Efficacy of caspofungin combined with amphotericin B against azole-resistant Candida albicans

Mohammad A. Hossain1, Guadalupe H. Reyes1, Lisa A. Long1, Pranab K. Mukherjee1 and Mahmoud A. Ghannoum1,2,*

1 Center for Medical Mycology, Department of Dermatology, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106-5028; 2 University Hospitals of Cleveland, Cleveland, OH 44106, USA

Received 25 September 2002; returned 1 December 2002; revised 26 February 2003; accepted 26 February 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The activity of caspofungin (CFG) combined with amphotericin B (AMB) against azole-resistant Candida albicans was evaluated in vitro (chequerboard) and in vivo (murine). CFG+AMB resulted in positive interactive effects in vitro (fractional inhibitory concentration index 0.75). Compared with untreated controls, CFG+AMB prolonged mouse survival (P = 0.006) and compared with AMB alone, CFG+AMB prolonged mouse survival (P = 0.36); however, the latter difference was not signficant. CFG+AMB treatment significantly reduced kidney cfu compared with untreated controls and CFG-treated groups (P <= 0.05 for both comparisons). In addition, this combination reduced brain cfu significantly compared with untreated controls and AMB-treated mice (P = 0.005 and 0.05, respectively).

Keywords: disseminated candidiasis, antifungal, therapy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Candidal infections are associated with the highest crude mortality compared with other nosocomial bloodstream pathogens.1 Therefore, there is a growing need for new approaches, such as combination therapy, to treat invasive candidiasis. The echinocandin caspofungin (CFG) is a new antifungal with activity against yeasts and moulds.2 Unlike amphotericin B (AMB), which binds to membrane sterols, CFG inhibits the fungal (1,3)-ß-D-glucan synthase enzyme complex that forms glucan polymers, a major fungal cell wall component.3 Our hypothesis is that an effective way to inhibit Candida albicans is by combining a cell wall active agent (CFG) with a membrane active drug (AMB).

This study evaluates the activity of CFG+AMB against an azole-resistant C. albicans isolate using in vitro and in vivo methodologies. No antagonistic interactions were observed between the two agents, which showed a trend towards additivity.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Caspofungin acetate (CFG, Merck Research Laboratories, Rahway, NJ, USA) and amphotericin B deoxycholate (AMB, Bristol-Myers Squibb, Princeton, NJ, USA) powders were dissolved in sterile distilled water. CFG was stored at –20°C and AMB at 4°C in the dark.

Candida albicans strain 12-99, a clinical isolate obtained from a patient for whom fluconazole therapy failed, was used. Antifungal susceptibility testing was performed according to M27-A methodology.4 Before each experiment, C. albicans was grown overnight at 37°C in Sabouraud dextrose broth (SDB; Difco Laboratories, Detroit, MI, USA). Blastospores were washed twice and suspended in sterile normal saline (NS; 0.85%); their number was determined using a haemocytometer, and confirmed by quantitative culturing.

A chequerboard technique was used to evaluate drug–drug interactions. Different concentrations of CFG and AMB in RPMI 1640 medium were combined into wells of microtitre plates so that the concentration of each agent increased simultaneously. Two rows, consisting of serial dilutions of the individual drugs alone, were also included. The highest concentrations of CFG and AMB used were 128 and 64 mg/L, respectively. Wells were inoculated with C. albicans (between 0.5 and 2.5 x 103 cfu/mL) and plates incubated at 35°C for 48 h. Growth in each well was observed visually. The fractional inhibitory concentration index (FICI) was calculated. The activity was expressed as synergic when the FICI was < 0.5 and antagonistic when the FICI was > 4.0; FICI < 1 was regarded as a positive and FICI > 1 as a negative interaction.

The Institutional Animal Care and Use Committee approved the protocol for our murine model. Six- to eight-week-old male, BALB/c mice (Charles River, Wilmington, MA, USA) were housed in cages (three mice or fewer/cage) and acclimatized for 5 days before fungal challenge. Each mouse was inoculated with 0.1 mL of C. albicans 12-99 (prepared in sterile NS from an overnight, 37°C, SDB culture) via the lateral tail vein. To determine the optimal challenge inoculum, three groups of mice (five mice/group) were infected intravenously with different concentrations of C. albicans (5 x 105, 1 x 106 or 3 x 106 blastospores).

In subsequent experiments, the animals were infected with 5 x 105 cells in 100 µL of NS. They were divided into seven mice/group and received their therapies intraperitoneally 2 h post-challenge. In preliminary experiments, drug dosages that were below an effective dose when used singly were determined. The following dosages were evaluated: CFG 0.0005, 0.001 and 0.002 mg/kg; and AMB 0.008, 0.016 and 0.032 mg/kg daily for 14 days. Based on these experiments, the following dosages were selected for subsequent drug–drug interaction studies: CFG 0.002 mg/kg and AMB 0.016 mg/kg. Efficacy was evaluated by monitoring survival and tissue burden. Animals were monitored daily for evidence of infection and its severity, and deaths were noted. Each experiment was performed twice (totalling 14 animals/group) to determine the survival pattern. For determination of tissue fungal burden, seven mice per group were killed; their kidneys and brain were removed aseptically, weighed and homogenized. Diluted samples of homogenates were cultured on agar plates (at 37°C for 48 h), and the number of cfu counted and expressed as cfu/g of tissue.

Differences in survival were assessed by the Kaplan–Meier method, while the mean cfu were compared using the Mann–Whitney U-test (mean ± S.E.). A P value of <0.05 was considered statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The MICs of fluconazole (FLC), itraconazole (ITC), AMB and CFG for C. albicans 12–99 were 64.0, 2.0, 1.0 and 0.06 mg/L, respectively, indicating resistance to FLC and ITC. Combining AMB with CFG resulted in two- and four-fold reductions in MICs of AMB and CFG, respectively. Also, a combination of CFG+AMB revealed a positive interaction (FICI value 0.75).

Experiments directed at determining the optimal challenge inoculum, showed that mice infected with 1 x 106, or 3 x 106 cells of C. albicans exhibited clinical signs of infection (e.g. reduced activity, ruffled hair) on day 2, and all died within 2–3 days. Mice inoculated with 5 x 105 cells showed signs of infection on day 4, and survived until day 7. Thus, 5 x 105 cells/animal was selected as the optimal challenge inoculum.

Treating animals with a CFG+AMB combination significantly prolonged survival compared with infected, untreated controls (P = 0.006) (Figure 1). Treatment of mice with AMB+CFG, even at low dosage (0.016 and 0.002 mg/kg), also tended to prolong survival. Survival rates for untreated controls, CFG, AMB and CFG+AMB groups at day 21 were 0%, 22%, 50% and 72%, respectively. Thus, combining CFG with AMB increased the survival rate compared with the control group. Although animals treated with a CFG+AMB combination survived longer than those treated with AMB alone (72% versus 50%, respectively) this difference was not significant (P = 0.36).



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Figure 1. Effect of antifungal therapy on survival of mice infected with C. albicans: solid circles, untreated controls; open circles, mice treated with CFG (0.002 mg/kg); open triangles, mice treated with AMB (0.016 mg/kg); open squares, mice treated with CFG+AMB. Time indicates the number of days the animals survived. The survival curve was prepared using the Kaplan–Meier method based on Logrank (Mantel–Cox) analysis; the cumulative data were generated from two experiments.

 
Table 1 compares the kidney and brain tissue burden following treatment with CFG or AMB alone, or CFG combined with AMB. Compared with untreated controls, the only treatment regimen that resulted in a reduction of cfu in the kidneys was CFG+AMB (P = 0.05). A comparison of brain tissue candidal load showed that treatment with CFG alone or CFG+AMB reduced the invasion of the brain (P values 0.05 and 0.005, respectively). No statistically significant differences in brain cfu were noted between mice treated with CFG alone or as part of a combination (P = 0.94).


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Table 1.  Effect of antifungal therapy on the tissue fungal burden of kidney and brain in mice infected with C. albicans
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our data demonstrated that no antagonism was noted in vitro or in vivo between the test drugs CFG and AMB. Franzot & Casadevall tested the combination against C. neoformans and showed synergic effects.5 Others demonstrated that the combination was synergic to additive against Aspergillus and Fusarium spp. isolates, whereas CFG alone was not effective against Fusarium. Importantly, similar to our data, no antagonism was seen with the CFG+AMB combination. Manavathu et al. reported a synergistic interaction by AMB with either micafungin (MFG) or CFG against A. fumigatus.6 To our knowledge, this is the first study to evaluate the effect of combining CFG+AMB in the treatment of haematogenously disseminated candidiasis caused by an azole-resistant C. albicans. Our data show that combination therapy resulted in prolongation of survival and a reduction in the tissue fungal burden. In agreement with our findings, Flattery et al., using a disseminated candidiasis mouse model, demonstrated that the CFG+AMB combination was synergic against C. albicans.7 A limitation of our study was that because the doses we selected were minimally effective, the difference in survival pattern and tissue fungal burden was marginal. This is often a problem when running in vivo combination studies. More strains, animals, doses and combinations would provide more biologically meaningful data.

The underlying mechanisms of synergic or additive effects for CFG+AMB are unknown. It is likely that inhibition of (1,3)-ß-D-glucan formation by CFG leads to cell wall damage. This would allow AMB easier access to the fungal cell membrane, where it binds to membrane ergosterol, resulting in pore formation and cell lysis.5 In this study, we focused on in vivo evaluation of the combined effects of CFG and AMB against an azole-resistant C. albicans strain. We did not include an azole-sensitive strain; such evaluation was beyond the scope of this study. Extending this work to cover more strains should be undertaken in the future.

In conclusion, our data demonstrate no evidence of antagonism between CFG and AMB when combined, and interactions in vivo and in vitro have tended to be favourable. Further work is needed to ascertain the clinical relevance of our findings.


    Acknowledgements
 
This study was supported by grants from Merck & Co., Inc. and NIH/NIAMS (AR-39750) to Skin Diseases Research Center, Department of Dermatology, Case Western Reserve University, Cleveland, OH, USA.


    Footnotes
 
* Corresponding author. Tel: +1-216-844-8580; Fax: +1-216-844-1076; E-mail: mag3@po.cwru.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Edmond, M. B., Wallace, S. E., McClish, D. K., Pfaller, M. A., Jones, R. N. & Wenzel, R. P. (1999). Nosocomial bloodstream infections in United States hospitals: a three-year analysis. Clinical Infectious Diseases 29, 239–44.[ISI][Medline]

2 . Abruzzo, G. K., Gill, C. J., Flattery, A. M., Kong, L., Leighton, C., Smith, J. G. et al. (2000). Efficacy of the echinocandin caspofungin against disseminated aspergillosis and candidiasis in cyclophosphamide-induced immunosuppressed mice. Antimicrobial Agents and Chemotherapy 44, 2310–8.[Abstract/Free Full Text]

3 . Hossain, M. A. & Ghannoum, M. A. (2000). New investigational antifungal agents for treating invasive fungal infections. Expert Opinion on Investigational Drugs 9, 1797–813.[ISI][Medline]

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

5 . Franzot, S. P. & Casadevall A. (1997). Pneumocandin L-743,872 enhances the activities of amphotericin B and fluconazole against Cryptococcus neoformans in vitro. Antimicrobial Agents and Chemotherapy 41, 331–6.[Abstract]

6 . Manavathu, E., Ganesan L. T., Cutright J. L. & Chandrasekar P. H. (2001). In vitro antifungal activity of voriconazole in two-drug combination with micafungin, caspofungin and amphotericin B. In Program and Abstracts of the Forty-first Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2001. Abstract J-125, p. 364. American Society for Microbiology, Washington, DC, USA.

7 . Flattery, A. M., Bartizal, K., Gill, C. J., Kong, L., Leighton, C., Pikounis, V. B. et al. (1998). Preclinical efficacy of MK-991 in combination with amphotericin B or fluconazole in mouse models of disseminated aspergillosis, candidiasis, and cryptococcosis. In Program and Abstracts of the Thirty-eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 1998. Abstract J-61, p. 468. American Society for Microbiology, Washington, DC, USA.