Antiviral Potential of Azelastine against Major Respiratory Viruses

Abstract

The Coronavirus Disease 2019 (COVID-19) pandemic and the subsequent increase in respiratory viral infections highlight the need for broad-spectrum antivirals to enable a quick and efficient reaction to current and emerging viral outbreaks. We previously demonstrated that the antihistamine azelastine hydrochloride (azelastine-HCl) exhibited in vitro antiviral activity against SARS-CoV-2. Furthermore, in a phase 2 clinical study, a commercial azelastine-containing nasal spray significantly reduced the viral load in SARS-CoV-2-infected individuals. Here, we evaluate the efficacy of azelastine-HCl against additional human coronaviruses, including the SARS-CoV-2 omicron variant and a seasonal human coronavirus, 229E, through in vitro infection assays, with azelastine showing a comparable potency against both. Furthermore, we determined that azelastine-HCl also inhibits the replication of Respiratory syncytial virus A (RSV A) in both prophylactic and therapeutic settings. In a human 3D nasal tissue model (MucilAir^TM-Pool, Epithelix), azelastine-HCl protected tissue integrity and function from the effects of infection with influenza A H1N1 and resulted in a reduced viral load soon after infection. Our results suggest that azelastine-HCl has a broad antiviral effect and can be considered a safe option against the most common respiratory viruses to prevent or treat such infections locally in the form of a nasal spray that is commonly available globally.

Keywords: RSV; antiviral; azelastine; drug repurposing; respiratory viruses.

Figure 1

In vitro efficacy of azelastine-HCl against coronaviruses. (A) Vero-TMPRSS2/ACE2 cells were infected with the B.1.1.529 variant of SARS-CoV-2 or (B) MRC-5 cells were infected with HCoV-229E simultaneously with the addition of (A) 0.05–12.5 μM or (B) 2–12 µM of azelastine. After 48 h (A) or 72 h (B) p.i., the viral count was determined using qPCR analysis. Graphs show percent inhibition of infection based on viral genome counts relative to the virus-only control expressed as the mean ± SEM from 3 independent experiments, each with (A) 3 or (B) 4 technical replicates. The curve was fitted, and EC₅₀ was calculated via nonlinear regression using GraphPad Prism.

Figure 2

In vitro antiviral effect of azelastine-HCl against RSV. HEp-2 cells were infected with RSV and concomitantly treated with 0.024–50 μM azelastine-HCl. After 24 h, the RSV-infected cells were stained with an RSV-specific recombinant monoclonal antibody followed by Alexa Fluor^TM Plus 488-labeled polyclonal goat anti-human IgG staining and the number of RSV-infected cells were counted using an ImmunoSpot^® analyzer. (A) A representative result of RSV-infected cells treated with azelastine-HCl, DMSO, or non-infected cells (w/o). (B) Percent inhibition of RSV infection in the presence of different concentrations of azelastine-HCl as a purified compound or (C) azelastine-HCl from the nasal spray Pollival^® calculated relative to the infection in virus-only cells (red circles). EC₅₀ was calculated via nonlinear regression using GraphPad Prism. The potential cytotoxic effect of azelastine-HCl on the HEp-2 cells was assessed using the MTT assay (black triangles). Graphs show the mean with 95% CI from 8 and 4 independent experiments for RSV inhibition and cell viability, respectively.

Figure 3

Antiviral effect of azelastine-HCl in an influenza H1N1 infection model on human nasal 3D tissue. (A) Experimental scheme. (B) H1N1 viral genome count from apical washes at 24 h p.i. measured using a quantitative PCR while the (C) IL-8 level and (D) RANTES level in basal tissue media after 48 and 96 h p.i. were measured with commercial ELISA kits. Mock indicates non-infected and non-treated tissues. The difference between groups was statistically analyzed with one-way ANOVA (multiple comparisons) using GraphPad Prism, and results were considered significant if p < 0.05. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns indicates non-significant difference. The graphs show the mean ± SD of the results from 3 tissue samples.