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, MIC₅₀, MIC₉₀ 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.

Introduction

Cryptococcus gattii is a basidiomycetous yeast. The disease caused by C. gattii usually results in pneumonia or dissemination to distant tissues, especially to the CNS.¹ While Cryptococcus neoformans primarily affects HIV-infected individuals worldwide, C. gattii appears to have a greater propensity to infect immunocompetent humans.²

Amphotericin B, with or without flucytosine, is the most widely used treatment for cryptococcal meningitis or complicated lung disease. Fluconazole is used either as the primary therapy for mild to moderate pulmonary involvement or as the consolidation and maintenance treatment.¹ If fluconazole is unavailable or contraindicated, voriconazole is an acceptable alternative.¹ It has been suggested that C. gattii presents higher pathogenicity than C. neoformans, which makes it necessary to improve and search for alternatives to current antifungals.³

T-2307 is a novel arylamidine developed by Toyama Chemical Co., Ltd (Tokyo, Japan). Clinical Phase I studies were successfully completed in the USA and no severe adverse effects were observed. Under both in vitro and in vivo conditions, T-2307 exhibits broad-spectrum antifungal activities against the majority of fungal pathogens, including Candida spp., C. neoformans and Aspergillus fumigatus.⁴ In this study, we evaluated the in vitro and in vivo antifungal activities of T-2307 against C. gattii in comparison with those of amphotericin B, fluconazole and voriconazole.

Materials and methods

In vitro study

Antifungal agents

T-2307 was synthesized by Toyama Chemical Co., Ltd (Tokyo, Japan), which also provided fluconazole solution for injection (2 mg/mL). Fluconazole powder was obtained by LKT Laboratories, Inc. (St Paul, MN, USA). Voriconazole and amphotericin B were commercially obtained from Pfizer Japan, Inc. (Tokyo, Japan) and Bristol-Myers KK (Tokyo, Japan), respectively.

Organisms

Fifteen clinical isolates of C. gattii were used and divided into four genotypes, namely VGI (n = 2), VGII (n = 2), VGIII (n = 9) and VGIV (n = 1), as well as another isolate with unidentified strains. C. gattii species identification was based on canavanine glycine bromothymol blue (CGB) agar⁵ and the majority of the strains were confirmed by molecular type using URA5 RFLP or MLST test.^6,7 The isolates were kindly provided by Dr J. Kwon-Chung (NIAID, NIH, Bethesda, MD, USA), Dr Yoshitsugu Miyazaki and Dr Hideaki Ohno (NIID, Tokyo, Japan).

Antifungal susceptibility studies

Broth microdilution testing was performed in accordance with CLSI standards M27-A3 and M27-S3,^8,9 using RPMI 1640 medium (Sigma–Aldrich Co., St Louis, MO, USA) adjusted to pH 7.0 with 0.165 M MOPS (Dojindo Laboratories, Kumamoto, Japan). The final inoculum concentration was ∼ 2.0 × 10³ cells/mL and microplates were incubated at 35°C for 72 h.

The MICs of T-2307, fluconazole and voriconazole were recorded as the lowest concentration at which a prominent decrease (∼ 50%) in turbidity was observed, relative to the turbidity of the growth control. The MIC of amphotericin B was read as the lowest concentration at which complete inhibition was observed. Candida parapsilosis (ATCC 22019) was included as a quality control strain in each test, as recommended by the CLSI.⁸ The MIC ranges, MIC₅₀s, MIC₉₀s and geometric mean (GM) MICs were determined. The MIC of T-2307 and the MICs of amphotericin B, fluconazole and voriconazole were compared using the non-parametric Dunnett’s multiple comparison test (Steel’s test) where statistical significance was set to P <0.05.

Time–kill studies

Time–kill studies were performed as described previously.¹⁰C. gattii YF2784 (VGII) was subcultured on an Sabouraud Dextrose Agar (SDA) plate and ∼ 1.0 × 10⁵ cells/mL in RPMI/MOPS with the test compounds were used as the inoculum, at concentrations equal to 1/4, 1, 4, 16 and 64 times the MIC. Test solutions were incubated at 35°C. At predetermined timepoints (0, 2, 4, 8, 24, 48 and 72 h) following inoculation, 100 μL aliquots were removed from each test solution and each dilution was streaked onto SDA plates for colony count determination. Following incubation at 35ºC for 21 days, the number of cfu on each plate was determined.

In vivo study

Animals

We used 4.5 week old, male, specific-pathogen-free Crlj:CDI (ICR) strain mice (Japan SLC Inc., Shizuoka, Japan) for the in vivo study. The mice were provided with food and water ad libitum.

Ethics

All animal experiments were performed in full compliance with the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, Washington, DC, 2011) as well as with all of the institutional regulations and guidelines for animal experimentation after pertinent review and approval by the Institutional Animal Care and Use Committee of Nagasaki University under protocol number 1206110994.

Pulmonary infection model

Mice were infected intranasally with C. gattii YF2784 at a target inoculum of ∼ 8.5 × 10³ cfu/mouse. T-2307, amphotericin B and fluconazole were administered subcutaneously, intravenously and orally, respectively, once daily for 7 days, starting either 2 h or 14 days post-infection.

Each group comprised eight mice and sterile saline was administered to control mice. On day 21 post-infection, the mice were euthanized by cutting the inferior vena cava under anaesthesia. The lungs and brain were extirpated and homogenized in 1 mL of sterile saline. Serial dilutions of the homogenate were prepared and 100 μL of each homogenate was spread onto SDA plates. After incubation at 35°C for 3 days, the colonies were counted and the viable counts (log number of cfu/organ) of the compound-treated group and control group were compared using the parametric Dunnett’s multiple comparison test where statistical significance was set to P <0.05. The therapeutic effect was also confirmed by histopathology in the maximum-dose group for each compound. The lungs were fixed in 10% neutral buffered formalin. Histopathological sections were prepared and stained with haematoxylin and eosin.

Results

Antifungal activity

The MIC ranges, MIC₅₀s, MIC₉₀s and GM MICs of the agents tested against the 15 C. gattii clinical isolates were as follows: T-2307, 0.0078–0.0625, 0.0313, 0.0625 and 0.0394 mg/L, respectively; amphotericin B, 1–1, 1, 1 and 1 mg/L; fluconazole, 1–16, 4, 16 and 5.53 mg/L; and voriconazole, 0.0625–0.25, 0.125, 0.25 and 0.109 mg/L. The MIC of T-2307 was significantly lower than those of amphotericin B (P <0.0001), voriconazole (P = 0.0003) and fluconazole (P <0.0001).

Time–kill study

The time–kill plots of T-2307, amphotericin B, fluconazole and voriconazole against C. gattii YF2784 are shown in Figure 1. T-2307 and amphotericin B showed concentration-dependent fungicidal activity with a reduction of >3 log₁₀ cfu/mL at 4 times the MIC or higher within 72 h. Fluconazole and voriconazole showed no fungicidal activity even at 64 times the MIC.

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.

In vivo efficacy

The therapeutic effects of T-2307, amphotericin B and fluconazole in a murine model of intranasal pulmonary infection caused by C. gattii YF2784 are shown in Figure 2(a). In this model, these agents were administered once daily for 7 days starting from 2 h post-infection. T-2307 at 2 mg/kg/day, amphotericin B at 1 mg/kg/day and fluconazole at 160 mg/kg/day significantly reduced the viable counts in the lungs and brain compared with the control group. Next, to evaluate the efficacy of the test compounds against an infection that had progressed, these agents were administered for 7 days starting at 14 days post-infection (Figure 2b). T-2307 at 1 mg/kg/day significantly reduced the viable counts in the lungs and brain compared with the control group. Amphotericin B did not reduce the viable counts in the lungs and brain even at 2 mg/kg/day. Fluconazole significantly reduced the viable counts in the brain at 160 mg/kg/day, but did not reduce the viable counts in the lungs even at 320 mg/kg/day. T-2307, unlike amphotericin B and fluconazole, prevented the progression of alveolar collapse caused by proliferation of C. gattii within the lungs (Figure 2c).

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.

Discussion

T-2307 exhibited potent in vitro antifungal activity against the 15 clinical isolates and showed concentration-dependent fungicidal activity. T-2307 selectively causes the collapse of mitochondrial membrane potential and disrupts the mitochondrial function in yeast.¹¹ Therefore, the mechanism of action of T-2307 could account for the fungicidal activity against C. gattii, a non-fermentative yeast.

To our knowledge, this is the first study to explore the efficacy of antifungal drugs in a progressed murine infection model of C. gattii.

Amphotericin B at 2 mg/kg/day, administered 14 days post-infection for 7 days, did not exhibit an obvious therapeutic effect in a progressed murine infection model. A high mortality rate has been reported in clinical trials utilizing lower doses of amphotericin B at 0.3–0.5 mg/kg even for the initial treatment of cryptococcal meningitis.¹² It has been reported in recent clinical trials that a lower mortality rate is associated with higher doses of amphotericin B (0.7–1 mg/kg) for cryptococcosis.^13,14 However, we could not increase the dose of amphotericin B above 2 mg/kg in this study as this was shown to be the maximum dosage tolerated by the mice.^15,16 We consider that the toxicity of amphotericin B at high doses would limit treatment efficacy.

The recommended dose of fluconazole for cryptococcosis is 400 mg/day, which corresponds to 75 mg/kg in mice in terms of the AUC.^17,18 However, fluconazole at a dose of 320 mg/kg did not exhibit any therapeutic effect in a progressed murine infection model.

Consistent with these results, the treatment of HIV-infected individuals with a cryptococcal infection with high-dose fluconazole does not demonstrate significant differences in survival rates.¹⁹

T-2307 showed potent activity in a progressed murine infection model, unlike amphotericin B and fluconazole. T-2307 can therefore be regarded as a promising new candidate for the treatment of cryptococcosis.