New Triazole NT-a9 Has Potent Antifungal Efficacy against Cryptococcus neoformans In Vitro and In Vivo

Abstract

In the past decades, the incidence of cryptococcosis has increased dramatically, which poses a new threat to human health. However, only a few drugs are available for the treatment of cryptococcosis. Here, we described a leading compound, NT-a9, an analogue of isavuconazole, that showed strong antifungal activities in vitro and in vivo NT-a9 showed a wide range of activities against several pathogenic fungi in vitro, including Cryptococcus neoformans, Cryptococcus gattii, Candida albicans, Candida krusei, Candida tropicalis, Candida glabrata, and Candida parapsilosis, with MICs ranging from 0.002 to 1 μg/ml. In particular, NT-a9 exhibited excellent efficacy against C. neoformans, with a MIC as low as 0.002 μg/ml. NT-a9 treatment resulted in changes in the sterol contents in C. neoformans, similarly to fluconazole. In addition, NT-a9 possessed relatively low cytotoxicity and a high selectivity index. The in vivo efficacy of NT-a9 was assessed using a murine disseminated-cryptococcosis model. Mice were infected intravenously with 1.8 × 10⁶ CFU of C. neoformans strain H99. In the survival study, NT-a9 significantly prolonged the survival times of mice compared with the survival times of the control group or the isavuconazole-, fluconazole-, or amphotericin B-treated groups. Of note, 4 and 8 mg/kg of body weight of NT-a9 rescued all the mice, with a survival rate of 100%. In the fungal-burden study, NT-a9 also significantly reduced the fungal burdens in brains and lungs, while fluconazole and amphotericin B only reduced the fungal burden in lungs. Taken together, these data suggested that NT-a9 is a promising antifungal candidate for the treatment of cryptococcosis infection.

Keywords: Cryptococcus neoformans; NT-a9; disseminated cryptococcosis; fungal burden; triazole compound.

FIG 1

Time-kill curves of C. neoformans strain H99 (A) and C. albicans strain SC5314 (B) treated with NT-a9 or fluconazole (FLC). Strains were grown in RPMI 1640 medium with a starting inoculum of 5 × 10⁵ CFU/ml. Aliquots were obtained at the predetermined time points, and serial dilutions were spread on SDA plates. Colony counts were determined after incubation. The results are calculated from two independent experiments.

FIG 2

Sterol analysis of C. neoformans cells by GC-MS. C. neoformans H99 was treated with NT-a9 or fluconazole (FLC) for 8 h. Cells were collected, and total sterols were extracted. Sterol compositions were analyzed by GC-MS. Data are shown as the mean values ± standard deviations from two independent experiments.

FIG 3

Cytotoxicity results for NT-a9 against HUVECs. A total of 1 × 10⁵ cells/ml of HUVECs in DMEM containing 10% fetal bovine serum (FBS) were seeded in 96-well tissue culture plates and incubated for 3 h for adhesion. After incubation, the supernatant was removed and fresh DMEM with different concentrations of NT-a9 was added. The plates were incubated for an additional 24 h at 37°C with 5% CO₂. After incubation, 10 μl of CCK-8 solution was added to each well and the plates were incubated at 37°C for 2 h. Cell viability was assessed by measuring absorbance at 450 nm. Data are shown as the mean values ± standard deviations for three independent experiments.

FIG 4

Survival curves for mice with disseminated cryptococcosis treated with antifungal agents. Mice were inoculated intravenously with C. neoformans H99 at 1.8 × 10⁶ CFU per animal and then treated with NT-a9, isavuconazole (ISA), fluconazole (FLC), or amphotericin B (AMB) (n = 10 mice per group). The differences in in vivo effects between high doses (A) and low doses (B) of NT-a9 were investigated. Antifungal monotherapy was initiated 2 h after inoculation and continued for 7 days. Mice were monitored without therapy until day 20. Amphotericin B was administered intraperitoneally, while other antifungal treatments were performed through oral gavage. ***, P < 0.001 versus the results for the control. One representative result out of two independent experiments is shown.

FIG 5

Fungal burdens in mice with disseminated cryptococcosis treated with antifungal agents. Mice were inoculated intravenously with C. neoformans H99 at 1.8 × 10⁶ CFU per animal and treated with NT-a9, fluconazole, or amphotericin B (n = 10 mice per group). Antifungal monotherapy was initiated 1 day after inoculation and continued for 3 days. Mice were humanely euthanized 1 day after the last treatment, and brain (A) and lung (B) samples were collected for fungal burden analysis. Amphotericin B was administered intraperitoneally, while other treatments were performed through oral gavage. Horizontal lines represent mean values, and whiskers represent standard deviations. ***, P < 0.001 versus the result for the control. One representative result out of two independent experiments is shown.