LDN-193189 4HCl

ACVR1 (IC50 = 5 nM); BMPR1A (IC50 = 30 nM); ALK2 (IC50 = 5 nM), ALK3 (IC50 = 30 nM)[1]

体外活性：LDN193189 有效抑制 BMP4 介导的 Smad1、Smad5 和 Smad8 激活，IC50 为 5 nM，并有效抑制 BMP I 型受体 ALK2 和 ALK3 的转录活性，IC50 分别为 5 nM 和 30 nM。此外，LDN193189 还显示出对由组成型活性 ALK2R206H 或 ALK2Q207D 突变蛋白诱导的转录活性的抑制作用。最近的一项研究表明，LDN-193189 可阻断人主动脉内皮细胞动脉粥样硬化过程中氧化 LDL 诱导的活性氧的产生。激酶检测：LDN193189是一种选择性BMP I型受体抑制剂，可有效抑制ALK2和ALK3（IC50分别=5 nM和30 nM），对ALK4、ALK5和ALK7作用较弱（IC50≥500 nM）。细胞测定：将生长的小鼠 PASMC 在六孔板中瞬时转染至 50% 汇合，使用 0.3 μg Id1 启动子荧光素酶报告基因构建体 (BRE-Luc) 结合 0.6 μg 表达组成型活性形式的 BMP I 型受体（caALK2、caALK3）的质粒或 caALK6)。对于两种报告质粒，均使用 0.2 μg pRL-TKRenilla 荧光素酶来控制转染效率。转染后 1 小时开始将 PASMC 与 LDN193189 (2 nM-32 μM) 或载体一起孵育。收获细胞提取物，并使用双荧光素酶测定试剂盒通过萤火虫与海肾荧光素酶活性的比率来量化相对启动子活性。

在出生后第 7 天 (P7) 开启 Ad.Cre 的条件 caALK2 转基因小鼠中，LDN-193189 (3 mg/kg ip) 导致左胫骨和腓骨周围轻度钙化，在 P13 时首次可见，并在 P15 时预防放射学损伤不会导致体重减轻或生长迟缓、自发性骨折、骨密度降低或行为异常。 LDN193189 通过抑制骨形态发生蛋白 (BMP)6 诱导的信号通路使斑马鱼胚胎背侧化，而不影响血管发育。在携带 PCa-118b 肿瘤的小鼠中，LDN-193189 治疗可减弱肿瘤生长并减少肿瘤中的骨形成。在 LDL 受体缺陷 (LDLR-/-) 小鼠中，LDN-193189 可有效抑制动脉粥样硬化的形成。此外，LDN-193189 还表现出对相关血管炎症、成骨活性和钙化的抑制作用。

碱性磷酸酶活性[1]
我们将C2C12细胞以每孔2000个细胞的速度接种到96孔板中，在补充有2%FBS的DMEM中。我们用BMP配体和LDN-193189或载体以四硅酸盐的形式处理孔。我们在50μl Tris缓冲盐水和1%Triton X-100中培养6天后收集细胞。我们将裂解物加入96孔板中的对硝基-苯基磷酸盐试剂中1小时，然后评估碱性磷酸酶活性（405nm处的吸光度）。我们使用与用于碱性磷酸酶测量的重复孔相同处理的重复孔，通过cell Titer Water One（在490nm处的吸光度）测量细胞活力和数量。

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Id1和纤溶酶原激活物抑制剂-1启动子萤光素酶报告基因测定[1]
我们使用Fugene6，用0.3μg Id1启动子荧光素酶报告子构建体（BRE-Luc30，由P.ten Dijke提供）和0.6μg表达组成型活性形式的BMP I型受体（caALK2、caALK3或caALK631，由K.Miyazono提供）的质粒在六孔板中瞬时转染生长至50%汇合的小鼠PASMC。为了评估激活素和TGF-βI型受体的功能，我们用0.3μg PAI1（纤溶酶原激活物抑制剂-1）启动子萤光素酶报告子构建体（CAGA-Luc32，由P.ten Dijke提供）与0.6μg表达I型受体组成型活性形式（caALK4、caALK5和caALK733）的质粒联合瞬时转染PASMC。对于两个报告质粒，我们使用0.2μg的pRL-TK雷尼拉萤光素酶来控制转染效率。我们在转染后1小时开始用LDN-193189（2 nM–32μM）或载体孵育PASMC。我们采集了细胞提取物，并用双荧光素酶测定试剂盒通过萤火虫与雷尼拉萤光素酶活性的比率来量化相对启动子活性。

细胞培养[1]
如前所述，我们从野生型和CAG-Z-EGFP-caALK2转基因小鼠中分离出PASMC，并将其在补充有10%FBS的RPMI培养基中培养。我们通过用Ad.Cre（50的多重感染）或Ad.GFP作为对照感染，然后培养3天并传代，在体外诱导表达条件caALK2的PASMC的重组。我们在补充谷氨酰胺和10%FBS的DMEM中培养C2C12肌成纤维细胞（美国典型培养物保藏中心）。我们用药物抑制剂预培养细胞10分钟，然后在37°C下将其暴露于BMP4、TGF-β或血小板衍生生长因子BB配体30分钟。[1]

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Smad1、Smad5和Smad8磷酸化的免疫印迹分析[1]
我们在SDS裂解缓冲液（62.5mM Tris-HCl（pH 6.8）、2%SDS、10%甘油、50mM二硫苏糖醇和0.01%溴酚蓝）中机械匀浆细胞提取物，通过SDS-PAGE分离蛋白质，用磷酸化Smad1、Smad5和Smad8特异性多克隆抗体、磷酸化Smad2或兔Smad1或Smad2特异性单克隆抗体免疫印迹，并用ECL-Plus观察免疫反应蛋白。

Conditionally-expressed, constitutively-active ALK2–transgenic mice[1]
The construction of mice expressing a single conditionally expressed allele of the gene encoding constitutively-active ALK2Q207D (CAG-Z-EGFP-caALK2) on a C57BL/6 background was previously described14. We obtained CAGGS-CreER mice, which express a tamoxifen-inducible Cre recombinase ubiquitously under the control of the cytomegalovirus immediate-early enhancer and the chicken β-actin promoter/enhancer20, from the Jackson Laboratory.

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Mice[3] In the first experiment, SCID mice are implanted with MDA-PCa-118b tumors. After 7 days when tumors reached measurable sizes, mice are injected with LDN-193189 (3 mg/kg) or with vehicle intraperitoneally twice a day. Tumor sizes and body weights are measured weekly. Mice are injected with calcein at three days and one day prior to sacrifice. Blood is collected and tumors are weighed. A portion of the tumors are fixed in formaldehyde for micro-computed tomography, using EVS CT, or further decalcified for bone histomorphometric analysis, using OsteoMeasure Analysis System, or flash frozen for RNA preparation. Osteocalcin in the mouse serum is determined by ELISA. In the second experiment, PCa-118b tumors are first digested with Accumax, and the isolated cells are plated overnight, resuspended in Matrigel in 1:1 ratio, and injected into SCID mice (1×106 cells/mouse) subcutaneously. Mice are treated with LDN-193189 five days post-injection.

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Rats[4] Male Sprague-Dawley (SD) rats, 8 weeks of age, weighing 200-220 g, are randomly assigned to one of seven experiment groups (n=6 per group). Rats are housed with free access to food and water under a natural 12/12 h day/night cycle. The Monocrotaline is administered (60 mg/kg) to rats by subcutaneous injection into the back region. The animal’s lungs are harvested at 28th day of the study after hemodynamic assessment. The Sildenafil group received daily intragastric administration of Sildenafil after the administration of MCT (60 mg/kg). The LDN-193189 group received daily intragastric administration of Sildenafil (50 mg/kg) and intraperitoneal injection of LDN-193189 (10 mg/kg). In other groups, the same volume saline is given.

Dissolved in DMSO and then diluted in water; 3 mg/kg; i.p. injection

Ad.Cre on P7 is injected into conditional caALK2–transgenic and wild-type mice

[1]. Nat Med.2008 Dec;14(12):1363-9.
[2]. Br J Pharmacol.2010 Sep;161(1):140-9.
[3]. Cancer Res, 2011, 71(15), 5194-5203.
[4]. Int J Clin Exp Pathol. 2014 Jul 15;7(8):4674-84.

Fibrodysplasia ossificans progressiva (FOP) is a congenital disorder of progressive and widespread postnatal ossification of soft tissues and is without known effective treatments. Affected individuals harbor conserved mutations in the ACVR1 gene that are thought to cause constitutive activation of the bone morphogenetic protein (BMP) type I receptor, activin receptor-like kinase-2 (ALK2). Here we show that intramuscular expression in the mouse of an inducible transgene encoding constitutively active ALK2 (caALK2), resulting from a glutamine to aspartic acid change at amino acid position 207, leads to ectopic endochondral bone formation, joint fusion and functional impairment, thus phenocopying key aspects of human FOP. A selective inhibitor of BMP type I receptor kinases, LDN-193189 (ref. 6), inhibits activation of the BMP signaling effectors SMAD1, SMAD5 and SMAD8 in tissues expressing caALK2 induced by adenovirus specifying Cre (Ad.Cre). This treatment resulted in a reduction in ectopic ossification and functional impairment. In contrast to localized induction of caALK2 by Ad.Cre (which entails inflammation), global postnatal expression of caALK2 (induced without the use of Ad.Cre and thus without inflammation) does not lead to ectopic ossification. However, if in this context an inflammatory stimulus was provided with a control adenovirus, ectopic bone formation was induced. Like LDN-193189, corticosteroid inhibits ossification in Ad.Cre-injected mutant mice, suggesting caALK2 expression and an inflammatory milieu are both required for the development of ectopic ossification in this model. These results support the role of dysregulated ALK2 kinase activity in the pathogenesis of FOP and suggest that small molecule inhibition of BMP type I receptor activity may be useful in treating FOP and heterotopic ossification syndromes associated with excessive BMP signaling.[4]

C₂₅H₂₆CL₄N₆

552.33

442.167

1062368-62-0

54613581

Typically exists as light yellow to yellow solids at room temperature

5.216

58.35

2

5

3

32

587

0

Cl[H].N1(C2C([H])=C([H])C(C3C([H])=NC4=C(C([H])=NN4C=3[H])C3=C([H])C([H])=NC4=C([H])C([H])=C([H])C([H])=C34)=C([H])C=2[H])C([H])([H])C([H])([H])N([H])C([H])([H])C1([H])[H]

PCCDKTWDGDFRME-UHFFFAOYSA-N

InChI=1S/C25H22N6.ClH/c1-2-4-24-22(3-1)21(9-10-27-24)23-16-29-31-17-19(15-28-25(23)31)18-5-7-20(8-6-18)30-13-11-26-12-14-30;/h1-10,15-17,26H,11-14H2;1H

4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline;hydrochloride

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)

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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