Division of Infectious Diseases, Department of Medicine, Wayne State University, 427 Lande Building, 550 E. Canfield, Detroit, MI 48201, USA
Received 3 December 2002; returned 23 January 2003; revised 7 March 2003; accepted 9 March 2003
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Abstract |
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Method: Conidial suspensions were prepared from 20 clinical isolates of A. fumigatus highly susceptible to itraconazole, voriconazole, posaconazole and ravuconazole (MIC-0 range 0.1251 mg/L), and caspofungin (MIC-0 range 3264 mg/L). The in vitro susceptibility of A. fumigatus to two-drug combinations of itraconazole, voriconazole, posaconazole and ravuconazole with caspofungin was evaluated by the fractional inhibitory concentration index (FICI) method.
Results: Two-drug combinations of caspofungin with itraconazole (FICI = 0.49 ± 0.04) or posaconazole (FICI = 0.32 ± 0.09) provided synergic interaction. On the other hand, ravuconazole (FICI = 0.61 ± 0.31) and voriconazole (FICI = 1.61 ± 0.42) in combination with caspofungin showed no interaction against A. fumigatus.
Conclusions: Our data show that the in vitro antifungal efficacies of combinations of members from two different classes are not always similar and hence are not predictable.
Keywords: triazoles, antifungal combination, caspofungin, Aspergillus fumigatus
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Introduction |
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Materials and methods |
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Voriconazole, itraconazole, posaconazole and ravuconazole were obtained from Pfizer Pharmaceuticals (New York, NY, USA), Janssen Pharmaceutica (Beerse, Belgium), Schering-Plough Research Institute (Kenilsworth, NJ, USA) and Bristol-Myers Squibb Institute for Medical Research (Princeton, NJ, USA), whereas caspofungin was obtained from Merck and Company (Rahway, NJ, USA), respectively. The triazoles were dissolved in dimethyl sulphoxide to obtain a stock solution of 1 g/L and stored as 0.25 mL lots at 20°C. Caspofungin was dissolved in sterile double-distilled water to obtain a concentration of 10 g/L and stored as 0.25 mL lots at 70°C. The frozen stocks of the antifungal agents were thawed at room temperature and used within 24 h.
Determination of fractional inhibitory concentration index (FICI)
Twenty clinical isolates of A. fumigatus obtained from the Microbiology Laboratory of the Detroit Medical Center (Detroit, MI, USA) were used in this study. Conidial suspensions from 7-day-old A. fumigatus cultures were prepared, standardized by haemocytometry and used as inoculum for susceptibility testing. The in vitro susceptibility of A. fumigatus to two-drug combinations of caspofungin with various triazoles was evaluated by the FICI method determined by two-dimensional chequerboard using the National Committee for Clinical and Laboratory Standards M38-A broth microdilution technique.7 This is the reference method for testing filamentous fungi with azoles, amphotericin B and flucytosine, and has not yet been validated for caspofungin. Pair-wise combinations of the required concentrations of caspofungin (antifungal A) and the required triazole (antifungal B) were prepared in two-fold increments in 0.1 mL RPMI 1640 containing 0.165 M MOPS buffer (pH 7.0). Eleven wells in the top row and seven wells in the first column contained various concentrations of caspofungin and the triazole alone, respectively. The well at the top left corner of the microdilution plate contained no drug (drug-free growth control). The other 77 wells in the microdilution plate contained combinations of various concentrations of caspofungin and the required triazole. To each well, 0.1 mL of fresh conidial suspension (2 x 104 conidia/mL) was added. The content of each well of the microdilution plate was mixed by repeated pipetting back and forth with a multichannel pipette. The plate was incubated at 35°C for 48 h and the MIC-0 was defined as the concentration of the drug that provided no visible growth as determined by the reduction of the tetrazolium compound 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) by the fungal cells.8 The microdilution plate well that represents the lowest concentration(s) of the drug(s) with no visible colour was taken as the end-point. Drug concentrations with a range of either 0.0151 mg/L (triazoles) or 0.06264 mg/L (caspofungin) were used for MIC determinations. The FICI was calculated by the formula: FICI = (Ac/Aa) + (Bc/Ba) where Ac and Bc are the MICs of drugs A and B in combination and Aa and Ba are the MICs of drugs A and B. The drug interactions were classified as synergic (FICI 0.5) and antagonistic (FICI > 4), and showing no interaction between FICI = >0.5 and 4.
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Results and discussion |
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The reason(s) for the differential in vitro activity of triazoles in two-drug combinations with caspofungin is at present only a matter of speculation. However, it is possible that the effect occurs only in vitro and has no in vivo correlation. The azoles may have additional less prominent mode(s) of action and such a minor mode of action is enhanced prominently in combination with caspofungin in the case of certain triazoles resulting in increased efficacy. Moreover, it is possible that the uptake of azoles is selectively interfered with or enhanced in the presence of caspofungin. Alternatively, it is possible that differential lipophilicity of the four triazoles may play a role in their differential interaction in combination with caspofungin. Itraconazole and posaconazole with their long hydrophobic aliphatic tail region will be more lipid soluble than voriconazole and ravuconazole. The lipophilic itraconazole and posaconazole interact synergically with caspofungin, a cyclic lipohexapeptide compound, whereas voriconazole and ravuconazole are unable to do so. These results indicate that the in vitro interactions of members from two drug classes are not always identical and hence are not predictable.
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Acknowledgements |
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Footnotes |
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References |
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