化合物信息

Description

Infection

Description

Posaconazole (Noxafil) is a sterol C14ɑ demethylase inhibitor with an IC50 of 0.25 nM.

Targets

IC₅₀

In Vitro

Posaconazole has potent trypanocidal activity. Amiodarone acts synergistically with Posaconazole. Posaconazole also affects and disrupts Ca²⁺ homeostasis in T. cruzi. Posaconazole blocks the biosynthesis of ergosterol, which is essential for parasite survival. Posaconazole has a clear, dose-dependent effect on proliferation of the epimastigote (extracellular) stages, with a minimal inhibitory concentration of 20 nM and an IC50 of 14 nM. Against the clinically relevant intracellular amastigote form of the parasite, Posaconazole is even more potent. Posaconazole has the minimal inhibitory concentration and IC50 values of 3 nM and 0.25 nM. ^[1] Posaconazole is active against isolates of Candida and Aspergillus spp. that exhibit resistance to Fluconazole, Voriconazole, and Amphotericin B and is much more active than the other triazoles against zygomycetes. ^[2]

In Vivo

Treatment of infected animals with amiodarone alone reduces parasitemia, increased survival 60 days pi (0% for untreated controls vs 40% for amiodarone-treated animals) and, when given in combination with Posaconazole, delays the development of parasitemia. ^[1] Coadministration of Posaconazole and Boost Plus increases drug exposure compared to the administration of Posaconazole alone in the fasted state. Food, particularly meals high in fat content, significantly increases Posaconazole bioavailability. Systemic exposure to Posaconazole increases 4- and 2.6-fold when it is consumed with a high-fat and nonfat meal, respectively. ^[3] Posaconazole and Amiodarone may constitute an effective anti-T. cruzi therapy with low side effect. ^[4] At twice-daily doses of ≥15 mg/kg of body weight, Posaconazole prolongs the survival of the mice and reduces tissue burden. ^[5]

Clinical Trials

Posaconazole has entered in a phase II clinical trial in the treatment of mycoses.

Features

Posaconazole is the most advanced candidate for the treatment of Chagas disease.

Cell Lines

Epimastigote form of T. cruzi amastigotes

Concentrations

0 nM -4 nM

Incubation Time

96 hours

Methods

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.

Animal Models

Female NMRI?IVIC mice with acute Chagas' disease

Formulation

Control

Doses

20 mg/kg/d

Administration

Oral