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 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。

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在人血浆凝血实验中，Nafamostat Mesylate 以剂量依赖性方式延长凝血酶原时间（PT）和活化部分凝血活酶时间（APTT）：浓度为0.5 μM时，PT较对照组延长约30%，APTT延长约45% [1]
- 在无细胞纤溶体系中，1 μM Nafamostat Mesylate 可抑制纤溶酶介导的纤维蛋白降解，抑制率约80%（通过纤维蛋白平板实验检测）[2]
- 在经脂多糖（LPS，1 μg/mL）诱导炎症的人脐静脉内皮细胞（HUVECs）中，10 μM Nafamostat Mesylate 可使肿瘤坏死因子-α（TNF-α）分泌减少约55%，白细胞介素-6（IL-6）分泌减少约48%（通过ELISA检测）[3]
- 在经血小板衍生生长因子（PDGF-BB，20 ng/mL）刺激的大鼠主动脉平滑肌细胞（RASMCs）中，5 μM Nafamostat Mesylate 可抑制细胞增殖，抑制率约60%（MTT法），并使细胞周期蛋白D1（cyclin D1）表达减少约52%（Western blot检测）[4]
- 在人胰腺癌细胞PANC-1中，20 μM Nafamostat Mesylate 处理48小时可诱导约35%的细胞凋亡（Annexin V-FITC/PI双染），并使剪切型caspase-3表达上调约2.1倍（Western blot检测）[5]
- 在感染SARS-CoV-2（感染复数MOI = 0.01）的Vero E6细胞中，1 μM Nafamostat Mesylate 可抑制病毒复制，抑制率约90%（通过qRT-PCR检测病毒RNA），并减少病毒空斑形成，抑制率约85% [6]
- 在过表达TMPRSS2和SARS-CoV-2刺突蛋白的HEK293T细胞中，0.1 μM Nafamostat Mesylate 可抑制刺突蛋白剪切，抑制率约75%（通过抗刺突蛋白抗体的Western blot检测）[7]

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。

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在大鼠动脉血栓模型（通过FeCl₃诱导颈动脉损伤建立）中，以0.3 mg/kg/h的剂量静脉输注Nafamostat Mesylate 2小时，血栓重量较溶剂对照组减少约65%；未观察到出血时间显著延长[1]
- 在小鼠弥散性血管内凝血（DIC）模型（通过腹腔注射10 mg/kg LPS建立）中，以1 mg/kg的剂量腹腔注射Nafamostat Mesylate，每6小时1次，持续24小时，血浆纤维蛋白降解产物（FDP）减少约50%，存活率从对照组的30%提升至70% [2]
- 在裸鼠胰腺癌异种移植模型（皮下注射1×10⁶个PANC-1细胞）中，以5 mg/kg的剂量腹腔注射Nafamostat Mesylate，隔天1次，持续3周，肿瘤体积较溶剂对照组减少约40%，肿瘤重量减少约35% [5]
- 在K18-hACE2转基因小鼠SARS-CoV-2感染模型（鼻内接种1×10⁵ PFU病毒）中，以10 mg/kg的剂量腹腔注射Nafamostat Mesylate，每日1次，持续5天，肺组织病毒载量减少约80%（qRT-PCR检测），肺部炎症减轻（中性粒细胞浸润减少约55%，HE染色检测）[7]

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[1]。

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凝血酶/Xa因子活性检测流程：将纯化的人凝血酶或Xa因子与显色底物（凝血酶用S-2238，Xa因子用S-2222）混合于Tris-HCl缓冲液（pH 7.4，含0.15 M NaCl）中。加入0.1 nM~100 nM的Nafamostat Mesylate，在37°C下孵育30分钟。检测405 nm处的吸光度以计算酶活性；通过与溶剂对照组比较确定抑制率，并采用Lineweaver-Burk双倒数作图法计算Ki值[1]
- SARS-CoV-2 Mpro活性检测流程：将重组SARS-CoV-2 Mpro与荧光底物（Dabcyl-KTSAVLQSGFRKME-Edans）混合于反应缓冲液（50 mM Tris-HCl pH 7.5，含1 mM EDTA）中。加入0.01~10 μM的Nafamostat Mesylate，在37°C下孵育1小时。检测荧光强度（激发波长355 nm，发射波长460 nm）以定量Mpro活性；通过将抑制率拟合至剂量-反应曲线计算IC50 [6]
- TMPRSS2活性检测流程：将重组人TMPRSS2与显色底物（S-2288）混合于HEPES缓冲液（pH 7.5，含10 mM CaCl₂）中。加入1~100 nM的Nafamostat Mesylate，在37°C下孵育45分钟。检测405 nm处的吸光度以评估TMPRSS2活性；通过剂量-反应分析确定IC50 [7]

细胞活力测定[8]
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。

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吉西他滨目前是胰腺癌的标准一线化疗药物。然而，由于吉西他滨诱导的核因子- kappab （NF-kappaB）活化，吉西他滨出现了化学耐药。我们之前报道了合成丝氨酸蛋白酶抑制剂Nafamostat mesilate抑制NF-kappaB激活并诱导胰腺癌细胞凋亡。本研究探讨Nafamostat mesilate是否能增强吉西他滨的抗癌作用。 材料和方法：通过电泳迁移位移法（体外）和免疫组化法（体内）研究p65在癌细胞中的位置，检测不同药物处理胰腺癌细胞中NF-kappaB的活化情况。流式细胞术检测药物对肿瘤细胞凋亡的影响。 结果：Nafamostat mesilate抑制吉西他滨诱导的NF-kappaB活化，增强吉西他滨诱导的细胞凋亡，抑制胰腺肿瘤生长。有趣的是，联合治疗改善了吉米他滨诱导的小鼠体重减轻。 结论：这种联合化疗可能是治疗胰腺癌的潜在新策略。[5]

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HUVEC炎症实验流程：将HUVECs在 endothelial cell培养基中培养至80%汇合。用1~20 μM Nafamostat Mesylate 预处理细胞1小时后，用1 μg/mL LPS刺激24小时。收集培养上清液，通过ELISA检测TNF-α和IL-6水平；裂解细胞后，用抗磷酸化NF-κB p65抗体进行Western blot分析，检测NF-κB活化情况[3]
- RASMC增殖实验流程：将RASMCs在含10%胎牛血清的DMEM培养基中培养至70%汇合，随后在无血清DMEM中同步化24小时。用1~10 μM Nafamostat Mesylate 和20 ng/mL PDGF-BB共同处理细胞48小时。加入MTT试剂检测细胞活力（570 nm吸光度）；裂解细胞后，用抗cyclin D1抗体进行Western blot分析[4]
- PANC-1细胞凋亡实验流程：将PANC-1细胞在含10%胎牛血清的RPMI 1640培养基中培养至60%汇合。用5~40 μM Nafamostat Mesylate 处理细胞48小时。收集细胞，用Annexin V-FITC和PI染色，通过流式细胞术定量凋亡率；裂解细胞后，用抗剪切型caspase-3抗体进行Western blot分析[5]
- Vero E6细胞SARS-CoV-2感染实验流程：将Vero E6细胞在含10%胎牛血清的DMEM培养基中培养至90%汇合。用MOI = 0.01的SARS-CoV-2感染细胞1小时，随后用0.1~5 μM Nafamostat Mesylate 处理48小时。从细胞上清液中提取病毒RNA，通过qRT-PCR（针对SARS-CoV-2 N基因的引物）定量病毒载量；结晶紫染色后计数病毒空斑[6]

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]

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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
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

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In male beagle dogs (10-12 kg) administered intravenous Nafamostat Mesylate at 1 mg/kg, the plasma elimination half-life (t₁/₂β) was ~15 minutes, and the total plasma clearance (CL) was ~2.5 L/h/kg [1]
- In rats, Nafamostat Mesylate was rapidly metabolized in the liver, with the main metabolite being 6-amino-2-naphthalenesulfonic acid (ANSA), which was excreted in urine (accounting for ~60% of the dose within 24 hours) [2]

In a 2-week repeated-dose toxicity study in rats, intravenous administration of Nafamostat Mesylate at 5 mg/kg/day 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, kidney, or heart [1]
- In the nude mouse pancreatic cancer xenograft model, intraperitoneal injection of Nafamostat Mesylate at 5 mg/kg every other day for 3 weeks did not cause weight loss (>5% of initial weight) or abnormal changes in white blood cell (WBC) or platelet counts [5]
- Nafamostat Mesylate showed no significant cytotoxicity in 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 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]

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Nafamostat Mesylate (FUT175) is a synthetic, low-molecular-weight serine protease inhibitor with rapid onset and short duration of action, clinically used in Japan for the treatment of disseminated intravascular coagulation (DIC) and pancreatitis [1,2]
- The anti-inflammatory effect of Nafamostat Mesylate is mediated by inhibiting NF-κB activation, thereby reducing the secretion of pro-inflammatory cytokines (e.g., TNF-α, IL-6) [3]
- In cardiovascular research, Nafamostat Mesylate inhibits smooth muscle cell proliferation by blocking PDGF-BB-mediated signaling, suggesting potential application in preventing restenosis after angioplasty [4]
- Nafamostat Mesylate exerts anticancer effects by inducing apoptosis of tumor cells via activation of the caspase-dependent pathway, particularly in pancreatic cancer models [5]
- Due to its ability to inhibit SARS-CoV-2 Mpro and TMPRSS2, Nafamostat Mesylate is a potential therapeutic agent for COVID-19, with in vitro and in vivo antiviral activity demonstrated in preclinical studies [6,7]
- Nafamostat Mesylate has a low risk of bleeding compared to other anticoagulants (e.g., heparin) due to its selective inhibition of coagulation proteases 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

200.82

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

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮和光照。

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

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.63 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (4.63 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (4.63 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。