MEK1 (IC50 = 14 nM); MEK1 (Kd = 99 nM); MEK2 (Kd = 530 nM)

NVP-BHG712 dose-dependently inhibited the autophosphorylation of EphRs in Hek293 cells transfected with various EphRs. When compared to EphB2, EphA2, EphB3, and NVP-BHG712, EphB4 exhibited a stronger inhibitory preference. AZD6244 inhibits the phosphorylation of ERK1/2 at IC50 concentrations below 40 nM and is not ATP-competitive. Inhibiting ERK1/2 and p90RSK phosphorylation, as well as elevating caspase-3 and caspase-7 cleavage and cleaved poly(ADP)ribose polymerase, all contribute to AZD6244's ability to stop the growth of primary HCC cells. Phosphatidylinositol 3-kinase, MEK5/ERK5 and p38 pathways are not significantly affected by AZD6244. Ras mutations and Raf mutations both affect AZD6244's sensitivity to breast cancer cell lines and NSCLC cell lines, respectively.

AZD6244 significantly inhibits phosphorylation of ERK1/2 in 2-1318, 5-1318, 26-1004 and 4-1318 xenografts and induces apoptosis in primary 2-1318 cells by activating the caspase pathway. At a dose of 100 mg/kg, AZD6244 could slow the growth of the tumor in the HT-29 xenograft, a colorectal tumor model with a B-Raf mutation; this tumor growth inhibition is superior to that of Gemcitabine. Apoptosis and the down-regulation of cell cycle regulators like cyclin D1, Cdc-2, CDK2 and 4, cyclin B1, and c-Myc are associated with increased apoptosis, which is why AZD6244 could inhibit the growth of HCC xenograft tumors in the absence of these factors.

MEK1. [3]
NH2-terminal hexahistidine tagged, constitutively active MEK1 (S218D, S222D ΔR4F; ref. 18) was expressed in baculovirus-infected Hi5 insect cells and purified by immobilized metal affinity chromatography, ion exchange, and gel filtration. The activity of MEK1 was assessed by measuring the incorporation of [γ-33P]phosphate from [γ-33P]ATP onto ERK2. The assay was carried out in a 96-well polypropylene plate with an incubation mixture (100 μL) composed of 25 mmol/L HEPES (pH 7.4), 10 mmol/L MgCl2, 5 mmol/L β-glycerolphosphate, 100 μmol/L sodium orthovanadate, 5 mmol/L DTT, 5 nmol/L MEK1, 1 μmol/L ERK2, and 0 to 80 nmol/L compound (final concentration of 1% DMSO). The reactions were initiated by the addition of 10 μmol/L ATP (with 0.5 μC k[γ-33P]ATP/well) and incubated at room temperature for 45 min. An equal volume of 25% trichloracetic acid was added to stop the reaction and precipitate the proteins. Precipitated proteins were trapped onto glass fiber B filter plates, excess labeled ATP was washed off with 0.5% phosphoric acid, and radioactivity was counted in a liquid scintillation counter. ATP dependence was determined by varying the amount of ATP in the reaction mixture. The data were globally fitted using SigmaPlot. Values were calculated using the following equation for noncompetitive inhibition: v = [Vmax × S / (1 + I / Ki)] / (Km + S).[3]

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ERK2. [3]
To measure inhibition of ERK2, the kinase activity of ERK2 was first activated by MEK1. Wild-type (WT) ERK2 containing an NH2-terminal hexahistidine tag was overexpressed in Escherichia coli and purified by immobilized metal affinity chromatography, ion exchange, and gel filtration. To activate WT ERK2, 2 mg WT ERK2 was mixed with 17 μg of constitutively active MEK1 in 4 mL of 25 mmol/L HEPES (pH 7.5) containing 1 mmol/L ATP. The reaction mixture was incubated at room temperature for 40 min, and the addition of two phosphates was confirmed by mass spectrometry. Activated WT ERK2 was further purified by ion exchange. ERK2 activity was assayed as described for constitutively active MEK, using 10 nmol/L activated ERK2. The substrate used was myelin basic protein at a concentration of 1 μmol/L.[3]

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MEK1 molecules are immunoprecipitated using an anti-MEK1 antibody. When recombinant ERK1 is activated by immuno-isolated MEK1 in a coupled assay with MBP as the end point, MEK kinase activity is calculated. Before being exposed to X-ray film, phosphorylated MBP is resolved on a 14% SDS-PAGE gel and vacuum-dried.

Cell Viability and Cell Proliferation[1]
Primary HCC cells were plated at a density of 2.0 × 104 per well in growth medium. After 48 h in growth medium, the cell monolayer was rinsed twice with MEM. Cells were treated with various concentrations of AZD6244 (0, 0.5, 1.0, 2.0, 3.0, and 4.0 μmol/L) for 24 or 48 h. Cell viability was determined by the 3-(4,5-dimethylthiazol-2y1)-2,5-diphenyltetrazolium bromide (MTT) assay (32). Cell proliferation was assayed using a bromodeoxyuridine kit (Roche) as described by the manufacturer. Experiments were repeated at least thrice, and the data were expressed as mean ± SE.[1]

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Detection of Apoptosis[1]
Primary HCC cells were grown in eight-chamber slides and treated with 0, 0.5, 1.0, 2.0, 3.0, and 4.0 μmol/L of AZD6244 in SRF medium for 24 h. Cells were fixed with PBS containing 4% formalin solution for 1 h at room temperature and washed with PBS. Apoptosis was detected by the terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) assay using the In situ Cell Death Detection kit (Roche) as described by the manufacturer. Apoptotic cells were then visualized under a fluorescent microscope equipped with a FITC filter. The labeling index was obtained by counting the number of positive cells among 500 cells per region. They were expressed as percentage values.[1]

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At a density of2.0 × 104, cells are seeded. The cells undergo two culture media rinses after 48 hours of incubation. AZD6244 is used to treat cells for 24 or 48 hours at various concentrations. The MTT assay uses 3-(4,5-dimethylthiazol-2y1)-2,5-diphenyltetrazolium bromide to measure the viability of cells. With the help of a bromodeoxyuridine kit, cell proliferation is measured.

HCC xenografts in mice homozygous for the SCID (severe combined immunodeficiency) mutation
50 or 100mg/kg
Administered via p.o.

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o investigate the effects of AZD6244 on HCC xenografts, AZD6244 was suspended in water at an appropriate concentration. Mice bearing HCC xenografts were p.o. given, twice a day, with either 100 μL of water (n = 12) or 50 mg (n = 12) or 100 mg (n = 12) of AZD6244 per kilogram of body weight for 21 days, starting from day 7 after tumor implantation. Growth of established tumor xenografts was monitored at least twice weekly by Vernier caliper measurement of the length (a) and width (b) of the tumor. Tumor volume was calculated as (a × b2)/2. Animals were sacrificed 3 h after the last dose of ADZ6244, and body and tumor weights were recorded, with the tumors harvested for analysis.[1]

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To study the effects of AZD6244 on caspase-3 activation and MEK1/2 phosphorylation, mice bearing HCC tumors (∼800 mm3) were treated with vehicle (n = 4) or 50 mg of AZD6244 per kilogram of body weight (n = 4) for 3 days as described above. Animals were sacrificed 3 h after the last dose, and tumors were harvested and frozen in liquid nitrogen for later analysis. Part of the tumor harvest was fixed in neutral buffer containing 10% formalin for immunohistochemistry.[1]

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HT-29 human colon carcinoma or BxPC3 human pancreatic tumor fragments were implanted s.c. in the flank of nude mice and allowed to grow to 100 to 150 mg. Mice (n = 10 per group) were randomized to treatment groups to receive vehicle (10 mL/kg and 10% ethanol/10% cremophor EL/80% D5W) or AZD6244/ARRY-142886 (10, 25, 50, or 100 mg/kg, oral, BID) on days 1 to 21. Tumors [(W2 × L) / L] were measured twice weekly. [3]

[1]. Targeted inhibition of the extracellular signal-regulated kinase kinase pathway with AZD6244 (ARRY-142886) in the treatment of hepatocellular carcinoma. Mol Cancer Therapy, 2007, 6 (1), 138-146 .

[2]. Identification of common predictive markers of in vitro response to the Mek inhibitor selumetinib (AZD6244; ARRY-142886) in human breast cancer and non-small cell lung cancer cell lines. Mol Cancer Thera, 2010, 9 (7), 1985-1994.

[3]. Biological characterization of ARRY-142886 (AZD6244), a potent, highly selective mitogen-activated protein kinase kinase 1/2 inhibitor. Clin Cancer Res, 2007, 13 (5), 1576-1583.

Selumetinib Sulfate is the sulfate salt of selumetinib, an orally active, small molecule with potential antineoplastic activity. Selumetinib is an ATP-independent inhibitor of mitogen-activated protein kinase kinase (MEK or MAPK/ERK kinase) 1 and 2. MEK 1 and 2 are dual specificity kinases that are essential mediators in the activation of the RAS/RAF/MEK/ERK pathway, are often upregulated in various cancer cells, and are drivers of diverse cellular responses, including proliferation. Inhibition of both MEK1 and 2 by selumetinib prevents the activation of MEK1/2 dependent effector proteins and transcription factors, thereby leading to an inhibition of cellular proliferation in various cancers.
See also: Selumetinib (has active moiety).

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Drug Indication
Koselugo as monotherapy is indicated for the treatment of symptomatic, inoperable plexiform neurofibromas (PN) in paediatric patients with neurofibromatosis type 1 (NF1) aged 3 years and above

C₁₇H₁₇BRCLFN₄O₇S

555.76

553.967

943332-08-9

Selumetinib;606143-52-6

16214875

Light yellow to yellow solid

4.601

5

10

6

32

604

0

O=C(C1C(NC2C(Cl)=CC(Br)=CC=2)=C(F)C2N=CN(C=2C=1)C)NOCCO.O=S(O)(O)=O

GRKFGZYYYYISDX-UHFFFAOYSA-N

InChI=1S/C17H15BrClFN4O3.H2O4S/c1-24-8-21-16-13(24)7-10(17(26)23-27-5-4-25)15(14(16)20)22-12-3-2-9(18)6-11(12)19;1-5(2,3)4/h2-3,6-8,22,25H,4-5H2,1H3,(H,23,26);(H2,1,2,3,4)

6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide;sulfuric acid

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 (4.50 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 (4.50 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 (4.50 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: 4% DMSO+30% PEG 300+5% Tween 80+ddH2O: 5mg/mL

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