Population pharmacokinetics and biodistribution of benznidazole in mice.

OBJECTIVES: To evaluate the population pharmacokinetics of different benznidazole treatment regimens and the drug's biodistribution in mice. METHODS: Two hundred mice were divided into five groups according to benznidazole dosing regimens: (1) 100 mg/kg/day for 20 days; (2) 100 mg/kg/day for 40 days; (3) 200 mg/kg/day for 20 days; (4) 40 mg/kg/day for 20 days; or (5) 40 mg/kg/day for 40 days. The mice were euthanized and blood, heart, liver, colon and brain were collected. Samples were prepared by liquid-liquid extraction and analysed by HPLC-diode-array detection. The pharmacokinetic analysis of benznidazole was evaluated via non-linear mixed-effects modelling using the NONMEN program. RESULTS: Our results demonstrate that mouse weight allometrically influences benznidazole clearance; the AUC curve and the highest plasma concentration are dose proportional; benznidazole does not influence its own metabolism; its tissue distribution is limited; and the standard treatment regimen for Chagas' disease in mice (100 mg/kg/day for 20 days) is inadequate from a pharmacokinetic standpoint, as are the other regimens tested in this study (100 mg/kg/day for 40 days, 200 mg/kg/day for 20 days and 40 mg/kg/day for 20 or 40 days). CONCLUSIONS: Benznidazole reformulations that allow better tissue penetration and plasma and tissue exposure should be evaluated to enable higher cure rates in both animals and patients. The population pharmacokinetic model developed here can allow optimization of the dosing regimen of benznidazole to treat experimental Chagas' disease. Determining appropriate treatment regimens in animals allows translation of these to clinical studies.

Effect of the preservative and temperature conditions on the stability of Leishmania infantum promastigotes antigens applied in a flow cytometry diagnostic method for canine visceral leishmaniasis.

The control of canine visceral leishmaniasis (CVL) is imperative, but euthanasia of seropositive dogs has been highly criticized. Commonly used, immunodiagnostic tests, including Dual-Path Platform®, enzyme-linked immunosorbent assay, and immunofluorescent antibody test, have failed at detecting asymptomatic dogs in endemic areas. In this context, new serological methods are needed. Flow cytometry serology has demonstrated potential as a test with excellent performance for CVL. In this study, we proposed to establish the best conditions for preserving Leishmania infantum promastigote antigens employed in this serology test. During 12 months of follow-up, promastigotes were maintained in different preservatives (phosphate-buffered saline with 3% fetal bovine serum, phenol 0.35%, thimerosal 0.01%, and formaldehyde 0.5%) and stored at 3 distinct temperatures (25 °C, 4 °C, and -20 °C). During the study period, the morphological characteristics of the promastigotes were assessed by flow cytometry according to the forward and side scatter parameters and also under optical microscopic analysis. Reactivity performance was evaluated as the percentage of positive fluorescent parasites in the sera of naturally infected and noninfected dogs. Microbiological analysis was performed at 2 time points, the first and sixth months, to rule out contamination of stored promastigotes. Taken together, our results indicated that the best conditions to preserve fixed L. infantum antigens were storage in formaldehyde at 4 °C. Promastigotes presented the best morphological profile, with appropriate antigenic stability even at 4 °C, in an inexpensive preservative for a long period of conservation.