Effects of low doses of benznidazole nanosystems in both acute and chronic experimental Chagas disease. A new promising treatment regimen.

CLAUDIO JAVIER SALOMON; EVA CAROLINA ARRUA; LAURA E. FICHERA; MARCELA S. RIAL; MARIA L. SCALISE

San Diego

Encuentro; AAPS Annual Meeting and Exposition; 2017

Effects of low doses of benznidazole nanosystems in both acute and chronic experimental Chagasdisease. A new promising treatment regimen.M. Rial1, C. Arrúa2, M. Scalise1, L. Fichera1, C. Salomon21Instituto Nacional de Parasitología Fatala Chabén, Argentina, 2Universidad de Rosario, ArgentinaPurposeChagas? disease, a neglected disease caused by the protozoan Trypanosoma cruzi and transmitted by triatomine bugs, is the mostprevalent parasitic disease in Latin America. Lately, and due to movement of infected people from endemic areas to non-endemicregions Chagas disease has been detected in North America, Europe and Japan, also. Benznidazole (BNZ) is one of the two availabledrugs for the treatment of Chagas disease. Although it is the drug of choice in many regions, BNZ has serious undesirable effectsincluding anorexia and weight loss, nausea, headache, nervous excitation and allergic dermatitis. Therefore, alternative treatmentsusing lower doses of BNZ is urgently required. The aim of this work to evaluate the efficacy of low doses of BNZ nanoparticlesduring the acute phase in mice infected with TcN that were immunosuppressed during the chronic stage of disease. Additionally,production of T. cruzi-specific antibodies and cardiac tissue inflammation in mice after treatment with this BNZ nanoformulationswere also investigated.MethodsBNZ nanoparticles were prepared by solvent diffusion method. BNZ (200 mg) was dissolved in ethanol (10 ml). The solution wasinjected (1 ml min−1) into water (20 ml) containing P188 (300 mg) under stirring (1000 rpm/60 min). The solution was stirred (500rpm) for 18 h at room temperature to allow solvent evaporation. Nanoparticles were recovered by centrifugation for 20 min (15000rpm), washed twice with distilled water, frozen overnight at -20 oC and freeze-dried (48 hours).Animal model.Five groups of ten one-month-old female C3H/HeN mice, were inoculated intraperitoneally with 1000 culture-derived trypomastigotesof the TcN. Infected mice were divided into the following groups (n=10): (1) infected mice without treatment, (2) infected micetreated with raw BNZ with daily doses of 50 mg/kg body weight for 30 days (2 to 32 dpi) (R-BNZ 50), (3) infected mice treated withBNZ nanoparticles for 30 days with daily doses of 50 mg/kg/day (BNZ-nps 50), (4) infected mice treated with BNZ nanoparticles for30 days with daily doses of 25 mg/kg/day (BNZ-nps 25), (5) infected mice treated with BNZ nanoparticles for 30 days with dailydoses of 10 mg/kg/day (BNZ-nps 10). R-BNZ and BNZ-nps were dispersed in olive oil and orally administered to mice, the controlgroup received olive oil alone.Induction of immunosuppression.After 60 days of inoculation, animals were immunosuppressed with three cycles of 50 mg of cyclophosphamide/kg of body weight,during four consecutive days, with an interval of 3 days between each cycle.Histopathological studies.Tissues from 8 different areas of the heart (left and right atria, upper and lower halves of each ventricular wall and septum) werescored according to the extension of inflammation.ResultsBNZ nanoparticlesFollowing the methodology of nanoprecipitation, small particles of about 63.3 ± 2.82 nm, with zeta potential of -18.30 ± 1.0, and asize distribution (polydispersity index) of 3.35 ± 0.1 were obtained.Course of infection.To determine whether BNZ nanoformulations treatments affect the course of infection and survival rate of C3H/HeN mice, suchnanoparticles were orally administered at 10, 25 and 50 mg/kg/d and compared with raw BNZ (R-BNZ) at a dose of 50 mg/kg/d. Allinfected mice treated with BNZ, no matter whether nanoparticle-formulated or raw BNZ, survived until the end of the experiment (92days p.i.). On the other hand, only 15% of the non-treated infected mice survived.Humoral immune responses specific for T. cruzi following chemotherapy.All mice treated with BNZ-nps at 25 mg/kg/day showed a decrease to T. cruzi-specific antibodies compared to titers of infectedcontrol mice. In particular, no antibodies could be detected in 50% of the animals at 3 months and 100 % at 6 months. With BNZ-npsapplied at 50 mg/kg/day all mice displayed negative titers in T. cruzi-specific antibodies at 3 months post-infection.HistopathologyAs expected, the myocardium of untreated infected mice showed extensive and multiple inflammatory foci of mononuclear cellinfiltrates and some necrotic areas with structural alterations and fibrotic foci. Parasite nests were absent in heart tissues of mice thatsurvived the acute stage. TcN-infected mice treated with raw BNZ (50 mg/kg/d) exhibited similar inflammatory damage than theuntreated infected group. In contrast, a significant decrease of inflammatory cells in heart tissue was observed after treatment withBNZ-nps at a dosage of 25 and 50 mg/kg/d.ConclusionBNZ nanoparticles could be used as an alternative to the conventionally used treatment regimen, where BNZ is applied at100mg/kg/day causing, very often, serious adverse side effects.