In vitro and in vivo antifungal activities of T-2307, a novel arylamidine, against Cryptococcus gattii: an emerging fungal pathogen

Abstract

Objectives: T-2307, a novel arylamidine, exhibits potent broad-spectrum activities against the majority of fungal pathogens. In this study, the antifungal activity of T-2307 against Cryptococcus gattii was evaluated in comparison with those of amphotericin B, fluconazole and voriconazole in vitro and in vivo .

Methods: The MICs for 15 clinical isolates were determined according to CLSI guidelines and time-kill studies were performed using C. gattii YF2784. In a murine model for intranasal pulmonary infection caused by C. gattii YF2784, the test compounds were administered once daily for 7 days from 2 h or 14 days post-infection. The viable counts in the lungs and brain were determined at 21 days post-infection.

Results: The MIC range, MIC 50 , MIC 90 and geometric mean MIC of T-2307 were 0.0078-0.0625, 0.0313, 0.0625 and 0.0394 mg/L, respectively. The MIC of T-2307 was significantly lower than those of fluconazole, voriconazole and amphotericin B. T-2307 showed concentration-dependent fungicidal activity at 4 times the MIC or higher. Administration of T-2307 at 2 mg/kg/day, amphotericin B at 1 mg/kg/day and fluconazole at 160 mg/kg/day from 2 h post-infection significantly reduced viable counts in the lungs and brain. However, when the administration was started 14 days post-infection, only T-2307 significantly reduced the viable counts in both the lungs and the brain at 1 mg/kg/day.

Conclusions: T-2307 shows excellent in vitro and in vivo antifungal activities against C. gattii and would be a promising new candidate for the treatment of cryptococcosis.

Figure 1

Time–kill curve of T-2307, amphotericin B, fluconazole and voriconazole against C. gattii YF2784. C. gattii YF2784 was adjusted to a starting inoculum of ∼ 1.0 × 10⁵ cells/mL in RPMI/MOPS with the test compounds, at concentrations equal to 1/4 (filled squares), 1 (open diamonds), 4 (filled diamonds), 16 (open squares) and 64 (filled triangles) times the MIC. The solid line indicates the control. Test solutions were incubated at 35 °C. The number of cfu was determined at 0, 2, 4, 8, 24, 48 and 72 h following inoculation. AMB, amphotericin B; FLC, fluconazole; VRC, voriconazole.

Figure 2

Therapeutic effect of T-2307, amphotericin B and fluconazole in a murine pulmonary infection model caused by C. gattii YF2784. Mice were infected by intranasally injecting 8.5 × 10³ cfu/mouse (n = 8). The agents were administered once daily for 7 days starting from 2 h (a) or 14 days (b) post-infection. On day 21 post-infection, the viable counts in the lungs and brain (log number of cfu/organ) were determined. The viable counts from the compound-treated group and control group were compared using the parametric Dunnett’s multiple comparison test. Asterisks indicate a significant difference when compared with the control (*P <0.05, **P <0.01 and ***P <0.001). (c) The therapeutic effect of daily administration for 7 days starting from 14 days post-infection was also confirmed by histopathology in the maximum-dose group for each compound. On day 21 post-infection, the lungs of the euthanized mice were extirpated. The lungs were fixed in 10% neutral buffered formalin. Histopathological sections were prepared and stained with haematoxylin and eosin. AMB, amphotericin B; FLC, fluconazole; sc, subcutaneously; iv, intravenously, po, orally.