Combinatory Treatment with Oseltamivir and Itraconazole Targeting Both Virus and Host Factors in Influenza A Virus Infection

Abstract

Influenza virus infections and their associated morbidity and mortality are a major threat to global health. Vaccination is an effective influenza prevention measure; however, the effectiveness is challenged by the rapid changes in the influenza virus genome leading to viral adaptation. Emerging viral resistance to the neuraminidase inhibitor oseltamivir limits the treatment of acute influenza infections. Targeting influenza virus‑host interactions is a new and emerging field, and therapies based on the combination of virus‑ and host‑directed drugs might significantly improve treatment success. We therefore assessed the combined treatment with oseltamivir and the repurposed antifungal drug itraconazole on infection of polarized broncho‑epithelial Calu-3 cells with pdm09 or Panama influenza A virus strains. We detected significantly stronger antiviral activities in the combined treatment compared to monotherapy with oseltamivir, permitting lower concentrations of the drug than required for the single treatments. Bliss independence drug interaction analysis indicated that both drugs acted independently of each other. The additional antiviral effect of itraconazole might safeguard patients infected with influenza virus strains with heightened oseltamivir resistance.

Keywords: additivity; combination therapy; drug repurposing; host‑directed therapy; influenza A virus; itraconazole; oseltamivir.

Figure 1

Establishment of polarized monolayers of the airway epithelial cell line Calu-3 cultured on semipermeable supports. (a) Transepithelial electrical resistance (TEER) was measured over the 4 days culture period. (b) 3D confocal microscopy image of polarized Calu-3 cells. To evaluate cell differentiation and polarization, the tight junction-associated protein ZO-1 (red signal) at the apical surface and the basolateral marker β-catenin (green signal) were immunostained. Nuclei were visualized with DAPI. Note the honeycomb-like ZO-1 pattern. Scale bar, 15 µm.

Figure 2

Analysis of the cytotoxicity of oseltamivir and itraconazole in single or combinatory treatment. Polarized Calu-3 monolayers were treated at the indicated concentrations with oseltamivir, itraconazole, or combinations for 24 h. Data represent mean percentages of viable cells ± SEM, with mean viability in control cells treated with the solvent DMSO (C) set to 100%. Cell death induced by staurosporine (ST) served as a positive control. n = 3, one-way ANOVA followed by Dunnett’s multiple comparison test. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 3

Analysis of the anti-influenza A virus (IAV) activity of oseltamivir and itraconazole. Polarized Calu-3 monolayers were infected with 0.1 MOI of (a,b) IAV H1N1pdm09 (pdm09) or (c,d) A/Panama/2007/99 (Panama) for 24 h. At 2 h p.i. cells were treated with oseltamivir (a,c) or itraconazole (b,d) at the indicated concentrations for 16 h. Data represent mean percentages of viral titers ± SEM, with mean virus titer in control cells (treated with the solvent DMSO) set to 100%; n = 3. LogIC₅₀ values were determined by fitting a non-linear regression model.

Figure 4

Individual virus titers obtained in all experimental samples. Viral titers obtained in cells infected with (a) pdm09 and (b) Panama are expressed as plaque-forming units (PFU) per mL with means and are presented on a logarithmic scale.

Figure 5

Analysis of the anti-IAV activity of oseltamivir and itraconazole in combinatory treatments. Polarized Calu-3 monolayers were infected with 0.1 MOI of (a,c) pdm09 or (b,d) Panama for 24 h. At 2 h p.i. cells were treated with combinations of oseltamivir and itraconazole at the indicated concentrations for 16 h. Data represent mean percentages ± SEM of inhibition, with mean virus titer in control cells (treated with the solvent DMSO) set to 100%; n = 3. LogEC₅₀ values were determined by fitting a non-linear regression model and were compared for shared logEC₅₀ values.

Figure 6

Analysis of the anti-IAV activity of combination treatment with oseltamivir and itraconazole. Polarized Calu-3 monolayers were infected with 0.1 MOI of (a) pdm09 or (b) Panama for 24 h. Oseltamivir and itraconazole were added at the indicated combinations 2 h p.i. Mean percentages of reduction in viral titers ± SEM, with mean virus titer in control cells (treated with the solvent DMSO) set to 100%; n = 3. Dotted line, 90% reduction in viral titer.

Figure 7

Analysis of the anti-IAV activity of combination treatments with oseltamivir and itraconazole. Polarized Calu-3 monolayers were infected with 0.1 MOI of (a) pdm09 or (b) Panama for 24 h. Oseltamivir and itraconazole were added at the indicated combinations 2 h p.i. For each oseltamivir concentration, mean percentages of reduction in viral titers ± SEM were compared to the respective oseltamivir treatment without itraconazole. Data are expressed as differences and 95% confidence intervals of the comparisons.

Figure 8

Overview of anti-IAV activities of combination treatments with oseltamivir and itraconazole. Polarized Calu-3 monolayers were infected with 0.1 MOI of (a) pdm09 or (b) Panama for 24 h. Oseltamivir and itraconazole were added at the indicated combinations 2 h p.i. Colored cells indicate ≥90% significant reduction in viral titers. Combinations with higher inhibitory activities than the respective individual drug concentrations are highlighted in yellow. Orange cells indicate inhibition ≥90% for combinations, with the individual drug treatment reaching inhibition ≤90%. Red cells indicate combinations leading to significant cytotoxicity. Asterisks indicate significant differences in antiviral activities when the reduction in viral titers found with the combined treatments was compared to the respective oseltamivir concentration.

Figure 9

Assessment of synergistic effects of combined treatment with oseltamivir and itraconazole. Bliss independence, Loewe additivity and ZIP reference models were used to assess drug interaction profiles for (a) pdm09 and (b) Panama virus infections. Bliss independence model, ΔE < 0 indicates synergism, ΔE > 0 indicates antagonism. Loewe and ZIP synergy scores, color scale bars indicate mode and strength of interaction.