JNK (Ki = 25-50 nM)

Comparing CC-401 to other closely related kinases, such as p38, extracellular signal-regulated kinase (ERK), inhibitor of B kinase (IKK2), protein kinase C, Lck, and zeta-associated protein of 70 kDa (ZAP70), JNK exhibits at least 40-fold selectivity. 1 to 5 μM of CC-401 specifically inhibits JNK in cell-based assays. A specific inhibitor of all three JNK isoforms, CC-401 is a small molecule. 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. In a dosage-dependent manner, CC-401 prevents sorbitol's induction of c-Jun phosphorylation. The phosphorylation of JNK, p38, or ERK caused by sorbitol is not inhibited by CC-401, though[1].

Bevazicumab, Oxaliplatin, and CC-401 treatments both slightly increase the staining of p-JNK when compared to the control, and the p-cJun content is significantly reduced in the CC-401-treated samples, indicating effective JNK inhibition. Combination treatments with CC-401 slightly increase DNA damage[2]. In comparison to the vehicle and no-treatment groups at days 14 and 21, proteinuria progresses more slowly during the CC-401 treatment period of days 7 to 24. Yet, CC-401-treated rats' proteinuria at day 21 is still more severe than it was at day 5 in terms of severity. As evidenced by an increase in serum creatinine, the vehicle and no-treatment groups experienced renal impairment by day 24. CC-401 therapy prevents this[3].

In DMEM/F12 media that has been supplemented with 10% FCS, 10 ng/mL EGF, and 10 μg/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 carried out, each with two replicates for each condition. For ELISA tests, HK-2 cells are seeded into 24-well plates, given time to adhere over night, cultured in DMEM/F12 with 0.5% FCS for 24 hours, then exposed to CC-401 or a vehicle for 60 minutes before being stimulated with 1 μM angiotensin II (AngII). 48 hours later, supernatants are collected, and a commercial ELISA kit is used to measure the presence of TGF-β1. Six replicates are used in each of three experiments[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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 (Please use freshly prepared in vivo formulations for optimal results.)