The TMPRSS2 Inhibitor Nafamostat Reduces SARS-CoV-2 Pulmonary Infection in Mouse Models of COVID-19

ABSTRACT The coronavirus disease 2019 (COVID-19) pandemic has caused significant morbidity and mortality on a global scale. The etiologic agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), initiates host cell entry when its spike protein (S) binds to its receptor, angiotensin-converting enzyme 2 (ACE2). In airway epithelia, the spike protein is cleaved by the cell surface protease TMPRSS2, facilitating membrane fusion and entry at the cell surface. This dependence on TMPRSS2 and related proteases suggests that protease inhibitors might limit SARS-CoV-2 infection in the respiratory tract. Here, we tested two serine protease inhibitors, camostat mesylate and nafamostat mesylate, for their ability to inhibit entry of SARS-CoV-2 and that of a second pathogenic coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). Both camostat and nafamostat reduced infection in primary human airway epithelia and in the Calu-3 2B4 cell line, with nafamostat exhibiting greater potency. We then assessed whether nafamostat was protective against SARS-CoV-2 in vivo using two mouse models. In mice sensitized to SARS-CoV-2 infection by transduction with human ACE2, intranasal nafamostat treatment prior to or shortly after SARS-CoV-2 infection significantly reduced weight loss and lung tissue titers. Similarly, prophylactic intranasal treatment with nafamostat reduced weight loss, viral burden, and mortality in K18-hACE2 transgenic mice. These findings establish nafamostat as a candidate for the prevention or treatment of SARS-CoV-2 infection and disease pathogenesis.

epithelia, we investigated their responses to several inhibitors during the early course of infection. We used primary cultures of well-differentiated human bronchial epithelia, which closely mimic the native surface airway epithelium in vivo. We tested camostat and nafamostat, bafilomycin A1 and chloroquine (both inhibitors of endosomal acidification), and E64d, an inhibitor of lysosomal cathepsins B and L. The inhibitors were added 1 h prior to infection with a multiplicity of infection (MOI) of 0.1 of each respective coronavirus, and 20 h later, we measured viral RNA abundance and the titer of virus released into airway surface liquid. In this setting, only camostat and nafamostat significantly inhibited infection of human airway epithelia ( Fig. 1A and B). Thus, both MERS-CoV and SARS-CoV-2 depend on cell surface serine proteases such as TMPRSS2 for entry into primary airway epithelial cells. We found that the SARS-CoV-2 inhibitory activity of camostat and nafamostat were lost when infections were performed in TMPRSS2-negative Vero E6 cells, suggesting that these compounds likely work through inhibition of TMPRSS2 protease activity rather than through direct virucidal action (see Fig. S1 in the supplemental material).
Nafamostat inhibits weight loss and virus burden in SARS-CoV-2-challenged mice. To assess whether i.n. nafamostat administration altered the course of SARS-CoV-2 infection, mice were transduced with Ad5-hACE2, followed by i.n. infection with SARS-CoV-2 (10 5 PFU/mouse). Animals received nafamostat (3 mg/kg, i.n.) at 2 h prior to infection, 1 day postinfection, or 3 days postinfection, and were monitored daily for weight loss (Fig. 3A). Nafamostat pretreatment abrogated SARS-CoV-2-induced weight loss (Fig. 3B). Weight loss was also significantly reduced in animals receiving nafamostat at 1 day postinfection (Fig. 3B). Consistent with these findings, lung viral loads were reduced at 1, 2, and 4 days postinfection in mice pretreated with nafamostat, whereas the reductions in lung viral titers were more modest in mice receiving nafamostat after SARS-CoV-2 challenge (Fig. 3C). Histopathological analysis of lung tissue from infected animals at 5 days postinfection suggests that nafamostat treatment reduced lung pathology in the infected mice, primarily in the animals receiving the pretreatment protocol ( Fig. 3D and E). These results indicate that in mice expressing hACE2 via Ad5-hACE2 transduction, nafamostat reduces SARS-CoV-2 infection severity, particularly when administered prior to or early in infection.
Nafamostat protects against SARS-CoV-2 infection in K18-hACE2 mice. We next tested nafamostat in K18-hACE2 mice (18). K18-hACE2 mice were pretreated with i.n. were measured by plaque assay, and viral RNA levels were assessed by real-time quantitative PCR (qPCR), as described in Materials and Methods. Viral titers and RNA levels are expressed relative to those for infected cells with vehicle treatment, and data are presented as mean 6 standard error (SE). Each data point represents an individual HAE donor. Log-transformed data were tested for significant differences from the vehicle control using one-way analysis of variance (ANOVA) followed by Dunnett's multiple-comparison test. *, P , 0.05; ****, P , 0.0001. (C, D) Calu-3 2B4 epithelial cells were preincubated in medium containing the indicated concentrations of camostat or nafamostat 1 h prior to infection. Cells were then infected with MERS-CoV or SARS-CoV-2 (MOI of 0.1) for 1 h and cultured overnight in medium containing the indicated inhibitor concentrations. At 20 h postinfection, viral RNA levels were quantified by real-time qPCR for MERS-CoV (C) or SARS-CoV-2 (D), as indicated. Data represent the mean 2 2DCT 6 SE (C T , threshold cycle). Logtransformed data were tested for statistically significant differences at each concentration using unpaired 2tailed t tests, corrected for multiple comparisons by the Holm-Sidak method. *, adjusted (Adj.) P , 0.05; **, Adj. P , 0.01 (n = 3 replicate wells per condition). (E) Calu-3 2B4 cells were incubated with increasing concentrations of camostat or nafamostat 1 h prior to infection (MOI of 0.1), using the same procedure as shown in panels C and D. The reduction in SARS-CoV-2 RNA at 20 h postinfection was assessed by 2 2DDCT method, using HPRT as a reference gene. Viral RNA levels are expressed relative to that for infected cells with vehicle treatment (n = 3 replicate wells per condition). Results represent two independent experiments. nafamostat (3 mg/kg) for 2 h, followed by SARS-CoV-2 challenge (2.5 Â 10 3 PFU/mouse) (Fig. 4A). Over a 14-day time course, nafamostat-treated mice lost less weight and exhibited significantly less mortality than vehicle-treated controls ( Fig. 4B and C). At 1 day postinfection, virus was detected in the lungs of vehicle-treated mice but was largely undetectable in tissue from nafamostat-treated mice (Fig. 4D). By 7 days postinfection, virus titers could be measured in both lung and brain in 50% of vehicle-treated mice, whereas no virus was detected in lung or brain tissue from mice receiving nafamostat (Fig. 4E).
These results suggest that nafamostat pretreatment significantly reduced viral loads over the course of SARS-CoV-2 infection. We examined the distribution of SARS-CoV-2positive cells in tissues from K18-hACE2 mice by immunostaining for viral antigen. In the lungs of vehicle-treated animals, infection was widespread throughout the cells of the small airways and alveoli by 7 days postinfection. In contrast, SARS-CoV-2-positive cells were far less abundant (though not entirely absent) in tissue from nafamostattreated mice ( Fig. 4F and G). Viral infection in the brain was more variable. While there were no virus-positive cells in either treatment group at 1 day postinfection, by 7 days postinfection, at least half of the vehicle-treated animals exhibited profound brain infection (see Fig. S2 in the supplemental material), generally mirroring the virus tissue titers. This finding of later onset of brain infection was previously reported (19,21,22). Infected cells were also found in the sinonasal cavity, with nafamostat-treated mice showing a trend toward fewer SARS-CoV-2-positive cells in the maxillary sinus and olfactory For results in panels B and C, data were tested for significant differences using one-way ANOVA followed by Tukey's multiple-comparison test. In all panels, data are presented as mean 6 SE. *, P , 0.05; **, P , 0.01; ****, P , 0.0001. LOD, limit of detection. Each experiment was performed once.
Nafamostat Inhibition of SARS-CoV-2 in Mice ® epithelium (Fig. S2). Very few lung lesions were observed in either treatment group (see Fig. S3 in the supplemental material), making it difficult to assess whether nafamostat treatment reduced lung disease severity in K18-hACE2 mice.

DISCUSSION
Here, we show that the serine protease inhibitors camostat and nafamostat potently reduce SARS-CoV-2 and MERS-CoV infection in well-differentiated primary cultures of airway epithelia, presumably by inhibiting the activity of cell surface serine proteases (such as TMPRSS2). Both camostat and nafamostat were previously shown to directly inhibit the enzymatic activity of TMPRSS2 and related serine proteases in biochemical assays (25), strongly suggesting that inhibition of TMPRSS2 catalytic activity is the primary mechanism for this effect. In contrast, we saw little or no effect from bafilomycin A1, chloroquine, or E64d, agents that alter pH and/or protease function in intracellular compartments, including endosomes and lysosomes. Our results agree with those of earlier studies (7, 8, 13-17, 26,27) and contribute to the growing consensus that fusion and K18-hACE2 mice were treated with i.n. nafamostat (3 mg/kg), and 2 h later they were infected i.n. with SARS-CoV-2 (2.5 Â 10 3 PFU/mouse). (B) Weight loss was monitored daily in nafamostat-and vehicle-treated mice (n = 9 mice/ group), and data were tested for significant differences at each day postinfection using unpaired 2-tailed t tests, corrected for multiple comparisons by the Holm-Sidak method. *, Adj. P , 0.05; **, Adj. P , 0.01 (C) Survival curves for nafamostat-and vehicle-treated mice. Weight loss and survival curve data represent results from two independent experiments. (D) Lung tissue virus titers measured at 1 day postinfection. No virus was detected in the brain for either (Continued on next page) Nafamostat Inhibition of SARS-CoV-2 in Mice ® entry at the plasma membrane is the preferred route of entry into cells of the respiratory tract. Our experiments in Calu-3 2B4 cells indicate that while both camostat and nafamostat are active against SARS-CoV-2 and MERS-CoV, nafamostat is more potent, a trend also observed in other in vitro studies (14,15). Importantly, we demonstrate the in vivo efficacy of nafamostat in reducing SARS-CoV-2 infection and pathogenesis. The protective effect of nafamostat was greatest when the drug was administered prior to viral infection. Pretreatment with nafamostat via the i.n. route either completely prevented or significantly reduced infection-induced weight loss, and substantially reduced viral loads throughout the ensuing course of illness, in Ad5-hACE2 transduced mice and K18-hACE2 mice. In Ad5-hACE2 transduced mice, although nafamostat treatment beginning at 1 day postinfection provided some protection, the results were less dramatic. These findings suggest that protease inhibitor treatment may provide its greatest clinical benefit when delivered prophylactically or in the early stages of infection, and that there may be a "treatment window" after which treatment no longer improves outcomes. We note that the course of SARS-CoV-2 infection in hACE2expressing mice proceeds more rapidly and on a shorter time course than in humans.
Further highlighting the importance of timing for the in vivo efficacy of nafamostat, we observed that the effectiveness of intranasal nafamostat increased as the time interval between nafamostat delivery and viral inoculation decreased (Fig. 2C). This likely reflects the relatively short half-life of nafamostat; early studies with nafamostat reported a plasma half-life of 8 min in rabbits and 1 min in dogs (28). Currently, there are no data regarding nafamostat stability in respiratory secretions following i.n. administration. It is possible that the fate of nafamostat is different in airway secretions than that in plasma, potentially contributing to the different outcomes observed via i.n. or i.p. routes in our study. It is also unknown how efficiently nafamostat is transported into airway secretions when delivered systemically, which may influence outcomes following i.n. versus i.p. administration. Pharmacokinetic studies are needed to better understand these aspects of nafamostat activity.
Based on their encouraging in vitro activity against SARS-CoV-2, both camostat and nafamostat are currently under evaluation as potential therapies for COVID-19 (https:// clinicaltrials.gov; NCT04652765, NCT04455815, NCT04353284, NCT04583592, NCT04470544, NCT04435015, NCT04608266, NCT04524663, NCT04750759, NCT04625114, NCT04730206, NCT04321096, NCT04355052, NCT04662073, NCT04681430, NCT04644705, NCT04657497, NCT04374019, NCT04418128, NCT04352400, NCT04390594, NCT04628143, NCT04623021, NCT04473053, NCT04483960). Both compounds are approved treatments for other medical conditions and are therefore attractive candidates for rapid drug repurposing. In Japan, camostat is approved for use in treatment of acute pancreatitis and postoperative reflux esophagitis (29), and it has a well-characterized safety profile. Camostat treatment was shown to improve survival in a mouse model of SARS-CoV infection (30). Nafamostat is marketed in Japan and South Korea for treatment of acute pancreatitis and disseminated intravascular coagulation (DIC). In vitro studies indicate that nafamostat does not cause cytotoxicity in cultured human endothelial or airway epithelial cells (14,(31)(32)(33), and a recent case report describing nafamostat administration in three elderly COVID-19 patients reported no adverse events (34). The proposed clinical trials generally involve systemic administration of nafamostat. In our studies with the Ad5-hACE2 mice, we observed that nafamostat reduced infection more effectively when delivered via the i.n. route, suggesting that it may be important to consider the route of administration when designing treatment regimens with FIG 4 Legend (Continued) treatment group at 1 day postinfection (n = 3 mice/group). LOD, limit of detection. (E) Virus titers in the lungs and brain 7 days postinfection (n = 4 mice/group). (F) Immunohistochemistry identified SARS-CoV-2-infected cells in lung tissue sections from vehicle-and nafamostat-treated mice at 1 and 7 days postinfection. Tissues were stained for the SARS-CoV-2 N protein (brown) and scored as described in Materials and Methods. Significant differences between vehicle-and nafamostat-treated mice at each time point were assessed by the Mann-Whitney test. In all panels, data are presented as mean 6 SE. (G) Representative images of lung tissue from vehicle-and nafamostat-treated K18-hACE2 mice at 1 and 7 days postinfection, immunostained for SARS-CoV-2 N protein (black arrows). Bar, 92 mm. Data presented in panels D to G represent one experiment. protease inhibitors in human patients. It is possible that nafamostat's therapeutic efficacy might be boosted by direct delivery to the airways as a nasal spray or inhaled aerosol. It is of note that nafamostat is a broad-spectrum protease inhibitor with effects on multiple biological processes, which has led to speculation that it may confer benefits beyond blocking SARS-CoV-2 entry. In particular, its anticoagulant properties may reduce or prevent COVID-19-related thrombotic complications. Nafamostat also inhibits proteases involved in inflammatory cascades and the complement system, which may dampen inflammation in severe COVID-19 cases.
In conclusion, we provide evidence that camostat and nafamostat potently inhibit SARS-CoV-2 and MERS-CoV infection in cultured human airway epithelia; nafamostat exhibited greater potency than camostat in reducing SARS-CoV-2 infection, suggesting that it may be a more attractive candidate for COVID-19 lung disease prevention or treatment. Nafamostat inhibited SARS-CoV-2 infection and improved disease outcomes in two COVID-19 mouse models. Our experiments in these animal models highlight the importance of route and timing of administration in the design of effective treatment regimens. These preclinical data support further investigation of protease inhibitors as antiviral prophylactic or therapeutic strategies for COVID-19. Cell culture. Primary human airway epithelia were prepared from bronchi as previously described (35). Briefly, epithelial cells were dissociated and seeded onto collagen-coated, semipermeable membranes with a 0.4-mm pore size (Costar Transwell, surface area, 0.33 cm 2 ; Corning) in 24-well plates maintained in Ultroser G (USG) medium at 37°C and 5% CO 2 . At 24 hours after seeding, the mucosal medium was removed, and cells were grown at the air-liquid interface. Only well-differentiated cultures (.3 weeks old; resistance, .1,000 X Á cm 2 ) were used in this study. Calu-3 2B4 cells were maintained in minimal essential medium (MEM) supplemented with 20% fetal bovine serum (FBS), 0.1 mM nonessential amino acids (NEAA), 1 mM sodium pyruvate, 2 mM L-glutamine, 1% penicillin and streptomycin, and 0.15% NaHCO 3 at 37°C with 5% CO 2 . Vero E6 cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, 0.1 mM NEAA, and 1% penicillin and streptomycin at 37°C in 5% CO 2 .

Inhibitors
Infections in airway epithelia. To investigate proteases required for MERS-CoV or SARS-CoV-2 infection in primary human airway epithelia, cells were incubated in medium (50 ml in the apical compartment and 500 ml basolaterally) containing bafilomycin A1 (50 nM), chloroquine (20 mM), E64d (25 mM), camostat (25 mM), or nafamostat (25 mM) at 37°C for 1 h. After 1 h of pretreatment, the apical medium was removed and replaced by medium containing MERS-CoV or SARS-CoV-2 (MOI of 0.1) in the presence of the indicated inhibitor/chemical for another 1 h incubation. The apical medium (containing unbound virus) was then removed, and cells were washed 2 times with phosphate-buffered saline (PBS) at the apical surface. At 20 h postinfection, the apical surface of infected cultures was rinsed with PBS to collect airway surface liquid (ASL), and titer was determined to verify the release of progeny virions into the ASL. Total cellular RNA was harvested in TRIzol reagent (Invitrogen, Waltham, MA).
To compare the efficacy of camostat and nafamostat against MERS-CoV or SARS-CoV-2 infection, Calu-3 2B4 cells were cultured in 96-well plates and pretreated with the indicated concentrations of inhibitors for 1 h. Cells were then infected with MERS-CoV or SARS-CoV-2 (MOI = 0.1) in the presence of the inhibitors for 1 h, followed by overnight incubation with the inhibitor. Total cellular RNA was harvested in TRIzol reagent at 20 h postinfection.
Transduction and infection of Ad5-hACE2 mice. Ad5-hACE2 was generated by the University of Iowa Viral Vector Core Facility. Six-to eight-week-old BALB/c mice were lightly anesthetized with ketamine-xylazine and transduced via the i.n. route with 2.5 Â 10 8 PFU of Ad5-hACE2 in 75 ml DMEM. At 5 days postransduction, mice were infected i.n. with SARS-CoV-2 (3 Â 10 3 or 1 Â 10 5 PFU, as indicated). To make a stock of nafamostat for in vivo studies, the compound was dissolved in H 2 O at a concentration of 10 mg/ml. This nafamostat stock (or H 2 O for vehicle control animals) was diluted in PBS prior to i.p. injection; for i.n. delivery, the nafamostat stock was diluted in DMEM and delivered as a liquid bolus in a total volume of 50 ml. After SARS-CoV-2 infection, mice were monitored and weighed daily. All work with SARS-CoV-2 was conducted in the biosafety level 3 (BSL3) Laboratory of the University of Iowa. All protocols were approved by the Institutional Animal Care and Use Committees of the University of Iowa.
Experiments with K18-hACE2 mice. Transgenic mice expressing human ACE2 under the control of the cytokeratin 18 promoter were previously reported (18). The 6-to 8-week-old mice used in these studies were obtained from the Jackson Laboratory [034860-B6.Cg-Tg(K18-ACE2)2Prlman/J] and are congenic on the C57BL/6 background.
SARS-CoV-2 plaque assay. Viral preps and lung or brain homogenate supernatants were serially diluted in DMEM. Vero E6 cells in 12-well plates were inoculated at 37°C in 5% CO 2 for 1 h with gentle Nafamostat Inhibition of SARS-CoV-2 in Mice ®