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

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.

Drug repurposing strategies in the development of potential antifungal agents

“…In addition, verapamil enhances the antifungal activity of tunicamycin or fluconazole against C. albicans during biofilm formation and pre-formed biofilms (Yu et al 2013 ). In A. fumigatus , the drug efflux pump was blocked and ergosterol content was decreased following treatment with verapamil and itraconazole simultaneously (Zeng et al 2019 ). Other CCBs, such as diltiazem, nicardipine, and nifedipine, have shown antifungal activity against C. albicans , C. glabrata , Ascomycetous, and Mucoralean fungi, either alone or in combination with antifungals (Bulatova and Darwish 2008 ; Homa et al 2017 ; Alnajjar et al 2018 ).…”

Combination of Miconazole and Domiphen Bromide Is Fungicidal against Biofilms of Resistant Candida spp

“…For example, Lafleur and colleagues (2013) showed that a suboptimal dose of fluconazole (0.005%) can be potentiated by 2-adamantanamine (AC17) in an in vivo guinea pig model of cutaneous candidiasis (68). Another study demonstrated a synergistic effect of verapamil and a suboptimal itraconazole dose in vivo, resulting in a reduced fungal lung burden in mice infected by Aspergillus fumigatus (69). To test the MCZ-DB combination in vivo, a formulation containing 1% MCZ nitrate and 0.144% DB was used.…”

Amlodipine Increases the Therapeutic Potential of Ravuconazole upon Trypanosoma cruzi Infection

“…Previous reports have indicated that ergosterol inhibitors can interact positively with antagonists of calcium channels against fungal organisms (26,27) and trypanosomatids (12)(13)(14). Here, we investigate if amlodipine, a CCB used clinically as an antihypertensive agent, could increase the anti-T. cruzi activity of ravuconazole in vitro and in vivo.…”

“…Amlodipine is among the best-studied agents that reverse resistance of Plasmodium to antimalarial agents (28), and accumulating evidence suggests that some CCBs can enhance the antifungal activity of azole derivatives by interfering with efflux pumps and/or influencing calcium signaling pathways (26,27). The drug has also exhibited broad-spectrum antibacterial activities and in vitro synergistic antimicrobial effects on multidrug-resistant microorganisms (29).…”