes valores: Silvio X10 cl1 - IC50 = 3,17 ±0,32 µM, Y - IC50 = 1,17 ±0,12 µM, Bug 2149 cl10 - IC50 = 1,81 ±0,18 µM e Colombiana - IC50 = 3,06 ±0,23 µM. Foram realizados cálculos de propriedades moleculares das estruturas tridimensionais dos compostos, seguido pela análise exploratória de dados por análise de agrupamentos hierárquicos (HCA) e análise de componentes principais (PCA), possibilitando o reconhecimento de padrões do conjunto. Considerando esta análise prévia, foram obtidos modelos QSAR com abordagem multivariada, aplicando algorítmo OPS e método de regressão por quadrados mínimos parciais, PLS. Os melhores modelos gerados foram obtidos considerando os compostos benzenos substituídos para as quatro cepas estudadas. Os descritores que mais influenciaram na análise foram o ClogP (coeficiente de partição) e cargas CHELPG. Considerando as informações obtidas, foram planejados e sintetizados quatro novos compostos com objetivo de obter compostos mais ativos e validar os modelos QSAR. Estes compostos apresentaram alta atividade frente a forma epimastigota das quatro cepas estudadas. Os compostos mais ativos foram avaliados quanto a citotoxicidade frente células LLC-MK2 e apresentaram seletividade até 25 vezes superior ao BZD. Estudos in vitro frente a forma amastigota da cepa Y em células U2OS foram realizados com metodologia fenotípica de análise de alto conteúdo (HCA') e os compostos apresentaram atividade até 64 vezes superior ao BZD e com seletividade de até 50 vezes superior a este fármaco. Quanto à determinação da atividade dos compostos frente às enzimas tripanotiona redutase (TcTR) e glutationa redutase (GR), os compostos analisados não apresentaram atividade relevante, indicando não ser este o mecanismo desta classe de compostos. Com finalidade de explorar outro possível mecanismo de ação dos compostos 5-nitrofurânicos, foi realizada a análise de potencial de redução da membrana mitocondrial, porém a morte parasitária não foi atribuída à despolarização da membrana em estudos simultâneos com iodeto de propídio

Chagas disease affects approximately 6-7 millions people worldwide, especially Latin America. The search for therapeutic alternatives for this disease is of great relevance to society, as current options are limited and there are only two available drugs: benznidazole (BZD) and nifurtimox. The nitroheterocyclic derivatives are considered bioactive compounds with increasing number of studies in the scientific community against its etiologic agent, Trypanosoma cruzi. In this sense, this work aims to identify derivatives of 5-nitrofuran with activity against different strains of T. cruzi, and to study possible mode of action of this compounds. This research involves computational studies to obtain models of quantitative structure-activity relationships (QSAR multivariate) that can help predict new structures with optimized pharmacological profile. In this work were carried out the design, synthesis and identification of 36 compounds with satisfactory results regarding the structural identification, purity and yield (approximately 70%). The determination of anti-T. cruzi activity in vitro of the compounds obtained was carried out with Silvio X10 cl1, Y, Bug 2149 CL10 and Colombiana strains of epimastigote form of the parasite. Most of the compounds examined showed greater capacity of growth inhibition of the parasite compared to the BZD (Silvio X10 CL1 - IC 50 = 29.16 ± 2.90 µM, Y - IC50 = 40.40 ± 3,37µM, 2149 CL10 Bug - IC 50 = 30.63 ± 3.21 µM, Colombiana - IC 50 = 47.91 ± 4.96 µM). The most active compound (BSF-35) showed the following values: Silvio X10 cl1 - IC 50 = 3.17 ± 0.32 uM, Y - IC 50 = 1.17 ± 0.12 µM, Bug 2149 CL10 - IC50 = 1, 81 ± 0.18 µM and Colombiana - IC 50 = 3.06 ± 0.23 µM. Calculations were performed for the molecular properties of three-dimensional structures of the compounds, followed by exploratory data analysis by hierarchical cluster analysis (HCA) and principal component analysis (PCA), allowing the recognition of the set. Considering this preliminary analysis were obtained QSAR models with multivariate approach, using OPS algorithm and regression method of partial least squares, PLS. The best generated models were obtained considering the benzyl substituted compounds for the four strains. The descriptors that most influenced the analysis were ClogP (partition coefficient) and CHELPG charges. Considering the information obtained, four new compounds were designed and synthesized to obtain more active compounds and validate QSAR models. These compounds showed high activity against epimastigote form of the four strains studied. The most active compounds were evaluated for cytotoxicity against LLC-MK2 cells and the compounds selectivity values were up to 25 times higher than BZD. In vitro studies against amastigote form of the Y strain in U2OS cells were performed with phenotypic method of high content analysis (HCA') and the compounds showed activity to 64 times higher than BZD and selectivity of up to 50 times. The activity of the compounds against trypanothione reductase enzymes (TcTR) and glutathione reductase (GR) showed no significant activity, indicating that this is not the mechanism of this class of compounds. In order to exploit another possible mechanism of action of 5-nitrofuran derivatives, analysis reduction of mitochondrial membrane potential was held, however the cell death was not attributed to membrane depolarization in simultaneous studies with propidium iodide

Estudo do mecanismo de resistência a compostos derivados da classe das tiossemicarbazonas em tripanosomatídeos, com ênfase na glicoproteína-P e na nitrorredutase do tipo I / Study class of compounds derived from the resistance mechanism of thiosemicarbazones in trypanosomatids, with emphasis on P-glycoprotein and type I nitroreductase

Os tratamentos disponíveis para a doença de Chagas e as leishmanioses não são eficientes e apresentam alta toxicidade. Diversos estudos mostram que há possibilidade de indução de resistência de Trypanosoma cruzi ao Benznidazol (BZ) o que pode interferir na eficácia do tratamento. O mesmo tem sido relatado com relação aos fármacos utilizados para o tratamento das leishmanioses, embora não exista um mecanismo de ação definido para a resistência a drogas nestes protozoários. Neste trabalho foram focalizados dois potenciais mecanismos: 1) atividade da glicoproteína-P (Pgp), uma proteína de membrana que atua como uma bomba de efluxo dependente de energia e associada ao fenótipo de resistência a múltiplas drogas (MDR); 2) a enzima nitrorredutase presente em T. cruzi (TcNTR), reponsável pela redução de nitroderivados, como BZ, para obter o efeito tripanocida. Na busca de novos compostos seletivos contra T. cruzi e Leishmania amazonensis, nosso grupo vem estudando derivados da classe das tiossemicarbazonas. Em estudos prévios foi observado que o derivado 4-N-(2-metoxi-estiril)-tiossemicarbazona (2-MEOTIO) foi o composto mais efetivo sobre diferentes formas de T. cruzi, enquanto 4-N-(4’-hidroxi-3’-metoxi-estiril)-tiossemicarbazona (3-MEOTIO) se mostrou o mais eficiente contra L. amazonensisO mecanismo de resistência a estes compostos foi avaliado, e nossos resultados mostram a participação da Pgp na resistência a 2-MEOTIO e BZ em T. cruzi, e a 3-MEOTIO em L. amazonensis...

The available drugs for the treatment of Chagas disease and leishmaniasisare not efficient and cause toxic side effects. Several studies show the possibilityof drug resistance induction to Benznidazol (BZ) in Trypanosoma cruzi, whichmay interfere with the treatment efficacy. The same has been observed regardingcompounds used to treat leishmaniasis, although more studies on drug resistancemechanism are needed. In the present study we focused on two potential drugresistance mechanisms: 1) P-glycoprotein (Pgp) activity, a membrane proteinwhich acts as an efflux pump energy-dependent and is associated with themultidrug resistance fenotype (MDR); 2) the enzyme nitroreductase (TcNTR)found in T. cruzi, which is responsible for the reduction of nitroheterocyclicderivatives, such as Bz and Nifurtimox, generating metabolites with trypanocidalactivity. In the search for new selective drugs for the treatment of Chagas diseaseand leishmaniasis, our group has been studying compounds from the class of thethiosemicarbazones. Previous studies showed that the 4-N-(2-methoxy-styryl)-thiosemicarbazone (2-MEOTIO) was the most efficient compound on differentforms of T. cruzi, whereas 4-N-(4’-hidroxy-3’-methoxy styryl)-thiosemicarbazone(3-MEOTIO) was the most active on Leishmania amazonensis. Here weevaluated the drug resistance mechanism to both thiosemicarbazone derivatives,as well as, to BZ which was used as reference drug for T. cruzi. Our results showthe participation of Pgp in the resistance to both 2-MEOTIO and BZ in T. cruzi, aswell as in the resistance in L. amazonensis to the compound 3-MEOTIO.Interestingly, in T. cruzi the participation of Pgp is related to its localization notonly in the plasma membrane but also in the mithocondrion...

Avaliação do potencial terapêutico de novos agentes tripanocidas sobre o Trypanosoma cruzi / Therapeutic potential of the evaluation of new trypanocidal agents on trypanosoma cruzi

O tratamento etiológico disponível para a doença de Chagas, causada peloparasito intracelular Trypanosoma cruzi, é baseado em dois nitroderivados,benzonidazol (Bz) e nifurtimox (Nif), ambos introduzidos empiricamente na práticaclínica há mais de 40 anos. Estes fármacos são considerados insatisfatóriosprincipalmente devido à (i) baixa eficácia, principalmente na fase crônica, (ii) efeitoscolaterais importantes, e (iii) ocorrência de linhagens de parasitas resistentes. Umdos atuais desafios desta doença negligenciada é o desenvolvimento de tratamentosalternativos mais efetivos e seletivos, constituindo o objetivo principal da presentedissertação. Assim, ensaios in vitro e in vivo foram conduzidos para avaliar aeficácia de diamidinas aromáticas (DAs) e arilimidamidas (AIAs), sobre T. cruzi. Noprimeiro artigo demonstramos a atividade de dez diamidinas sobre formastripomastigotas, na faixa micromolar, sem redução significativa da viabilidade dacélula hospedeira. Três DAs com anéis externos benzimidazólicos N-metiladosapresentaram diferenças na atividade tripanocida, sendo o composto DB2247, commeta-N-metilação em ambos os anéis, o mais ativo e também o de mais rápidaação. Todavia, nenhum do compostos testados foi ativo sobre amastigotasintracelulares. No segundo artigo avaliamos atividade anti-T. cruzi de oito novasAIAs. Nossos dados mostram que seis destes compostos foram inativos sobreambas formas evolutivas do parasito. As duas AIAs que apresentaram efeito sobreas formas tripomastigotas foram 18SAB075 e 16DAP005, que exibiram aindaexcelente ação in vitro sobre formas intracelulares, com eficácia similar ao Bz...

The available etiologic treatment of Chagas disease, caused by theintracellular parasite Trypanosoma cruzi, is based on two nitroderivatives,benznidazole (Bz) and Nif, both introduced empirically in the clinical practice for over40 years ago. These drugs are considered unsatisfactory mainly due to their (i) lowefficacy, mainly in the chronic phase, (ii) severe side effects, and (iii) occurrence ofresistant parasite strains. One of the main challenges of this neglected disease is thedevelopment of more effective and selective therapies, which is our main objective.Thus, in vitro and in vivo studies were conducted to evaluate the efficacy of aromaticamidines (DAs) and arylimidamides (AIAs) against T. cruzi. In the first paper, wedemonstrated the activity of ten novel diamidines at micromolar range againstagainst trypomastigotes, without significant loss in host cell viability. In this study wefound that three diamidines with N-methylated benzimidazoles outer rings displayeddifferent trypanocidal activities, being the compound DB2247, with meta-Nmethylationin both rings, the most active with also a faster trypanocidal action.However, none of the diamidines were active against intracellular amastigotes. In thesecond paper, we evaluated the efficacy of eight novel AIAs against T. cruzi. Ourdata showed that six out of the eight studied compounds were inactive against bothevolutive forms of the parasite. The only two AIAs that were active, 18SAB075 and16DAP005, displayed outstanding in vitro effect with efficacy similar to that of Bzagainst intracellular forms. In this way, the compound 18SAB075, which had anexcellent selective index for bloodstream trypomasitgotes (SI > 106), was moved toin vivo tests for acute toxicity and parasite efficacy...

Experiencia terapéutica en un centro para la enfermedad de Chagas / Therapeutic experience in a Chagas disease center

American Trypanosomiasis or Chagas Disease is a major public health problem, endemic in the American continent since prehistoric times. Its natural course is towardschronicity in the immunocompetent host, often leading to severe cardiopathy or bowelinvolvement. Pharmacologic therapy is restricted to two drugs and only one of themis currently available in Chile. Both have poor effectiveness in the chronic stages ofthe disease and cause frequent adverse reactions. Many physicians avoid their use,despite published evidences about the usefulness. We herein report the experienceof our Center in the treatment of Chronic Chagas Disease in adults using the drugnifurtimox, emphasizing its degree of acceptability and its secondary effects.

Evaluation of the ELISA-F29 test as an early marker of therapeutic efficacy in adults with chronic Chagas disease / Avaliação do tratamento tripanossomicida em coorte de adultos chagásicos crônicos através de técnicas sorológicas convencionais e ELISA-F29

This work compared the time at which negative seroconversion was detected by conventional serology (CS) and by the ELISA-F29 test on a cohort of chronic chagasic patients treated with nifurtimox or benznidazole. A retrospective study was performed using preserved serum from 66 asymptomatic chagasic adults under clinical supervision, and bi-annual serological examinations over a mean follow-up of 23 years. Twenty nine patients received trypanocide treatment and 37 remained untreated. The ELISA-F29 test used a recombinant antigen which was obtained by expressing the Trypanosoma cruzi flagellar calcium-binding protein gene in Escherichia coli. Among the untreated patients, 36 maintained CS titers. One patient showed a doubtful serology in some check-ups. ELISA-F29 showed constant reactivity in 35 out of 37 patients and was negative for the patient with fluctuating CS. The treated patients were divided into three groups according to the CS titers: in 13 they became negative; in 12 they decreased and in four they remained unchanged. ELISA-F29 was negative for the first two groups. The time at which negativization was detected was significantly lower for the ELISA-F29 test than for CS, 14.5 ± 5.7 and 22 ± 4.9 years respectively. Negative seroconversion was observed in treated patients only. The results obtained confirm that the ELISA-F29 test is useful as an early indicator of negative seroconversion in treated chronic patients.

Este trabalho comparou os tempos de soroconversão negativos obtidos pela sorologia convencional (CS) e teste ELISA-F29 em uma coorte de pacientes chagásicos crônicos tratados com nifurtimox ou benznidazol. Um estudo retrospectivo foi realizado com soro preservado de 66 adultos chagásicos assintomáticos com acompanhamento clínico e sorológico semestral ao longo de um seguimento médio de 23 anos. 29 pacientes receberam tratamento tripanossomicida e 37 outras permaneceram sem tratamento. O teste ELISA-F29 usou um antígeno recombinante obtido por expressão do gene de uma proteína flagelar de Trypanosoma cruzi de ligação de cálcio em Escherichia coli. Entre os pacientes não tratados, 36 mantiveram os títulos da CS. Um paciente apresentou sorologia duvidosa em alguns controles. ELISA-F29 apresentou reatividade constante em 35/37 e foi negativo no paciente com CS flutuante. Os pacientes tratados foram agrupados de acordo com os títulos da CS, em três grupos: 13 tornaram-se negativos, 12 diminuíram e quatro permaneceram inalterados. ELISA-F29 foi negativo nos dois primeiros grupos. O tempo de negativização foi significativamente menor para o teste ELISA-F29 do que para CS (14,5 ± 5,7 e 22 ± 4,9 anos, respectivamente). A soroconversão negativa foi observada somente nos pacientes tratados. Os resultados obtidos confirmam que o teste ELISA-F29 é útil como um indicador precoce de soronegativação em pacientes crônicos tratados.