Serine Protease; Granzyme; I-kappaBalpha
Nafamostat targets a wide range of serine proteases, which are involved in various physiological and pathological processes. Its primary clinical targets include enzymes of the coagulation and fibrinolytic systems, such as thrombin, factor Xa, and factor XIIa. It also inhibits the kallikrein-kinin system, the complement system, and pancreatic proteases like trypsin. Furthermore, it inhibits the activation of protease-activated receptors (PARs). Nafamostat's mechanism of action involves covalent, reversible binding to the active site of these serine proteases, effectively blocking their proteolytic activity. It also promotes endothelium-dependent vasorelaxation via the Akt-eNOS dependent pathway, and its metabolites inhibit channel-activating proteases such as CAP1 (prostasin).

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 vitro, Nafamostat is a potent inhibitor of various serine proteases. It shows strong, selective inhibition of trypsin, kallikrein, plasmin, and thrombin, as well as enzymes of the complement system. Its anticoagulant activity is primarily due to the inhibition of thrombin and factor Xa. Nafamostat also inhibits the activation of protease-activated receptors (PARs). Its ability to inhibit TMPRSS2, a host cell serine protease essential for SARS-CoV-2 spike protein priming, has made it a subject of interest as a potential antiviral agent. The compound is also noted for its ability to promote endothelium-dependent vasorelaxation through the Akt-eNOS pathway in cellular models.

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]
In vivo, Nafamostat is primarily used for its anticoagulant properties during hemodialysis. Its short half-life and rapid metabolism make it suitable for this application, as it provides effective anticoagulation within the extracorporeal circuit without significant systemic effects. It has also been shown to attenuate ischemia-reperfusion-induced renal injury in animal models. Nafamostat has been used clinically in Japan for the treatment of acute pancreatitis and DIC. Recently, its potential as an antiviral agent has been explored in vivo, with studies suggesting it can inhibit SARS-CoV-2 replication. However, resistance can emerge through mutations in target serine proteases.

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].
For in vitro cell-free enzyme assays, Nafamostat's inhibitory activity against its target proteases can be measured using chromogenic or fluorogenic substrates specific to each enzyme. For example, to measure its inhibition of thrombin, a chromogenic substrate like S-2238 can be used. The assay involves incubating varying concentrations of Nafamostat with a fixed concentration of the purified enzyme (e.g., thrombin) and its substrate. The rate of substrate cleavage, which is proportional to enzyme activity, is monitored spectrophotometrically (at 405 nm for chromogenic substrates). The IC50 value, which represents the concentration of Nafamostat required to inhibit 50% of the enzyme activity, is calculated from a dose-response curve.

Cell Viability Assay
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.
For cellular assays, the effect of Nafamostat on the Akt-eNOS pathway can be studied in endothelial cells. Cells are treated with various concentrations of Nafamostat for a defined period, and the phosphorylation levels of Akt and eNOS are measured by Western blot using phospho-specific antibodies. This indicates the compound's ability to promote endothelium-dependent vasorelaxation. To study its antiviral activity, cells susceptible to SARS-CoV-2 infection (e.g., Vero E6 cells) are treated with Nafamostat prior to or during viral infection. After incubation, the level of viral replication is quantified by measuring viral RNA via qPCR or by plaque assay, and the EC50 (half-maximal effective concentration) for inhibiting viral entry is determined.

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.[2]
For in vivo studies, Nafamostat is typically administered intravenously due to its peptidic nature and poor oral bioavailability. In animal models of ischemia-reperfusion injury, such as renal or cardiac models, Nafamostat is administered as a bolus or continuous infusion before the ischemic event. Endpoints include measurement of infarct size, serum biomarkers of organ damage (e.g., creatinine, BUN for kidney injury), and histological analysis of tissue damage. In models of acute pancreatitis, Nafamostat is administered to reduce pancreatic enzyme activity and inflammation. For antiviral efficacy, it can be administered to SARS-CoV-2-infected animal models, with endpoints including viral load in lung tissue, inflammatory cytokine levels, and survival rate.

Absorption, Distribution and Excretion
Nafamostat (NM) has two metabolites, p-guanidinobenzoic acid (PGBA) and 6-amidinyl-2-naphthol (AN), which are excreted via the kidneys. Nafamostat can accumulate in the kidneys. Metabolism/Metabolites Nafamostat 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.
Nafamostat mesilate has a molecular formula of C19H17N5O2·CH4O3S and a molecular weight of 539.6 g/mol for the mesilate salt. The free base has a molecular weight of 347.37 g/mol. It is supplied as a white to off-white powder. For in vitro studies, it is typically dissolved in DMSO or water to prepare stock solutions. For in vivo administration, it is formulated in saline or other suitable buffers. Storage is recommended at -20°C, protected from light, to ensure stability. As a synthetic compound, its purity is typically >98%.

Nafamostat has a well-established clinical safety profile, particularly as an anticoagulant during hemodialysis. Common side effects are related to its anticoagulant activity and can include bleeding complications. It is generally well-tolerated, but caution is advised in patients with a high risk of bleeding. In preclinical toxicology studies, the compound has demonstrated a relatively low toxicity profile at therapeutic doses. However, like all anticoagulants, the potential for hemorrhage is a significant concern, and its use requires careful monitoring of coagulation parameters. Its short half-life is a key safety feature, allowing for rapid reversal of its anticoagulant effect if needed.

[1]. Thromb Haemost.1996 Jan;75(1):76-82;
[2]. Eur J Pharmacol.2006 Jan 13;530(1-2):172-8.

Nafamostat belongs to the benzoic acid and guanidine derivatives. It is a synthetic serine protease inhibitor and, due to its basicity, is usually formulated with hydrochloric acid. It has been used in clinical trials for the prevention of liver transplantation and reperfusion syndrome. In Asian countries, Nafamostat is approved for the treatment of patients receiving continuous renal replacement therapy for acute kidney injury as an anticoagulant. Nafamostat is 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, and 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 into trypsin and the inflammatory cascade it triggers. Nafamostat can also reduce the activity of epithelial sodium channels (ENaC) and increase airway mucus clearance. ENaC activity is elevated in patients with cystic fibrosis. Furthermore, Nafamostat can inhibit the activity of transmembrane serine protease 2 (TMPRSS2). TMPRSS2 is a host cell serine protease that mediates the entry of influenza virus and coronavirus into cells, thereby inhibiting viral infection and replication. Drug Indications For the treatment of patients with disseminated intravascular coagulation, hemorrhagic lesions, and bleeding tendencies, as an anticoagulant. It can prevent thrombosis during extracorporeal circulation in patients receiving continuous renal replacement therapy and extracorporeal membrane oxygenation (ECMO). Mechanism of Action Nafamostat mesylate inhibits the activation of multiple enzyme systems, such as the coagulation and fibrinolytic systems (thrombin, Xa, and XIIa), the kallikrein-kinin system, the complement system, trypsin, and protease-activating receptors (PARs). Nafamostat inhibits lipopolysaccharide-induced nitric oxide production, apoptosis, and interleukin (IL)-6 and IL-8 levels in cultured human trophoblast cells. Studies have shown that it plays an antioxidant role in TNF-α-induced reactive oxygen species (ROS) production.

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Activation of humoral and cytokine kines during inflammation increases the risk of postoperative bleeding and multi-organ injury after cardiopulmonary bypass (CPB).
We now compare the effects of heparin alone combined with nafamostat mesylate (NM, a protease inhibitor that specifically inhibits trypsin-like enzymes) in a CPB-mimicking loop. NM significantly inhibited the release of platelet β-thromboglobulin (β-TG) at 60 and 120 minutes. There was no difference in platelet count. In the loop with NM added, ADP-induced platelet aggregation was reduced due to the direct effect of NM on platelet function. NM significantly inhibited the release of neutrophil elastase; at 120 minutes, the plasma elastase-α1-antitrypsin complex concentration in the NM group was 0.16 μg/mL, compared to 1.24 μg/mL in the control group. NM completely inhibited the formation of complexes between C1 inhibitors and kallikrein and FXIIa. NM did not alter complement activation markers (C1-C1-inhibitor complex and C5b-9) or thrombin formation indicators (F1.2). However, at 120 minutes, thrombin activity, as measured by fibrin peptide A release, was significantly reduced. The data suggest that complement activation during cardiopulmonary bypass is poorly correlated with neutrophil activation, and kallikrein or FXIIa, or both, may be more important agonists. The ability of NM to inhibit the release of two important contact system proteins, as well as platelet and neutrophil release, suggests its potential to suppress inflammatory responses during clinical cardiopulmonary bypass. [1]

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Pharmacodynamics
Nafamostat is a rapid-acting proteolytic inhibitor used during hemodialysis to prevent fibrinogen from being hydrolyzed into fibrin by competitively inhibiting multiple serine proteases, including thrombin. It can improve acute pancreatitis, prevent thrombosis during cardiopulmonary bypass, and has anti-inflammatory effects in vitro. One study showed that Nafamostat exerts a neuroprotective effect in ischemic brain injury through its antithrombin activity.
Nafamostat mesilate is an approved drug in several countries, including Japan, for use as an anticoagulant during hemodialysis and for the treatment of acute pancreatitis and disseminated intravascular coagulation (DIC). It is not FDA-approved for use in the United States but is available as an investigational agent. Its mechanism of action as a potent, broad-spectrum serine protease inhibitor makes it a valuable drug for conditions involving dysregulated protease activity. Beyond its clinical uses, it is a widely used research tool for studying coagulation, inflammation, and viral entry mechanisms. Its ability to inhibit TMPRSS2 has garnered significant interest in the context of COVID-19 research.

C19H17N5O2

347.37

347.138

C, 65.69; H, 4.93; N, 20.16; O, 9.21

81525-10-2

Nafamostat mesylate;82956-11-4;Nafamostat hydrochloride;80251-32-7;Nafamostat formate salt-13C6

4413

Solid powder

1.4±0.1 g/cm3

637.2±65.0 °C at 760 mmHg

339.1±34.3 °C

0.0±1.9 mmHg at 25°C

1.694

1.93

4

4

5

26

552

0

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

MQQNFDZXWVTQEH-UHFFFAOYSA-N

InChI=1S/C19H17N5O2/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/h1-10H,(H3,20,21)(H4,22,23,24)

6-Amidino-2-naphthyl 4-guanidinobenzoate

Nafamostat free base; Nafamostat [INN]; Nafamstat; (6-carbamimidoylnaphthalen-2-yl) 4-(diaminomethylideneamino)benzoate; CHEMBL273264; Y25LQ0H97D; p-Guanidinobenzoic acid ester with 6-hydroxy-2-naphthamidine; Nafamostat; FUT-175; FUT 175; FUT175.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: >10 mM

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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