JNK (Ki = 25-50 nM)

将 CC-401 与其他密切相关的激酶进行比较，例如 p38、细胞外信号调节激酶 (ERK)、B 激酶抑制剂 (IKK2)、蛋白激酶 C、Lck 和 70 kDa zeta 相关蛋白 (ZAP70)、JNK表现出至少 40 倍的选择性。在基于细胞的检测中，1 至 5 μM 的 CC-401 特异性抑制 JNK。 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 以剂量依赖性方式阻止山梨醇诱导 c-Jun 磷酸化。然而，CC-401 不会抑制山梨醇引起的 JNK、p38 或 ERK 磷酸化[1]。

与对照相比，贝伐单抗、奥沙利铂和 CC-401 治疗均略微增加了 p-JNK 的染色，并且 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 联合治疗会略微增加 DNA 损伤[2]。与第 14 天和第 21 天的载体组和未治疗组相比，CC-401 治疗第 7 天至第 24 天期间蛋白尿进展得更慢。然而，CC-401 治疗组大鼠在第 21 天的蛋白尿仍然更严重就严重性而言，比第 5 天时要严重。血清肌酐升高证明，载体组和未治疗组在第 24 天出现肾损伤。CC-401 治疗可以防止这种情况发生[3]。

在补充有 10% FCS、10 ng/mL EGF 和 10 μg/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。进行了三个实验，每个实验针对每种条件进行了两次重复。对于 ELISA 测试，将 HK-2 细胞接种到 24 孔板中，给予贴壁过夜的时间，在含有 0.5% FCS 的 DMEM/F12 中培养 24 小时，然后暴露于 CC-401 或载体 60 分钟，然后进行用 1 μM 血管紧张素 II (AngII) 刺激。 48小时后，收集上清液，并使用商业ELISA试剂盒来测量TGF-β1的存在。三个实验中每个实验均使用六次重复[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 weekly intraperitoneal injections of 5 mg/kg Oxaliplatin for two weeks. The CC-401 treatment group is injected intraperitoneally 25 mg/kg for every 3 days. 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 day 7 or day 24 following injection of the anti-GBM serum, additional groups of untreated rats are put to death as a control. On days 5, 14, and 21, urine is collected from the animals while they are kept in metabolic cages for 22 hours. When someone dies, blood is drawn. Urinary protein and serum creatinine are both 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.

C22H24N6O

388.47

388.20

C, 68.02; H, 6.23; N, 21.63; O, 4.12

395104-30-0

CC-401 hydrochloride;1438391-30-0

Solid powder

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

XDJCLCLBSGGNKS-UHFFFAOYSA-N

InChI=1S/C22H24N6O/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)(H,23,24,27)

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

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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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。