Efficacy of micafungin against deep-seated candidiasis in cyclophosphamide-induced immunosuppressed mice

Mochiyoshi Ninomiya1, Hiroshige Mikamo1,2,*, Kaori Tanaka2, Kunitomo Watanabe2 and Teruhiko Tamaya1

1 Department of Obstetrics and Gynecology, Gifu University Graduate School of Medicine; 2 Division of Anaerobe Research, Life Science Research Center, Gifu University, 1-1 Yanagido, Gifu City, Gifu 501-1194, Japan


* Corresponding author. Tel: +81-58-230-6552; Fax: +81-58-230-6551; Email: mikamo{at}cc.gifu-u.ac.jp

Received 19 March 2004; returned 25 May 2004; revised 14 November 2004; accepted 15 December 2004


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: We investigated the effects of fluconazole and micafungin for the therapy of deep-seated candidiasis in a cyclophosphamide-induced immunosuppressed mouse model.

Methods: We used the experimental model of intraperitoneal fungal abscess caused by Candida albicans, as described previously.

Results and conclusions: Micafungin efficacy was equal to that of fluconazole in one-tenth dosage even in peritonitis. We also assessed the short-term (24 h) and long-term (8 days) therapeutic effects after the end of therapy. Although the therapeutic effect of fluconazole was similar to that of micafungin at 24 h after the end of therapy, the effect of micafungin was superior to that of fluconazole at 8 days after the end of therapy.

Keywords: micafungin , fluconazole , experimental infection , Candida albicans


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The increase in immunocompromised hosts as a result of the increased use of immunosuppressive agents in organ transplantations, aggressive anticancer chemotherapy, the spread of human immunodeficiency virus infection, and improved life-saving medical techniques necessitating indwelling catheters, has led to a substantial increase in the occurrence of serious fungal infections.1 In the surgical fields, fungal peritonitis is one of the major deep-seated fungal infections.2 The main causative organism for fungal peritonitis is Candida species, and non-albicans systemic candidiasis, such as Candida glabrata, Candida tropicalis, Candida krusei and Candida parapsilosis, has recently increased in fungal peritonitis.2,3 Although fluconazole has been used against fungal peritonitis, the development of new antifungal agents that are effective against non-albicans species has been expected in clinical practice, since the efficacy of fluconazole against C. glabrata, etc. is low.4

Micafungin, which is one of the parenteral echinocandins,5 has also been shown to have good antifungal and fungicidal activity against non-albicans species,68 and the clinical use of micafungin has recently increased.

To evaluate the clinical utility of micafungin against deep-seated fungal infections in patients with neutropenia, we investigated the efficacy of micafungin in the mouse model of intraperitoneal abscesses caused by Candida albicans.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antifungal agents

The antifungal agents used in this study were fluconazole (Pfizer Central Research, Sandwich, UK), and micafungin (Fujisawa Pharmaceutical Co., Osaka, Japan). The compounds were dissolved in dimethyl sulphoxide, and the final concentration of dimethyl sulphoxide was less than 1% of the total volume of medium. Both antifungal agents were given subcutaneously at 8 or 16, and 0.8 or 1.6 mg/kg of body weight for fluconazole and micafungin, respectively.

Organism

In this study, we used C. albicans isolated from a patient with pelvic peritonitis at the Gifu University Hospital. MIC was determined by the broth microdilution method based on the standard method for antifungal susceptibility testing proposed by the NCCLS with RPMI 1640 medium (Nissui Pharmaceutical Co., Tokyo, Japan).9

Animals

Female ICR mice (specific pathogen free, 4 weeks old) were purchased from Nippon Bio-Supply Center (Tokyo, Japan). The mice were used for the experiments after an acclimatization period of 6 days. Mice were given food and water ad libitum throughout the experiments. The experimental protocol was approved by the local Ethics Review Committee for animal experimentation of Gifu University School of Medicine.

Experimental mouse infection

The infection model was performed as described previously.10 Briefly, the autoclaved caecal content was prepared from meat-fed rats (Wistar, female, weighing 150 g, purchased from the Nippon Bio-Supply Center) according to the method of Weinstein et al.11,12 The test strain was cultivated for 48 h on Sabouraud dextrose agar plates (Becton Dickinson, Sparks, MD, USA), and the resultant colonies were suspended in Sabouraud liquid broth modified antibiotic medium 13 (Becton Dickinson). One hundred microlitres of the mixture consisting of a fungal suspension and equal amount of autoclaved caecal content was injected into mice intraperitoneally, using a disposable sterile tuberculin syringe with a 23-gauge needle.

For the experiment on the influence of immunosuppression on efficacy against disseminated candidiasis, mice received cyclophosphamide (Acros Organics, Springfield, NJ, USA) at 200 mg/kg administered intraperitoneally 4 days before and 1 day after infection.13

Fungal suspension (C. albicans: 2.5 x 106 cfu/mouse) was inoculated into the peritoneal space of 50 mice, 4 days (96 h) after the intraperitoneal injection of cyclophosphamide.

Treatment regimen

The antifungal agents were administered once daily for 4 days, starting at 1 h after infection. On the basis of the fact that micafungin efficacy has been shown to be equal to that of fluconazole in one-tenth dosage in a previous study,13 we set up the regimens as follows: (i) once daily with fluconazole 8 mg/kg of body weight; (ii) once daily with fluconazole 16 mg/kg of body weight; (iii) once daily with micafungin 0.8 mg/kg of body weight; or (iv) once daily with micafungin 1.6 mg/kg of body weight.

Assessment of therapeutic efficacy

We assessed the short-term and long-term therapeutic effects after the end of therapy. The mice were humanely terminated using inhaled ether and dissected to find intra-abdominal tumours at 24 h (1 day) and 8 days after the end of therapy. The number of C. albicans in each abscess was also determined by a quantitative culture technique. When abdominal tumorous lesions were found, they were removed and placed into a sterilized Petri dish. All such tumours were confirmed to contain purulent discharge through incision and considered as abscesses. A whole abscess was put into 1 mL of Sabouraud liquid broth modified antibiotic medium 13 (Becton Dickinson), and vortexed with sterilized glass beads. The homogenate was diluted by serial 10-fold dilution, and the diluent was spread onto Sabouraud dextrose agar. After 96 h of incubation, the number of fungi was calculated, and viable cell counts expressed as common logarithms. In this manner, the lower level of detection of viable fungal number was found to be 20 cells per abscess. When there were no colonies on Sabouraud dextrose agar plates, we assumed them to be sterile abscesses. The therapeutic efficacies were assessed by three criteria: the number of abscesses formed, the number of abscesses containing viable fungi, and the viable fungal count per abscess. These experiments were carried out twice, and the reproducibility was confirmed.

Statistical analyses

The results of the experiments were statistically analysed using the Student's t-test. P values below 0.05 were considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The MICs of fluconazole and micafungin for C. albicans were 0.0313 and 0.0156 mg/L, respectively.

Figure 1 shows the therapeutic effects of fluconazole and micafungin, with regard to the number of abscesses formed, the number of abscesses containing viable fungi, and the viable fungal count per abscess at 24 h (1 day) and 8 days after the end of therapy. The therapeutic effect of micafungin 8 days after the end of therapy was superior to that of fluconazole. There was a significant difference (P < 0.001) between fluconazole- and micafungin-treated groups at 8 days after the end of therapy, whereas there was no significant difference (P > 0.05) at 24 h after the end of therapy, for the abscesses formed, abscesses containing viable fungi, and the viable fungal count per abscess. We also obtained a significant difference between fluconazole- and micafungin-treated groups in the second experiment.



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Figure 1. The number of abscesses formed, the number of abscesses containing viable fungi, and the viable fungal count per abscess at 24 h and 8 days after the end of therapy. The left and right columns show the results at 24 h (1 day) and 8 days after the end of therapy, respectively. There are significant differences between fluconazole- and micafungin-treated groups (*P < 0.001).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The frequency of fungal peritonitis has been increasing in recent times because of progress in medicine.24 However, even though appropriate experimental infection models have been developed,10 few studies on fungal peritonitis have been conducted. We investigated the therapeutic efficacy of micafungin, one of the echinocandins, against peritonitis, using the experimental intraperitoneal abscess model caused by Candida species as described previously.10 This study showed that the therapeutic effect of micafungin was equal to that of fluconazole in one-tenth dosage even in peritonitis.

There have been few reports that compared the therapeutic effects at various time points after the end of treatment in any experimental infection models.13,14 We assessed the therapeutic effect of antifungals at 24 h and 8 days after the end of therapy. Interestingly, a significant difference was recognized in therapeutic effect by the time of the assessment in this study. Both fluconazole and micafungin appeared to be effective at 24 h after the end of therapy, whereas recurrent fungal infection may have occurred in part in the fluconazole-treated group. The result may be caused by micafungin being a fungicidal agent against Candida species, whereas fluconazole is fungistatic against Candida species.6 We hypothesize that it would be better to use the fungicidal agent micafungin in the therapy of fungal peritonitis, especially in highly immunosuppressed patients.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Lamagni, T. L., Evans, B. G., Shigematsu, M. et al. (2001). Emerging trends in the epidemiology of invasive mycoses in England and Wales (1990–9). Epidemiology and Infection 126, 397–414.[CrossRef][ISI][Medline]

2 . Mikamo, H., Kawazoe, K., Sato, Y. et al. (1996). Pelvic abscess and fungemia caused by Candida glabrata. Journal of Infection and Chemotherapy 2, 294–6.

3 . Mikamo, H., Ninomiya, M. & Tamaya, T. (2003). Tuboovarian abscess caused by Candida glabrata in a febrile neutropenic patient. Journal of Infection and Chemotherapy 9, 257–9.[CrossRef][Medline]

4 . Mikamo, H., Sato, Y., Hayasaki, Y. et al. (2000). Current status and fluconazole treatment of pelvic fungal gynecological infections. Chemotherapy 46, 209–12.[CrossRef][ISI][Medline]

5 . Wiederhold, N. P. & Lewis, R. E. (2003). The echinocandin antifungals: an overview of the pharmacology, spectrum and clinical efficacy. Expert Opinion on Investigational Drugs 12, 1313–33.[CrossRef][ISI][Medline]

6 . Denning, D. W. (2003). Echinocandin antifungal drugs. Lancet 362, 1142–51.[CrossRef][ISI][Medline]

7 . Mikamo, H., Sato, Y. & Tamaya, T. (2000). In vitro antifungal activity of FK463, a new water-soluble echinocandin-like lipopeptide. Journal of Antimicrobial Chemotherapy 46, 485–7.[Abstract/Free Full Text]

8 . Ostrosky-Zeichner, L., Rex, J. H., Pappas, P. G. et al. (2003). Antifungal susceptibility survey of 2,000 bloodstream Candida isolates in the United States. Antimicrobial Agents and Chemotherapy 47, 3149–54.[Abstract/Free Full Text]

9 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard M27-A. NCCLS, Villanova, PA, USA.

10 . Mikamo, H., Hua, Y. X., Hayasaki, Y. et al. (2000). Effect of fluconazole on viable cell count in experimental intraperitoneal Candida abscesses. Journal of Infection and Chemotherapy 6, 144–7.[CrossRef][Medline]

11 . Weinstein, W. M., Onderdonk, A. B., Bartlett, J. G. et al. (1974). Experimental intra-abdominal abscesses in rats: development of an experimental model. Infection and Immunity 10, 1250–5.[ISI][Medline]

12 . Weinstein, W. M., Onderdonk, A. B., Bartlett, J. G. et al. (1975). Antimicrobial therapy of experimental intraabdominal sepsis. Journal of Infectious Diseases 132, 282–6.[ISI][Medline]

13 . Ikeda, F., Wakai, Y., Matsumoto, S. et al. (2000). Efficacy of FK463, a new lipopeptide antifungal agent, in mouse models of disseminated candidiasis and aspergillosis. Antimicrobial Agents and Chemotherapy 44, 614–8.[Abstract/Free Full Text]

14 . Maesaki, S., Hossain, M. A., Miyazaki, Y. et al. (2000). Efficacy of FK463, a (1, 3)-ß-D-glucan synthase inhibitor, in disseminated azole-resistant Candida albicans infection in mice. Antimicrobial Agents and Chemotherapy 44, 1728–30.[Abstract/Free Full Text]





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