Combination therapy of itraconazole and an acylhydrazone derivative (D13) for the treatment of sporotrichosis in cats

Abstract

Acylhydrazone (AH) derivatives represent a novel category of anti-fungal medications that exhibit potent activity against Sporothrix sp., both in vitro and in a murine model of sporotrichosis. In this study, we demonstrated the anti-fungal efficacy of the AH derivative D13 [4-bromo-N'-(3,5-dibromo-2-hydroxybenzylidene)-benzohydrazide] against both planktonic cells and biofilms formed by Sporothrix brasiliensis. In a clinical study, the effect of D13 was then tested in combination with itraconazole (ITC), with or without potassium iodide, in 10 cats with sporotrichosis refractory to the treatment of standard of care with ITC. Improvement or total clinical cure was achieved in five cases after 12 weeks of treatment. Minimal abnormal laboratory findings, e.g., elevation of alanine aminotransferase, were observed in four cats during the combination treatment and returned to normal level within a week after the treatment was ended. Although highly encouraging, a larger and randomized controlled study is required to evaluate the effectiveness and the safety of this new and exciting drug combination using ITC and D13 for the treatment of feline sporotrichosis.

Importance: This paper reports the first veterinary clinical study of an acylhydrazone anti-fungal (D13) combined with itraconazole against a dimorphic fungal infection, sporotrichosis, which is highly endemic in South America in animals and humans. Overall, the results show that the combination treatment was efficacious in ~50% of the infected animals. In addition, D13 was well tolerated during the course of the study. Thus, these results warrant the continuation of the research and development of this new class of anti-fungals.

Keywords: Sporothrix; acylhydrazone; anti-fungal therapy; cats; fungal infection; itraconazole; sporotrichosis; terbinafine.

Fig 1

Effect of D13 on sporothrix Biofilm. Sporothrix yeasts were cultured in RPMI 1640 medium together with cat’s claw fragments and incubated at 37°C for 3 days. Following this, the claws were washed 3 x and subsequently treated with 10 µg/mL of D13 or ITC for another 3 days before being prepared for SEM analysis. In the biofilm control groups, both the ATCC strain of S. brasiliensis and the clinically isolated (C.I 17522) strain displayed abundant yeasts and hyphae with a consistently uniform and intact surface. In contrast, cells from both strains treated with either D13 or ITC exhibited slight growth and irregularities on their surfaces.

Fig 2

Fungal load of Sporothrix biofilm on cat claw fragments. Mature Sporothrix biofilms grown for 3 days were treated with D13 or ITC at 10 µg/mL. After 3 days of incubation, the claws were washed and suspended in RMPI/PenStrep 1%. Claws were sonicated and vortexed to drop the cells from the biofilm. Aliquots were placed on brain heart infusion plates and incubated at 37°C for up to 3 days. Floating bars represent the mean minimum to maximum values of three independent experiments. Statistical analysis was performed by one-way analysis of variance and Sidak’s multiple comparison test. **P < 0,0028, ****P < 0,0001. ns, not significant.

Fig 3

Case 1. The cat presented no improvement of the cutaneous lesions on the head and on the thoracic limbs upon treatment with ITC and KI. After adding D13, the cat significantly improved, and clinical cure was achieved after 8 months of treatment with ITC, KI, and D13 combination

Fig 4

Case 2. The cat presented no improvement of the cutaneous and nasal mucosal lesions upon treatment with ITC, TRB, and KI. After adding D13, the cat significantly improved and clinical cure was achieved after 12 weeks of treatment with ITC, TRB, KI, and D13 combination.

Fig 5

Case 3. The cat presented no improvement of the cutaneous and nasal mucosal lesions upon treatment with ITC. After adding D13, the cat significantly improved, and clinical cure was achieved after 12 weeks of treatment with ITC and D13 combination.

Fig 6

Case 4. The cat presented no improvement of the cutaneous lesions in the cephalic region and left cervical region upon treatment with ITC. After adding D13, the cat significantly improved, and clinical cure was achieved after 12 weeks of treatment with ITC and D13 combination.

Fig 7

Case 5. The cat presented no improvement of the tumor-like lesion and of an ulcerated lesion on the nasal region upon treatment with ITC. After adding D13, the cat significantly improved, even though clinical cure was not achieved after 4 weeks of treatment with ITC and D13 combination.