Posaconazole (SCH 56592)

The epimastigote form of the parasite is cultivated in liver infusion tryptose medium, supplemented with 10% new born calf serum at 28 °C with strong (120 rpm) agitation. Cultures are initiated at a cell density of 2 × 10⁶ epimastigotes/mL, and Posaconazole is added at a cell density of 0.5−1.0 × 10⁷ epimastigotes/mL. Cell densities are measured by using an electronic particle counter as well as by direct counting with a hemocytometer. Cell viability is followed by Trypan blue exclusion, using light microscopy. Amastigotes are cultured in Vero cells maintained in minimal essential medium supplemented with 1% fetal calf serum in a humidified atmosphere (95% air−5% CO₂) at 37 °C. Cells are infected with 10 tissue culture-derived trypomastigotes per cell for 2 hours and then washed three times with phosphate-buffered saline (PBS) to remove nonadherent parasites. Fresh medium with and without Posaconazole is added, and the cells are incubated for 96 hours with a medium change at 48 hours. The percent of infected cells and the numbers of parasites per cell are determined directly using light microscopy, and a statistical analysis of the results is carried out. IC50 values are calculated by nonlinear regression, using the program GraFit. Fractional inhibitory concentrations (FIC) are calculated. Cytoplasmic free Ca²⁺ concentrations in control and drug-treated extracellular epimastigotes are determined by fluorimetric methods using Fura-2. Subcellular Ca²⁺ levels and mitochondrial membrane potentials are monitored on individual Vero cells infected with T. cruzi amastigotes by using time-scan confocal microscopy. Briefly, Vero cells heavily infected (72 hours) with T. cruzi amastigotes are plated onto 22 × 40 mm glass coverslips (0.15 mm thickness) and incubated simultaneously with 10 μM cell-permeant Rhod-2 and 10 μg/mL Rhodamine-123 for 50 minutes at 37 °C in culture medium and then washed and incubated with Ringer's solution, with or without amiodarone. Under the conditions used fluorescence of Rhod-2 comes mainly from intracellular Ca²⁺-rich compartments, like mitochondria, since its low affinity for Ca²⁺ limits its fluorescence in the Ca²⁺-poor cytoplasm of the Vero cells or amastigotes. Rhodamine-123 is a mitochondrion-specific cationic dye, which distributes across the inner mitochondrial membranes strictly according to their membrane potential.