AZD6244 (Selumetinib)

(Synonyms

ARRY-142886

)

Technical Data:

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M.Wt: 457.68
Formula: C₁₇H₁₅BrClFN₄O₃
Solubility: DMSO
Purity: >99%
Storage: at -20℃ 2 years
CAS No.: 606143-52-6

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Applications & Customer's Feedback of AZD6244 (Selumetinib):

AZD6244 was purchased from Selleck. Data from British Journal of Haematology; 149: 537–549.

INA-6 MM cells were treated with AS703026 or AZD6244 for 2 d, followed by [3H]thymidine uptake assay. PBMCs isolated from normal donors (n = 3) were incubated with M-CSF and RANKL, in the presence or absence of AS703026 or AZD6244 for 14 d. The TRAP assay was performed to measure the formation of multinuclear osteoclast cells (OC).
AZD6244 was purchased from Selleck. Data from Cancer Res, 2010 June; 70:4709-4718.

AZD6244 enhanced FOXO3a expression and induced suppression of cancer cell proliferation. A, tumor volume of the HCT116 xenografts treated with Placebo or AZD6244 was measured for 21 d. The tumor sections of four individual DMSO or AZD6244-treated HCT116 xenografts were subjected to immunohistochemistry with a FOXO3a antibody. Relative percentages of nuclear FOXO3a expression of individual xenograft tumors from B were analyzed and the mean values of FOXO3a expression in Placebo or AZD6244-treated group were indicated as bars.
AZD6244 was purchased from Selleck. Data from Cancer Res, 2010 June; 70:4709-4718.

lysates from various cancer cell lines: breast cancer (MDA-MB-435), colon cancer (HCT116, SW620, and HT29), and melanoma (WM793) treated with DMSO or AZD6244 (10 μmol/L) for 4 h were subjected to immunoblotting with the indicated antibodies.
AZD6244 was supplied by Selleck. Data was provided by Dr Jong-In Park of Medical College of Wisconsin.

B-RafV600E mutated melanoma line,A375, was treated with different doses of AZD6244 for 1hour or 24 hours.Cell lysates were analyzed by Western Blotting to determine the levels of phosphorylated ERK1/2(Perk1/2).
AZD6244 was purchased from Selleck. Data from Nature 2010.November;468:968-972

A,ERK phosphorylation in A375 expressing indicated ORFs following treatment with DMSO or 1 mM of PLX4720, RAF265, CI-1040 or AZD6244. B,ERK phosphorylation in A375 expressing indicated ORFs following treatment with DMSO, PLX4720 (1 mM) or PLX4720 in combination with CI-1040 or AZD6244 (all 1 mM).
AZD6244 was purchased from Selleck. Data from PNAS 2009.December;106.20411-20416.

Ex vivo and functional characterization of MEK1(P124L). (A) AZD6244-mediated growth inhibition ex vivo of treatment-naïve BRAFV600E melanoma cells (black and blue) or cells cultured from an AZD6244-resistant metastatic focus (red). (B) ERK phosphorylation (p-ERK) andMEKphosphorylation (p-MEK) are shown following treatment with increasing concentrations of AZD6244 in treatment-naïve or AZD6244-resistant melanoma cells cultured ex vivo. The tubulin loading control (-tubulin) is also shown. (C) AZD6244 growth inhibition curves of parental A375 (solid black), A375 cells expressing MEK-DD (grey), wild-type MEK1 (hatched black), or MEK1(P124L) (red) are shown. In each instance n6 anderrorstandard deviation. (D) p-ERK and p-MEK are shown following treatment with increasing AZD6244 concentrations in the cell lines described in C, Above. The -tubulin control is also shown.
AZD6244 was purchased from Selleck. Data from Clin Cancer Res, 2010 December;16:6029-6039.

A and B, clonogenic assays in 2 melanoma cell lines (YUVON and YUSIK) treated with NVPBEZ235 (BEZ) and AZD6244 (AZD) alone and in combination. Combinations were more effective in inhibiting colony formation at lower concentrations than either drug alone.
AZD6244 was purchased from Selleck. Data from Nature , 2010 December; 468:973-977.

Survival curves for isogenic cell line pairs and melanoma cultures treated with the indicated AZD6244 concentration for 72 h.

Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. ------ Ramin Nazarian,Hubing Shi,et al. Nature 2010 December;468:973-977

COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. ------ Cory M. Johannessen, Jesse S. Boehm,et al. Nature 2010;468:968-972

Vertical Targeting of the Phosphatidylinositol-3 Kinase Pathway as a Strategy for Treating Melanoma. ------ Saadia A. Aziz, Lucia B. Jilaveanu,et al. Clin Cancer Res 2010 December;16:6029-6039

Activation of FOXO3a Is Sufficient to Reverse Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase Inhibitor Chemoresistance in Human Cancer. ------ Jer-Yen Yang,Chun-Ju Chang,et al. Cancer Res 2010;70:4709-4718

Blockade of the MEK/ERK signalling cascade by AS703026, a novel selective MEK1/2 inhibitor, induces pleiotropic anti-myeloma activity in vitro and in vivo. ------ Kihyun Kim,Sun-Young Kong,et al. Brit J Haematol 2010;149:537–549

MEK1 mutations confer resistance to MEK and B-RAF inhibition. ------ Caroline M. Emery, Krishna G. Vijayendrana,et al. PNAS 2009;106:20411-20416

Combinatorial Treatments That Overcome PDGFRb-Driven Resistance of Melanoma Cells to V600EB-RAF Inhibition. ------ Hubing Shi,Xiangju Kong,et al. Cancer Res 2011.August;71:5067-74

p53 Rescue through HDM2 Antagonism Suppresses Melanoma Growth and Potentiates MEK Inhibition. ------ Zhenyu Ji, Ching Ni Njauw et al. J Invest Dermatol 2011.October;ahead of print

Strong negative feedback from Erk to Raf confers robustness to MAPK signalling. ------ Raphaela Fritsche-Guenther,Franziska Witzel,et al. Mol Syst Biol 2011.May;7:489

Down-regulation of mitogen-inducible gene 6, a negative regulator of EGFR, enhances resistance to MEK inhibition in KRAS mutant cancer cells. ------ Yoon YK,Kim HP,et al. Cancer Lett. 2012 Mar;316:77-84.

The Akt Inhibitor MK2206 Synergizes, but Perifosine Antagonizes, the BRAFV600E Inhibitor PLX4032 and the MEK1/2 Inhibitor AZD6244 in the Inhibition of Thyroid Cancer Cells. ------ Liu R, Liu D, Xing M. J Clin Endocrinol Metab. 2011 Nov;ahead of print

Biological Activity of AZD6244 (Selumetinib):

AZD6244 (Selumetinib) is highly potent to inhibit MEK1 with IC50 of 14nmol. AZD6244 is a not competitive with ATP and inactivates the ERK1/2 phosphorylation with IC50 concentrations below 40 nmol. AZD6244 also inhibits the growth of primary HCC cells through inhibition of ERK1/2 and p90RSK phosphorylation, accompanied with elevation of the cleavage of caspase-3 and caspase-7, and cleaved poly(ADP)ribose polymerase.^[1] AZD6244 is sensitive to raf mutations in breast cancer cell lines and ras mutations in NSCLC cell lines. ^[2]AZD6244 had little effects on the p38, c-Jun-NH2-kinase, phosphatidylinositol 3-kinase, and MEK5/ERK5 pathways. In vivo studies showed that AZD6244 significantly inhibits phosphorylation of ERK1/2 in 2-1318, 5-1318, and 26-1004 4-1318 xenografts and induces apoptosis in primary 2-1318 cells by activating the caspase pathway. ^[1] AZD6244 could inhibit the tumor growth in HT-29 xenograft, which is a colorectal tumor model carrying a B-Raf mutation, at a dose of 100mg/kg and the TGI of AZD6244 is better than it of gemcitabine. ^[3] Otherwise AZD6244 could inhibit HCC xenografts tumor growth, which associated with increased apoptosis and down-regulation of cell cycle regulators, including cyclin D1, cdc-2, cyclin-dependent kinases 2 and 4, cyclin B1, and c-Myc. [4] AZD6244 could be used to treat with many cancers including colorectal, NSCLC, pancreatic and breast. AZD6244 is current in Phase II clinical trial against NSCLC. AZD6244 is originally developed by Array BioPharma and latterly by Astra Zeneca.

Protocol:

Kinase Assay:
NH2-terminal hexahistidine tagged, constitutively active MEK1 is expressed in baculovirus-infected Hi5 insect cells and purified by immobilized metal affinity chromatography, ion exchange, and gel filtration. The activity of MEK1 is assessed by measuring the incorporation of [γ-33P]phosphate from [γ-33P]ATP onto ERK2. The assay is 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 AZD6244 (dissolved in 1% DMSO). The reactions are 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 is added to stop the reaction and precipitate the proteins. Precipitated proteins are trapped onto glass fiber B filter plates, excess labeled ATP is washed off with 0.5% phosphoric acid, and radioactivity is counted in a liquid scintillation counter. ATP dependence is determined by varying the amount of ATP in the reaction mixture. ^[3]

Cell Assay:
Tumor cells are seeded at a density of 2.0 × 104. After 48h incubation, the cells are rinsed twice with culture media. Cells are treated with various concentrations of AZD6244 for 24 or 48 h. Cell viability is determined by the 3-(4,5-dimethylthiazol-2y1)-2,5-diphenyltetrazolium bromide (MTT) assay.
Cell proliferation is assayed using a bromodeoxyuridine kit. ^[1]

Animal Studies:
AZD6244 is suspended in water and administered to tumor bearing mice by p.o. with 50/100mg AZD6244 per kg of body weight after tumor implanting. Tumor size is measured by Vernier caliper. Tumor volume is calculated as (length × width2)/2. Animals are sacrificed 3 h after the last dose of ADZ6244, and body and tumor weights ae recorded, with the tumors harvested for analysis. ^[1]

References on AZD6244 (Selumetinib):

[1] Huynh H, et al, Mol Cancer Therapy, 2007, 6 (1), 138-146

[2] Garon EB, et al, Mol Cancer Thera, 9 (7), pp. 1985-1994.

[3] Yeh TC, et al, Clin Cancer Res, 2007, 13 (5), 1576-1583.

[4] Huynh H, et al, Mol Cancer Ther, 2007, 6 (9), 2468-2476.

Quality Control:

MSDS
Batch S100804: H-NMR COA
Batch S100807: H-NMR  HPLC COA
Batch S100808: H-NMR  HPLC COA
Batch S100809: H-NMR  HPLC COA
Batch S100810: H-NMR HPLC COA
Batch S100811: H-NMR  COA