Potent synergic effect between ibuprofen and azoles on Candida resulting from blockade of efflux pumps as determined by FUN-1 staining and flow cytometry

Objectives: Resistance to antifungals often relates to efflux pumps exporting drugs; several modulators may block them, reverting resistance. Verapamil, beta-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. H-3-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 H-3-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.