Second Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan
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Abstract |
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Introduction |
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This study evaluates the in-vitro and in-vivo activities of SCH56592, a new triazole antifungal compared with fluconazole against C. neoformans.
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Materials and methods |
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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.
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Results |
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Discussion |
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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.
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Notes |
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References |
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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, 32630.[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, 1803.[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, 23101.[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, 28268.[Abstract]
6 . The Guidelines of the Laboratory Animal Center for Biomedical Research, Nagasaki University School of Medicine. (1995).
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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.
Received 14 December 1998; returned 19 April 1999; revised 28 May 1999; accepted 16 August 1999