Abstract
Nafamostat, a synthetic serine protease inhibitor, has been used for the treatment of inflammatory diseases such as pancreatitis. Recently, an increasing number of studies have shown the promising antiviral effects of nafamostat for the treatment of coronavirus disease-19 (COVID-19). This study aimed to develop a novel liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis and to characterize the pharmacokinetics of nafamostat in rats. Nafamostat in the rat plasma was extracted by solid phase extraction, and 13C6-nafamostat was used as an internal standard. The quantification limit of nafamostat in the rat plasma was 0.5 ng/mL. The LC-MS/MS method was fully validated and applied to characterize the pharmacokinetics of nafamostat in rats. Following intravenous injection (2 mg/kg), nafamostat in the plasma showed a multiexponential decline with an average elimination half-life (t1/2) of 1.39 h. Following oral administration of nafamostat solutions (20 mg/kg) in 10% dimethyl sulfoxide (DMSO) and in 10% DMSO with 10% Tween 80, nafamostat was rapidly absorbed, and the average oral bioavailability was 0.95% and 1.59%, respectively. The LC-MS/MS method and the pharmacokinetic information of nafamostat could be helpful for the further preclinical and clinical studies of nafamostat.
Highlights
Nafamostat, a potent serine protease inhibitor, acts as a fast-acting proteolytic inhibitor, which inhibits a wide spectrum of proteases
The S protein is cleaved into subunits, S1 and S2, by cellular proteases, and S1 allows SARS-CoV-2 to directly bind to the peptidase domain of the angiotensin-converting enzyme 2 (ACE2) receptor
An liquid chromatography (LC)-MS/MS assay for the determination of nafamostat in the rat plasma was developed and validated
Summary
Nafamostat, a potent serine protease inhibitor, acts as a fast-acting proteolytic inhibitor, which inhibits a wide spectrum of proteases. An increasing number of studies have shown the potent antiviral activities of nafamostat on coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [7–11]. S2 is further cleaved by a host type 2 transmembrane serine protease 2 (TMPRSS2), resulting in the fusion between the viral envelope and cell membrane and viral entry into human cells. Nafamostat has been shown to inhibit SARS-CoV-2 entry into host cells and block SARS-CoV-2 infection of human lung cells [7]. Nafamostat is expected to bind spontaneously and stably to the catalytic center of TMPRSS2 and inhibit its proteolytic processing of the S protein [7,10,11,14,15]. In a recently completed Phase 2 clinical trial (NCT04623021), no significant clinical improvement was observed in hospitalized patients with moderate to severe COVID-19 pneumonia by the addition of nafamostat to standard-of-care [16]. A retrospective observational study failed to find an association between nafamostat mesylate and in-hospital mortality in patients with COVID-19 [17]
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