Effect of protein binding on the activity of voriconazole alone or combined with anidulafungin against Aspergillus spp. using a time-kill methodology

Abstract

Objectives: the aims of the study were to explore the activity of total and free (according to protein binding) maximal concentrations achieved in serum after multiple doses of voriconazole 400/200 mg and anidulafungin 200/100 mg against Aspergillus fumigatus and Aspergillus flavus and the human albumin or serum effects on antifungal activity. Material and methods: Time-kill curves were performed with two A. fumigatus and two A. flavus strains at voriconazole and anidulafungin Cmax concentrations using different media: a) RPMI broth (Cmax-RPMI); b) RPMI with human serum (Cmax-HS), and c) RPMI with human albumin (Cmax-HAlb). In parallel, free-drug (fCmax) concentrations considering theoretical protein binding were performed in RPMI broth. Aspergillus metabolic activity was measured by the XTT reduction assay. Results: Voriconazol or voriconazole plus anidulafungin reduced >88.4% the metabolic activity of Aspergillus sp. at Cmax-RPMI and fCmax after 48 h of exposition. Anidulafungin alone showed poor metabolic reductions (<80.1% at Cmax- RPMI and <15% at fCmax). Anidulafungin activity, but not voriconazole activity alone or combined decreased in presence of HS or HAlb (more pronounced in A. flavus strains and HAlb). However, anidulafungin Cmax-HS or Cmax-HAlb against A. fumigatus strains were significantly more active (p<0.05) than fCmax in RPMI. These species and culture medium-dependent impact of human protein binding in the activity of anidulafungin was related to macroscopic and microscopic differences among mycelial mat grown in RPMI, HS or HAlb in whose XTT retention was different. Conclusions: Synergism could not be demonstrated due to the high activity showed by voriconazole. Protein binding has not impact on voriconazole activity and this impact is considerably less than predicted by free concentration extrapolated from theoretical binding rate on anidulafungin. The XTT colorimetric assay needs to be standardized for use with Aspergillus spp. since without DMSO extraction the activity of echinocandins in a free-human protein RPMI medium could be overestimated.