Preclinical Evaluation of Acylhydrazone SB-AF-1002 as a Novel Broad-Spectrum Antifungal Agent

Abstract

The incidence of invasive fungal infections is rising due to the increase in susceptible populations. Current clinically available drugs have therapeutic limitations due to toxicity, a narrow spectrum of activity, and, more importantly, the consistent rise of fungal species that are intrinsically resistant or that develop resistance due to prolonged therapy. Thus, there is an urgent need for new broad-spectrum antifungal agents with low toxicity and a novel mechanism of action. We previously reported a new class of potent antifungal compounds, acylhydrazones, that target the fungal sphingolipid pathway. Based upon our initial lead molecules, (E)-N'-(5-bromo-2-hydroxybenzylidene)-2-methylbenzohydrazide and D13, we performed a structure-activity relationship study, synthesizing ca. 300 new compounds. Of these, 5 compounds were identified to be the most promising for further studies, based on their broad-spectrum activity and low toxicity in mammalian cells lines. Among these top 5 lead compounds, we report here the impressive in vivo activity of 2,4-dibromo-N'-(5-bromo-2-hydroxybenzylidene)benzohydrazide (SB-AF-1002) in several models of systemic fungal infection. Our data show that SB-AF-1002 is efficacious and outperforms current standard-of-care drugs in models of invasive fungal infections, such as cryptococcosis, candidiasis, and aspergillosis. Specifically, animals treated with SB-AF-1002 not only survived the infection but also showed a clearing of fungal cells from key organs. Moreover, SB-AF-1002 was very effective in an aspergillosis model as a prophylactic therapy. SB-AF-1002 also displayed acceptable pharmacokinetic properties in mice, similar to those of the parent compound, D13. These results clearly indicate that our novel acylhydrazones constitute a new class of highly potent and efficacious antifungal agents which warrant further development for the treatment of invasive fungal infections.

Keywords: Aspergillus; Candida; Cryptococcus; acylhydrazone; antifungal agents; invasive fungal infections.

FIG 1

Structure of SB-AF-1002.

FIG 2

(a) Survival of mice infected intranasally with 5 × 10⁵ C. neoformans cells and treated with 20 mg/kg/day of the compound through gavage. #, P = 0.0001 for SB-AF-1002 versus no drug; *, P = 0.0007 for SB-AF-1002 versus fluconazole (Fluco). (b and c) Endpoint numbers of CFU in the brain and lungs of mice that survived in the experiment whose results are presented in panel a. (d) Survival of mice infected intranasally with 5 × 10⁵ C. neoformans cells with treatment starting at 5 days after infection with 20 mg/kg/day of compounds through gavage. #, P = 0.0001 for SB-AF-1002 versus no drug; *, P = 0.0016 for SB-AF-1002 versus fluconazole. (e and f) Endpoint numbers of CFU in the lungs and brains of mice that survived in the experiment whose results are presented in panel d.

FIG 3

(a) Survival of mice infected intranasally with 5 × 10⁵ C. neoformans cells and treated daily with increasing concentrations of SB-AF-1002 (05, 5, 20, and 40 mg/kg) through gavage, starting at day 5 postinfection. #, P < 0.0001 for SB-AF-1002 at 40 mg/kg versus no drug; *, P = 0.0005 for SB-AF-1002 at 20 mg/kg versus no drug; ^, P = 0.0016 for SB-AF-1002 at 5 mg/kg versus no drug; ✬, P = 0.136 for SB-AF-1002 at 0.5 mg/kg versus no drug. (b and c) Endpoint numbers of CFU in the lungs and brains of the mice that survived in the experiment whose results are presented in panel a.

FIG 4

(a) Survival of mice infected intravenously with 10⁵ C. albicans cells and starting a daily treatment on the day of infection with increasing concentration of SB-AF-1002 through gavage. There were no statistically significant differences between the groups. (b) Endpoint numbers of CFU in the left (L) and right (R) kidneys of the surviving mice treated with SB-AF-1002 at 20, 40, or 80 mg/kg. No CFU were observed in the left kidneys. (c) Survival of mice infected intravenously with 10⁵ C. albicans cells and treated with 3 mg/kg/day of SB-AF-1002 intravenously. *, P = 0.0003 for SB-AF-1002 versus no drug. (d) Endpoint numbers of CFU in the left and right kidneys of the surviving mice in the experiment whose results are presented in panel c.

FIG 5

(a) Survival of mice infected intranasally with 5 × 10⁵ A. fumigatus conidia. Animals were immunosuppressed (IS) at 1 day prior to infection and treated with increasing concentrations of SB-AF-1002 (20, 40, and 80 mg/kg/day) or with voriconazole (Vori) at 20 mg/kg/day through gavage starting on the same day of infection. *, P = 0.0012 for SB-AF-1002 versus no drug. (b) Endpoint numbers of PFU in the lungs of mice that survived in the experiment whose results are presented in panel a. (c) Survival of mice infected intranasally with 5 × 10⁵ A. fumigatus conidia and treated with SB-AF-1002 at 40 mg/kg/day or voriconazole at 20 mg/kg/day starting at 5 days before infection. Animals were immunocompromised at 1 day prior to infection. *, P < 0.0001 for SB-AF-1002 versus no drug; #, P = 0.013 for SB-AF-1002 versus voriconazole; ^, P = 0.0269 for voriconazole versus no drug. (d) Endpoint numbers of PFU in the lungs of mice that survived the experiment whose results are presented in panel c.

FIG 6

Pharmacokinetic studies of SB-AF-1002 administered intravenously (A) or orally (B) using a single dose of 1 mg/kg or 20 mg/kg, respectively.