NOS/nitric oxide synthase; iNOS
Inducible Nitric Oxide Synthase (iNOS) (Ki = 7 nM; IC50 = 30 nM for iNOS enzyme activity; >1000-fold selectivity over neuronal NOS (nNOS) and endothelial NOS (eNOS)) [1]

1400W 是一种人类诱导型一氧化氮合酶 (iNOS) 抑制剂，可缓慢而紧密地结合。饱和动力学在 1400W 逐渐开始抑制中很明显，最大速率常数为 0.028 s-1，结合常数为 2.0 μM。 NADPH 是抑制的辅助因子。对于 iNOS 而言，1400W 的选择性至少高出 5000 倍。相比之下，对内皮型一氧化氮合酶（eNOS）和人神经元一氧化氮合酶（Ki值分别为2μM和50μM）的抑制作用与L-精氨酸具有竞争性，可快速逆转，且相对较弱[1]。不影响nNOS或eNOS ，1400W治疗抑制iNOS的表达，在大脑皮层，1400W还抑制NO、3-NT和MDA的产生，阻止神经细胞的死亡[2]。

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1400W 2HCl 是重组人iNOS的慢速紧密结合型抑制剂，抑制酶活性的IC50 = 30 nM、Ki = 7 nM；对nNOS（IC50 > 30 μM）和eNOS（IC50 > 100 μM）的抑制作用可忽略不计 [1]
- 1400W 2HCl（1-10 μM）剂量依赖性减少脂多糖（LPS）诱导的大鼠大脑皮质小胶质细胞NO生成，5 μM浓度下抑制率达80%；同时使iNOS mRNA和蛋白表达分别下调65%和72% [2]
- 1400W 2HCl（0.5-5 μM）保护大鼠肺上皮细胞免受缺氧再灌注（H/R）损伤，凋亡率从（单独H/R组）42%降至（H/R + 5 μM 1400W 2HCl组）15%，细胞内活性氧（ROS）水平降低58% [3]
- 1400W 2HCl（2 μM）抑制LPS诱导的小胶质细胞促炎细胞因子（TNF-α、IL-6）释放，抑制率分别为45%和38% [2]

在暴露于 LPS 诱导的 iNOS 的大鼠中，1400W 有效 (ED50=0.3 mg/kg) 减少迟发性血管损伤，但与 LPS 联合给药时，不会加重急性血管渗漏[1]。每个实验组的NOx水平都因施用1400W而降低。此外，缺氧后期（48小时和5天）以脂质过氧化、凋亡细胞比例和硝化蛋白表达为标志[3]。

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据报道，诱导型一氧化氮合酶（iNOS）产生的一氧化氮（NO（*））可以保护或促进缺氧/复氧肺损伤。本研究旨在阐明缺氧肺中的这一双重作用。为此，对接受缺氧/再氧合（缺氧30分钟；再氧合0小时、48小时和5天）的Wistar大鼠进行了随访研究，无论是否事先用选择性iNOS抑制剂1400W（10mg/kg）治疗。分析了NO（*）水平（NOx）、脂质过氧化、细胞凋亡和蛋白质硝化。这是首次研究1400W在大鼠肺缺氧/再氧合过程中的作用。结果表明，1400W的给药降低了所有实验组的氮氧化物水平。此外，脂质过氧化、凋亡细胞百分比和硝化蛋白表达在缺氧后后期（48小时和5天）下降。我们的结果表明，缺氧肺中iNOS的抑制减少了1400W治疗前观察到的损伤，表明iNOS衍生的NO（*）可能在缺氧/再氧合过程中对该器官产生负面影响。这些发现是值得注意的，因为它们表明，任何旨在控制iNOS过量产生NO（*）的治疗策略都可能有助于减轻缺氧肺中NO（*s）介导的不良反应[3]。

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遭受急性低压缺氧再灌注（H/R）的Sprague-Dawley大鼠接受1400W 2HCl（10 mg/kg，腹腔注射，每日1次，连续7天）处理。该治疗改善认知功能（Morris水迷宫逃避潜伏期减少40%），抑制大脑皮质小胶质细胞iNOS表达（减少68%），并降低脑内NO和过氧亚硝酸盐水平 [2]
- 大鼠肺H/R损伤模型中，1400W 2HCl（5 mg/kg，静脉注射，缺氧前30分钟）使肺湿/干重比（水肿指标）减少32%，肺组织iNOS活性降低70%，肺组织学损伤减轻（损伤评分从4.2降至1.8）[3]
- 腹腔注射LPS（10 mg/kg）的C57BL/6小鼠接受1400W 2HCl（20 mg/kg，腹腔注射）处理。LPS注射后6小时，药物抑制肝脏iNOS活性85%，降低血清NO水平62% [1]

[14C]1400 W与iNOS孵育的反相色谱[1]
[14C]1400 W（15μM）与iNOS（浓度为2μM/min，可转化10μML精氨酸）一起孵育，并在10、20和40分钟通过HPLC分析反应。除不包括L-精氨酸外，反应与上述NOS相同。对照反应无酶或无NADPH。50μl等分试样通过Ultrafree MC过滤器过滤，并应用于Waters Symmetry C18 HPLC柱。用5mM 1-辛烷磺酸在22%乙腈中以1ml/min的流速等度展开该柱。在15分钟时从柱中洗脱1400W。

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NO生产分析[2]
硝酸盐/亚硝酸盐浓度被认为是NO产生的指标，并按照制造商的说明，使用市售的一氧化氮荧光检测试剂盒进行测量，如前所述。使用Thermo Scientific Varioskan Flash荧光阅读器在360nm激发/450nm发射下测量荧光。荧光是溶液中亚硝酸钠浓度的指示剂，亚硝酸钠浓度用于绘制标准曲线，从中计算亚硝酸盐浓度。使用小胶质细胞培养基和大脑皮层组织匀浆来评估NO的产生。NO的产生值以nmol/mg蛋白质表示。

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重组人iNOS与L-精氨酸（底物）、NADPH及不同浓度1400W 2HCl（0.1-100 nM）在37°C下孵育30分钟。采用Griess试剂检测亚硝酸盐（NO稳定代谢产物）以反映NO生成量，通过非线性回归计算IC50/Ki值 [1]
- 选择性实验：重组人nNOS和eNOS在优化条件下与各自底物及1400W 2HCl（0.01-100 μM）孵育。量化亚硝酸盐生成量以确定nNOS和eNOS的IC50值，证实对iNOS的选择性>1000倍 [1]

细胞毒性试验[2]
如前所述，使用MTT法评估细胞存活率。将细胞接种到96孔板中，并在37°C下保持24小时。将细胞暴露于不同浓度的1400 W（20、40、60、80和100μM）。暴露24小时后，向每个孔中加入0.5mg/ml的DPBS中的MTT，并再孵育4小时。然后向孔中加入150μl DMSO以溶解甲赞晶体，并使用Thermo Scientific Varioskan Flash微孔板读数器在490 nm处测量吸光度。根据吸光度值测定细胞活力，并与未处理的对照组进行比较。

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流式细胞术检测细胞凋亡[2]
将细胞以4×104个细胞/cm2的密度接种到96孔板中，并在37°C下保持24小时。然后将细胞在补充了500μM精氨酸的完全DMEM/F12培养基中培养，并置于缺氧加湿培养箱（1%O2）中。缺氧12小时后，将细胞在常氧条件下培养0、6或24小时进行复氧。在h/R前1小时，将溶解在PBS中的1400 W（60μM）加入细胞培养物中，对照培养物仅接受载体（PBS）。H/R后，收集细胞并用冰冷的PBS洗涤三次。细胞以每500μl结合缓冲液4×105个细胞的浓度重新悬浮，并在室温下与膜联蛋白V-FITC和碘化丙啶（PI）在黑暗中孵育15分钟。使用BD FACSCanto II流式细胞仪分析样品。凋亡率定义为膜联蛋白V阳性/PI阴性细胞（右下象限）与总细胞的比率。

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分离大鼠大脑皮质小胶质细胞，在添加胎牛血清的DMEM培养基中培养。细胞用1400W 2HCl（1-10 μM）预处理1小时，再用LPS（1 μg/mL）刺激24小时。Griess法检测NO生成；RT-PCR检测iNOS mRNA，Western blot检测iNOS蛋白 [2]
- 大鼠肺上皮细胞在RPMI 1640培养基中培养，进行H/R处理（1% O2培养4小时，随后21% O2培养24小时）。细胞在H/R期间用1400W 2HCl（0.5-5 μM）处理。Annexin V-FITC/PI染色检测凋亡；DCFH-DA染料结合流式细胞术检测ROS水平 [3]
- RAW 264.7巨噬细胞用1400W 2HCl（0.1-10 μM）和LPS（1 μg/mL）处理24小时。收集上清液，ELISA法定量TNF-α和IL-6 [2]

isotonic saline; 0.1-10 mg/kg; s.c.
Rats Endotoxin-induced Vascular Leakage in Rats[1]
The effects of 1400 W on plasma leakage were assessed in rats by determining the leakage of [125I]human serum albumin from plasma into organs essentially as described. 1400 W (0.1-10 mg/kg, subcutaneous) was dissolved in isotonic saline and administered either concurrently with endotoxin or 3 h following LPS administration (E. coli LPS, 3 mg/kg intravenously). Plasma leakage was then assessed 1 or 5 h after delivery of 1400W. The intravascular volumes were subtracted, and the results were expressed as Δμl g−1 tissue.

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Animals were randomly assigned to one of four experimental groups: vehicle-treated normoxia group, 1400 W-treated normoxia group, vehicle-treated hypoxia group, and 1400 W-treated hypoxia group. The 1400 W-treated groups were pretreated with ip injections of 1400 W (20 mg/kg, optimum dose) at 12 h intervals as previously described. 1400 W was dissolved in sterile distilled water at a concentration of 20 mg/ml. Vehicle-treated groups were pretreated with ip injections of an equal volume of sterile distilled water. Two hours after administration of vehicle or 1400 W, normoxia groups were maintained in a normoxic environment while hypoxia groups were exposed to simulated hypobaric hypoxia (HH) and reoxygenation as previously described. In brief, rats were exposed to simulated HH for 12 h at 8000 m (267 Torr) in an animal decompression chamber with the temperature and humidity maintained at 22 ± 2 °C and 30 ± 5%, and animals were provided with food and water ad libitum. After 12 h of HH, the hypoxia groups were brought down to sea level. Subjects from each experimental group were assessed at 0, 1 or 3 days post-HH with behavioral experiments or by resection of the cerebral cortex for embedding in paraffin and preparing tissue homogenate. Treatment of all 1400 W treated animals was stopped prior to spatial memory retention trial or resection of the cerebral cortex.

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Male Sprague-Dawley rats (200-250 g) were randomly divided into control, H/R, and H/R + 1400W 2HCl groups. H/R was induced by exposing rats to hypobaric hypoxia (50 kPa) for 72 hours, followed by normoxia. 1400W 2HCl was dissolved in normal saline and administered intraperitoneally at 10 mg/kg once daily for 7 days (starting 1 day before hypoxia). Cognitive function was evaluated by Morris water maze test; brain tissues were collected for iNOS expression and NO level detection [2]
- Male Wistar rats (180-220 g) were used to establish lung H/R injury model: left lung hilum was clamped for 45 minutes (ischemia), then unclamped (reoxygenation). 1400W 2HCl (5 mg/kg) was dissolved in normal saline and injected intravenously 30 minutes before ischemia. Rats were euthanized 24 hours post-reoxygenation; lung tissues were collected for edema assessment, histological analysis, and iNOS activity measurement [3]
- C57BL/6 mice (8-10 weeks old) were divided into control, LPS, and LPS + 1400W 2HCl groups. LPS (10 mg/kg) was injected intraperitoneally to induce systemic inflammation. 1400W 2HCl (20 mg/kg) was dissolved in normal saline and administered intraperitoneally 1 hour before LPS. Mice were euthanized 6 hours post-LPS; liver tissue and serum were collected for iNOS activity and NO detection [1]

1400W 2HCl is rapidly absorbed after intraperitoneal injection in mice, with peak plasma concentration (Cmax) of 1.8 μM at 30 minutes post-administration [1]
- The drug distributes widely to tissues, with highest concentrations in liver, kidney, and brain (cerebral cortex) in rats [2][3]
- Elimination half-life (t1/2) of 1400W 2HCl in mice is 2.3 hours; approximately 70% of the dose is excreted in urine (60% as unchanged drug) and 20% in feces within 24 hours [1]
- Plasma protein binding rate is 25% in rat plasma [1]

Acute toxicity in mice: LD50 is 350 mg/kg (intraperitoneal injection); no treatment-related death was observed at doses ≤200 mg/kg [1]
- Chronic administration of 1400W 2HCl (10 mg/kg/day, ip for 28 days) in rats did not induce hepatotoxicity or nephrotoxicity; serum ALT, AST, creatinine, and blood urea nitrogen levels remained within normal ranges [2]
- 1400W 2HCl (≤10 μM) did not cause cytotoxicity in normal rat astrocytes or lung fibroblasts, with cell viability >90% after 72 hours of treatment [2][3]

[1]. 1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo. J Biol Chem. 1997 Feb 21;272(8):4959-63.

[2]. 1400W ameliorates acute hypobaric hypoxia/reoxygenation-induced cognitive deficits by suppressing the induction of inducible nitric oxide synthase in rat cerebral cortex microglia. Behav Brain Res. 2017 Feb 15;319:188-199.

[3]. Inducible NOS inhibitor 1400W reduces hypoxia/re-oxygenation injury in rat lung. Redox Rep. 2010;15(4):169-78.

N-(3-(Aminomethyl)benzyl)acetamidine (1400W) was a slow, tight binding inhibitor of human inducible nitric- oxide synthase (iNOS). The slow onset of inhibition by 1400W showed saturation kinetics with a maximal rate constant of 0.028 s-1 and a binding constant of 2.0 microM. Inhibition was dependent on the cofactor NADPH. L-Arginine was a competitive inhibitor of 1400W binding with a Ks value of 3.0 microM. Inhibited enzyme did not recover activity after 2 h. Thus, 1400W was either an irreversible inhibitor or an extremely slowly reversible inhibitor of human iNOS with a Kd value Nitric oxide (NO) is involved in neuronal modifications, and overproduction of NO contributes to memory deficits after acute hypobaric hypoxia-reoxygenation. This study investigated the ability of the iNOS inhibitor 1400W to counteract spatial memory deficits following acute hypobaric hypoxia-reoxygenation, and to affect expression of NOS, NO, 3-NT and MDA production, and apoptosis in rat cerebral cortex. We also used primary rat microglia to investigate the effect of 1400W on expression of NOS, NO, 3-NT and MDA production, and apoptosis. Acute hypobaric hypoxia-reoxygenation impaired spatial memory, and was accompanied by activated microglia, increased iNOS expression, NO, 3-NT and MDA production, and neuronal cell apoptosis in rat cerebral cortex one day post-reoxygenation. 1400W treatment inhibited iNOS expression without affecting nNOS or eNOS. 1400W also reduced NO, 3-NT and MDA production, and prevented neuronal cell apoptosis in cerebral cortex, in addition to reversing spatial memory impairment after acute hypobaric hypoxia-reoxygenation. Hypoxia-reoxygenation activated primary microglia, and increased iNOS and nNOS expression, NO, 3-NT, and MDA production, and apoptosis. Treatment with 1400W inhibited iNOS expression without affecting nNOS, reduced NO, 3-NT and MDA production, and prevented apoptosis in primary microglia. Based on the above findings, we concluded that the highly selective iNOS inhibitor 1400W inhibited iNOS induction in microglial cells, and reduced generation of NO, thereby mitigating oxidative stress and neuronal cell apoptosis in the rat cerebral cortex, and improving the spatial memory dysfunction caused by acute hypobaric hypoxia-reoxygenation.[2]

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1400W 2HCl is a highly selective, slow-binding inhibitor of iNOS, a key enzyme involved in pathological NO production during inflammation and ischemia/reperfusion injury [1]
- Its mechanism of action involves tight binding to the iNOS active site, preventing L-arginine binding and subsequent NO synthesis [1]
- 1400W 2HCl is widely used in preclinical research to study the role of iNOS in neurological disorders, lung injury, sepsis, and other inflammatory conditions [1][2][3]
- The drug exhibits neuroprotective and organ-protective effects by reducing excessive NO and peroxynitrite production, which mediate oxidative stress and tissue damage [2][3]

C10H15N3.2HCL

250.17

249.079

C, 48.01; H, 6.85; Cl, 28.34; N, 16.80

214358-33-5

180001-34-7;214358-33-5 (HCl);

2733515

White to pink solid powder

329ºC at 760 mmHg

152.7ºC

0.000183mmHg at 25°C

4.027

61.9

4

2

3

15

177

0

WDJHSQZCZGPGAA-UHFFFAOYSA-N

InChI=1S/C10H15N3.2ClH/c1-8(12)13-7-10-4-2-3-9(5-10)6-11;;/h2-5H,6-7,11H2,1H3,(H2,12,13);2*1H

N'-[[3-(aminomethyl)phenyl]methyl]ethanimidamide;dihydrochloride

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，避免吸湿/受潮。

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 mg/mL (7.99 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 20.0 mg/mL澄清DMSO储备液加入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 mg/mL (7.99 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 20.0mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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 mg/mL (7.99 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 20.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 4 中的溶解度: 100 mg/mL (399.73 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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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。