SD-208

TGF-βRI (ALK5) (IC50 = 48 nM)

在人 LN-308 和鼠 SMA-560 神经胶质瘤细胞中，SD-208 增加免疫原性，同时阻断组成型、TGF-β 诱发和细胞生长 [1]。在体外，SD-208 通过阻断 TGF-β 诱导的 Smad2 和 Smad3 磷酸化来促进迁移、侵袭和上皮间质转分化 [2]。此外，SD-208 消除了 TGF-β 对体外新生内膜平滑肌样细胞 (SMLC) 迁移和增殖的刺激影响[3]。

使用 SD-208（1 mg/mL，口服）治疗时，携带 SMA-560 神经胶质瘤的小鼠的中位生存期显着延长[1]。 SD-208（60 mg/kg/d，口服）可防止同基因 129S1 小鼠中原发性 R3T 肿瘤的形成，并减少肺转移的数量和大小[2]。 SD-208在小鼠同种异体主动脉移植模型中成功抑制移植动脉硬化（TA）内膜增生的发展[3]。

---

口服SD-208治疗同基因R3T或4T1荷瘤小鼠抑制了原发性肿瘤的生长以及转移的数量和大小。相比之下，SD-208未能抑制无胸腺裸鼠中R3T肿瘤的生长或转移。[2]

---

与赋形剂治疗的对照组相比，SD-208治疗组的内膜增生显著减少（与对照组相比40 mg/kg和60 mg/kg SD-208分别减少32%和48%[n=5]，p<0.05）。SD-208在移植物中分别将SMLC增殖和内膜胶原的产生减少了21%和75%。SD-208也消除了TGF-β对SMLC增殖和迁移的促进作用，但不影响TGF-β对体外VSMCs的抑制作用。CTGF是TGF-β下游的一种蛋白质，在体外和体内，SD-208抑制Smad3磷酸化后，CTGF被下调。此外，我们发现，移植后2周，SMLCs中的内源性Smad3上调，8周时比VSMCs高64%。 结论：这些结果表明，SD-208可以有效减少小鼠主动脉移植模型中TA内膜增生的形成。[3]

转化生长因子β报告物测定。[1]
使用pGL2 3TP-Luc或pGL3 SBE-2-Luc（17）报告基因质粒通过报告分析评估细胞内TGF-β信号传导。pGL2 3TP-Luc构建体包含一个合成启动子，该启动子由插入三个佛波酯反应元件下游的TGF-β反应性纤溶酶原激活物抑制剂1启动子片段组成。pGL3 SBE-2-Luc报告基因包含Smad结合元件GTCTAGAC的两个拷贝。使用FuGene转染LN-308和SMA-560细胞。转染后24小时，细胞在含血清的SD-208培养基中预处理12小时（1μmol/L）。然后再加入TGF-β1（5 ng/mL）16小时。将细胞裂解并转移到LumiNunc板上，使用萤光素酶测定底物在LumimatPlus中测量发光。对于T细胞检测，使用Nucleofector装置和细胞类型特异性人T细胞Nucleofeptor试剂盒，将5×106个新鲜分离的PBL与4.5μg pGL2-3TP-Luc或pGL3-SBE-2-Luc报告基因质粒和0.5μg pRL-CMV共转染。在核转染后4小时加入IL-2（50单位/mL），用SD-208预处理细胞1小时，然后再加入TGF-β1（5 ng/mL）16小时。使用Firelite双发光报告基因测定法依次测定萤火虫和肾形莲子荧光素酶的各自活性。从萤火虫荧光素酶的测量中获得的计数相对于pRL-CMV进行了归一化。

增殖。[1]
胶质瘤细胞在SD-208（1μmol/L）存在或不存在的情况下培养48小时。用[甲基-3H]胸苷（0.5μCi）对细胞进行最后24小时的脉冲处理并收获，在液体闪烁计数器中测定掺入的放射性

---

上皮间质转分化。[2]
为了评估亚细胞F-actin纤维的分布，用PBS洗涤细胞，并用缓冲福尔马林固定10分钟。用PBS洗涤后，用0.1%（v/v）的Triton X-100在PBS中透化细胞5分钟，然后在20°C的黑暗中与0.165μmol/L的Alexa Fluor 488偶联的鬼笔环肽和1%（w/v）的牛血清白蛋白在PBS中孵育20分钟。对于E-钙粘蛋白免疫染色，用PBS洗涤细胞，用预冷至-20°C的甲醇固定5分钟。然后将风干的载玻片与5%（v/v）山羊血清在室温下孵育20分钟，然后与2μg/mL小鼠单克隆抗E-cadherin抗体在2.5%（v/v”）山羊血清中在室温下温育1小时。然后用PBS洗涤细胞3×5分钟，然后在黑暗中用2μg/mL罗丹明偶联的山羊抗小鼠IgG孵育45分钟。在这两种情况下，用PBS洗涤染色皿3×5 min，使用Vectashield安装介质安装，并使用配备MTI电荷耦合器件相机的蔡司落射荧光显微镜（型号090477）进行观察。[2]

---

蛋白质印迹分析。[2]
为了检测Smad蛋白，在蛋白酶抑制剂（完整的迷你蛋白酶抑制剂鸡尾酒片）存在下，使用由150 mmol/L NaCl、10 mmol/L Tris-HCl（pH 8.0）、1 mmol/L EDTA、1 mmol/L EGTA和1%（v/v）Triton X-100组成的缓冲液在4°C下原位裂解半流细胞培养物30分钟。如前所述，对细胞裂解物进行蛋白质印迹分析（42）。使用我们自己的兔抗pSmad2和抗pSmad3抗体以1:1000的稀释度检测活化的Smad2（pSmad2）和活化的Smad3（pSmad3）。分别使用1:500稀释的兔抗Smad2、兔抗Smad3和小鼠抗Smad4抗体检测总Smad2、Smad3和Smad4。[2]

---

体外细胞迁移和侵袭试验。[2]
对于迁移分析，通过向上室和下腔室中加入0.5 mL细胞培养基，然后在37°C下孵育2小时，平衡未涂覆的聚对苯二甲酸乙二醇酯轨道蚀刻膜（24孔插入；孔径，8μm）插入物。对于侵袭试验，通过向上部腔室中加入0.5 mL温（37°C）培养基，然后在37°C下孵育2小时，对BD生物涂层生长因子降低基质凝胶侵袭腔室（24孔插入；孔径，8μm）进行再水化。对于这两种测定，去除了用于平衡的培养基，并在上室中放置了105个细胞。将TGF-β1（100 pmol/L，2.5 ng/mL）、SD-093（1μmol/L）、两种药物或仅载体添加到上下腔室。在37°C下孵育24小时后，吸出悬浮细胞，用PBS洗涤插入物两次，用棉签刮除膜的上表面，去除附着在插入物顶部的细胞。迁移到插入物下侧的细胞被固定并使用DiffQuick染色试剂盒染色。每个孔中10个100×100μm的随机正方形中的细胞在×200放大倍数下使用每个测定条件的四个孔进行计数，结果表示为细胞数/mm2。

Survival Studies In vivo.[1]
VM/Dk cice of 6 to 12 weeks of age were used for the survival experiments. The experiments were performed according to the German animal protection law. Groups of eight mice were anesthesized before all intracranial procedures and placed in a stereotaxic fixation device. A burr hole was drilled in the skull 2 mm lateral to the bregma. The needle of a Hamilton syringe was introduced to a depth of 3 mm. SMA-560 cells [5 × 103 cells] resuspended in a volume of 2 μL of PBS were injected into the right striatum. Three days later, the mice were allowed to drink SD-208 at 1 mg/mL in deionized water. The mice were observed daily and, in the survival experiments, sacrificed on development of neurologic symptoms.[1]

---

Animal experiments. [2]
For in vivo studies, SD-208 was suspended in 1% (w/v) methylcellulose in water. Pharmacokinetic analysis. [2]
Descriptive pharmacokinetic variables were determined by standard model-independent methods. Noncompartmental analysis was done using WinNonlin version 4.0.1. Because individual mice were not used to describe a full profile, variables were calculated using mean data. Samples with SD-208 concentration below quantifiable limits (10 ng/mL) were assigned the value of 0 for the analysis. Nominal time points were used for all calculations. AUC(0-8) is the area under the plasma concentration-time curve from time 0 to 8 hours for animals dosed with SD-208.

---

BALB/c (H-2(d)) donor aortas were transplanted into C57BL/6 (H-2(b)) recipients, and the mice then received different doses (40 or 60 mg/kg) of SD-208 or control vehicle by daily gavage for 8 weeks. The grafts were analyzed by histology and morphometry at 1, 2, 4, 6 and 8 weeks after transplantation. The effects of TGF-β and SD-208 on neointimal smooth muscle-like cell (SMLC) and vascular smooth muscle cell (VSMC) proliferation and migration were evaluated, and the expression levels of Smad3, P-Smad3, connective tissue growth factor (CTGF) and collagen I were determined by in vitro experiments.[3]

Dissolved in water; 1 mg/mL; p.o.

VM/Dk mice bearing SMA-560 tumors

[1]. SD-208, a novel transforming growth factor beta receptor I kinase inhibitor, inhibits growth and invasiveness and enhances immunogenicity of murine and human glioma cells in vitro and in vivo. Cancer Res. 2004 Nov 1;64(21):7954-61.

[2]. Inhibition of growth and metastasis of mouse mammary carcinoma by selective inhibitor of transforming growth factor-beta type I receptor kinase in vivo. Clin Cancer Res. 2006 Jul 15;12(14 Pt 1):4315-30.

[3]. Inhibition of intimal hyperplasia in murine aortic allografts by the oral administration of the transforming growth factor-beta receptor I kinase inhibitor SD-208. J Heart Lung Transplant. 2014 Jun;33(6):654-61.

The cytokine transforming growth factor (TGF)-beta, by virtue of its immunosuppressive and promigratory properties, has become a major target for the experimental treatment of human malignant gliomas. Here we characterize the effects of a novel TGF-beta receptor (TGF-betaR) I kinase inhibitor, SD-208, on the growth and immunogenicity of murine SMA-560 and human LN-308 glioma cells in vitro and the growth of and immune response to intracranial SMA-560 gliomas in syngeneic VM/Dk mice in vivo. SD-208 inhibits the growth inhibition of TGF-beta-sensitive CCL64 cells mediated by recombinant TGF-beta1 or TGF-beta2 or of TGF-beta-containing glioma cell supernatant at an EC(50) of 0.1 mumol/L. SD-208 blocks autocrine and paracrine TGF-beta signaling in glioma cells as detected by the phosphorylation of Smad2 or TGF-beta reporter assays and strongly inhibits constitutive and TGF-beta-evoked migration and invasion, but not viability or proliferation. Peripheral blood lymphocytes or purified T cells, cocultured with TGF-beta-releasing LN-308 glioma cells in the presence of SD-208, exhibit enhanced lytic activity against LN-308 targets. The release of interferon gamma and tumor necrosis factor alpha by these immune effector cells is enhanced by SD-208, whereas the release of interleukin 10 is reduced. SD-208 restores the lytic activity of polyclonal natural killer cells against glioma cells in the presence of recombinant TGF-beta or of TGF-beta-containing glioma cell supernatant. The oral bioavailability of SD-208 was verified by demonstrating the inhibition of TGF-beta-induced Smad phosphorylation in spleen and brain. Systemic SD-208 treatment initiated 3 days after the implantation of SMA-560 cells into the brains of syngeneic VM/Dk mice prolongs their median survival from 18.6 to 25.1 days. Histologic analysis revealed no difference in blood vessel formation, proliferation, or apoptosis. However, animals responding to SD-208 showed an increased tumor infiltration by natural killer cells, CD8 T cells, and macrophages. These data define TGF-beta receptor I kinase inhibitors such as SD-208 as promising novel agents for the treatment of human malignant glioma and other conditions associated with pathological TGF-beta activity.[1]

---

Purpose: Transforming growth factor-beta (TGF-beta) suppresses tumor development by inhibiting cellular proliferation, inducing differentiation and apoptosis, and maintaining genomic integrity. However, once tumor cells escape from the tumor-suppressive effects of TGF-beta, they often constitutively overexpress and activate TGF-beta, which may promote tumor progression by enhancing invasion, metastasis, and angiogenesis and by suppressing antitumor immunity. The purpose of this study was to test this hypothesis using TGF-beta pathway antagonists. Experimental design: We examined the effects of selective TGF-beta type I receptor kinase inhibitors, SD-093 and SD-208, on two murine mammary carcinoma cell lines (R3T and 4T1) in vitro and in vivo. Results: Both agents blocked TGF-beta-induced phosphorylation of the receptor-associated Smads, Smad2 and Smad3, in a dose-dependent manner, with IC50 between 20 and 80 nmol/L. TGF-beta failed to inhibit growth of these cell lines but stimulated epithelial-to-mesenchymal transdifferentiation, migration, and invasiveness into Matrigel in vitro. These effects were inhibited by SD-093, indicating that these processes are partly driven by TGF-beta. Treatment of syngeneic R3T or 4T1 tumor-bearing mice with orally given SD-208 inhibited primary tumor growth as well as the number and size of metastases. In contrast, SD-208 failed to inhibit R3T tumor growth or metastasis in athymic nude mice. Moreover, in vitro anti-4T1 cell cytotoxic T-cell responses of splenocytes from drug-treated animals were enhanced compared with cells from control animals. In addition, SD-208 treatment resulted in a decrease in tumor angiogenesis. Conclusion: TGF-beta type I receptor kinase inhibitors hold promise as novel therapeutic agents for metastatic breast cancer.[2]

---

Background: Transforming growth factor-beta (TGF-β) plays a significant role in the pathogenesis of the intimal hyperplasia of transplant arteriosclerosis (TA). The aim of this study was to evaluate the efficacy of an oral inhibitor of TGF-β receptor I kinase (SD-208) on the development of TA.[3]

C17H10CLFN6

352.75

352.063

C, 57.88; H, 2.86; Cl, 10.05; F, 5.39; N, 23.82

627536-09-8

10316032

Light yellow to yellow solid powder

1.5±0.1 g/cm3

460.4±45.0 °C at 760 mmHg

232.2±28.7 °C

0.0±1.1 mmHg at 25°C

1.717

2.69

76.48

1

7

3

25

437

0

ClC1C([H])=C([H])C(=C(C=1[H])C1=NC2C(C(=N1)N([H])C1C([H])=C([H])N=C([H])C=1[H])=NC([H])=C([H])N=2)F

BERLXWPRSBJFHO-UHFFFAOYSA-N

InChI=1S/C17H10ClFN6/c18-10-1-2-13(19)12(9-10)15-24-16-14(21-7-8-22-16)17(25-15)23-11-3-5-20-6-4-11/h1-9H,(H,20,22,23,24,25)

2-(5-chloro-2-fluorophenyl)-N-4-pyridinyl-4-pteridinamine

SD-208; SD 208; 2-(5-chloro-2-fluorophenyl)-N-(pyridin-4-yl)pteridin-4-amine; SD 208; SD208; 2-(5-chloro-2-fluorophenyl)-N-pyridin-4-ylpteridin-4-amine; CHEMBL238125; MFCD11519969; TGF-β RI Kinase Inhibitor V; SD208;

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: 0.91 mg/mL (2.58 mM) 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), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 9.1 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。

---

配方 2 中的溶解度: 0.91 mg/mL (2.58 mM) 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), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 9.1 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。

---

View More

配方 3 中的溶解度: ≥ 0.91 mg/mL (2.58 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 9.1 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

---

配方 4 中的溶解度: 1% methylcellulose:8 mg/mL

---

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。