Perspectives for a new drug candidate for Chagas disease therapy

Chagas disease (CD), a neglected tropical illness caused by the protozoan Trypanosoma cruzi, affects more than 6 million people mostly in poor areas of Latin America. CD has two phases: an acute, short phase mainly oligosymptomatic followed to the chronic phase, a long-lasting stage that may trigger cardiac and/or digestive disorders and death. Only two old drugs are available and both present low efficacy in the chronic stage, display side effects and are inactive against parasite strains naturally resistant to these nitroderivatives. These shortcomings justify the search for novel therapeutic options considering the target product profile for CD that will be presently reviewed besides briefly revisiting the data on phosphodiesterase inhibitors upon T. cruzi.

Mechanism of action of glycyrrhizin against Plasmodium falciparum

Extracts of the plant Glycyrrhiza glabra (licorice) are used in traditional medicine to treat malaria. The main active components are the saponin glycyrrhizin (GLR) and its active metabolite glycyrrhetinic acid (GA) which both display activities against Plasmodium falciparum. We have identified three main mechanisms at the origin of their anti-plasmodial activity: (i) drug-induced disorganisation of membrane lipid rafts, (ii) blockade of the alarmin protein HMGB1 and (iii) potential inhibition of the detoxifying enzyme glyoxalase 1 (GLO-1) considered as an important drug target for malaria. Our analysis shed light on the mechanism of action of GLR against P. falciparum.

Efficacy of sertraline against Trypanosoma cruzi: an in vitro and in silico study

Drug repurposing has been an interesting and cost-effective approach, especially for neglected diseases, such as Chagas disease. Methods: In this work, we studied the activity of the antidepressant drug sertraline against Trypanosoma cruzi trypomastigotes and intracellular amastigotes of the Y and Tulahuen strains, and investigated its action mode using cell biology and in silico approaches. Results: Sertraline demonstrated in vitro efficacy against intracellular amastigotes of both T. cruzi strains inside different host cells, including cardiomyocytes, with IC50 values between 1 to 10 µM, and activity against bloodstream trypomastigotes, with IC50 of 14 µM. Considering the mammalian cytotoxicity, the drug resulted in a selectivity index of 17.8. Sertraline induced a change in the mitochondrial integrity of T. cruzi, resulting in a decrease in ATP levels, but not affecting reactive oxygen levels or plasma membrane permeability. In silico approaches using chemogenomic target fishing, homology modeling and molecular docking suggested the enzyme isocitrate dehydrogenase 2 of T. cruzi (TcIDH2) as a potential target for sertraline. Conclusions: The present study demonstrated that sertraline had a lethal effect on different forms and strains of T. cruzi, by affecting the bioenergetic metabolism of the parasite. These findings provide a starting point for future experimental assays and may contribute to the development of new compounds.(AU)

Impact of autologous whole blood administration upon experimental mouse models of acute Trypanosoma cruzi infection

Background: Autologous whole blood (AWB) administration is described as alternative/complementary medical practice widely employed in medical and veterinary therapy against infections, chronic pathologies and neoplasias. Our aim is to investigate in vivo biological effect of AWB using healthy murine models under the course of Trypanosoma cruzi acute infection. Methods: The first set of studies consisted of injecting different volumes of AWB and saline (SAL) into the posterior region of quadriceps muscle of healthy male Swiss mice under distinct therapeutic schemes evaluating: animal behavior, body and organ weight, hemogram, plasmatic biochemical markers for tissue damage and inflammatory cytokine levels and profile. To assess the impact on the experimental T. cruzi infection, different schemes (prior and post infection) and periods of AWB administration (from one up to 10 days) were conducted, also employing heterologous whole blood (HWB) and evaluating plasma cytokine profile. Results: No major adverse events were observed in healthy AWB-treated mice, except gait impairment in animals that received three doses of 20 L AWB in the same hind limb. AWB and SAL triggered an immediate polymorphonuclear response followed by mononuclear infiltrate. Although SAL triggered an inflammatory response, the kinetics and intensity of the histological profile and humoral mediator levels were different from AWB, the latter occurring earlier and more intensely with concomitant elevation of plasma IL-6. Inflammatory peak response of SAL, mainly composed of mononuclear cells with IL-10, was increased at 24 h. According to the mouse model of acute T. cruzi infection, only minor decreases ( 30%) in the parasitemia levels were produced by AWB and HWB given before and after infection, without protecting against mortality. Rises in IFN-gamma, TNF-alpha and...

Impact of autologous whole blood administration upon experimental mouse models of acute Trypanosoma cruzi infection

Autologous whole blood (AWB) administration is described as alternative/complementary medical practice widely employed in medical and veterinary therapy against infections, chronic pathologies and neoplasias. Our aim is to investigate in vivo biological effect of AWB using healthy murine models under the course of Trypanosoma cruzi acute infection. Methods: The first set of studies consisted of injecting different volumes of AWB and saline (SAL) into the posterior region of quadriceps muscle of healthy male Swiss mice under distinct therapeutic schemes evaluating: animal behavior, body and organ weight, hemogram, plasmatic biochemical markers for tissue damage and inflammatory cytokine levels and profile. To assess the impact on the experimental T. cruzi infection, different schemes (prior and post infection) and periods of AWB administration (from one up to 10 days) were conducted, also employing heterologous whole blood (HWB) and evaluating plasma cytokine profile. Results: No major adverse events were observed in healthy AWB-treated mice, except gait impairment in animals that received three doses of 20 µL AWB in the same hind limb. AWB and SAL triggered an immediate polymorphonuclear response followed by mononuclear infiltrate. Although SAL triggered an inflammatory response, the kinetics and intensity of the histological profile and humoral mediator levels were different from AWB, the latter occurring earlier and more intensely with concomitant elevation of plasma IL-6. Inflammatory peak response of SAL, mainly composed of mononuclear cells with IL-10, was increased at 24 h. According to the mouse model of acute T. cruzi infection, only minor decreases (< 30%) in the parasitemia levels were produced by AWB and HWB given before and after infection, without protecting against mortality. Rises in IFN-gamma, TNF-alpha and IL-6 were detected at 9 dpi in all infected animals as compared to uninfected mice but only Bz displayed a statistically significant diminution (p= 0.02) in TNF-alpha levels than infected and untreated mice. Conclusions: This study revealed that the use of autologous whole blood (AWB) in the acute model employed was unable to reduce the parasitic load of infected mice, providing only a minor decrease in parasitemia levels (up to 30%) but without protecting against animal mortality. Further in vivo studies will be necessary to elucidate the effective impact of this procedure.(AU)

The biological in vitro effect and selectivity of aromatic dicationic compounds on Trypanosoma cruzi

Trypanosoma cruzi is a parasite that causes Chagas disease, which affects millions of individuals in endemic areas of Latin America. One hundred years after the discovery of Chagas disease, it is still considered a neglected illness because the available drugs are unsatisfactory. Aromatic compounds represent an important class of DNA minor groove-binding ligands that exhibit potent antimicrobial activity. This study focused on the in vitro activity of 10 aromatic dicationic compounds against bloodstream trypomastigotes and intracellular forms of T. cruzi. Our data demonstrated that these compounds display trypanocidal effects against both forms of the parasite and that seven out of the 10 compounds presented higher anti-parasitic activity against intracellular parasites compared with the bloodstream forms. Additional assays to determine the potential toxicity to mammalian cells showed that the majority of the dicationic compounds did not considerably decrease cellular viability. Fluorescent microscopy analysis demonstrated that although all compounds were localised to a greater extent within the kinetoplast than the nucleus, no correlation could be found between compound activity and kDNA accumulation. The present results stimulate further investigations of this class of compounds for the rational design of new chemotherapeutic agents for Chagas disease.

In vitro and in vivo experimental models for drug screening and development for Chagas disease

Chagas disease, a neglected illness, affects nearly 12-14 million people in endemic areas of Latin America. Although the occurrence of acute cases sharply has declined due to Southern Cone Initiative efforts to control vector transmission, there still remain serious challenges, including the maintenance of sustainable public policies for Chagas disease control and the urgent need for better drugs to treat chagasic patients. Since the introduction of benznidazole and nifurtimox approximately 40 years ago, many natural and synthetic compounds have been assayed against Trypanosoma cruzi, yet only a few compounds have advanced to clinical trials. This reflects, at least in part, the lack of consensus regarding appropriate in vitro and in vivo screening protocols as well as the lack of biomarkers for treating parasitaemia. The development of more effective drugs requires (i) the identification and validation of parasite targets, (ii) compounds to be screened against the targets or the whole parasite and (iii) a panel of minimum standardised procedures to advance leading compounds to clinical trials. This third aim was the topic of the workshop entitled Experimental Models in Drug Screening and Development for Chagas Disease, held in Rio de Janeiro, Brazil, on the 25th and 26th of November 2008 by the Fiocruz Program for Research and Technological Development on Chagas Disease and Drugs for Neglected Diseases Initiative. During the meeting, the minimum steps, requirements and decision gates for the determination of the efficacy of novel drugs for T. cruzi control were evaluated by interdisciplinary experts and an in vitro and in vivo flowchart was designed to serve as a general and standardised protocol for screening potential drugs for the treatment of Chagas disease.

Inhibition of Trypanosoma cruzi proline racemase affects host-parasite interactions and the outcome of in vitro infection

Proline racemase is an important enzyme of Trypanosoma cruzi and has been shown to be an effective mitogen for B cells, thus contributing to the parasite's immune evasion and persistence in the human host. Recombinant epimastigote parasites overexpressing TcPRAC genes coding for proline racemase present an augmented ability to differentiate into metacyclic infective forms and subsequently penetrate host-cells in vitro. Here we demonstrate that both anti T. cruzi proline racemase antibodies and the specific proline racemase inhibitor pyrrole-2-carboxylic acid significantly affect parasite infection of Vero cells in vitro. This inhibitor also hampers T. cruzi intracellular differentiation.

Clássicos em doença de Chagas: histórias e perspectivas / Classics in Chagas disease: histories and perspectives

Em 1909, Carlos Chagas anunciou a descoberta de uma nova doença: a tripanossomíase humana, doença de Chagas. A descoberta representou não apenas uma contribuição inovadora para o campo da medicina tropical, em particular os estudos sobre as doenças parasitárias, mas também mostrou a realidade sanitária e social do interior do país, assolado pelas endemias rurais. Cem anos depois nos deparamos com uma valiosa oportunidade de reflexão sobre os múltiplos sentidos e implicações da comemoração do grande feito de Carlos Chagas, que, tal como a biografia do cientista, associam ciência, saúde pública e projetos para a nação. Comemorar, lembrar junto, significa, assim, refletir sobre as várias temporalidades imbricadas nesta data: a memória de um passado que engendou o presente e convida a pensar os desafios do futuro. A história, tecendo os fios de um tempo construído por tantos indivíduos e gerações ao longo destes cem anos, apresenta-se como caminho para congregar todos aqueles que se associam a este legado e pretendem compartilhá-lo entre as novas gerações. Diante deste cenário e do atual quadro epidemiológico de uma enfermidade que ainda afeta milhões de pessoas em toda a América Latina, evidencia-se a importância de se articular ações que permitam suprir as lacunas relacionadas ao conhecimento da doença de Chagas e às medidas efetivas para tratamento e controle. Os textos reunidos neste livro constituem um importante marco nesse sentido. Clássicos em Doença de Chagas: história e perspectivas no cenário da descoberta lança nova luz sobre os estudos da doença a partir da reunião de 15 artigos científicos comentados por renomados pesquisadores, especialmente convidados para esta homenagem. Estamos diante de um valioso acervo documental que esperamos possa estimular o avanço do conhecimento e, assim, contribuir para a busca de soluções dos problemas sociais e de saúde relacionados á enfermidade de Chagas.

Contribuição do estudo de Zigman Brener (1961) para a terapia da doença de Chagas / Contribution of the study of Zigman Brener (1961) for the treatment of Chagas disease

Revisões históricas aos avanços científicos para o controle da doença, o Simpósio Internacional Comemorativo do Centenário da Descoberta da Doença de Chagas (1909-2009).

O futuro da pesquisa em doença de chagas e o legado de Carlos Chagas / The future of research on Chagas disease and the legacy of Carlos Chagas

Revisões históricas aos avanços científicos para o controle da doença, o Simpósio Internacional Comemorativo do Centenário da Descoberta da Doença de Chagas (1909-2009).

In vitro measurement of enzymatic markers as a tool to detect mouse cardiomyocytes injury

Primary cultures of cardiomyocytes represent a useful model for analyzing cardiac cell biology as well as pathogenesis of several cardiovascular disorders. Our aim was to standardize protocols for determining the damage of cardiac cells cultured in vitro by measuring the creatine kinase and its cardiac isotype and lactate dehydrogenase activities in the supernatants of mice cardiomyocytes submitted to different protocols of cell lysis. Our data showed that due to its higher specificity, the cardiac isotype creatine kinase was the most sensitive as compared to the others studied enzymatic markers, and can be used to monitor and evaluate cardiac damage in in vitro assays.

Estudos sobre componentes da fase aguda da infecção experimental por Trypanosoma cruzi: invasão parasita-célula hospedeira e inibidores de protease da família das Alfa-Macroglobulinas / Studies on components of the acute phase of the experimental infection for Trypanosoma cruzi: invasion parasite-cell hostess and inhibitors of protease of the family of Alfa-Macroglobulinas

O Trypanosoma cruzi é o agente etiológico da doença de Chagas, uma importante parasitose em diversos países da América Latina. O coração é um dos principais órgãos afetados, nas fases aguda e crônica. No primeiro bloco de nosso trabalho analisamos a expressão de um receptor para resíduos de manose/fucose em cardiomiócitos, antes e durante a sua infecção in vitro pelo parasita. Nesse estudos aplicamos um sistema rápido de quantificação da carga parasitária de culturas de células infectadas pelo T. cruzi, por meio de um ensaio Elisa, previamente padronizado nesta tese para essa finalidade... No segundo bloco, estudamos a expressão in vivo dos mensageiros para duas proteínas da família das alfa-macroglobulinas e do seu sistema receptor, e sua atividade antes e após a infecção, correlacionado-os com alguns parâmetros parasitológicos e histopatológicos. Analisamos comparativamente duas linhagens isogênicas de camundongos: C3H e C57BL/6 que, infectados com 104 parasitas da cepa Y, apresentaram parasitemia e mortalidade semelhantes. Observamos que a transcrição e síntese de alfa-macroglobulina apresenta níveis basais maiores em animais C3H que em B6, que são ainda mais acentuados durante a infecção pelo T. cruzi. Verificamos também que baixos níveis de A2M se associam a maiores e mais rápidos níveis de parasitismo no coração. Além disso, observamos que animais B6 não infectados apresentam uma maior transcrição e expressão de A2MR/LRP que animais C3H, o que é sugestivo de um melhor sistema de ôclearanceö de AM nos animais B6, em relação aos animais da linhagem C3H. Durante a infecção pelo T. cruzi observamos diferenças na transcrição e expressão de A2MR/LRP no fígado e no coração de animais B6 e C3H, possivelmente em razão de diferenças na natureza e concentração de citocinas; a hipótese de que a disponibilidade de TNF-gama e TGF-B possa ser regulada pela diferente expressão de AM nestes animais ainda precisa ser investigada. Embora o conjunto de resultados nos sugiram que a A2M pode realmente ser um modulador da invasão celular, em especial no coração, esse papel não é decisivo para o prognóstico de sobrevida do animal infectado. Isso provavelmente será influenciado mais pelo balanço de citocinas pró-inflamatórias (TNF-gama e TGF-B) e imunes (IFN-gama), as quais em concentrações adequadas e controladas concorreriam para a resistência do animal na fase aguda, associada ou não ao desenvolvimento de patologia na fase crônica.