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.

New safe and effective treatments for Chagas disease (CD) are urgently needed. Current chemotherapy options for CD have significant limitations, including failure to uniformly achieve parasitological cure or prevent the chronic phase of CD, and safety and tolerability concerns. Fexinidazole, a 2-subsituted 5-nitroimidazole drug candidate rediscovered following extensive compound mining by the Drugs for Neglected Diseases initiative and currently in Phase I clinical study for the treatment of human African trypanosomiasis, was evaluated in experimental models of acute and chronic CD caused by different strains of Trypanosoma cruzi. We investigated the in vivo activity of fexinidazole against T. cruzi, using mice as hosts. The T. cruzi strains used in the study were previously characterized in murine models as susceptible (CL strain), partially resistant (Y strain), and resistant (Colombian and VL-10 strains) to the drugs currently in clinical use, benznidazole and nifurtimox. Our results demonstrated that fexinidazole was effective in suppressing parasitemia and preventing death in infected animals for all strains tested. In addition, assessment of definitive parasite clearance (cure) through parasitological, PCR, and serological methods showed cure rates of 80.0% against CL and Y strains, 88.9% against VL-10 strain, and 77.8% against Colombian strain among animals treated during acute phase, and 70% (VL-10 strain) in those treated in chronic phase. Benznidazole had a similar effect against susceptible and partially resistant T. cruzi strains. Fexinidazole treatment was also shown to reduce myocarditis in all animals infected with VL-10 or Colombian resistant T. cruzi strains, although parasite eradication was not achieved in all treated animals at the tested doses. Fexinidazole is an effective oral treatment of acute and chronic experimental CD caused by benznidazole-susceptible, partially resistant, and resistant T. cruzi. These findings illustrate the potential of fexinidazole as a drug candidate for the treatment of human CD. This study describes the in vivo activity of fexinidazole against Trypanosoma cruzi, the protozoan parasite causing Chagas disease, using mice infected with parasite strains with varying susceptibility to benznidazole, the standard treatment for Chagas. Fexinidazole and benznidazole were shown to have similar activity against benznidazolesusceptible and partially resistant T. cruzi strains (CL and Y), but fexinidazole had potent activity against benznidazole-resistant strains (VL-10 and Colombian). Fexinidazole treatment resulted in parasitological cure during acute disease phase in 88.9% of mice infected with the VL-10 strain and 78% with Colombian strain; benznidazole treatment did not result in cure in animals infected with these strains. Fexinidazole treatment was also shown to reduce myocarditis in all VL-10- and Colombian-infected animals, although parasite eradication was not achieved in all treated animals. These data demonstrate that it is possible to achieved better cure rates with fexinidazole in these experimental infection models than what is achieved with the standard benznidazole at the doses tested in this animal study benznidazole treatment regimen.

Chronic myocarditis is the main pathological finding associated with Chagas disease—related morbidity. Concentrations of CCL2, CCL3, tumor necrosis f

Current chemotherapy for Chagas disease is unsatisfactory due to its limited efficacy, particularly in the chronic phase, with frequent side effects that can lead to treatment discontinuation. Combined therapy is envisioned as an ideal approach since it may improve treatment efficacy whilst decreasing toxicity and the likelihood of resistance development. We evaluated the efficacy of posaconazole in combination with benznidazole on Trypanosoma cruzi infection in vivo. Benznidazole and posaconazole were administered individually or in combination in an experimental acute murine infection model. Using a rapid treatment protocol for 7 days, the combined treatments were more efficacious in reducing parasitemia levels than the drugs given alone, with the effects most evident in combinations of sub-optimal doses of the drugs. Subsequently, the curative action of these drug combinations was investigated, using the same infection model and 25, 50, 75 or 100 mg/kg/day (mpk) of benznidazole in combination with 5, 10 or 20 mpk of posaconazole, given alone or concomitantly for 20 days. The effects of the combination treatments on parasitological cures were higher than the sum of such effects when the drugs were administered separately at the same doses, indicating synergistic activity. Finally, sequential therapy experiments were carried out with benznidazole or posaconazole over a short interval (10 days), followed by the second drug administered for the same period of time. It was found that the sequence of benznidazole (100 mpk) followed by posaconazole (20 mpk) provided cure rates comparable to those obtained with the full (20 days) treatments with either drug alone, and no cure was observed for the short treatments with drugs given alone. Our data demonstrate the importance of investigating the potential beneficial effects of combination treatments with marketed compounds, and showed that combinations of benznidazole with posaconazole have a positive interaction in murine models of Chagas disease. In this study, we investigated the efficacy of posaconazole in combination with benznidazole against Trypanosoma cruzi acute infections in mice, to support the potential clinical evaluation of such combination therapy for Chagas disease. The curative action of benznidazole/posaconazole combinations was explored in an established acute infection model with the Y strain in which benznidazole and posaconazole treatments induced a 70% and 80% cure rate, respectively, when administered alone at optimal doses. When tested in combination, a 80% to 90% cure rate was detected in mice receiving 25, 50 or 75 mpk of benznidazole, plus 5 or 10 mpk of posaconazole, while treatment with the sub-optimal doses of the drugs given alone induced only 0–43% cures, indicating synergistic effects. Finally, sequential short (10 days) treatments with benznidazole (100 mpk) followed by posaconazole (20 mpk) led to an 80% cure rate, comparable with full-length treatments with either drug given alone, while no cures were observed for short treatments with single drugs. Our results demonstrate that it is possible to achieve the same or better therapeutic effect using lower dosages of posaconazole and benznidazole in combination, decreasing treatment costs and potential toxicity.

In this work, we investigated the in vivo activity of ravuconazole against the Y and Berenice-78 Trypanosoma cruzi strains using acutely infected dogs as hosts. Ravuconazole was well tolerated, as no significant side effects were observed during the treatment using 6.0 mg/kg twice a day (12 mg/kg/day) for up to 90 days. In all treated animals, parasitemia was permanently suppressed by the first day of treatment, independently of the parasite strain. Cultures of blood obtained posttreatment were negative for 90% of the animals, confirming that the drug induced a marked reduction in the parasite load. The results of PCR tests for T. cruzi in blood performed 1 month posttreatment were consistently negative for three of five and two of five animals infected with the Y and Berenice-78 strains, respectively. All ravuconazole-treated dogs consistently had negative serological test results during and until 30 days after treatment, regardless of the therapeutic scheme used. However, after the end of treatment, an increase in specific antibody levels was observed in all treated animals, although the antibody levels were always significantly lower than those of the nontreated control dogs. Despite being unable to induce a parasitological cure, ravuconazole treatment led to significant reductions in the levels of gamma interferon expression and lesions in cardiac tissues in animals infected with the Y strain, while the level of interleukin-10 mRNA expression increased. We conclude that ravuconazole has potent suppressive but not curative activity in the canine model of acute Chagas' disease, probably due to its unfavorable pharmacokinetic properties (half-life, 8.8 h). The longer half-life of ravuconazole in humans (4 to 8 days) makes it a promising drug for assessment for use as chemotherapy in human Chagas' disease.