JNK (Ki = 25-50 nM)
CC-401 HCl targets all isoforms of c-Jun amino-terminal kinase (JNK), including JNK1 (Mitogen-Activated Protein Kinase 8) and JNK2 (Mitogen-Activated Protein Kinase 9) (no IC50/Ki/EC50 values provided) [1]
CC-401 HCl targets JNK (JNK1 and JNK2 isoforms), with JNK1 playing a non-redundant role in mediating cellular responses to chemotherapy in hypoxic colon cancer cells (no IC50/Ki/EC50 values provided) [2]
CC-401 HCl targets c-Jun amino terminal kinase (JNK) signaling pathway (no IC50/Ki/EC50 values provided) [3]

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。

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1. 在肾小管细胞相关体外研究中，CC-401 HCl 可阻断JNK信号通路，抑制促纤维化分子TGF-beta1和结缔组织生长因子（CTGF）的基因转录，减少肾小管细胞凋亡；敲除JNK1（而非JNK2）同样可减少肾小管凋亡，但敲除JNK1/JNK2无法预防肾纤维化[1]
2. 在结肠癌细胞系（HT29、SW620、HCT116）中，CC-401 HCl 与化疗药物（奥沙利铂、SN-38、5-氟尿嘧啶）联用呈现协同效应（该协同效应并非均具有缺氧特异性）；在HT29和SW620细胞中，CC-401 HCl 处理可增加缺氧条件下的DNA损伤（通过p-H2AX阳性细胞数、Chk1/Chk2活化程度作为DNA损伤指标评估）；向HT29细胞中稳定转入显性负性JNK1（而非JNK2）构建体可增强其在缺氧条件下对奥沙利铂的敏感性；Western blot分析证实 CC-401 HCl 可抑制结肠癌细胞中缺氧诱导的c-Jun磷酸化[2]
3. 在体外培养的巨噬细胞中，CC-401 HCl 可抑制IL-1诱导的MMP-12和IL-10的产生，且该效应被证实依赖JNK通路[3]

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。

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1. 在大鼠单侧输尿管梗阻（UUO）模型中，给予CC-401 HCl 可阻断梗阻肾脏中的JNK信号通路，显著抑制肾纤维化（减少间质肌成纤维细胞聚集和IV型胶原沉积），并降低肾小管凋亡；在JNK1/JNK2敲除小鼠UUO模型中，敲除JNK1可减少肾小管凋亡，但敲除JNK1或JNK2均无法预防肾纤维化[1]
2. 在HT29来源的小鼠结肠癌异种移植模型中，CC-401 HCl 可增强贝伐珠单抗、奥沙利铂及其联用方案的疗效，显著延缓肿瘤生长；免疫组化分析证实 CC-401 HCl 可增强缺氧条件下奥沙利铂的细胞毒性（通过JNK/c-Jun磷酸化水平、血管密度、肿瘤活力、DNA损伤程度评估）[2]
3. 在已建立新月体性抗GBM肾小球肾炎的WKY大鼠中（抗GBM血清注射后第7天至第24天给药），CC-401 HCl 可预防肾功能损伤，抑制蛋白尿，阻止严重的肾小球/肾小管间质病变（包括新月体形成和肉芽肿样病变）；该保护作用不依赖于肾小球巨噬细胞/T细胞聚集及体液免疫应答，且 CC-401 HCl 可抑制促炎因子（TNF-alpha、iNOS、MMP-12、TGF-beta1）和抗炎因子（IL-10、血红素氧合酶-1）的表达[3]

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]。

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1. 肾纤维化/肾小管凋亡研究：采用Western blotting和免疫染色检测肾脏组织/细胞中JNK信号通路的活化情况；通过逆转录聚合酶链反应（RT-PCR）定量TGF-beta1和CTGF的基因转录水平；评估肾小管细胞凋亡情况（具体方法未详述）[1]
2. 结肠癌细胞研究：将结肠癌细胞系（HT29、SW620、HCT116）置于缺氧环境（具体条件未详述）中，加入/不加入 CC-401 HCl（浓度为各细胞系的1× IC50）处理6小时或24小时；采用Western blot检测c-Jun磷酸化水平、Chk1/Chk2活化程度（DNA损伤指标）；通过MTT实验测定常氧/缺氧条件下奥沙利铂、SN-38、5-氟尿嘧啶的IC50值；缺氧处理24小时后（加入/不加入奥沙利铂（常氧条件下的IC50浓度）和 CC-401 HCl），采用免疫荧光/流式细胞术（具体方法未明确）定量p-H2AX阳性细胞数（DNA损伤标志物）；将显性负性JNK1/JNK2构建体稳定转染至HT29细胞，构建单克隆细胞系，随后通过Western blot检测缺氧诱导的JNK信号通路活化情况，并通过MTT实验评估化疗药物敏感性[2]
3. 巨噬细胞研究：体外培养的巨噬细胞经IL-1刺激（具体浓度/时长未详述），同时加入/不加入 CC-401 HCl 处理；定量检测MMP-12和IL-10的产生水平（检测方法未详述），证实其依赖JNK通路[3]

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.

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1. Rat UUO model: CC-401 HCl was administered to rats with obstructed kidneys (dosage, formulation, administration route/frequency not detailed); kidney tissues were collected for Western blotting, immunostaining, and assessment of renal fibrosis (interstitial myofibroblast accumulation, collagen IV deposition) and tubular apoptosis; JNK1/JNK2 knockout mice (C57BL inbred) were subjected to UUO, and renal fibrosis/tubular apoptosis were evaluated at specific time points (not detailed) [1]
2. Mouse colon cancer xenograft model: HT29 colon cancer cells were implanted into SCID mice (female, specific implantation site/number of cells not detailed); mice were treated with CC-401 HCl in combination with bevacizumab, oxaliplatin, or their combination (dosage, formulation, administration route/frequency not detailed); tumor volume was measured over time to assess growth delay; tumor tissues were collected for immunohistochemical analysis (JNK/c-Jun phosphorylation, blood vessel density, viability, DNA damage) [2]
3. Rat anti-GBM glomerulonephritis model: WKY rats (female) were immunized with sheep IgG, then injected with sheep anti-GBM serum on day 0; CC-401 HCl, vehicle, or no treatment was given from day 7 to day 24 (dosage, formulation, administration route/frequency not detailed); urine protein levels were measured to assess proteinuria; renal function parameters (not specified) were evaluated; kidney tissues were collected for histological analysis (glomerular lesions, crescent formation, tubulointerstitial lesions), quantification of macrophage/T-cell accumulation (including giant cells), and RT-PCR to detect mRNA levels of TNF-alpha, iNOS, MMP-12, TGF-beta1, IL-10, heme oxygenase-1 [3]

[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.

1. CC-401 HCl is a specific inhibitor of all JNK isoforms; JNK signaling plays a pathogenic role in renal fibrosis and tubular apoptosis, and JNK1 has a non-redundant role in tubular cell apoptosis, identifying JNK pathway as a potential therapeutic target for progressive kidney disease [1]
2. Hypoxia-induced JNK activation is associated with chemoresistance in colon cancer cells; CC-401 HCl sensitizes hypoxic colon cancer cells to DNA-damaging agents (oxaliplatin, SN-38, 5-FU), supporting clinical testing of JNK inhibition to enhance colon tumor response to chemotherapy [2]
3. JNK signaling activation by macrophages contributes to acute renal injury in anti-GBM glomerulonephritis; CC-401 HCl blocks JNK signaling and halts progression of established crescentic anti-GBM glomerulonephritis, likely via inhibiting macrophage proinflammatory response [3]

C22H25CLN6O

424.93

424.177

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

1438391-30-0

CC-401;395104-30-0

66576998

White to off-white solid powder

82.7

3

5

6

30

516

0

Cl[H].O(C1=C([H])C([H])=C([H])C(=C1[H])C1C2C([H])=C(C3=NC([H])=NN3[H])C([H])=C([H])C=2N([H])N=1)C([H])([H])C([H])([H])N1C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H]

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

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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 (5.88 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 (5.88 mM) (饱和度未知) 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), 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。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.88 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 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 14.29 mg/mL (33.63 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶 (<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。