Serine Protease; Granzyme; I-kappaBalpha
Nafamostat Mesylate (FUT175) is a broad-spectrum serine protease inhibitor, targeting thrombin (Ki = 2.3 nM), factor Xa (Ki = 7.0 nM), plasmin (Ki = 2.7 nM), and kallikrein (Ki = 4.7 nM) [1]
- Nafamostat Mesylate inhibits factor XIIa (Ki = 15 nM) and activated protein C (APC, Ki = 34 nM) [2]
- Nafamostat Mesylate inhibits the main protease (Mpro, 3CLpro) of SARS-CoV-2 with an IC50 of 0.31 μM [6]
- Nafamostat Mesylate inhibits transmembrane protease serine 2 (TMPRSS2) with an IC50 of 14.5 nM, a key enzyme for SARS-CoV-2 spike protein cleavage [7]

Nafamostat mesilate significantly prevents platelet beta-thromboglobulin (beta TG) from being released after 60 and 120 minutes. Neutrophil elastase is not significantly released when using napamostat mesilate (NM); at 120 minutes, the plasma elastase-alpha 1-antitrypsin complex is 0.16 mg/mL in the NM group and 1.24 mg/mL in the control group. The formation of complexes between C1 inhibitor and FXIIa and kallikrein is entirely inhibited by napamostat mesilate.[1]
Nafamostat mesilate inhibits a number of proteases that could play a significant role in the pathogenesis of disseminated intravascular coagulation (DIC).At an IC50 of 0.1 μM, napamostat mesilate inhibits the activity of the TF-F.VIIa mediated-F.Xa extrinsic pathway in a concentration-dependent manner.[2]
Nafamostat mesilate inhibits the initial-phase transient component of biphasic ASIC3 currents in a concentration-dependent manner with an IC50 value of approximately 2.5 mM.[3]

---

In human plasma-based coagulation assays, Nafamostat Mesylate prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT) in a dose-dependent manner: at 0.5 μM, PT was prolonged by ~30% and APTT by ~45% compared to control [1]
- In a cell-free fibrinolytic system, 1 μM Nafamostat Mesylate inhibited plasmin-mediated fibrin degradation by ~80%, as measured by fibrin plate assay [2]
- In human umbilical vein endothelial cells (HUVECs) treated with lipopolysaccharide (LPS, 1 μg/mL) to induce inflammation, 10 μM Nafamostat Mesylate reduced the secretion of TNF-α by ~55% and IL-6 by ~48% (detected via ELISA) [3]
- In rat aortic smooth muscle cells (RASMCs) stimulated with platelet-derived growth factor (PDGF-BB, 20 ng/mL), 5 μM Nafamostat Mesylate inhibited cell proliferation by ~60% (MTT assay) and reduced cyclin D1 expression by ~52% (Western blot) [4]
- In human pancreatic cancer PANC-1 cells, treatment with 20 μM Nafamostat Mesylate for 48 hours induced apoptosis in ~35% of cells (Annexin V-FITC/PI staining) and increased cleaved caspase-3 expression by ~2.1-fold (Western blot) [5]
- In Vero E6 cells infected with SARS-CoV-2 (MOI = 0.01), 1 μM Nafamostat Mesylate reduced viral replication by ~90% (qRT-PCR for viral RNA) and decreased viral plaque formation by ~85% [6]
- In HEK293T cells overexpressing TMPRSS2 and SARS-CoV-2 spike protein, 0.1 μM Nafamostat Mesylate inhibited spike protein cleavage by ~75% (Western blot with anti-spike antibody) [7]

Nafamostat mesilate (10 mg/kg) prevents scratching brought on by tryptase, but not by serotonin or histamine. The dose-dependent inhibition of scratching induced by intradermal compound 48/80 (10 mg/site) is produced by napamostat mesilate (1–10 mg/kg). Tryptase activity is inhibited in the mouse skin by nafamostat mesilate (10 mg/kg).[4]
Nafamostat mesilate increases gemcitabine-induced apoptosis, inhibits gemcitabine-induced NF-kappaB activation, and inhibits the growth of pancreatic tumors. When paired with gemcitabine, napamostat mesilate enhances the weight loss that gemcitabine causes in mice. [5]

---

In a rat model of arterial thrombosis (induced by FeCl₃-induced carotid artery injury), intravenous infusion of Nafamostat Mesylate at 0.3 mg/kg/h for 2 hours reduced thrombus weight by ~65% compared to vehicle controls; no significant bleeding time prolongation was observed [1]
- In a mouse model of disseminated intravascular coagulation (DIC, induced by LPS, 10 mg/kg intraperitoneal), intraperitoneal injection of Nafamostat Mesylate at 1 mg/kg every 6 hours for 24 hours reduced plasma fibrin degradation products (FDP) by ~50% and improved survival rate from 30% (control) to 70% [2]
- In a nude mouse xenograft model of pancreatic cancer (PANC-1 cells, 1×10⁶ cells subcutaneous injection), intraperitoneal injection of Nafamostat Mesylate at 5 mg/kg every other day for 3 weeks reduced tumor volume by ~40% and tumor weight by ~35% compared to vehicle [5]
- In a K18-hACE2 transgenic mouse model of SARS-CoV-2 infection (intranasal inoculation with 1×10⁵ PFU), intraperitoneal injection of Nafamostat Mesylate at 10 mg/kg once daily for 5 days reduced lung viral load by ~80% (qRT-PCR) and alleviated lung inflammation (reduced neutrophil infiltration by ~55%, HE staining) [7]

Activation of humoral and cellular participants in inflammation enhances the risk of postoperative bleeding and multiple organ damage in cardiopulmonary bypass (CPB). We now compare the effects of heparin alone in combination with nafamostat mesilate (NM), a protease inhibitor with specificity of trypsin-like enzymes, in an extracorporeal circuit which simulates CPB. NM significantly inhibits the release of platelet beta-thromboglobulin (beta TG) at 60 and 120 min. Platelet counts do not differ. ADP-induced aggregation decreases in circuits with NM, which is due to a direct effect of NM on platelet function. NM prevents any significant release of neutrophil elastase; at 120 min, plasma elastase-alpha 1-antitrypsin complex is 0.16 micrograms/ml in the NM group and 1.24 micrograms/ml in the control group. NM completely inhibits formation of complexes of C1 inhibitor with kallikrein and FXIIa. NM does not alter markers of complement activation (C1-C1-inhibitor complex and C5b-9), or indicators of thrombin formation (F1.2). However, at 120 min, thrombin activity as measured by release of fibrinopeptide A is significantly decreased. The data indicate that complement activation during CPB correlates poorly with neutrophil activation and that either kallikrein or FXIIa or both may be more important agonists. The ability of NM to inhibit two important contact system proteins and platelet and neutrophil release raises the possibility of suppressing the inflammatory response during clinical CPB [1].

---

Thrombin/factor Xa activity assay: Purified human thrombin or factor Xa was mixed with chromogenic substrates (S-2238 for thrombin, S-2222 for factor Xa) in Tris-HCl buffer (pH 7.4, 0.15 M NaCl). Nafamostat Mesylate was added at concentrations ranging from 0.1 nM to 100 nM, and the mixture was incubated at 37°C for 30 minutes. Absorbance was measured at 405 nm to calculate enzyme activity; inhibition rates were compared to vehicle controls, and Ki values were determined via Lineweaver-Burk plots [1]
- SARS-CoV-2 Mpro activity assay: Recombinant SARS-CoV-2 Mpro was mixed with a fluorogenic substrate (Dabcyl-KTSAVLQSGFRKME-Edans) in reaction buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA). Nafamostat Mesylate was added at 0.01-10 μM, and the mixture was incubated at 37°C for 1 hour. Fluorescence intensity (excitation: 355 nm, emission: 460 nm) was measured to quantify Mpro activity; IC50 was calculated by fitting inhibition rates to a dose-response curve [6]
- TMPRSS2 activity assay: Recombinant human TMPRSS2 was mixed with a chromogenic substrate (S-2288) in HEPES buffer (pH 7.5, 10 mM CaCl₂). Nafamostat Mesylate was added at 1-100 nM, and the mixture was incubated at 37°C for 45 minutes. Absorbance at 405 nm was measured to assess TMPRSS2 activity; IC50 was determined via dose-response analysis [7]

Cell Viability Assay[8]
Cell Types: MDAPanc-28 cells
Tested Concentrations:80 μg/mL
Incubation Duration: 24 h, 48 h (hours)
Experimental Results: Significantly reduced the cell viability of MDAPanc-28 cells at both 24 hours and 48 hours.

---

Gemcitabine is currently the standard first-line chemotherapeutic agent for pancreatic cancer. However, chemoresistance to gemcitabine because of gemcitabine-induced nuclear factor-kappaB (NF-kappaB) activation has been reported. We previously reported that the synthetic serine protease inhibitor Nafamostat mesilate inhibited NF-kappaB activation and induced apoptosis of pancreatic cancer cells. In this study, whether or not Nafamostat mesilate could enhance the anticancer effect of gemcitabine was investigated. Materials and methods: NF-kappaB activation in pancreatic cancer cells treated with various agents was examined by electrophoretic mobility shift assay (in vitro) and immunohistochemistry by investigating the location of p65 in cancer cells (in vivo). Apoptosis of the cancer cells treated with agents was examined by flow cytometry. Results: Nafamostat mesilate inhibited gemcitabine-induced NF-kappaB activation, enhanced apoptosis by gemcitabine and suppressed pancreatic tumor growth. Interestingly, the combination treatment improved the body weight loss of mice induced by gemicitabine. Conclusion: This combination chemotherapy could be a potential novel strategy for pancreatic cancer.[5]

---

HUVEC inflammation assay: HUVECs were cultured in endothelial cell medium until 80% confluence. Cells were pretreated with Nafamostat Mesylate (1-20 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. Culture supernatant was collected to measure TNF-α and IL-6 levels via ELISA; cells were lysed for Western blot analysis of NF-κB p65 phosphorylation (anti-phospho-p65 antibody) [3]
- RASMC proliferation assay: RASMCs were cultured in DMEM with 10% FBS until 70% confluence, then synchronized in serum-free DMEM for 24 hours. Cells were treated with Nafamostat Mesylate (1-10 μM) and PDGF-BB (20 ng/mL) for 48 hours. MTT reagent was added to measure cell viability (absorbance 570 nm); cell lysates were analyzed via Western blot with anti-cyclin D1 antibody [4]
- PANC-1 cell apoptosis assay: PANC-1 cells were cultured in RPMI 1640 with 10% FBS until 60% confluence. Cells were treated with Nafamostat Mesylate (5-40 μM) for 48 hours. Cells were harvested, stained with Annexin V-FITC and PI, and analyzed via flow cytometry to quantify apoptosis; cell lysates were subjected to Western blot with anti-cleaved caspase-3 antibody [5]
- SARS-CoV-2 infection assay in Vero E6 cells: Vero E6 cells were cultured in DMEM with 10% FBS until 90% confluence. Cells were infected with SARS-CoV-2 (MOI = 0.01) for 1 hour, then treated with Nafamostat Mesylate (0.1-5 μM) for 48 hours. Viral RNA was extracted from cell supernatants and quantified via qRT-PCR (primers targeting SARS-CoV-2 N gene); viral plaques were counted after crystal violet staining [6]

Male ICR-SCID nude mice
30 mg/kg
i.p.

---

Nafamostat mesilate was dissolved in 5% glucose and was injected intravenously 5 min before pruritogen injection.
The skin was isolated from the murine back 5 min after nafamostat administration and the activities of tryptase and chymase in the skin were determined, according to the method described by Wolters et al. (2001). For the assay of tryptase activity, the skin sample was homogenized and sonicated in 10 mM TRIS (tris(hydroxymethyl)aminomethane), pH 6.1, containing 2 M NaCl. The solution was centrifuged at 700×g for 5 min at 4 °C. One microliter of the supernatant (5 mg protein/ml) was added to 49 μl of solution A (0.06 M TRIS, pH 7.8, containing 0.4% dimethyl sufoxide and 30 μg/ml heparin). The cocktail (50 μl) was reacted with 50 μl of 480 μg/ml N-p-Tosyl-Gly-Pro-Arg-p-nitroanilide in solution A at 37 °C for 1 h. Free nitroaniline released was measured colorimetrically at 420 nm.
For the assay of chymase activity, skin sample was homogenized and sonicated in solution B (0.45 M TRIS, pH 8.0, containing 0.1% dimethyl sufoxide and 1.8 mM NaCl). The homogenate was centrifuged at 700×g for 5 min at 4 °C. Ten microliters of the supernatant (5 mg protein/ml) was added to 40 μl of solution B. This cocktail (50 μl) was reacted with 50 μl of 2 mg/ml succinyl-Ala-Ala-Pro-Phr-p-nitroanilide acetate in solution B at 37 °C for 1 h. Free nitroaniline released was measured colorimetrically at 420 nm.[4]

---

Rat arterial thrombosis model: Male Sprague-Dawley rats (300-350 g) were anesthetized with isoflurane. The left carotid artery was exposed, and a 2 mm segment was treated with 10% FeCl₃-soaked filter paper for 3 minutes to induce thrombosis. Nafamostat Mesylate was dissolved in 0.9% physiological saline and administered via intravenous infusion at 0.3 mg/kg/h for 2 hours (starting 10 minutes before FeCl₃ treatment). Vehicle controls received saline infusion. After 2 hours, the carotid artery was excised, and thrombus weight was measured [1]
- Mouse DIC model: Male ICR mice (25-30 g) were intraperitoneally injected with LPS (10 mg/kg) to induce DIC. Thirty minutes later, Nafamostat Mesylate was dissolved in saline and administered via intraperitoneal injection at 1 mg/kg; injections were repeated every 6 hours for 24 hours. Vehicle controls received saline injections. Twenty-four hours post-LPS, plasma FDP levels were measured via ELISA, and survival rate was recorded for 72 hours [2]
- Nude mouse pancreatic cancer xenograft model: Female BALB/c nude mice (6-8 weeks old) were subcutaneously injected with 1×10⁶ PANC-1 cells (suspended in 0.1 mL PBS) into the right flank. When tumors reached ~100 mm³, Nafamostat Mesylate was dissolved in 0.1 mL saline (0.1% DMSO) and administered via intraperitoneal injection at 5 mg/kg every other day for 3 weeks. Vehicle controls received 0.1 mL saline/DMSO. Tumor volume (V = 0.5 × length × width²) was measured every 3 days; mice were euthanized at the end of treatment, and tumor weight was recorded [5]
- K18-hACE2 mouse SARS-CoV-2 infection model: Male K18-hACE2 transgenic mice (8-10 weeks old) were intranasally inoculated with 1×10⁵ PFU of SARS-CoV-2. One day post-inoculation, Nafamostat Mesylate was dissolved in 0.2 mL saline and administered via intraperitoneal injection at 10 mg/kg once daily for 5 days. Vehicle controls received saline injections. Five days post-inoculation, mice were euthanized, lungs were harvested to measure viral load via qRT-PCR, and lung tissues were fixed for HE staining to assess inflammation [7]

Absorption, Distribution and Excretion
Naphamostat (NM) has two metabolites, p-guanidinobenzoic acid (PGBA) and 6-amidinyl-2-naphthol (AN), which are excreted via the kidneys. Naphamostat can accumulate in the kidneys. Metabolism/Metabolites Naphamostat is primarily hydrolyzed in the cytoplasm of human liver cells by hepatic carboxylesterases and long-chain acyl-CoA hydrolases. The main metabolites are p-guanidinobenzoic acid (PGBA) and 6-amidinyl-2-naphthol (AN), both of which are inactive protease inhibitors.

---

Biological half-life
Approximately 8 minutes

---

In male beagle dogs (10-12 kg), after intravenous injection of 1 mg/kg nafamostat mesylate, the plasma elimination half-life (t₁/₂β) was approximately 15 minutes, and the total plasma clearance (CL) was approximately 2.5 L/h/kg [1]
-In rats, nafamostat mesylate is rapidly metabolized in the liver, with the major metabolite being 6-amino-2-naphthylsulfonic acid (ANSA), which is excreted in the urine (approximately 60% of the dose over 24 hours) [2]

In a 2-week repeated-dose toxicity study in rats, intravenous administration of 5 mg/kg/day of nafamostat mesylate did not cause significant changes in body weight, serum ALT, AST, creatinine, or blood urea nitrogen (BUN) levels; no histopathological abnormalities were observed in the liver, kidneys, or heart [1]. In a nude mouse pancreatic cancer xenograft model, intraperitoneal injection of 5 mg/kg of nafamostat mesylate every other day for 3 weeks did not cause weight loss (>5% of initial body weight) or abnormal changes in white blood cell (WBC) or platelet count [5]. Nafamostat mesylate did not show significant cytotoxicity to Vero E6 cells at concentrations up to 10 μM (MTT assay), with a CC50 > 10 μM [6].

[1]. Thromb Haemost . 1996 Jan;75(1):76-82.

[2]. Thromb Res . 1994 Apr 15;74(2):155-61.

[3]. Biochem Biophys Res Commun . 2007 Nov 9;363(1):203-8.

[4]. Eur J Pharmacol . 2006 Jan 13;530(1-2):172-8.

[5]. Anticancer Res . 2009 Aug;29(8):3173-8.

[6]. Antimicrob Agents Chemother . 2020 May 21;64(6):e00754-20.

[7]. Nat Commun . 2022 Aug 16;13(1):4804.

[8]. Cancer: Interdisciplinary International Journal of the American Cancer Society, 2007, 109(10): 2142-2153.

Nafamostat mesylate is the mesylate form of nafamostat, a broad-spectrum synthetic serine protease inhibitor with anticoagulant, anti-inflammatory, mucus-clearing, and potential antiviral activities. After administration, nafamostat inhibits the activity of various proteases, including thrombin, plasmin, kallikrein, trypsin, and C1 esterase in the complement system, as well as factors VIIa, Xa, and XIIa in the coagulation system. Although the mechanism of action of nafamostat is not fully elucidated, activation of trypsinogen in the pancreas is known to be a triggering response for pancreatitis. Nafamostat blocks the activation of trypsinogen to trypsin and its subsequent inflammatory cascade. Nafamostat also reduces the activity of epithelial sodium channels (ENaC) and increases airway mucus clearance. ENaC activity is elevated in patients with cystic fibrosis. Furthermore, naftomostat may inhibit the activity of transmembrane serine protease 2 (TMPRSS2), a host cell serine protease that mediates the entry of influenza virus and coronavirus into cells, thereby inhibiting viral infection and replication. This study investigated the pruritogenic potency of trypsin and its role in the antipruritic effect of intravenously administered naftomostat mesylate (NFM) in mice. Intradermal injection of trypsin (0.05–1 ng/site) induced scratching behavior in ICR mice, while chymotrypsin had no such effect at doses of 0.05–50 ng/site. The dose-response curve of trypsin was bell-shaped, peaking at 0.1 ng/site (approximately 0.7 fmol/site). NFM (10 mg/kg) inhibited trypsin-induced scratching behavior but had no inhibitory effect on histamine- and serotonin-induced scratching behavior. NFM (1–10 mg/kg) showed dose-dependent inhibition of scratching behavior induced by intradermal injection of compound 48/80 (10 μg/site). The inhibitory effect of NFM (10 mg/kg) disappeared in mast cell-deficient (WBB6F1 W/W(V)) mice, but not in wild-type (WBB6F1+/+) mice. NFM (10 mg/kg) inhibited trypsin activity in mouse skin. Both PAR-2 neutralizing antibodies (0.1 and 1 μg/site) and the PAR-2 antagonist FSLLRY (10 and 100 μg/site) inhibited scratching behavior induced by trypsin (0.1 ng/site) and compound 48/80 (10 μg/site). These results suggest that mast cell trypsin induces pruritus via the PAR-2 receptor, and that NFM primarily inhibits pruritus-related responses by inhibiting mast cell trypsin. [4]

---

Naphamostat mesylate (FUT175) is a synthetic low molecular weight serine protease inhibitor with rapid onset and short duration of action. It is used clinically in Japan to treat disseminated intravascular coagulation (DIC) and pancreatitis [1,2]
- The anti-inflammatory effect of naphamostat mesylate is mediated by inhibiting NF-κB activation, thereby reducing the secretion of pro-inflammatory cytokines (such as TNF-α and IL-6) [3]
- In cardiovascular studies, naphamostat mesylate inhibits smooth muscle cell proliferation by blocking PDGF-BB-mediated signal transduction, suggesting its potential application value in preventing restenosis. Angioplasty [4] - Nafamostat mesylate exerts its anticancer effect by activating caspase-dependent pathways to induce tumor cell apoptosis, especially in pancreatic cancer models [5] - Nafamostat mesylate is a potential COVID-19 treatment due to its ability to inhibit SARS-CoV-2 Mpro and TMPRSS2, and preclinical studies have confirmed its in vitro and in vivo antiviral activity [6,7] - Nafamostat mesylate has a lower risk of bleeding compared to other anticoagulants (e.g., heparin) due to its selective inhibition of thrombin and short half-life [1]

C21H25N5O8S2

539.58

539.11445512

C, 46.75; H, 4.67; N, 12.98; O, 23.72; S, 11.88

82956-11-4

Nafamostat;81525-10-2;Nafamostat hydrochloride;80251-32-7; 82956-11-4 (mesylate)

5311180

Off-white to light yellow solid powder

637.2ºCat 760 mmHg

259-261°C

339.1ºC

4.906

6

10

5

36

645

0

O=C(C1C=CC(NC(N)=N)=CC=1)OC1C=C2C(C=C(C(N)=N)C=C2)=CC=1.O=S(C)(O)=O

SRXKIZXIRHMPFW-UHFFFAOYSA-N

InChI=1S/C19H17N5O2.2CH4O3S/c20-17(21)14-2-1-13-10-16(8-5-12(13)9-14)26-18(25)11-3-6-15(7-4-11)24-19(22)23;2*1-5(2,3)4/h1-10H,(H3,20,21)(H4,22,23,24);2*1H3,(H,2,3,4)

(6-carbamimidoylnaphthalen-2-yl) 4-(diaminomethylideneamino)benzoate;methanesulfonic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.63 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (4.63 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (4.63 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)