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 (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
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.

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.[2]

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.

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

Naphamostat 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, naphamostat is approved for the treatment of patients receiving continuous renal replacement therapy for acute kidney injury as an anticoagulant. Naphamostat is a broad-spectrum synthetic serine protease inhibitor with anticoagulant, anti-inflammatory, mucus-clearing, and potential antiviral activities. After administration, naphamostat 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 naphamostat is not fully elucidated, activation of trypsinogen in the pancreas is known to be a triggering response for pancreatitis. Naphamostat blocks the activation of trypsinogen into trypsin and the inflammatory cascade it triggers. Naphamostat 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, naphamostat 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 Naphamostat 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 min, 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 generation indicators (F1.2). However, at 120 min, 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 that it may suppress inflammatory responses during clinical cardiopulmonary bypass. [1]

C19H19CL2N5O2

420.2925

419.091

80251-32-7

Nafamostat mesylate;82956-11-4;Nafamostat;81525-10-2

13050562

Solid powder

5.123

6

4

5

28

552

0

Cl[H].Cl[H].O(C(C1C([H])=C([H])C(=C([H])C=1[H])/N=C(\N([H])[H])/N([H])[H])=O)C1C([H])=C([H])C2C([H])=C(/C(=N/[H])/N([H])[H])C([H])=C([H])C=2C=1[H]

GKGJACPQHBIISL-UHFFFAOYSA-N

InChI=1S/C19H17N5O2.2ClH/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);2*1H

(6-carbamimidoylnaphthalen-2-yl) 4-(diaminomethylideneamino)benzoate;dihydrochloride

FUT-175; Nafamostat (hydrochloride); Nafamostat hydrochloride; 80251-32-7; (6-carbamimidoylnaphthalen-2-yl) 4-(diaminomethylideneamino)benzoate;dihydrochloride; Nafamostathydrochloride; SCHEMBL3302794; Nafamostat HCl; 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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)