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. 2015 Jun 8;10(6):e0128794.
doi: 10.1371/journal.pone.0128794. eCollection 2015.

The Intranasal Application of Zanamivir and Carrageenan Is Synergistically Active against Influenza A Virus in the Murine Model

Martina Morokutti-Kurz  1 , Marielle König-Schuster  1 , Christiane Koller  1 , Christine Graf  1 , Philipp Graf  1 , Norman Kirchoff  1 , Benjamin Reutterer  1 , Jan-Marcus Seifert  1 , Hermann Unger  2 , Andreas Grassauer  1 , Eva Prieschl-Grassauer  1 , Sabine Nakowitsch  1
Affiliations

The Intranasal Application of Zanamivir and Carrageenan Is Synergistically Active against Influenza A Virus in the Murine Model

Martina Morokutti-Kurz et al. PLoS One. .

Abstract

Background: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.

Principal findings: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection.

Conclusion: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.

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Conflict of interest statement

Competing Interests: The authors have read the journal's policy and declare that authors Martina Morokutti-Kurz, Marielle König-Schuster, Christiane Koller, Christine Graf, Philipp Graf, Norman Kirchoff, Benjamin Reutterer, Jan-Marcus Seifert, Andreas Grassauer, Eva Prieschl-Grassauer and Sabine Nakowitsch are employed by Marinomed Biotechnologie GmbH. Authors Hermann Unger, Andreas Grassauer and Eva Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Andreas Grassauer and Eva Prieschl-Grassauer are inventors of the patent #WO2009027057; “Antiviral composition comprising a sulfated polysaccharide”. Marielle König-Schuster, Christiane Koller, Andreas Grassauer and Eva Prieschl-Grassauer are inventors of the patent #WO2011076367 “Synergistic antiviral composition and use thereof”. Both patents are held by Marinomed Biotechnologie GmbH and are related to the content of the manuscript. A product containing a combination of carrageenan and Zanamivir (related to the content of the manuscript) is under development at Marinomed Biotechnologie GmbH. There are no marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Isobologram of compound interaction.
Comparison of the combination of different doses of both compounds necessary to reach 50% replication inhibition of (A) H7N7 and (B) H1N1(09)pdm (◆) to a model of dose additivity that would represent a curve progression of 1 (□). Dose response was tested with an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. On the x-axis the concentration of Zanamivir and on the y-axis the concentration of carrageenan is presented. The concentrations (determined as mean of 3 replicates) are given as the fraction of the respective IC50 values of the different viruses with the particular compound (IC50 = 1).
Fig 2
Fig 2. Therapeutic efficacy in influenza H7N7 lethally infected mice.
(A) Mice (n = 10 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05.
Fig 3
Fig 3. Therapeutic efficacy in influenza H1N1(09)pdm lethally infected mice.
Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan and Zanamivir (1 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant.
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Grants and funding

This work was co-funded by the Austrian Science Promotion Agency (FFG) project number 824211. https://www.ffg.at. Marinomed Biotechnologie GmbH provided support in the form of salaries for authors Martina Morokutti-Kurz, Marielle König-Schuster, Christiane Koller, Christine Graf, Philipp Graf, Norman Kirchoff, Benjamin Reutterer, Jan-Marcus Seifert, Andreas Grassauer, Eva Prieschl-Grassauer and Sabine Nakowitsch. Authors Hermann Unger, Andreas Grassauer and Eva Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript.