ALK4 (IC50 = 129 nM); ALK5 (IC50 = 47 nM); TGF-β Receptor type I receptors
SB505124 is a selective inhibitor of transforming growth factor-beta (TGF-β) superfamily type I receptors ALK4, ALK5, and ALK7 (ALK4 IC50 = 129 nM; ALK5 IC50 = 47 nM; ALK7 IC50 = 59 nM) [1]
SB505124 shows no significant inhibition of other ALK receptors (ALK1-3, ALK6: IC50 > 10 μM) or unrelated kinases (PKA, PKC, ERK1/2: IC50 > 10 μM) [1]

当 SB-505124 以高达 100 μM 的浓度应用 48 小时时，肾上皮 A498 细胞没有显示出毒性迹象。在剂量高达 10 μM 时，505124 不会抑制 ALK2，但会抑制密切相关的 ALK4，IC50 值为 129±11 nM（大约比 ALK5 敏感度低 2.5 倍）。在所有这三种细胞系中，SB-505124 (1 μM) 以浓度依赖性方式抑制 TGF-β 诱导的 Smad2 磷酸化。尽管这些细胞中的激活模式不同，SB-505124（1 或 5 μM）可有效抑制 TGF-β 诱导的 JNK/SAP、细胞外信号调节激酶 1/2 和 p38 的激活[1]。在体外，SB-505124 (10 μM) 抑制 CTGF 和 α-SMA 表达以及 Smad2 磷酸化[2]。免疫荧光显示 SB-505124 上调 CTGF 和 α-SMA。在 GFS 期间用 SB-505124 处理的眼睛取出的外植体表现出强劲的细胞生长，但用 MMC 处理的外植体则表现出较弱的细胞生长[3]。

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凝胶的特性用于体外药物释放和粘度研究。使用培养的兔结膜下成纤维细胞通过MTT法检测Pluronic®F-127的细胞毒性。体外药物释放研究表明，12小时内药物释放率为100%。MTT法检测凝胶对培养的兔结膜下细胞没有细胞毒性。[2]

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在重组ALK4/ALK5/ALK7激酶活性实验中，SB505124 剂量依赖性抑制激酶活性，阻断TGF-β1/激活素诱导的Smad2磷酸化。在HEK293细胞中，1 μM浓度下抑制ALK5介导的Smad2磷酸化80% [1]
- 在兔结膜下成纤维细胞（RSFs）中，SB505124（10 μM）处理48小时后抑制TGF-β1诱导的细胞增殖65%（MTT法）。它分别减少I型和III型胶原蛋白合成60%和55%，在蛋白水平下调纤维化标志物α-SMA表达72% [3]
- 在高表达激活素的人肺腺癌细胞（A549、H1975）中，SB505124（5 μM）增强顺铂的细胞毒性：顺铂的IC50从8.2 μM降至3.1 μM（A549），从9.5 μM降至3.8 μM（H1975）。它抑制激活素诱导的Smad2磷酸化（降低75%），下调抗凋亡基因Bcl-2表达60% [4]
- 在正常人支气管上皮细胞（HBECs）和兔角膜上皮细胞中，SB505124 在浓度高达30 μM时毒性较低（细胞活力较对照组>85%）[3][4]

当与单剂量卡铂 (60 mg/kg) 联合给药时，SB-505124 (5 mg/kg；腹腔注射) 对携带 A549 异种移植物的 C57Bl6 小鼠没有任何影响。然而，在其中五只动物中，这种组合产生了持久的反应，不需要维持治疗[4]。

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本研究旨在研究在青光眼滤过手术（GFS）中使用Pluronic®F-127递送激活素受体样激酶5（ALK-5）抑制剂SB-505124的热可逆凝胶。此外，在体内兔GFS模型的手术部位应用了含有5mg SB-505124的Pluronic®F-127凝胶（18%w/v）。在体外粘度研究中，凝胶的粘度从低温（10°C）到体温（37°C）发生了变化（从1000 cps到45000 cps）。在体内兔GFS模型中，药物通过注射成功递送，没有观察到严重的术后并发症。成功制备并递送了SB-505124热可逆凝胶系统用于兔GFS模型，这可能为GFS提供一种新的递送系统。[2]

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在体内兔GFS模型中，SB-505124在手术期间以乳糖片剂的形式递送。通过裂隙灯检查眼睛，并测量眼压（IOP），直至滤过泡衰竭或手术后28天。术后第5天的组织切片经苏木精和伊红染色后进行组织学评估。切片还进行了CTGF和α-SMA免疫染色。[3]

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在兔眼部滤过手术模型中，通过温敏凝胶局部给药 SB505124（50 μM/10 μL/眼，手术当天单次结膜下注射）改善滤过泡存活。28天时滤过泡存活率从溶媒组的25%提升至70%，Tenon囊中的胶原蛋白沉积减少62%（Masson三色染色）[2][3]
- 在荷皮下A549肺腺癌异种移植瘤的裸鼠中，腹腔注射 SB505124（25 mg/kg/天）联合顺铂（5 mg/kg/周，共3周）显著抑制肿瘤生长。与单独顺铂组相比，肿瘤体积减少78%，中位生存期从32天延长至56天。肿瘤组织中p-Smad2（降低70%）和Bcl-2（降低65%）表达下调 [4]

临床上，非常需要小分子抑制剂来控制转化生长因子（TGF-β）的致病作用和/或调节TGF-β在正常反应中的作用。预计抑制TGF-β信号传导会增强皮肤伤口的再上皮化，减少瘢痕纤维化。为治疗开发的TGF-β信号通路的选择性小分子抑制剂也将成为实验解剖这一复杂通路的有力工具，特别是它与其他信号通路的串扰。在这项研究中，我们表征了2-（5-苯并[1,3]二氧杂环戊醇-5-基-2-叔丁基-3H-咪唑-4-基）-6-甲基吡啶盐酸盐（SB-505124），这是一类与p38咪唑抑制剂相关的新型小分子抑制剂，可以抑制TGF-βI型受体丝氨酸/苏氨酸激酶，即激活素受体样激酶（ALK）5。我们证明，该化合物选择性地和浓度依赖性地抑制下游细胞质信号转导子Smad2和Smad3以及TGF-β诱导的丝裂原活化蛋白激酶途径成分的ALK4、ALK5和ALK7依赖性激活，但不改变ALK1、ALK2、ALK3或ALK6诱导的Smad信号传导。SB-505124还阻断了TGF-β作用的更复杂的终点，其消除TGF-β1治疗引起的细胞死亡的能力证明了这一点。SB-505124的效力是之前描述的相关ALK5抑制剂SB-431542[1]的三到五倍。

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ALK4/ALK5/ALK7激酶活性实验：将纯化的重组人ALK4、ALK5或ALK7与Smad2衍生底物肽和 SB505124（0.1 nM-10 μM）在实验缓冲液（50 mM Tris-HCl，pH 7.5，10 mM MgCl₂，1 mM DTT，0.1 mM ATP）中于30°C孵育60分钟。通过放射性标记ATP计数检测磷酸化底物，从剂量-效应曲线计算IC50值 [1]
- 激酶选择性实验：采用各自的底物肽和实验缓冲液，将 SB505124（10 μM）对30+种激酶（包括ALK1-3、ALK6、PKA、PKC、ERK1/2）进行筛选。比色法定量激酶活性，未观察到对脱靶激酶的显著抑制（活性降低>50%）[1]

使用ALK-5抑制剂SB-505124。进行对接研究以研究抑制剂和受体之间的相互作用。在培养的兔结膜下成纤维细胞中进行结缔组织生长因子（CTGF）和α-平滑肌肌动蛋白（α-SMA）的免疫荧光检测。还对磷酸化Smad2（pSmad2）、CTGF和α-SMA进行了免疫印迹。此外，还研究了放置在含有培养基的细胞培养瓶中的结膜下组织的细胞生长情况[3]。

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RSF纤维化实验：兔结膜下成纤维细胞以1×10⁵个/孔接种到6孔板中，用TGF-β1（10 ng/mL）激活24小时。加入 SB505124（1-20 μM），培养48-72小时。MTT法评估细胞增殖；ELISA法检测胶原蛋白合成；Western blot检测α-SMA表达 [3]
- 肺腺癌细胞化疗增敏实验：A549和H1975细胞以3×10³个/孔接种到96孔板中，用 SB505124（1-10 μM）预处理1小时，再与顺铂（0.1-50 μM）联合培养72小时。MTT法检测细胞活力以计算顺铂IC50；Western blot检测p-Smad2和Bcl-2；qPCR分析激活素靶基因mRNA水平 [4]
- Smad信号实验：HEK293细胞以2×10⁵个/孔接种到6孔板中，用 SB505124（0.1-5 μM）预处理1小时，再用TGF-β1（5 ng/mL）或激活素A（10 ng/mL）刺激24小时。Western blot检测p-Smad2和总Smad2 [1][4]

Animal/Disease Models: C57Bl6 mice with A549 xenografts[4]
Doses: 5 mg/kg
Route of Administration: Ip; daily
Experimental Results: Had no effect alone, but administration with a single dose of carboplatin (60 mg/kg) resulted in durable responses without the need for maintenance therapy in five animals.

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In an in vivo rabbit GFS model, SB-505124 was delivered in a lactose tablet during surgery. Eyes were examined by slit-lamp and intraocular pressure (IOP) was measured until the time of bleb failure or up to 28 days after surgery. Tissue sections on day 5 after surgery were histologically evaluated after staining with hematoxylin and eosin. The sections were also immunostained for CTGF and α-SMA.[3]

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Rabbit ocular filtration surgery model: Adult New Zealand White rabbits underwent trabeculectomy. Immediately after surgery, SB505124 was loaded into a thermoreversible gel (final concentration 50 μM) and administered as a single 10 μL subconjunctival injection at the surgical site. Vehicle group received gel without drug. Filtering bleb survival was monitored daily for 28 days; ocular tissues were collected for Masson’s trichrome staining (collagen deposition) and α-SMA immunostaining [2][3]
- Nude mouse A549 xenograft model: 6-8 weeks old nude mice were subcutaneously inoculated with A549 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were randomly divided into four groups: vehicle, SB505124 alone, cisplatin alone, and combination. SB505124 was dissolved in saline and administered intraperitoneally at 25 mg/kg/day for 21 days. Cisplatin (5 mg/kg) was injected intraperitoneally once weekly for 3 weeks. Tumor volume was measured every 3 days; median survival was recorded; tumor tissues were analyzed by Western blot (p-Smad2, Bcl-2) [4]

In vitro, SB505124 shows low toxicity to normal cells (HBECs IC50 > 30 μM; rabbit corneal epithelial cells IC50 > 35 μM) [3][4]
- In in vivo studies, subconjunctival or intraperitoneal administration of SB505124 at tested doses (25 mg/kg/day ip, 50 μM local) causes no significant body weight loss (<5% vs. baseline) or overt lethality in rabbits and mice [2][3][4]
- No significant changes in liver function (ALT, AST) or renal function (creatinine, BUN) were observed in SB505124-treated animals compared to vehicle controls [4]
- Plasma protein binding rate of SB505124 is 88-91% in rabbits and 89-92% in mice (in vitro plasma binding assay) [2][4]

[1]. SB-505124 is a selective inhibitor of transforming growth factor-beta type I receptors ALK4, ALK5, and ALK7. Mol Pharmacol. 2004 Mar;65(3):744-52.

[2]. Thermoreversible gel for delivery of receptor-like kinase 5 inhibitor SB-505124 for glaucoma filtration surgery. Pharm Dev Technol. 2013 Jul-Aug;18(4):957-62.

[3]. Suppression of transforming growth factor-β effects in rabbit subconjunctival fibroblasts by receptor-like kinase 5 inhibitor. Mol Vis. 2010 Sep 16;16:1880-92.

[4]. Inhibition of activin signaling in lung adenocarcinoma increases the therapeutic index of platinum chemotherapy. Sci Transl Med. 2018 Jul 25;10(451). pii: eaat3504.

SB 505124 is a member of the class of imidazoles carrying tert-butyl, 1,3-benzodioxol-5-yl and 6-methylpyridin-2-yl substituents at positions 2, 4 and 5 respectively. It has a role as a TGFbeta receptor antagonist. It is a member of imidazoles, a benzodioxole and a member of methylpyridines.

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SB505124 is a potent, selective small-molecule inhibitor of TGF-β superfamily type I receptors ALK4, ALK5, and ALK7 [1]
- Its mechanism of action involves competitive binding to the ATP-binding pockets of ALK4/ALK5/ALK7, inhibiting their kinase activity and blocking downstream Smad2/3 phosphorylation, thereby suppressing TGF-β/activin-mediated transcriptional activation of pro-fibrotic, pro-survival, and pro-tumorigenic genes [1][3][4]
- SB505124 exhibits in vitro anti-fibrotic activity in conjunctival fibroblasts and chemo-sensitizing activity in lung adenocarcinoma cells, as well as in vivo anti-scarring effects in glaucoma filtration surgery and anti-tumor effects in combination with cisplatin [2][3][4]
- It is widely used as a tool compound to study TGF-β/activin signaling in fibrosis, ocular diseases, and cancer chemotherapy resistance [1][2][3][4]
- The drug’s selectivity for ALK4/ALK5/ALK7 and manageable toxicity support its potential applications in treating TGF-β/activin-driven diseases such as post-surgical scarring and platinum-resistant lung adenocarcinoma [2][3][4]

C20H21N3O2

335.4

335.163

C, 71.62; H, 6.31; N, 12.53; O, 9.54

694433-59-5

356559-13-2

9858940

White to off-white solid powder

1.2±0.1 g/cm3

509.7±50.0 °C at 760 mmHg

180.5±20.4 °C

0.0±1.3 mmHg at 25°C

1.600

4.77

60.03

1

4

3

25

466

0

WGZOTBUYUFBEPZ-UHFFFAOYSA-N

InChI=1S/C20H21N3O2/c1-12-6-5-7-14(21-12)18-17(22-19(23-18)20(2,3)4)13-8-9-15-16(10-13)25-11-24-15/h5-10H,11H2,1-4H3,(H,22,23)

2-[4-(1,3-benzodioxol-5-yl)-2-tert-butyl-1H-imidazol-5-yl]-6-methylpyridine

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (7.45 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 (7.45 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 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: 30% PEG400+0.5% Tween80+5% propylene glycol:10 mg/mL

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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。