ATS 2021 will feature presentations of original research from accepted abstracts. Mini Symposia and Thematic Poster Sessions are abstract based sessions.
Please use the form below to browse scientific abstracts and case reports accepted for ATS 2021. Abstracts presented at the ATS 2021 will be published in the Online Abstract Issue of the American Journal of Respiratory and Critical Care Medicine, Volume 203, May 3, 2021.
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SARS-CoV-2 Infection and ENaC Activity Are Both Inhibited by Nafamostat Applied Apically on Human Airway Epithelial Cells in Air-Liquid Interface Culture
Session Title
TP91 - TP091 EPIDEMIOLOGY AND TRANSLATIONAL ADVANCES IN SARS-COV-2
Abstract
A3782 - SARS-CoV-2 Infection and ENaC Activity Are Both Inhibited by Nafamostat Applied Apically on Human Airway Epithelial Cells in Air-Liquid Interface Culture
Author Block: L. Kirkpatrick1, J. Millard1, R. Hall2, P. J. Cole2, S. Constant3; 1Ensysce Biosciences Inc., La Jolla, CA, United States, 2Covistat, La Jolla, CA, United States, 3Epithelix, Geneva, Switzerland.
Background:
Nafamostat (NF) is a small molecule drug with activity against a range of serine proteases (SP) expressed by human airway epithelial cells (HAEC) including transmembrane protease serine 2 (TMPRSS2) and prostasin. NF is used in Asia as an i.v. anti-coagulant and has an established safety profile. NF, soluble in aqueous solutions, can be delivered by inhalation to the airways. It is currently being repurposed for treatment of both SARS-CoV-2 (CoV) infection and Cystic Fibrosis.
CoV binding and entry into HAEC requires host proteases, particularly TMPRSS2, which cleaves the viral spike protein. NF is a potent inhibitor of TMPRSS2.
Excessive airway epithelia sodium channel (ENaC) activity in CF causes failure to clear purulent secretions. Full ENaC activity requires an inhibitory peptide domain to be cleaved from the ENaC gamma subunit by membrane bound SP, particularly prostasin. This study reports safety of NF on HAEC, its efficacy and potency to inhibit CoV infection, and the IC50 of NF and its time course to inhibit ENaC in HAEC.
Methods:
HAEC cultured in 3D MucilAirTM tissue model were exposed to NF 0.002 to 20μM for up to 72h and evaluated for cytotoxicity and inflammatory effects. To study infectivity, CoV viral inoculum was added apically for 1h. Cells were exposed to NF 0.2 to 20μM for up to 72h. Apical washes taken at 48 and 72h were measured for viral RNA by qRT-PCR. ENaC activity in HAEC was measured as amiloride-dependent decrease in short-circuit current. Time course and dose dependency of NF induced decrease in ENaC activity was studied.
Results:
Conclusions:
1. No detrimental effects of NF up to 20 μM applied to surface of HAEC.
2. Anti-viral effects of NF on CoV infection of HAEC consistent with inhibition of TMPRSS2 and prevention of viral entry via ACE2 receptor (Yamamoto et al. Antimicrob. Agents & Chemotherapy 2016, 60 p6532).
3. NF induced inhibition of ENaC in HAEC is consistent with data in pig airways (Hall & Cole Pediatr. Pulmonol. 2017, 52S2, p239) where NF caused decreased ENaC activity and increased ciliary transport of secretions with long duration of action.
4. Inhibition of CoV infection and ENaC activity by NF occurs over a similar dose range suggesting the potential for a double benefit of NF inhalation in CoV infection; i.e. prevention of viral entry to airway epithelial cells, and increased removal of mucus and viral particles from the lung/nose by up-regulated ciliary clearance.
Author Block: L. Kirkpatrick1, J. Millard1, R. Hall2, P. J. Cole2, S. Constant3; 1Ensysce Biosciences Inc., La Jolla, CA, United States, 2Covistat, La Jolla, CA, United States, 3Epithelix, Geneva, Switzerland.
Background:
Nafamostat (NF) is a small molecule drug with activity against a range of serine proteases (SP) expressed by human airway epithelial cells (HAEC) including transmembrane protease serine 2 (TMPRSS2) and prostasin. NF is used in Asia as an i.v. anti-coagulant and has an established safety profile. NF, soluble in aqueous solutions, can be delivered by inhalation to the airways. It is currently being repurposed for treatment of both SARS-CoV-2 (CoV) infection and Cystic Fibrosis.
CoV binding and entry into HAEC requires host proteases, particularly TMPRSS2, which cleaves the viral spike protein. NF is a potent inhibitor of TMPRSS2.
Excessive airway epithelia sodium channel (ENaC) activity in CF causes failure to clear purulent secretions. Full ENaC activity requires an inhibitory peptide domain to be cleaved from the ENaC gamma subunit by membrane bound SP, particularly prostasin. This study reports safety of NF on HAEC, its efficacy and potency to inhibit CoV infection, and the IC50 of NF and its time course to inhibit ENaC in HAEC.
Methods:
HAEC cultured in 3D MucilAirTM tissue model were exposed to NF 0.002 to 20μM for up to 72h and evaluated for cytotoxicity and inflammatory effects. To study infectivity, CoV viral inoculum was added apically for 1h. Cells were exposed to NF 0.2 to 20μM for up to 72h. Apical washes taken at 48 and 72h were measured for viral RNA by qRT-PCR. ENaC activity in HAEC was measured as amiloride-dependent decrease in short-circuit current. Time course and dose dependency of NF induced decrease in ENaC activity was studied.
Results:
Conclusions:
1. No detrimental effects of NF up to 20 μM applied to surface of HAEC.
2. Anti-viral effects of NF on CoV infection of HAEC consistent with inhibition of TMPRSS2 and prevention of viral entry via ACE2 receptor (Yamamoto et al. Antimicrob. Agents & Chemotherapy 2016, 60 p6532).
3. NF induced inhibition of ENaC in HAEC is consistent with data in pig airways (Hall & Cole Pediatr. Pulmonol. 2017, 52S2, p239) where NF caused decreased ENaC activity and increased ciliary transport of secretions with long duration of action.
4. Inhibition of CoV infection and ENaC activity by NF occurs over a similar dose range suggesting the potential for a double benefit of NF inhalation in CoV infection; i.e. prevention of viral entry to airway epithelial cells, and increased removal of mucus and viral particles from the lung/nose by up-regulated ciliary clearance.
