JNK (Ki = 25-50 nM)

CC-401 是一种小分子，是所有三种 JNK 亚型的特异性抑制剂。当药物 CC-401 以竞争性方式结合 JNK 中的 ATP 结合位点时，转录因子 c-Jun 的 N 端激活结构域被阻止磷酸化。利用 HK-2 人肾小管上皮细胞系的渗透压，在体外检查了该抑制剂的特异性。

CC-401 治疗第 7 天至第 24 天减缓了蛋白尿的进展，与第 14 天和第 21 天的未治疗组和赋形剂组相比，蛋白尿明显减轻。与第 5 天的蛋白尿相比，蛋白尿的严重程度仍然有所增加在第 21 天，CC-401 治疗的大鼠出现蛋白尿。第 24 天，媒介物组和未治疗组表现出肾损伤，血清肌酐增加证明了这一点。使用 CC-401 治疗可以阻止这种情况发生。与对照相比，贝伐单抗和奥沙利铂治疗适度增加了 p-JNK 的染色，并且用 CC-401 处理的样品中 p-cJun 含量显着降低，表明 JNK 抑制有效。与 CC-401 联合治疗时，DNA 损伤略有增加。

CC-401 是一种有效、特异性、第二代 ATP 竞争性蒽吡唑酮 c-Jun N 末端激酶 (JNK) 抑制剂，具有潜在的抗肿瘤活性。它的 Ki 介于 25 至 50 nM 之间，是所有三种 JNK 形式的强抑制剂。它限制了山梨醇引起的 c-Jun 剂量依赖性磷酸化。然而，CC-401 无法阻止山梨醇引起的 JNK、p38 或 ERK 磷酸化。 Celgene 公司创造了特定的 JNK 抑制剂 CC-401，作为 JNK 活性磷酸化形式中 ATP 结合位点的竞争性抑制剂。与其他相关激酶相比，CC-401 对 JNK 的选择性至少高出 40 倍。

在补充有 10% FCS、10 ng/mL EGF 和 10 g/mL 牛垂体提取物的 DMEM/F12 培养基中培养人 HK-2 近端肾小管上皮细胞。将细胞接种到六孔板中并使其粘附过夜。第二天，将培养基更换为仅补充 0.5% FCS 的 DMEM/F12，此时细胞已汇合。用在柠檬酸（pH 5.5）中制备的 CC-401 处理汇合细胞，柠檬酸在添加 300 mM 山梨糖醇前 1 小时添加。 30 分钟后使用尿素-RIPA 缓冲液收获细胞。共有三个实验，每个实验针对每个条件有两个重复。 48小时后，收集上清液并使用商业ELISA试剂盒测试TGF-β1含量。每个实验在三种不同条件下使用六次重复[1]。

Mice: Female adult severe combined immunodeficient mice (C.B.17 SCID), which are 8–10 weeks old, are used to evaluate the effectiveness of CC-401 in inhibiting JNK signaling in anti-angiogenic and Oxaliplatin combination therapy in a mouse xenograft model. HT29 cells (1×106 cells) are subcutaneously injected into the left flank of the mice to produce tumors. To treat the mice with bevacizumab, oxaliplatin, CC401, and the proper combinations of bevacizumab, oxaliplatin, and CC-401, the tumors were divided into eight groups of eight mice each when they reached a size of about 200 mm3. The intraperitoneal injection of 5 mg/kg of bevacizumab is given to mice in the bevacizumab treatment group every three days for 21 days. The Oxaliplatin treatment group receives 2 weeks of intraperitoneal injections of 5 mg/kg Oxaliplatin each week. Every three days, 25 mg/kg of the CC-401 treatment group receives an intraperitoneal injection. The combination treatment groups are given Bevacizumab (5 mg/kg every 3 days), Oxaliplatin (5 mg/kg every week for 2 weeks), and CC-401 (25 mg/kg every 3 days). In the control group, intraperitoneal saline is administered. Every three days, the body's weight and tumor volume are measured. The tumor volume is determined. The time difference between control and treated tumors to grow from 200 to 800 mm3 is used to calculate the tumor growth delay. In order to calculate the tumor growth delay, mice were given treatments until the tumor volume reached 800 mm3. Mice are sacrificed for immunohistochemistry on day 9 after treatments for tumor processing and staining.
Rats: Female WKY rats weighing 180–220 g are employed. Injections of sheep anti-rat GBM serum are administered intravenously five days later (referred to as day 0), after groups of nine or ten rats have received subcutaneous injections of 5 mg of sheep IgG in Freund's complete adjuvant. In this study, treatment with CC-401 (200 mg/kg/b.i.d. by oral gavage) or the control (sodium citrate) is started seven days after anti-GBM serum administration and continued twice daily until the animals are killed on day 24. At days 7 or 24 after receiving an injection of anti-GBM serum, additional groups of untreated rats are put to death. On days 5, 14, and 21, urine is collected from animals that have spent 22 hours in metabolic cages. At the time of death, blood is collected. Urinary and serum creatinine and protein levels are analyzed.

[1]. A pathogenic role for c-Jun amino-terminal kinase signaling in renal fibrosis and tubular cell apoptosis. J Am Soc Nephrol. 2007 Feb;18(2):472-84.

[2]. Inhibition of JNK Sensitizes Hypoxic Colon Cancer Cells to DNA-Damaging Agents. Clin Cancer Res. 2015 Sep 15;21(18):4143-52.

[3]. Blockade of the c-Jun amino terminal kinase prevents crescent formation and halts established anti-GBM glomerulonephritis in the rat. Lab Invest. 2009 Apr;89(4):470-84.

C22H25CLN6O

424.93

424.18

C, 62.18; H, 5.93; Cl, 8.34; N, 19.78; O, 3.77

1438391-30-0

CC-401;395104-30-0

Solid powder

C1CCN(CC1)CCOC2=CC=CC(=C2)C3=NNC4=C3C=C(C=C4)C5=NC=NN5.Cl

OIBVXKYKWOUGAO-UHFFFAOYSA-N

InChI=1S/C22H24N6O.ClH/c1-2-9-28(10-3-1)11-12-29-18-6-4-5-16(13-18)21-19-14-17(22-23-15-24-27-22)7-8-20(19)25-26-21;/h4-8,13-15H,1-3,9-12H2,(H,25,26)

3-[3-(2-piperidin-1-ylethoxy)phenyl]-5-(1H-1,2,4-triazol-5-yl)-1H-indazole;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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.88 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.88 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.88 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 14.29 mg/mL (33.63 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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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。