In-vitro and in-vivo activities of SCH56592 against Cryptococcus neoformans

Mohammad Ashraf Hossain, Shigefumi Maesaki, Kotaro Mitsutake, Hiroshi Kakeya, Eisuke Sasaki, Kazunori Tomono, Takayoshi Tashiro and Shigeru Kohno*,

Second Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in-vitro and in-vivo activities of SCH56592, a triazole antifungal agent, against Cryptococcus neoformans were studied. MIC90s for 16 strains of C. neoformans measured by microdilution method (NCCLS M27-A) were 1 mg/L of SCH56592, 16 mg/L of fluconazole, 32 mg/L of flucytosine, and 0.5 mg/L of amphotericin B. In a murine model of pulmonary cryptococcosis, 10 mg/kg of SCH56592 was more effective than fluconazole. The fungal burden of the lung of animals treated with SCH56592 was significantly reduced (7.40 ± 0.21 log10 cfu/g), as compared with fluconazole (7.77 ± 0.07 log10 cfu/g) and control (7.79 ± 0.1 log10 cfu/g) (P < 0.01). For C. neoformans-infected mice following 7 days treatment with 10 mg/kg of SCH56592 there was a higher concentration in lung (3.36 ± 0.62 ng/ml) than in plasma (2.16 ± 0.86 ng/mL), and this was maintained for 12 h after administration.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The diversity of fungal pathogens appears to be related to increases in the number of susceptible individuals: HIV infection, bone marrow and organ transplant recipients, cancer patients receiving chemotherapy and the critically ill. Cryptococcosis is a major cause of illness and death among patients infected with HIV. Most authorities favour an initial period of combination therapy with amphotericin B for acute cryptococcosis; however, use of amphotericin B is limited by its adverse effects. The triazoles, especially fluconazole, play a role in both initial and subsequent maintenance therapy for cryptococcosis. Long term or repeated therapy with fluconazole in patients with oropharyngeal candidosis has induced resistant-strains,1 although there are few reports of Cryptococcus neoformans.2 SCH5659,2 a new triazole, has been reported to have activity against various medically important fungi including Candida albicans and other Candida spp.3,4

This study evaluates the in-vitro and in-vivo activities of SCH56592, a new triazole antifungal compared with fluconazole against C. neoformans.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sixteen strains of C. neoformans var. neoformans, serotype A, isolated from HIV-negative patients at Nagasaki University Hospital, were used for the in-vitro susceptibility study. Identification and serotyping of C. neoformans were performed as mentioned previously.5 The yeasts were subcultured on Sabouraud Dextrose Agar (SDA) (Becton Dickinson & Co., Cockeysville, MD, USA) plates at 30°C for 48 h.

Antifungal agents used were fluconazole (Pfizer Inc., Sandwich, UK), amphotericin B (Bristol Myers Squibb K.K., Tokyo, Japan), flucytosine (Sigma Chemical Co., St Louis, MO, USA), and SCH56592 (Schering-Plough K. K., Osaka, Japan). The compounds were dissolved in dimethylsulphoxide (DMSO) at a final concentration not more than 1% of the total volume of medium. MICs were determined by microdilution using 96-well plate according to the NCCLS method (M27-A). The plates were incubated at 35°C for 72 h in the presence of the antifungal agents and the lowest concentration of drug that prevented visible growth was considered the MIC.

The protocol for in-vivo experiments was approved by the ethics review committee of our institution, and the guidelines of the Laboratory Animal Center for Biomedical Research, Nagasaki University School of Medicine, were followed.6 Six-week-old, BALB/c male mice (Charles River Inc., Yokohama, Japan) (10 mice in each group) were inoculated according to the procedure described in our previous report.7 Briefly, while under general anaesthesia with intraperitoneal Nembutal, 1x 105 cells of C. neoformans (YC-11), in 50 µL of sterile normal saline was inoculated intratracheally. The final concentrations of the test drugs were adjusted to 10 mg/kg bodyweight in 0.4% hydroxypropyl-ß-cyclodexrin. From day 1 until day 7 after inoculation, 100 µL of fluconazole, SCH56592 or 0.4% hydroxypropyl-ß-cyclodextrin (as untreated control) was administered od orally through a gavage needle.

For mycological studies, mice were killed 14 days after inoculation. Lungs and brains were suspended in normal saline and homogenized by Polytron homogenizer. Serial 10-fold diluted suspensions (50 µL) in sterile normal saline were inoculated on to SDA, incubated at 35°C for 48 h and the colonies counted. Data were expressed as mean ± S.D. Statistical analysis for differences in survival distributions were based on a generalized Wilcoxon test. Differences between fungal burdens of lungs and brains were compared by t-test. A P value < 0.05 was considered statistically significant.

In the pharmacokinetic study, after drug administration on day 7, blood was collected by cardiac puncture with heparin-rinsed injectors, and decanted to Eppendorf tubes. Lungs were excised, perfused with normal saline via the pulmonary artery, and homogenized with 50% ethanol. Plasma and supernatants of lung homogenates were preserved at –20°C until analysis. Drug concentrations in plasma and supernatants were measured by HPLC. Limits of quantification of SCH56592 were 15 ng/mL for plasma and 90 ng/mL for lung.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The MICs of SCH56592 for the 16 strains of C. neoformans are summarized in Table I. MIC for YC-11 strain was 0.5 mg/L of SCH56592, 4 mg/L of fluconazole, 4 mg/L of flucytosine and 0.5 mg/L of amphotericin B. As shown in the Figure, SCH56592-treated mice survived longer than the control and the fluconazole-treated mice. There was a slower death rate in the SCH56592-treated mice and after 25 days all the mice receiving SCH56592 were alive compared with only two in the fluconazole group. By 35 days only two mice had survived. Table II indicates the tissue fungal burden 2 weeks after inoculation. Both SCH56592 and fluconazole inhibited cell growth in lung and brain tissue, but the cell counts in lungs from SCH56592-treated mice were significantly lower than those of the fluconazole-treated mice and controls. Table III shows the concentrations of SCH56592 in plasma and lung tissue in the infected mice. The peak of plasma concentration was 5.03 ng/mL at 2 h after administration which decreased to 0.31 ng/mL 24 h after administration. The concentration in lung tissue elevated rapidly (7.78 ng/mL in post-administration) and remained higher than plasma until 12 h after administration (3.36 ng/mL).


View this table:
[in this window]
[in a new window]
 
Table I. In-vitro activities of SCH56592 against 16 clinical isolates of Cryptococcus neoformans
 


View larger version (10K):
[in this window]
[in a new window]
 
Figure. Survival rate of mice with experimental pulmonary cryptococcosis treated with 0.4% hydroxypropyl-ß-cyclodextrin ({circ}), fluconazole (•) or SCH56592 ({blacksquare}) (dose: 10 mg/kg). Ten mice were included in each group (P< 0.05 compared with 0.4% hydroxypropyl-ß-cyclodextrin and fluconazole by generalized Wilcoxon test).

 

View this table:
[in this window]
[in a new window]
 
Table II. Number of Cryptococcus neoformans recovered from the lung and brain of mice with pulmonary cryptococcosis after 7 days treatment with SCH56592 or fluconazole
 

View this table:
[in this window]
[in a new window]
 
Table III. Concentrations of SCH56592 in plasma and lung tissue after oral administration in murine pulmonary cryptococcosis
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
SCH56592 is a new triazole antifungal agent with demonstrated in-vivo and in-vitro activity against a broad range of both yeast of filamentous fungi, including Aspergillus,Blastomyces, Candida, Coccidioides, Cryptococcus,Fusarium, Trichosporon, Rizopus spp. and other opportunistic fungal pathogens. In experimental animal models, SCH56592 has a long serum elimination half-life and sustained tissue concentrations after a single dose. In a rising single-dose trial in healthy volunteers, plasma concentrations of SCH56592 were dose proportional from 50 to 800 mg and slowly eliminated with a half-life of approximately 25 h. In the same rising dose study, SCH56592 was demonstrated to be safe and well tolerated with no SCH56292-related toxicity identified.

In the present study, the MIC range of SCH56592 was very narrow and efficacy was better than that of fluconazole. In a murine model of pulmonary cryptococcosis, SCH56592 also showed better efficacy than fluxonazole at equivalent doses. The difference in the in-vivo efficacy seemed to reflect the results of the in-vitro susceptibility study. However, C. neoformans was still viable in the lung after treatment indicating that SCH56592 may not be fungicidal at a dosage of 10 mg/kg in mice. As C. neoformans is an intracellular pathogen, outcome in pulmonary cryptococcosis may depend on the ratio of intra- and extra-cellular concentration of the drugs, and further study may be needed to determine the levels of SCH56592 in alveolar macrophages.

As higher concentrations were observed in lung as compared with plasma, SCH56592 may be beneficial in treating pulmonary crptococcosis. Improved efficacy may be expected with bd administration of SCH56592 based on the results of pharmacokinetic study. In conclusion, SCH56592 is a promising new agent in the treatment of pulmonary cryptococcocis.


    Notes
 
* Corresponding author. Tel: +81-95-849-7271; Fax: +81-95-849-7285; E-mail: sk1227{at}net.nagasaki-u.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Johnson, E. M., Warnock, D. W., Luker, J., Porter, S. R. & Scully, C. (1995). Emergence of azole drug resistance in Candida species from HIV-infected patients receiving prolonged fluconazole therapy for oral candidosis. Journal of Antimicrobial Chemotherapy 35, 103–14.[Abstract]

2 . Lamb, D. C., Corran, A., Baldwin, B. C., Kwon-Chung, J. & Kelly, S. L. (1995). Resistant P45091A1 activity in azole antifungal tolerant. Cryptococcus neoformans from AIDS patients. FEBS Letters 368, 326–30.[ISI][Medline]

3 . Galgiani, J. N. & Lewis, M. L. (1997). In-vitro studies of activities of the antifungal triazole SCH56592 and itraconazole against Candida albicans, Cryptococcus neoformans, and other pathogenic yeasts. Antimicrobial Agents and Chemotherapy 41, 180–3.[Abstract]

4 . Law, D., Moore, C. B. & Denning, D. W. (1997). Activity of SCH56592 compared with those of fluconazole and itraconazole against Candida spp. Antimicrobial Agents and Chemotherapy 41, 2310–1.[Abstract]

5 . Tanaka, K., Miyazaki, T., Maesaki, S., Mitsutake, K., Kakeya, H., Yamamoto, Y. et al. (1996). Detection of Cryptococcus neoformans gene in patients with pulmonary cryptococcosis. Journal of Clinical Microbiology 34, 2826–8.[Abstract]

6 . The Guidelines of the Laboratory Animal Center for Biomedical Research, Nagasaki University School of Medicine. (1995).

7 . Hossain, M. A., Maesaki, S., Kakeya, H., Noda, T., Yanagihara, K., Sasaki, E. et al. (1998). Efficacy of NS-718, a novel lipid nanosphere-encapsulated amphotericin B, against Cryptococcus neoformans. Antimicrobial Agents and Chemotherapy 42,1722 –5.[Abstract/Free Full Text]

Received 14 December 1998; returned 19 April 1999; revised 28 May 1999; accepted 16 August 1999