Serine Protease; Granzyme; I-kappaBalpha

体外活性：Nafamostat 甲磺酸盐在 60 分钟和 120 分钟时显着抑制血小板 β-血栓球蛋白 (β TG) 的释放。甲磺酸萘莫司他 (NM) 可防止中性粒细胞弹性蛋白酶的显着释放； 120 分钟时，NM 组血浆弹性蛋白酶-α 1-抗胰蛋白酶复合物为 0.16 mg/mL，对照组为 1.24 mg/mL。 Nafamostat mesilate 完全抑制 C1 抑制剂与激肽释放酶和 FXIIa 形成复合物。 Nafamostat mesilate 抑制多种蛋白酶，这些蛋白酶可能在弥散性血管内凝血 (DIC) 的病理生理学中发挥重要作用。Nafamostat mesilate 以浓度依赖性方式抑制外在途径活性（TF-F.VIIa 介导的 F.Xa 生成），IC50 为 0.1 μM 。 Nafamostat mesilate 对双相 ASIC3 电流的初始相瞬态分量产生浓度依赖性抑制，IC50 值约为 2.5 mM。细胞测定：在通过ELISA评估NF-κB活化时，联合组PANC-1细胞核提取物中NF-κB p65的浓度在统计学上低于奥沙利铂组(p<0.0001)。与核 NF-κB 水平一样，Western blot 分析显示联合组磷酸化 IκBa 水平显着低于奥沙利铂组 (p=0.037)。换句话说，FUT-175 在体外通过抑制 IκBa 磷酸化来抑制奥沙利铂诱导的 NF-κB 激活。

Nafamostat mesilate (10 mg/kg) 抑制类胰蛋白酶诱导的抓挠，但不抑制组胺和血清素诱导的抓挠。 Nafamostat mesilate (1-10 mg/kg) 对皮内化合物 48/80（10 mg/位点）引起的抓挠产生剂量依赖性抑制。 Nafamostat mesilate (10 mg/kg) 可抑制小鼠皮肤中的类胰蛋白酶活性。 Nafamostat mesilate 抑制吉西他滨诱导的 NF-κB 激活，增强吉西他滨引起的细胞凋亡并抑制胰腺肿瘤生长。甲磺酸萘莫司他联合吉西他滨可改善吉西他滨引起的小鼠体重减轻。

炎症中体液和细胞参与者的激活会增加体外循环术后出血和多器官损伤的风险。我们现在在模拟体外循环的体外回路中比较单独使用肝素与甲磺酸那法莫司他酯（NM）的效果，后者是一种具有胰蛋白酶样酶特异性的蛋白酶抑制剂。NM在60分钟和120分钟时显著抑制血小板β-血栓球蛋白（β-TG）的释放。血小板计数没有差异。ADP诱导的NM回路聚集减少，这是由于NM对血小板功能的直接影响。NM可防止中性粒细胞弹性蛋白酶的任何显著释放；在120分钟时，NM组的血浆弹性蛋白酶α1-抗胰蛋白酶复合物为0.16微克/毫升，对照组为1.24微克/毫升。NM完全抑制C1抑制剂与激肽释放酶和FXIIa复合物的形成。NM不改变补体激活的标志物（C1-C1抑制剂复合物和C5b-9）或凝血酶形成的指标（F1.2）。然而，在120分钟时，通过纤维蛋白肽A的释放测量的凝血酶活性显著降低。数据表明，CPB期间的补体激活与中性粒细胞激活相关性较差，激肽释放酶或FXIIa或两者都可能是更重要的激动剂。NM抑制两种重要接触系统蛋白以及血小板和中性粒细胞释放的能力增加了在临床CPB期间抑制炎症反应的可能性

细胞活力测定
细胞类型：MDAPanc-28细胞
测试浓度：80μg/mL
培养时间：24小时、48小时
实验结果：在24小时和48小时显著降低MDAPanc-28细胞的细胞活力。

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

Nafamostat is a member of benzoic acids and a member of guanidines.
Nafamostat is a synthetic serine protease inhibitor that is commonly formulated with hydrochloric acid due to its basic properties. It has been used in trials studying the prevention of Liver Transplantation and Postreperfusion Syndrome. The use of nafamostat in Asian countries is approved as an anticoagulant therapy for patients undergoing continuous renal replacement therapy due to acute kidney injury.
Nafamostat is 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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Drug Indication
Used as an anticoagulant in patients with disseminative blood vessel coagulation, hemorrhagic lesions, and hemorrhagic tendencies. It prevents blood clot formation during extracorporeal circulation in patients undergoing continuous renal replacement therapy and extra corporeal membrane oxygenation.

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Mechanism of Action
Nafamostat mesilate inhibits various enzyme systems, such as coagulation and fibrinolytic systems (thrombin, Xa, and XIIa), the kallikrein–kinin system, the complement system, pancreatic proteases and activation of protease-activated receptors (PARs). Nafamostat inhibits lipopolysaccharide-induced nitric oxide production, apoptosis, and interleukin (IL)-6 and IL-8 levels in cultured human trophoblasts. It is shown to act as an antioxidant in TNF-α-induced ROS production.

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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]

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Pharmacodynamics
Nafamostat is a fast-acting proteolytic inhibitor used during hemodialysis to prevent the proteolysis of fibrinogen into fibrin by competitively inhibiting several serine proteases including thrombin. It improves acute pancreatitis and prevents blood clot formation during extracorporeal circulation and has an anti-inflammatory effect in vitro. A study suggets that nafamostat has a neuroprotective role during ischemia-induced brain injury from antithrombin activity.

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

138.07

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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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