Serine Protease; Granzyme; I-kappaBalpha

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]

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]

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].

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.

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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]

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

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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]

Absorption, Distribution and Excretion
Two metabolites of NM, p-guanidinobenzoic acid (PGBA) and 6-amidino-2-naphthol (AN), are renally excreted. Nafamostat accumulates in the kidneys.

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Metabolism / Metabolites
Nafamostat is mainly hydrolyzed by hepatic carboxyesterase and long-chain acyl-CoA hydrolase in human liver cytosol. Main metabolites are p-guanidinobenzoic acid (PGBA) and 6-amidino-2-naphthol (AN) as inactive protease inhibitors.

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Biological Half-Life
Approximately 8 minutes

[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 salt form of nafamostat, a broad-spectrum, synthetic serine protease inhibitor, with anticoagulant, anti-inflammatory, mucus clearing, and potential antiviral activities. Upon administration, nafamostat inhibits the activities of a variety of proteases, including thrombin, plasmin, kallikrein, trypsin, and Cl 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 understood, trypsinogen activation in the pancreas is known to be a trigger reaction in the development of pancreatitis. Nafamostat blocks the activation of trypsinogen to trypsin and the inflammatory cascade that follows. Nafamostat may also decrease epithelial sodium channel (ENaC) activity and increase mucus clearance in the airways. ENaC activity is increased in cystic fibrosis. In addition, nafamostat may inhibit the activity of transmembrane protease, serine 2 (TMPRSS2), a host cell serine protease that mediates viral cell entry for influenza virus and coronavirus, thereby inhibiting viral infection and replication.

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The pruritogenic potency of tryptase and its involvement in anti-pruritic effect of intravenous nafamostat mesilate (NFM) were studied in mice. An intradermal injection of tryptase (0.05-1 ng/site) elicited scratching in ICR mice, while chymase was without effects at doses of 0.05-50 ng/site. The dose-response curve of tryptase action was bell-shaped and the effect peaked at 0.1 ng/site (approximately 0.7 fmol/site). NFM (10 mg/kg) inhibited scratching induced by tryptase but not by histamine and serotonin. NFM (1-10 mg/kg) produced the dose-dependent inhibition of scratching induced by intradermal compound 48/80 (10 microg/site). The inhibition by NFM (10 mg/kg) was abolished in mast cell-deficient (WBB6F1 W/W(V)) mice, but not in wild-type (WBB6F1 +/+) mice. NFM (10 mg/kg) suppressed tryptase activity in the mouse skin. Proteinase-activated receptor-2 (PAR-2) neutralizing antibody (0.1 and 1 microg/site) and the PAR-2 antagonist FSLLRY (10 and 100 microg/site) inhibited scratching induced by tryptase (0.1 ng/site) and compound 48/80 (10 microg/site). These results suggest that mast cell tryptase elicits itch through PAR-2 receptor and that NFM inhibits itch-associated responses mainly through the inhibition of mast cell tryptase.[4]

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.

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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.

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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.

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