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, a small molecule that is a specific inhibitor of all three JNK isoforms. The N-terminal activation domain of the transcription factor c-Jun is prevented from being phosphorylated when the drug CC-401 binds the ATP binding site in JNK in a competitive manner. Using osmotic stress on the HK-2 human tubular epithelial cell line, the specificity of this inhibitor is examined in vitro.

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1. In renal tubular cell-related in vitro studies, CC-401 HCl blocked JNK signaling, suppressed gene transcription of profibrotic molecules TGF-beta1 and connective tissue growth factor (CTGF), and reduced tubular cell apoptosis; genetic deletion of JNK1 (but not JNK2) also reduced tubular apoptosis, while JNK1/JNK2 deletion did not protect against renal fibrosis [1]
2. In colon cancer cell lines (HT29, SW620, HCT116), CC-401 HCl combined with chemotherapeutic drugs (oxaliplatin, SN-38, 5-FU) showed synergistic effects (not always hypoxia-specific); in HT29 and SW620 cells, CC-401 HCl treatment increased DNA damage under hypoxia (assessed by p-H2AX positive cells and activation of Chk1/Chk2 as DNA damage indicators); stable introduction of dominant negative JNK1 (but not JNK2) construct into HT29 cells enhanced sensitivity to oxaliplatin under hypoxia; Western blot analysis confirmed CC-401 HCl inhibited hypoxia-induced c-Jun phosphorylation in colon cancer cells [2]
3. In cultured macrophages, CC-401 HCl inhibited IL-1-induced MMP-12 and IL-10 production, which was confirmed to be JNK-dependent [3]

CC-401 treatment from days 7 to 24 slows the progression of proteinuria, which is significantly reduced compared to the no-treatment and vehicle groups at days 14 and 21. In contrast to proteinuria at day 5, there is still an increase in the severity of proteinuria in CC-401-treated rats at day 21. At day 24, the vehicle and no-treatment groups exhibited renal impairment as evidenced by an increase in serum creatinine. Treatment with CC-401 stops this from happening. In comparison to the control, bevazicumab and oxaliplatin treatments moderately increased the staining of p-JNK, and the p-cJun content was significantly lower in the samples treated with CC-401, indicating effective JNK inhibition. In combination treatments with CC-401, DNA damage is slightly increased.

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1. In the rat unilateral ureteral obstruction (UUO) model, administration of CC-401 HCl blocked JNK signaling in obstructed kidneys, significantly inhibited renal fibrosis (reduced interstitial myofibroblast accumulation and collagen IV deposition), and decreased tubular apoptosis; in JNK1/JNK2 knockout mice UUO model, JNK1 deletion reduced tubular apoptosis but neither JNK1 nor JNK2 deletion protected against renal fibrosis [1]
2. In HT29-derived mouse xenograft models of colon cancer, CC-401 HCl potentiated the efficacy of bevacizumab, oxaliplatin, and their combination, resulting in greater tumor growth delay; immunohistochemical analysis confirmed CC-401 HCl enhanced oxaliplatin-induced cytotoxicity under hypoxia (assessed by JNK/c-Jun phosphorylation, blood vessel density, tumor viability, and DNA damage) [2]
3. In WKY rats with established crescentic anti-GBM glomerulonephritis (treated from day 7 to day 24 post anti-GBM serum injection), CC-401 HCl prevented renal impairment, suppressed proteinuria, and inhibited severe glomerular/tubulointerstitial lesions (including crescent formation and granulomatous-like lesions); the protective effect was independent of glomerular macrophage/T-cell accumulation and humoral immune response, and CC-401 HCl inhibited expression of proinflammatory (TNF-alpha, iNOS, MMP-12, TGF-beta1) and anti-inflammatory (IL-10, heme oxygenase-1) molecules [3]

CC-401 is a potent, specific, second generation and ATP-competitive anthrapyrazolone c-Jun N terminal kinase (JNK) inhibitor with potential antineoplastic activity. It has a Ki of between 25 and 50 nM and is a strong inhibitor of all three JNK forms. It limits the dosage-dependent phosphorylation of c-Jun brought on by sorbitol. However, CC-401 is unable to stop the phosphorylation of JNK, p38, or ERK that is brought on by sorbitol. Celgene Corporation created the specific JNK inhibitor, CC-401, as a competitive inhibitor of the ATP binding site in the active, phosphorylated form of JNK. When compared to other related kinases, CC-401's selectivity for JNK is at least 40 times higher.

In DMEM/F12 media that has been supplemented with 10% FCS, 10 ng/mL EGF, and 10 g/mL bovine pituitary extract, human HK-2 proximal tubular epithelial cells are cultured. Cells are seeded into six-well plates and allowed to adhere over night. The following day, the medium is changed to DMEM/F12 supplemented with only 0.5% FCS, and the cells are confluent by this point. Confluent cells are treated with CC-401 prepared in citric acid (pH 5.5), which is added 1 hour before 300 mM sorbitol is added. Cells are harvested using urea-RIPA buffer 30 minutes later. There are three experiments, each with two replicates for each condition. After 48 hours, supernatants are collected and tested for TGF-β1 content using a commercial ELISA kit. Six replicates are used in each experiment across three different conditions[1].

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1. For renal fibrosis/tubular apoptosis studies: Western blotting and immunostaining were used to detect JNK signaling activation in kidney tissues/cells; reverse transcriptase polymerase chain reaction (RT-PCR) was applied to quantify gene transcription levels of TGF-beta1 and CTGF; apoptosis of tubular cells was assessed (specific methods not detailed) [1]
2. For colon cancer cell studies: Colon cancer cell lines (HT29, SW620, HCT116) were exposed to hypoxia (specific conditions not detailed) with/without CC-401 HCl (at 1× IC50 for each cell line) for 6 or 24 hours; Western blot analysis was performed to detect c-Jun phosphorylation, Chk1/Chk2 activation (DNA damage indicators); MTT assays were used to determine IC50 values of oxaliplatin, SN-38, 5-FU in normoxic/hypoxic conditions; immunofluorescence/flow cytometry (method not specified) was used to quantify p-H2AX positive cells (DNA damage marker) after 24-hour hypoxia with/without oxaliplatin (IC50 for oxic condition) and CC-401 HCl; stable transfection of dominant negative JNK1/JNK2 constructs into HT29 cells was conducted to generate monoclonal cell lines, followed by Western blot to assess hypoxia-induced JNK signaling and MTT assays for chemotherapeutic drug sensitivity [2]
3. For macrophage studies: Cultured macrophages were stimulated with IL-1 (specific concentration/duration not detailed), with/without CC-401 HCl treatment; production of MMP-12 and IL-10 was quantified (detection method not detailed) to confirm JNK-dependence [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.
\nRats: 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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\n1. 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]
\n2. 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]
\n3. 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 subtypes; the JNK signaling pathway plays a pathogenic role in renal fibrosis and renal tubular cell apoptosis, and JNK1 plays an irreplaceable role in renal tubular cell apoptosis, which suggests that the JNK pathway is a potential therapeutic target for progressive kidney disease [1]
2. Hypoxia-induced JNK activation is associated with chemotherapy resistance in colon cancer cells; CC-401 HCl can make hypoxic colon cancer cells sensitive to DNA damaging agents (oxaliplatin, SN-38, 5-FU), supporting the development of clinical trials of JNK inhibitors to enhance the chemotherapy response of colon tumors [2]
3. Macrophage activation of the JNK signaling pathway can lead to acute kidney injury in anti-GBM glomerulonephritis; CC-401 HCl can block the JNK signaling pathway and prevent the progression of already formed glomerular crescents in anti-GBM glomerulonephritis, the mechanism of which may be through inhibiting the pro-inflammatory response of macrophages [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

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

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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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 (Please use freshly prepared in vivo formulations for optimal results.)