Clinical and therapeutic developments in fungal infection

Saturday, March 31, 2012, 15:18 - 15:30
Effective reversion of fluconazole resistance by ibuprofen in an animal model
S. Costa-de-Oliveira*, I. M Miranda, E. Ricardo, A. Silva-Dias, A. G Rodrigues, C. Pina-Vaz (Porto, PT)
Objectives: Ibuprofen was found to be an efficient reverter of in vitro fluconazole resistance due to overexpression of efflux pumps1,2; however its in vivo effect is still unproven.The aim of our study was to evaluate in an animal model the effect of ibuprofen associated to fluconazole in the treatment of an invasive infection by a resistant C. albicans isolate.
Methods: A C. albicans resistant (R) strain to fluconazole was obtained by subculturing with serial concentrations of fluconazole a susceptible strain (S) during 30 days. Minimal inhibitory concentrations (MIC) to fluconazole was determined in the presence of 100µg/ml of ibuprofen (IBU), an efflux pump blocker1,2.
Comparative transcriptome analysis between the S and the induced resistant strain (R) incubated with and without ibuprofen (RI) was performed using C. albicans DNA microarrays from Agilent Technologies.
The in vivo study was carried out according to the murine candidiasis model. Female BALB/c mice were infected with 5x105 cells in 0.1 ml of sterile saline via the lateral tail vein with the S strain (three groups) or the R strain (three groups). Antifungal therapy was administered intraperitoneally with FLC or IBU or FLC+IBU on both groups 3 hours after microbial challenge and repeated once a day for a total of four days. The kidney fungal burden was determined.
Results: Ibuprofen decreased azole MIC values, the R phenotype changing to S. Microarray analysis identified 836 and 1517 with differential expression in R and RI strains, respectively. The R strain showed overexpression of CDR11, ERG251, CDR4 and the transcription factor UPC2. In the RI and in the S strains those genes were down regulated.
FLC showed to be effective only in the treatment of the infection by the S strain, reducing dramatically the fungal burden. Interestingly, in mice infected with the R strain but treated with FLC + IBU, a significant decrease in the fungal burden was observed. In the absence of FLC, IBU did not display antifungal activity per se.
Conclusions: The in vivo synergic effect between fluconazole and ibuprofen demonstrated herein may represent a hopeful future approach for a better management of antifungal resistance conferred by efflux pump overexpression.
1. Pina-Vaz, C., et al. J Antimicrob Chemother 2005, 4: 678-85

Antifungal activity of ibuprofen alone and in combination with fluconazole against Candida species.
Pina-Vaz C, Sansonetty F, Rodrigues AG, Martinez-De-Oliveira J, Fonseca AF, Mårdh PA.
Source
Department of Microbiology, Porto School of Medicine, University of Porto, Portugal. micfam@ip.pt
Abstract
Ibuprofen, a non-steroidal anti-inflammatory drug, exhibited antimicrobial activity against Candida albicans and non-albicans strains. At 10 mg/ml, ibuprofen showed a rapid cidal activity against exponential growth phase C. albicans, accompanied by rapid and extensive leakage of intracellular K+, permeation to propidium iodide, lysis of spheroplasts and severe membrane ultrastructural alterations. These results indicate that the killing of Candida cells is due to direct damage to the cytoplasmic membrane. At 5 mg/ml, ibuprofen inhibited growth; however, it did not kill the yeasts and did not directly affect the cytoplasmic membrane. Evaluation of yeast metabolic vitality with the fluorescent probe FUN-1 showed that growth inhibition induced by the fungistatic drug concentration was due to metabolic alterations. The combination of ibuprofen with fluconazole resulted in synergic activity with eight of the 12 Candida strains studied, including four of the five fluconazole-resistant strains. The MICs of fluconazole for the fluconazole-resistant strains decreased 2-128-fold when the drug was associated with ibuprofen. When in combination with fluconazole, MICs for ibuprofen decreased by up to 64-fold for all the 12 strains studied. These results point to the practicability of using ibuprofen, alone or in combination with azoles, in the treatment of candidosis, particularly when applied topically, taking advantage of the drug's antifungal and anti-inflammatory properties.

Ibuprofen may possess a marked therapeutic potential, particularly due to its ability to revert resistance to fluconazole. The serum
concentration of ibuprofen needed to achieve an anti-inflammatory
effect27 is lower than the concentration needed to obtain an antifungal effect.9
This concentration is thus sufficient to induce the
blockade of efflux pumps and to revert resistance to azoles. The
anti-inflammatory and analgesic properties of ibuprofen might also
represent an additional advantage. Additionally, our study opens
new perspectives for treatment of candidosis by rehabilitating an
important drug like fluconazole.

Potent synergic effect between ibuprofen and azoles on Candida resulting from blockade of efflux pumps as determined by FUN-1 staining and flow cytometry
Cidália Pina-Vaz1,2,*, Acácio Gonçalves Rodrigues1,2, Sofia Costa-de-Oliveira1, Elisabete Ricardo1 and Per-Anders Mårdh3
+ Author Affiliations

1Department of Microbiology, Porto Faculty of Medicine, Alameda Prof. Hernani Monteiro, 4200 Porto, Portugal; 2IPATIMUP—Institute of Pathology and Molecular Immunology of Porto University, Porto, Portugal; 3Department of Obstetrics and Gynecology, University Hospital, Lund, Sweden
*Corresponding author. Tel: +351-91-9358514; Fax: +351-229962096; E-mail: cpinavaz@yahoo.com
Received April 29, 2005.
Revision received May 24, 2005.
Revision received June 15, 2005.
Accepted June 23, 2005.

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Abstract

Objectives: Resistance to antifungals often relates to efflux pumps exporting drugs; several modulators may block them, reverting resistance. Verapamil, β-oestradiol and progesterone, known efflux pump inhibitors of human neoplastic cells, and ibuprofen were tested as potential modulators of resistance of Candida spp.

Methods: Forty-two clinical isolates of Candida (38 fluconazole-resistant), two ATCC type strains and two C. albicans strains with known mechanisms of fluconazole resistance were incubated with subinhibitory concentrations of the modulators. After exposure, MICs of fluconazole, itraconazole and voriconazole were re-determined. Simultaneously, yeasts exposed to modulators were stained with FUN-1 and analysed by flow cytometry. 3H-labelled itraconazole was also used to study efflux in the presence and absence of modulators.

Results: Fluconazole MICs decreased in most strains after exposure to modulators, including control strains with documented efflux overexpression. No significant MIC variation was noticed for: all C. krusei strains tested, for the resistant strain by target change, for susceptible strains, and for a very few other clinical isolates. Reverted resistant phenotypes showed cross-resistance to itraconazole and to voriconazole, which was also reverted by the modulators. For these strains, an increase in FUN-1 staining and increased accumulation of 3H-labelled itraconazole were noticed after incubation with modulators.

Conclusions: Resistance related to overexpression of efflux pumps was common among clinical isolates and could be reverted by the assayed modulators, particularly ibuprofen. The mechanism of resistance in all tested C. krusei and in a few other strains seems, however, to be of a different nature. Ibuprofen is a promising compound in association with azoles, deserving future clinical trials. FUN-1 proved to be a good marker of efflux in Candida.