CM were maintained at 37��C in an atmosphere of 5% CO2 and air and the medium replaced every 24 h. Then, untreated and treated infected CM were fixed and stained with Giemsa solution and the mean number of infected host cells and of parasites per infected cells scored as reported [12]. Only characteristic parasite nuclei Erlotinib cancer and kinetoplasts were counted as surviving parasites since irregular structures could mean parasites undergoing death. The drug activity was estimated by calculating the infection index (II – percentage of infected cells times the average number of intracellular amastigotes per infected host cell) [12]. All assays in vitro were run at least twice in duplicates. Mice acute toxicity NOAEL (No Observed Adverse Effect Level) was evaluated using male and female Swiss Webster mice (20�C23 g).

On day 1, one female and one male mice were treated with DB1965 under two therapeutic schemes: (i) injection via intraperitoneal (ip) every 2 h, with increasing doses, starting at 25 mg/kg up to 400 mg/kg and (ii) administration by ip or per oral (p.o) at doses ranging 25�C400 mg/kg [11]. Additionally, male mice (n=5 for each group) were treated with 20 daily consecutive doses with vehicle (only DMSO and water), 5 mg/kg DB1965 (ip), 50 mg/kg Bz (p.o) and with both combined compounds (5 mg/kg DB1965 ip+50 mg/kg Bz p.o). In all schemes, mice were inspected for toxic and sub-toxic symptoms according to OECD guidelines (Organization for Economic Co-operation and Development). Forty-eight hours after compound injection, the NOAEL values were determined [11].

Mice infection and treatment schemes Male Swiss mice were obtained from the Funda??o Oswaldo Cruz (FIOCRUZ) animal facilities (Rio de Janeiro, Brazil). Mice were housed at maximum 8 per cage and kept in a conventional room at 20�C24��C under a 12/12 h light/dark cycle. The animals were provided with sterilized water and chow ad libitum. Infection was performed by ip injection of 104 bloodstream trypomastigotes. The animals (18�C21 g) were divided into the following groups (at least five mice per group): uninfected (non-infected and non-treated); untreated (infected with T. cruzi but treated only with vehicle); and treated (infected and treated – ip and p.o – with 12.5 up to 100 mg/kg/day DB1965 or with 100 mg/kg/day benznidazole). For DB1965 treatment, mice received 0.1 mL ip injection or 0.

2 mL oral dose, starting at the 5 dpi followed by (i) for 5, (ii) 10 or (iii) 20 consecutive daily doses. For Bz treatment, infected mice received Cilengitide 0.2 mL oral dose (gavage) following the same therapeutic schemes as above described. Thirty days after compound administration, about 1000 ��L of blood were collected from the heart of anesthetized mice and then 500, 200 and 250 ��L were used for PCR, hemoculture and biochemical analysis, respectively [11].