Antifungal activity of Acylhydrazone derivatives against Sporothrix spp

Abstract

Sporotrichosis is an emerging mycosis caused by members of the genus Sporothrix The disease affects humans and animals, particularly cats, which plays an important role in the zoonotic transmission. Feline sporotrichosis treatment options include itraconazole (ITC), potassium iodide and amphotericin B, drugs usually associated with deleterious adverse reactions and refractoriness in cats, especially when using ITC. Thus, affordable, non-toxic and clinically effective anti-Sporothrix agents are needed. Recently, acylhydrazones (AH), molecules targeting vesicular transport and cell cycle progression, exhibited a potent antifungal activity against several fungal species and displayed low toxicity when compared to the current drugs. In this work, the AH derivatives D13 and SB-AF-1002 were tested against Sporothrix schenckii and Sporothrix brasiliensis Minimal inhibitory concentrations of 0.12 - 1 μg/mL were observed for both species in vitro D13 and SB-AF-1002 showed an additive effect with itraconazole. Treatment with D13 promoted yeast disruption with release of intracellular components, as confirmed by transmission electron microscopy of S. brasiliensis exposed to the AH derivatives. AH-treated cells displayed thickening of the cell wall, discontinuity of the cell membrane and an intense cytoplasmic degeneration. In a murine model of sporotrichosis, treatment with AH derivatives was more efficient than ITC, the drug of choice for sporotrichosis. The results of the preliminary clinical study in cats indicate that D13 is safe and has potential to become a therapeutic option for sporotrichosis when associated to ITC. Our results expand the antifungal broadness of AH derivatives and suggest that these drugs could be exploited to combat sporotrichosis.

FIG 1

Effect of AH derivatives on Golgi apparatus and related morphology and distribution. Control or AH-treated yeasts of S. brasiliensis (A) and S. schenckii (B) were incubated with NBD-Cer. DIC, differential interference contrast; Uvitex, chitin layer (blue fluorescence); NBD-Cer, Golgi and Golgi-derived compartments (green fluorescence). (C) Cellular fluorescence intensity of each treatment. The inset in panel A shows a merged labeling of S. brasiliensis with a small hole in the chitin layer, depicting the point of extravasation of intracellular material (white arrow). Bars: black, 10 μm; white, 2 μm.

FIG 2

Ultrastructural analysis of Sporothrix brasiliensis treated with AH derivatives. S. brasiliensis ATCC 5110 cells treated for 48 h with subinhibitory concentrations of AH derivatives were analyzed by TEM. (A to C) Untreated cells exhibited compact and homogeneous cell wall, intact plasma membrane, and organelles. (D to F) Yeast treated with D13 (0.25 μg/ml) showed intense intracellular damage, vesicle accumulation, and discontinuous plasma membrane (white arrow). (G to I) Cells treated with SB-AF-1002 (0.12 μg/ml) exhibited cytoplasm without well-defined organelles and cell wall detachment (black arrow). Bars, 500 nm (A, B, D, E, G, and H) and 200 nm (C, F, and I).

FIG 3

In vivo test 1. S. brasiliensis (5 × 10⁶ cells/25 μl) was injected into the left hind paw of C57BL/6 mice. Treatments with D13 and itraconazole were performed intravenously (0.5 mg/kg/day/per tail). The images shown here correspond to days 1 to 35 of the treatment started after 25 days of infection. Mice treated with D13 showed the best recovery and did not show any lesions or signs of disease during the 3 weeks of observation after the end of treatment.

FIG 4

In vivo test 2. (A) Mice infected with S. brasiliensis ATCC 5110 by subcutaneous injection in the hind paw were treated with AH derivatives and itraconazole. For D13 and SB-AF-1002, the treatment was performed intravenously (0.5 mg/kg/day/per tail) and by gavage (2 mg/kg/day). Itraconazole was administered intravenously as a control treatment. Mice were anesthetized with xylazine and ketamine before intravenous treatment. (B) Survival of mice subcutaneously infected with S. brasiliensis yeasts. Mice were treated daily after 15 days, when the lesions were developed, with the different drugs (orally or i.v.). ITC (i.v.) and PBS (i.v.) were used as controls.

FIG 5

Weight and paw diameter evaluation in mice infected with S. brasiliensis. Infection in C57BL/6 mice was performed subcutaneously (5 × 10⁶ cells/25 μl). Lesions were visualized 15 days after infection, and the antifungal treatment was initiated by gavage or intravenously. Once the last mouse from the control group died, the survivors of the other groups were monitored for 6 more days of treatment. The weight of the mice (A) and the diameter of the inflamed paw (B) of each tested group were monitored.