In vivo efficacy of pitavastatin combined with itraconazole against Aspergillus fumigatus in silkworm models

Abstract

Azole resistance in Aspergillus fumigatus is a worldwide concern and new antifungal drugs are required to overcome this problem. Statin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, has been reported to suppress the growth of A. fumigatus, but little is known about its in vivo antifungal effect against A. fumigatus. In this study, we evaluated the in vivo efficacy of pitavastatin (PIT) combined with itraconazole (ITC) against azole-susceptible and azole-resistant strains with silkworm models. Prolongation of survival was confirmed in the combination-therapy (PIT and ITC) group compared to the no-treatment group in both azole-susceptible and azole-resistant strain models. Furthermore, when the azole-susceptible strain was used, the combination-therapy resulted in a higher survival rate than with ITC alone. Histopathological analysis of the silkworms revealed a reduction of the hyphal amount in both azole-susceptible and azole-resistant strain models. Quantitative evaluation of fungal DNA by qPCR in azole-susceptible strain models clarified the reduction of fungal burden in the combination-therapy group compared with the no-treatment group and ITC-alone group. These results indicate that the efficacy of PIT was enhanced when combined with ITC in vivo. As opposed to most statins, PIT has little drug-drug interaction with azoles in humans and can be used safely with ITC. This combination therapy may be a promising option as an effective treatment in clinical settings in the future. IMPORTANCE Azole resistance among A. fumigatus isolates has recently been increasingly recognized as a cause of treatment failure, and alternative antifungal therapies are required to overcome this problem. Our study shows the in vivo efficacy of PIT combined with ITC against A. fumigatus using silkworm models by several methods including evaluation of survival rates, histopathological analysis, and assessment of fungal burden. Contrary to most statins, PIT can be safely administered with azoles because of less drug-drug interactions, so this study should help us to verify how to make use of the drug in clinical settings in the future.

Keywords: Aspergillus fumigatus; azoles; checkerboard; histopathological analysis; in vivo efficacy; qPCR; silkworm; statin; survival.

Fig 1

Ergosterol synthesis pathway in Aspergillus fumigatus. Azoles inhibit Cyp51 and statins inhibit Hmg1 on the ergosterol synthesis pathway.

Fig 2

Evaluation of survival rates. The survival rates of silkworms were compared among no-treatment (n = 10), ITC-alone (n = 10), PIT-alone (n = 10), and a combination-therapy group (n = 10). We also made Mock (n = 5) as an uninfected group. The experiments were performed twice, and the results were shown as mean values. (a) Infection with azole-resistant strain (IFM63224). Combination therapy of PIT and ITC prolonged the survival compared to no treatment. (b) Infection with azole-susceptible strain (IFM64301). Combination therapy of PIT and ITC prolonged the survival compared to no treatment and ITC alone. Each symbol indicates the following: ns, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Fig 3

Histopathological analysis of silkworms infected with A. fumigatus. Thin sections were stained with hematoxylin-eosin staining and Grocott’s silver stain. (a) Normal histology in silkworm without infection. (b) Infection with azole-resistant strain (IFM63224). The amount of hyphae was reduced around the fat body and in the midgut of the combination-therapy group compared with the no-treatment group. (c) Infection with azole-susceptible strain (IFM64301). The amount of hyphae in the combination-therapy group was reduced around the fat body and in the midgut in comparison to the no-treatment group; it was also reduced around the fat body in comparison to the ITC-alone group.

Fig 4

Evaluation of fungal burden using qPCR. (a and b) Fungal burden in whole bodies of silkworms was measured by qPCR, after infection with each diluted concentration of inoculum: initial concentration (n = 2), 10 × diluted concentration (n = 2), 100 × diluted concentration (n = 2). The initial concentration group (n = 2) was also assessed as controls. Fungal burden was confirmed quantitatively in an inoculum-dose-dependent manner by infection with IFM63224 (a) and IFM64301 (b). (c and d) Fungal burden was measured after infection and treatment: no-treatment (n = 2), ITC-alone (n = 2), combination-therapy (n = 2). (c) Infection with IFM63224. Combination therapy failed to reduce the fungal burden significantly compared with the no-treatment group. (d) Infection with IFM64301. Fungal burden in whole bodies was reduced significantly in the combination-therapy group compared with the no-treatment group and ITC-alone group. In the graphs (a and b) and (c and d), fungal burden of the initial concentration and no-treatment groups was taken as one, respectively. Mean values of two larvae in each group were shown. Bars indicate standard deviations. Each of the symbols indicate the following: ns, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.