Trypanosoma cruzi Secreted Cyclophilin TcCyP19 as an Early Marker for Trypanocidal Treatment Efficiency.

Cyclophilins (CyPs) are a family of enzymes involved in protein folding. Trypanosoma cruzi, the causative agent of Chagas disease, has a 19-kDa cyclophilin, TcCyP19, that was found to be secreted in parasite stages of the CL Brener clone and recognized by sera from T. cruzi-infected mice and patients. The levels of specific antibodies against TcCyP19 in T. cruzi-infected mice and subjects before and after drug treatment were measured by an in-house enzyme linked immunosorbent assay (ELISA). Mice in the acute and chronic phase of infection, with successful trypanocidal treatments, showed significantly lower anti-TcCyP19 antibody levels than untreated mice. In children and adults chronically infected with T. cruzi, a significant decrease in the anti-TcCyP19 titers was observed after 12 months of etiological treatment. This decrease was maintained in adult chronic patients followed-up 30-38 months post-treatment. These results encourage further studies on TcCyP19 as an early biomarker of trypanocidal treatment efficiency.

In vitro studies and preclinical evaluation of benznidazole microparticles in the acute Trypanosoma cruzi murine model.

Chagas disease is a serious parasitic infection caused by Trypanosoma cruzi. Unfortunately, the current chemotherapeutic tools are not enough to combat the infection. The aim of this study was to evaluate the trypanocidal activity of benznidazole-loaded microparticles during the acute phase of Chagas infection in an experimental murine model. Microparticles were prepared by spray-drying using copolymers derived from esters of acrylic and methacrylic acids as carriers. Dissolution efficiency of the formulations was up to 3.80-fold greater than that of raw benznidazole. Stability assay showed no significant difference (P > 0.05) in the loading capacity of microparticles for 3 years. Cell cultures showed no visible morphological changes or destabilization of the cell membrane nor haemolysis was observed in defibrinated human blood after microparticles treatment. Mice with acute lethal infection survived 100% after 30 days of treatment with benznidazole microparticles (50 mg kg-1 day-1). Furthermore, no detectable parasite load measured by quantitative polymerase chain reaction and lower levels of T. cruzi-specific antibodies by enzyme-linked immunosorbent assay were found in those mice. A significant decrease in the inflammation of heart tissue after treatment with these microparticles was observed, in comparison with the inflammatory damage observed in both infected mice treated with raw benznidazole and untreated infected mice. Therefore, these polymeric formulations are an attractive approach to treat Chagas disease.

Effectiveness of the repurposed drug isotretinoin in an experimental murine model of Chagas disease.

Benznidazole and nifurtimox are the drugs currently used for the treatment of Chagas disease, however its side effects may affect patient adherence. In the search for new alternative therapies, we previously identified isotretinoin (ISO), an FDA-approved drug widely used for the treatment of severe acne through a drug repurposing strategy. ISO shows a strong activity against Trypanosoma cruzi parasites in the nanomolar range, and its mechanism of action is through the inhibition of T. cruzi polyamine and amino acid transporters from the Amino Acid/Auxin Permeases (AAAP) family. In this work, a murine model of chronic Chagas disease (C57BL/6 J mice), intraperitoneally infected with T. cruzi Nicaragua isolate (DTU TcI), were treated with different oral administrations of ISO: daily doses of 5 mg/kg/day for 30 days and weekly doses of 10 mg/kg during 13 weeks. The efficacy of the treatments was evaluated by monitoring blood parasitemia by qPCR, anti-T. cruzi antibodies by ELISA, and cardiac abnormalities by electrocardiography. No parasites were detected in blood after any of the ISO treatments. The electrocardiographic study of the untreated chronic mice showed a significant decrease in heart rate, while in the treated mice this negative chronotropic effect was not observed. Atrioventricular nodal conduction time in untreated mice was significantly longer than in treated animals. Mice treated even with ISO 10 mg/kg dose every 7 days, showed a significant reduction in anti-T. cruzi IgG levels. In conclusion, the intermittent administration of ISO 10 mg/kg would improve myocardial compromise during the chronic stage.

Elucidating the impact of low doses of nano-formulated benznidazole in acute experimental Chagas disease.

BACKGROUND: Chagas disease is a neglected parasitic infection caused by the protozoan Trypanosoma cruzi (T. cruzi) that affects more than 6 million people, mainly in Latin America. Benznidazole is still the drug of choice in many countries to treat it in spite of its dosage regimen and adverse side effects such as such as allergic dermatitis, peripheral neuropathy and anorexia. Thus, novel, safer, and more efficacious treatments for such neglected infection are urgently required. METHODOLOGY: In this study, the efficacy of orally administered low doses of benznidazole (BNZ) nanoparticles was evaluated during the acute phase in mice infected with T. cruzi Nicaragua (TcN) that were immunosuppressed during the chronic stage of the disease. Moreover, the production of T. cruzi-specific antibodies, cardiac tissue inflammation and reactive oxygen species generation by Vero cells treated with both BNZ nanoparticles (BNZ-nps) and raw BNZ (R-BNZ) were also evaluated. PRINCIPAL FINDINGS: T. cruzi infected mice treated with 10, 25 or 50 mg/kg/day of BNZ-nps survived until euthanasia (92 days post infection (dpi)), while only 15% of infected untreated mice survived until the end of the experiment. PCR analysis of blood samples taken after induction of immunosuppression showed that a dosage of 25 mg/kg/day rendered 40% of the mice PCR-negative. The histological analysis of heart tissue showed a significant decrease in inflammation after treatments with 25 and 50 mg/kg/day, while a similar inflammatory damage was observed in both infected mice treated with R-BNZ (50 mg/kg/day) and untreated mice. In addition, only BNZ-nps treated mice led to lower levels of T. cruzi-specific antibodies to 50-100%. Finally, mammalian Vero cells treated with BNZ-nps or R-BNZ lead to a significant increase in ROS production. CONCLUSIONS: Based on these findings, this research highlights the in-vitro/in-vivo efficacy of nanoformulated BNZ against T. cruzi acute infections in immunosuppressed and non-immunosuppressed mice and provides further evidence for the optimization of dosage regimens to treat Chagas disease.

Promising Efficacy of Benznidazole Nanoparticles in Acute Trypanosoma cruzi Murine Model: In-Vitro and In-Vivo Studies.

The aim of this study was to evaluate the effectiveness of benznidazole nanoparticles (BNZ-nps) on trypomastigote forms and on intracellular infection in mammalian cells and primary cardiac myocyte cells. Its effectiveness was also evaluated on acute Trypanosoma cruzi Nicaragua mice infection. Trypomastigotes from culture were treated with different concentrations of BNZ-nps to determine the drug concentration that lyses 50% of trypomastigotes (LC50). Infected mammalian cells were incubated with different concentrations of BNZ-nps to determine the percentage of amastigote inhibition. C3H/HeN mice with lethal acute infection were treated with 10, 25, and 50 mg/kg/day of BNZ-nps for 30 and 15 days to control the survival rate of animals. BNZ-nps having a mean particle size of 63.3 nm, a size distribution of 3.35, and a zeta potential of -18.30 were successfully prepared using poloxamer 188 as a stabilizer. BNZ-nps 25 and 50 µg/mL showed no significant differences in the percentage of inhibition of infected mammalian cells. Infected mice treated with BNZ-nps (50, 25, and 10 mg/kg/day) for 30 days and with BNZ-nps (50 and 25 mg/kg/day) for 15 days presented a 100% survival, whereas the animals treated with 10 mg/kg/day for 15 days of BNZ-nps showed a 70% survival rate. The results obtained demonstrate, for the first time, that benznidazole nanoparticles are a useful and attractive approach to treat Chagas disease in infected mice.