Anti-viral and Anti-inflammatory Effects of Camostat and Nafamostat on Influenza Virus and Coronavirus Infections in Human Airway Cells and the Mouse Lungs

Mutsuo Yamaya^1,2,3* and Hidekazu Nishimura^2
*Correspondence: Mutsuo Yamaya, Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan, Japan, Email:

Keywords

Airway epithelial cells • Bronchial asthma • Coronavirus • Influenza virus • Transmembrane protease serine 2

Abbreviations

COPD: Chronic Obstructive Pulmonary Disease, CD13: aminopeptidase N, HAT: Human Trypsin-like Protease, HCoV: Human Coronavirus, HNE cell: Human Nasal Epithelial cell, HTE cell: Human Tracheal Epithelial cell, IL: Interleukin, TMPRSS2: Transmembrane Protease Serine S1 Member 2, TNF: Tumor Necrosis Factor, Vero E6 cell: African Green Monkey Kidney cell.

About the Study

Influenza viruses resistant to neuraminidase inhibitors and baloxavir have been identified, and several patients with influenza virus infection have died despite intensive drug treatment. Anti-viral drugs against seasonal coronaviruses have not been developed. Furthermore, because of the worldwide outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, effective drugs are needed to treat patients with Coronavirus Disease 2019 (COVID-19), even though some drugs have already been developed. Therefore, further development of anti-influenza and anti-coronavirus drugs is needed. Influenza viruses and coronaviruses are activated by proteases, including Transmembrane Protease Serine S1 Member (TMPRSS) 2 expressed in airway epithelial cells [1]. Here, we report the findings of our studies on the effects of the protease inhibitors camostat and nafamostat on the proliferation of influenza viruses and seasonal coronaviruses and on the viral infectioninduced production of inflammatory cytokines.

Effects of protease inhibitors on influenza virus infection

Infection with influenza virus induces bronchitis, pneumonia and pulmonary fibrosis and exacerbates bronchial asthma and Chronic Obstructive Pulmonary Disease (COPD). We identified that the TMPRSS2 and Human Trypsin-like Protease (HAT; TMPRSS11D), which are known to cleave HA0 and activate influenza virus [2], were expressed at the cell membrane and in the cytoplasm of cultured Human Tracheal Epithelial (HTE) cells, and mRNA transcripts encoding TMPRSS2, TMPRSS4 and TMPRSS11D were detectable in these cells (Table 1) [3]. The protease inhibitor camostat, which has been used for the treatment of pancreatitis, reduced the amounts of pandemic (A/Sendai-H/ 108/2009/(H1N1) pdm09) and seasonal (A/ New York/ 55/2004(H3N2)) type A influenza virus strains in the supernatant and/or viral RNA in cells (Table 1) [3]. Camostat reduced the cleavage of the influenza virus precursor protein HA0 into the subunit HA1. Camostat also reduced the concentrations of Interleukin (IL)-6 and Tumor Necrosis Factor (TNF)-α in the supernatant. The mRNA expression levels of TMPRSS2, TMPRSS4 and TMPRSS11D were not affected by camostat. Other types of protease inhibitors, including gabexate and aprotinin, also reduced the viral titers and RNA levels in HTE cells, and aprotinin reduced the concentration of TNF-α in the supernatant. These findings suggest that the clinically used protease inhibitor camostat may reduce influenza virus RNA levels in primary HTE and Human Nasal Epithelial (HNE) cells and the titers of pandemic (A/Sendai-H/108/2009/(H1N1) pdm09) and seasonal (A/New York/55/2004(H3N2)) type A influenza virus strains; it also reduced the secretion of inflammatory cytokines, including IL-6 and TNF-α, into the supernatants of cells infected with the pandemic influenza virus strain (Table 1) [4]. HNE and HTE cells exhibited mRNA and/or protein expression of TMPRSS2, TMPRSS4, and TMPRSS11D. Pretreatment with nafamostat reduced the cleavage of the precursor protein HA0 of the pandemic influenza virus strain into the subunit HA1 in HTE cells and reduced the number of acidic endosomes in both HTE and HNE cells in which influenza virus RNA enters the cytoplasm. Additionally, nafamostat (30 mg/kg/day, intraperitoneal administration) reduced the levels of the pandemic influenza virus strain (A/Hyogo/YS/2011 (H1N1) pdm09) in mouse lung washes. These findings suggest that nafamostat may also inhibit influenza virus replication in human airway epithelial cells and the mouse lungs, and reduce infectioninduced airway inflammation by modulating cytokine production replication and infection-induced airway inflammation.

Pretreatment with nafamostat, which has been used for the treatment of disseminated intravascular coagulation, reduced viral also cause the common cold, bronchitis, and pneumonia and exacerbate bronchial asthma and COPD. HCoV-229E binds to the aminopeptidase N (CD13) receptor and enters cells partly via cellsurface pathways using TMPRSS2 [1]. To examine the effects of protease inhibitors on the replication of HCoV-229E, we pretreated primary cultures of HNE cells with camostat or nafamostat and then infected the cells with HCoV-229E; the viral titers in the Airway Surface Liquid (ASL) of the cells were examined. Pretreatment with camostat or nafamostat reduced the titers of HCoV-229E (Table 1) [5] in the ASL of cultured HNE cells. Furthermore, a significant amount of the TMPRSS2 protein was detected in the ASL of HNE cells. These findings suggest that the protease inhibitors camostat and nafamostat may also reduce HCoV-229E replication in human airway epithelial cells.

Conclusion

We demonstrated that camostat and nafamostat reduced the proliferation of influenza viruses and a seasonal coronavirus in human airway cells and the mouse lungs. Wang, et al. also demonstrated that nafamostat reduced SARS-CoV-2 replication by inhibiting TMPRSS2 in African Green Monkey Kidney (Vero E6) cells [6]. These findings suggest that SARS-CoV-2 enters partly via cell-surface pathways using TMPRSS2. In contrast to the effects observed in the in vitro studies, the clinical efficacy of nafamostat in the treatment of COVID-19 is still uncertain, and anti-viral drugs for influenza viruses and seasonal coronaviruses have not been developed. Thus, new types of protease inhibitors without adverse effects are expected to be developed.

Acknowledgement

Camostat mesilate, sivelestat and gabexate mesilate were provided by Ono Yakuhin Co., Ltd. This study was supported by a Grant-in-Aid for Exploratory Research from the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 25293189 and Ono Yakuhin Co., Ltd.

Conflict of Interest

The authors have no conflicts of interest.

References

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