Department of Microbiology, School of Medicine, University of Seville, Spain
Received 16 November 2004; returned 15 December 2004; revised 21 January 2005; accepted 26 January 2005
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Abstract |
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Methods: The intracellular penetration of voriconazole into PMNs was evaluated by a radiometric assay. The effect of cell viability, environmental conditions, metabolic inhibitors and membrane stimulation was also studied. The intracellular activity was determined by incubation of PMNs containing intracellular blastospores in the presence of voriconazole for 3 h.
Results: The uptake of voriconazole by PMNs was rapid and not saturable. The cellular to extracellular concentration (C/E) ratio for voriconazole was 8.5±1.3. Voriconazole was rapidly released from loaded PMNs. The uptake of voriconazole was not affected by environmental temperature and cell viability. Neither the external pH nor the metabolic inhibitors affected the uptake of voriconazole. The ingestion of opsonized zymosan, but not of opsonized Candida spp., significantly decreased the levels of PMN-associated voriconazole. At the extracellular concentrations evaluated, voriconazole did not affect the intracellular survival of Candida.
Conclusions: Voriconazole reached high intracellular concentrations within human PMNs. The uptake was rapid and not saturable but it did not affect the intracellular killing of Candida spp.
Keywords: Candida , azoles , accumulation
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Introduction |
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Fluconazole has been a convenient and effective treatment for candidiasis. However, with extensive use of fluconazole, strains of Candida albicans have emerged that are resistant to fluconazole.2 In addition, some Candida spp., such as Candida krusei, are intrinsically resistant to fluconazole.3
Voriconazole, a new triazole agent, is commonly used for treatment of invasive aspergillosis; moreover this drug is also active against many fluconazole-resistant Candida spp.4 There is little information on the ability of this antifungal agent to penetrate and concentrate within PMN cells.
The purpose of this study was to evaluate the intracellular penetration of voriconazole into human PMNs. Additionally, the possible mechanism involved in the membrane transportation of this antimicrobial agent was also evaluated. Finally, the correlation between intracellular penetration and activity against Candida spp. within human PMNs was also studied.
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Materials and methods |
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Further studies to elucidate the mechanism of voriconazole uptake by PMNs were performed, as described previously.6 The influence of cell viability, environmental temperature, pH and metabolic inhibitors was evaluated.
In a series of experiments, voriconazole uptake by human PMNs was measured after stimulation of cells with phorbol myristate acetate (PMA) and after phagocytosis of either opsonized zymosan or opsonized C. albicans (strain CNMCL1017, fluconazole-susceptible; and strain CNMCL1518, fluconazole-resistant) and C. krusei ATCC 6258.7
The efflux of PMN-associated voriconazole was also studied. PMNs were incubated for 20 min at 37°C with voriconazole (extracellular concentration of 2 mg/L), collected by centrifugation and rapidly resuspended in antifungal agent-free medium. Cell-associated voriconazole was quantified at various time intervals after the removal of the extracellular antifungal agent.
The partition coefficients of voriconazole were determined by the modified method of Nikaido.8
To evaluate the intracellular activities of antifungal agents, a previously described method was used.6 C. albicans (a fluconazole-susceptible strain and a fluconazole-resistant strain) and C. krusei were used for killing assays. C. albicans strains were kindly provided by Dr M. Cuenca-Estrella, Centro Nacional de Microbiología, Madrid. Susceptibility studies were performed using the colorimetric microdilution test (Yeastone®; Trek Diagnostic Systems Ltd, UK). The MICs of voriconazole for C. albicans (fluconazole-susceptible and fluconazole-resistant) and C. krusei were 0.06, 0.12 and 1 mg/L, respectively.
Yeast suspension and PMNs were incubated for 60 min. After this, extracellular blastospores were removed by differential centrifugation. Cells were then suspended in RPMI medium. At this time, different concentrations of antifungal agents (1, 2 and 5 mg/L) were added, and the cells reincubated for 3 h at 37°C. Cells were lysed in distilled water, and samples were diluted and pour plated onto Sabouraud agar. Colonies were counted after 48 h of incubation at 37°C. The data were expressed as percentages of Candida surviving compared with levels in controls (without antifungal agent).
All data were expressed as means ± SD. Differences among groups were compared by analysis of variance, to assess statistical significance at P < 0.05.
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Results and discussion |
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In summary, voriconazole penetrates into human phagocytic cells, reaching intracellular concentrations several times higher than extracellular concentrations and greater than those described for other azoles. Using our assay system, the intracellular survival of Candida spp., however, is not affected by voriconazole.
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References |
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