ETB (endothelin receptor type B) (Ki = 16 pM)

在 ETA 浓度为 19 μM 和 ETB 浓度为 16 PM 时，Ki, IRL-1620(TFA) 是最强大和特异性的 ETB 配体 (KiETA/KiETB=120,000) [1]。 IRL-1620(TFA) ET-3 的 KiETA/KiETB 比率为 1,900，对 ETB 受体的选择性比其高 60 倍 [1]。

在豚鼠中，IRL-1620 (TFA) (1-100 nM) 会导致气管收缩。大约 28 nM 是引起 60 mM KCI 诱导的 30% 收缩所需的 IRL-1620 有效浓度 [1]。 IRL-1620 (TFA) (1-100 nM) 可提高大鼠主动脉血管内皮 ([Ca]E) 中的胞质 Ca2+ 并放松去甲肾上腺素刺激的张力，同时对静息肌张力影响极小 [1]。 IRL-1620 (TFA) 增加血管生成和神经源性重塑，同时改善水迷宫任务的习得（学习）和记忆（记忆）。在接受 IRL-1620 治疗的大鼠中，Aβ 引起的认知障碍显着减轻。比较 IRL-1620 治疗与媒介物治疗，发现 VEGF 识别的血管数量有所增加 [2]。

合成了一系列内皮素(ET)-1 c端线性肽及其n α-琥珀酰（Suc）类似物，并检测了它们在猪肺膜上与内皮素受体（ETA）和内皮素受体（ETB）两种亚型的结合亲和力。通过对ETA （1.9 μM）和ETB （16 pM）受体的Ki值判断，合成的类似物中，IRL 1620的Suc-[Glu9， ala11,15]- et -1（8-21）是ETB受体最有效和特异性最强的配体。IRL 1620对ETB受体的选择性是ET-3的60倍。IRL 1620(10−9-10−7 M)诱导豚鼠气管收缩的效力与ET-1或ET-3相当，表明IRL 1620是一种有效的ETB受体激动剂。[１]

Western blot法检测外周血内皮素b、VEGF和NGF [2]
Western blotting检测脑内ETB、血管内皮和神经元生长因子水平。动物被斩首，大脑被快速冷冻并保存在- 80°C。组织在RIPA缓冲液中匀浆（20 mM Tris-HCl pH 7.5, 120 mM NaCl, 1.0% Triton X100, 0.1% SDS， 1%脱氧胆酸钠，10%甘油，1 mM EDTA和1倍蛋白酶抑制剂）。以溶解形式分离蛋白质，并使用Folin-Ciocalteu试剂测定浓度（Lowry et al., 1951）。蛋白质（60 μg）在Laemmli样品缓冲液中变性，10% SDS-PAGE溶解，转移到硝化纤维素膜上。然后在室温下，用5% BSA （w/v）在TBST （10 mM Tris, 150 mM NaCl, 0.1% Tween 20）中阻断膜30分钟。用兔多克隆抗乙脑(1:1000；Abcam, Cambridge， MA， USA)，抗vegf抗体（1:1000）和抗ngf抗体（1:50 00）在4°C下过夜，然后与山羊抗兔IgG，辣根过氧化物酶偶联（HRP）二抗（1:2000）在室温下孵育1.5 h。β-肌动蛋白（1:10 000）和β-微管蛋白（1:2000）作为加载对照。

Animals were randomly divided into five groups (six rats per group) (i) Sham, (ii) Aβ + Vehicle, (iii) Aβ + IRL 1620, [iv] Aβ + BQ788 (v) Aβ + BQ788 + IRL 1620. Aβ1–40 was administered intracerebroventricularly (i.c.v.) (20 μg in three equally divided doses i.e., 6.67 μg was injected three times for a total of 20-μg dose) on day 1, 7, and 14. We have used Aβ (1–40) because it is highly soluble compared to Aβ (1–42) and induces endothelial dysfunction of both cerebral and systemic blood vessels in addition to memory deficit (Weller et al., 1998, Niwa et al., 2000, Smith et al., 2004). Specific ETB receptor agonist, IRL 1620 (5 μg/kg) and specific ETB receptor antagonist, BQ788 (1 mg/kg) were administered intravenously (i.v.) on day 8. IRL 1620 was administered on day 8 three times at a dose of 5 μg/kg, i.v. at 2-h intervals between each injection. BQ788 was administered at a dose of 1-mg/kg, i.v., 15 min prior to administration of either vehicle or IRL 1620. The doses of IRL 1620 and BQ788 were based on preliminary studies and previous work conducted in our laboratory (Leonard et al., 2011, Leonard et al., 2012). [2]
IRL 1620[N-Succinyl-[Glu9, Ala11,15] endothelin 1] and BQ788 were dissolved in sterile saline and all the solutions were freshly prepared before the injections.

[1]. A potent and specific agonist, Suc-[Glu9,Ala11,15]-endothelin-1(8-21), IRL 1620, for the ETB receptor. Biochem Biophys Res Commun. 1992 Apr 30;184(2):953-9.

[2]. Stimulation of endothelin B receptors by IRL-1620 decreases the progression of Alzheimer's disease. Neuroscience. 2015 Aug 20;301:1-11.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by severe cognitive impairment that ultimately leads to death. Endothelin (ET) and its receptors have been considered as therapeutic targets for AD. Recent studies in our lab have shown that stimulation of ETB receptors provide significant neuroprotection following Aβ1-40 administration. It is possible that IRL-1620 may be neuroprotective due to angiogenesis. However, the effect of IRL-1620 on neurovascular remodeling following Aβ1-40 administration has not been established. The purpose of this study was to determine the effect of stimulation of ETB receptors by IRL-1620 on vascular and neuronal growth factors after Aβ1-40 administration. Rats were treated with Aβ1-40 (day 1, 7 and 14) in the lateral cerebral ventricles using stereotaxically implanted cannula and received three intravenous injections of IRL-1620 (an ETB agonist), and/or BQ788 (an ETB antagonist) at 2-h interval on day 8; experiments were performed on day 15. Rats were sacrificed for estimation of brain ETB receptors, vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) expression using immunofluorescence and Western blot. In the Morris swim task, amyloid-β (Aβ)-treated rats showed a significant (p<0.0001) impairment in spatial memory. Rats treated with IRL-1620 significantly (p<0.001) reduced the cognitive impairment induced by Aβ. BQ788 treatment completely blocked IRL-1620-induced improvement in cognitive impairment. IRL-1620 treatment enhanced the number of blood vessels labeled with VEGF compared to vehicle treatment. Additionally, cells showed increased (p<0.001) positive staining for NGF in IRL-1620-treated animals. ETB, VEGF and NGF protein expression significantly (p<0.001) increased in the brain of IRL-1620-treated rats as compared to vehicle. Pretreatment with BQ788 blocked the effects of IRL-1620, thus confirming the role of ETB receptors in the neurovascular remodeling actions of IRL-1620. Results of the present study demonstrate that IRL-1620 improves both acquisition (learning) and retention (memory) on the water maze task and enhances angiogenic and neurogenic remodeling. These findings indicate that the ETB receptor may be a novel therapeutic target for AD and other neurovascular degenerative disorders.[2]

C88H118F3N17O29

1934.97

142569-99-1 (IRL-1620)

{Suc}-Asp-Glu-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp-Ile-Ile-Trp

{Suc}-DEEAVYFAHLDIIW

White to off-white solid powder

(2S,5S,8S,11S,14S,17S,20S,23S,26S,29S,32S,35S,38S,41S)-17-((1H-imidazol-5-yl)methyl)-2-((1H-indol-3-yl)methyl)-23-benzyl-5,8-di((S)-sec-butyl)-35,38-bis(2-carboxyethyl)-11,41-bis(carboxymethyl)-26-(4-hydroxybenzyl)-14-isobutyl-29-isopropyl-20,32-dimethyl-4,7,10,13,16,19,22,25,28,31,34,37,40,43-tetradecaoxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42-tetradecaazahexatetracontanedioic acid TFA salt

IRL-1620 TFA; sovateltide TFA; sovateltidum; Endothelin-1 (8-21), succinyl-(glu(9),ala(11,15))-; Succinyl-(glu(9),ala(11,15))-endothelin-1 (8-21); Succinyl-(glutamyl(9)-alanyl(11,15))-endothelin-1 (8-21); IRL1620 TFA.

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)

DMSO :~50 mg/mL (~25.84 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；
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;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。