Abstract

Back ground: Enveloped viruses invade cells by fusing the viral envelope with the cell membrane. Most viral fusion proteins require specific host protease(s) to activate their fusion activity. Many influenza viruses and severe acute respiratory syndrome associated coronaviruses use transmembrane serine protease TMPRSS2 for activation. Protease inhibitor nafamostat suppresses TMPRSS2, thereby interfering with the viral infection in-vitro. However, no successful application of nafamostat for the treatment of respiratory viral infection has been reported. This is because no method has been established to deliver nafamostat to the respiratory epithelium. Additionally, many coronaviruses have another infectious pathway, in which the virus is engulfed in endosome and activated by endosomal protease(s) and acidification. Ammonium chloride is known to block this pathway in-vitro , by interfering with the endosomal acidification. The present study has done to explore the method to safely deliver these reagents by assessing whether adverse effects occur when the reagents are administered to the respiratory epithelium in mice.

Methods: To ass ess adverse effects, inbred mice were intranasally administered the reagents 2~20 μL/day for a week under anesthesia. Mice were daily observed and change in the body weight was used as a health status barometer. At the end of experiment, the serum biochemical examination was done.

Results: The solution of 200 μM nafamostat and 74 mM ammonium chloride could be intranasally administered 20 μL/day for 1 week to adult C57BL/6 mice without any visible adverse effects. Biochemical data on these mice were within the normal range.

Conclusion: Since 1 μM nafamostat and 50 mM ammonium chloride are known to efficiently suppress the viral invasion to cell in-vitro, nafamostat is highly expected to show inhibitory effect in the virus-infected mice, and ammonium chloride may be also available to treat the virus-infected mice. The present study encourages future researches in infected mice and to apply these reagents for the clinical treatment.

Keywords: Nafamostat; Protease inhibitor; Respiratory viral infection; SARS-CoV-2