In vitro and in vivo Efficacy of a Synergistic Combination of Itraconazole and Verapamil Against Aspergillus fumigatus.

Qiuqiong Zeng,Zheng Zhang,Peiying Chen,Ling Lu,Nanbiao Long,Hong Sang

doi:10.3389/fmicb.2019.01266

Abstract

The incidence of aspergillosis continues to rise sharply, while the progress made in expanding the antifungal drug arsenal remains extremely slow, indicating an urgent need for new strategies. Previous studies have shown that the calcium signaling pathway, which is evolutionarily conserved in mammals and fungi, is involved in regulating the tolerance of azoles in fungi. In this study, we performed a preliminary screening among various combinations of different clinical calcium channel blockers and different antifungal drugs. We found that the combination of itraconazole and verapamil showed the best synergistic effect against Aspergillus fumigatus. Thereafter, using the checkboard assays we observed synergistic effects of the combination treatment against most of the A. fumigatus strains tested, including itraconazole-sensitive and itraconazole-resistant strains, with a fractional inhibitory concentration index (FICI) < 0.5. Furthermore, we showed that verapamil strongly decreased the cytosolic calcium transients following itraconazole stimulation by an aequorin-mediated method. Moreover, verapamil influenced the efflux of rhodamine 6G, an azole mimic substance. An ergosterol assay revealed that verapamil alone had no effect on ergosterol biosynthesis, but the combination of itraconazole and verapamil treatment decreased the ergosterol level. Further murine assays were performed using a luciferase-probed bioluminescence imaging method. Drug combination therapy reduced lung burden and improved survival rate. In conclusion, verapamil is a promising candidate to enhance the antifungal activity of itraconazole against A. fumigatus. In addition, our study suggests the effectiveness of an emerging approach based on bioluminescence imaging in monitoring the efficacy of drug combination therapy for invasive aspergillosis.

Highlights

Aspergillus fumigatus is a ubiquitous and opportunistic filamentous fungal pathogen that can cause invasive, chronic, and allergic aspergillosis
To find the drug combination that displays the best synergism, we initially performed a preliminary screening by comparing the antifungal effects among three representative Calcium channel blockers (CCBs) and three commonly used antifungal drugs against the clinical A. fumigatus isolate Afc03
The combination of VER and ITC reduced the diameters of the colonies of A. flavus and A. terreus, but no differences were observed in A. niger with the combination compared to ITC alone. These preliminary screening results suggested that the combination of VER and ITC was the best candidate to show a synergistic effect against A. fumigatus strains among the representative CCBs and antifungal drugs tested

Summary

Introduction

Aspergillus fumigatus is a ubiquitous and opportunistic filamentous fungal pathogen that can cause invasive, chronic, and allergic aspergillosis. Invasive aspergillosis is one of the most important life-threatening fungal infections and mainly affects immunocompromised hospitalized patients, such as patients with hematological malignancies or AIDS and solid organ or hematopoietic stem-cell transplant (HSCT) recipients (Morgan et al, 2005; Rubio et al, 2009; Taccone et al, 2015; Koehler et al, 2017; Zilberberg et al, 2018). This condition has a very high mortality rate ranging from 40 to 90% (Montagna et al, 2013). Mining existing agents that can enhance the efficacy of antifungal drugs is a promising approach to improve the drug susceptibility of A. fumigatus

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