MEK1; MEK2

Pimasertib (5, 0.5, and 0.1 μM) specifically inhibits ERK1/2 activation in MM cells that are cultured alone or with BMSCs. Pimasertib's IC50 values, which range from 0.005 to 2 M, show that it inhibits the growth of MM cell lines in a dose-dependent manner. Pimasertib has IC50 values of 10 nM, 5 nM, and 200 nM for INA-6, U266, and H929 cells, respectively. Both apoptosis and the cell cycle profile are modified by pimasertib. The BM microenvironment is the target of pimasertib's action on MM cells[1]. In D-MUT cells that are resistant to cetuximab, pimasertib (10 mol/L) inhibits ERK pathway, proliferation, and transformation[2]. In contrast to each drug used alone, pimasertib significantly increases the likelihood that RPMI-7951 cells will undergo apoptosis when combined with PLX4032. To achieve outcomes comparable to those of PLX4032 and Pimasertib combined therapy, Pimasertib works in synergy with small interfering RNA-mediated downregulation of BRAF[3].

Pimasertib (15, 30 mg/kg) significantly slows tumor growth in CB17 SCID mice bearing a human H929 MM xenograft[1]. Pimasertib (10 mg/kg, p.o.) inhibits the growth of tumors that are resistant to cetuximab due to a mutation in the K-ras gene[2].

AS703026 is dissolved in 2.5% DMSO. Activated diphosphorylated MEK (pp-MEK) assays contained 40 M 33P-γATP (AppKm 8.5 MμM, 0.5 nM human-activated MEK1 or MEK2, and 1 M kinase-dead ERK2 (AppKm 0.73 μM). All tests are conducted in a buffer containing 20 mM HEPES (pH 7.2), 5 mM 2-mercaptoethanol, 0.15 mg/mL BSA, and 10 mM MgCl2. For all assays, the final 33P- ATP concentration is 0.02 μCi/μL. After 40 minutes, pp-MEK kinase reactions are halted by transferring 30 μL of the reaction mixture to Durapore 0.45-μm filters plates containing 12.5% TCA. Filters are dried before being read on a TopCount using liquid scintilant. For IC50, concentration response data are examined. The IC50 of initially unphosphorylated MEK (u-MEK) is calculated by preincubating 0.2 nM recombinant human MEK1 or MEK2 with vehicle or with AS703026 for 40 minutes in reaction buffer. By adding a final concentration of 20 nM B-RafV600E and 30 μM ATP for 10 minutes, phosphorylation/activation is started. The B-Raf inhibitor SB590885 is then added (final concentration 100 nM), quenching B-Raf activity, and the MEK kinase activity is measured by adding 1 μM KD-ERK2 and 0.02 μCi/μL 33P-ATP in reaction buffer. By transferring 30μL of the reaction mixture to a Durapore filter plate and reading as usual, the kinase reactions are stopped after 90 minutes.

Both [3H]thymidine incorporation and MTT dye absorbance measurements are used to determine the inhibitory effects of study compounds on MM cell growth and survival. In 96-well plates, cells are cultured for 3 days (MM cell lines) or 5 days (patient MM cells) at a density of 104 cells per well in triplicates and 2-5×105 cells per well. For the [3H]thymidine incorporation assay, cells are pulsed with 0.5 μCi (0.0185 MBq)/well [3H]thymidine for 6 h (cell lines), harvested onto glass fiber filters, and counted in a β-scintillation counter. Due to the patient's MM cells' low DNA synthesis, they are pulsed with 2 μCi/well [3H]thymidine and their DNA synthesis is measured over the last 36 hours of culture.

H929 (4×106 cells) are subcutaneously injected into CB17 severe combined immunodeficiency (SCID) mice in 100 μL RPMI-1640 medium. Pimasertib (15 or 30 mg/kg) or the control vehicle alone was administered orally twice daily to the mice, which had palpable tumors (about 130 mm3) by the third week after cell injection. Every other day, calipers are used to measure the tumor's size in two dimensions, and the tumor's volume is computed. When an animal's quality of life is significantly compromised, its tumors grow to a volume of 2 cm3, it becomes moribund, it exhibits paralysis, or it becomes moribund. GraphPad Prism version 4.03 for Windows is used to plot changes in tumor formation in mice treated with control medication vs. pimasertib. Utilizing specific monoclonal (m) antibodies, immunoblotting and immunochemistry analyses of tumors are performed. Abs. Leica IM50 Image Manager is used to take pictures, Leica DM LB research microscope is used for image analysis, and Adobe Photoshop Software 7.0 is used for post-processing.

[1]. 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. Br J Haematol, 2010, 149(4), 537-549.

[2]. The MEK1/2 inhibitor AS703026 circumvents resistance to the BRAF inhibitor PLX4032 in human malignant melanoma cells. Am J Med Sci. 2013 Dec;346(6):494-8.

[3]. MEK1/2 inhibitors AS703026 and AZD6244 may be potential therapies for KRAS mutated colorectal cancer that is resistant to EGFR monoclonal antibody therapy. Cancer Res, 2011, 71(2), 445-453.

N-[(2S)-2,3-dihydroxypropyl]-3-(2-fluoro-4-iodoanilino)-4-pyridinecarboxamide is a pyridinecarboxamide.
Pimasertib is under investigation in clinical trial NCT01378377 (Combination Trial of Pimasertib (MSC1936369B) With Temsirolimus).
Pimasertib is an orally bioavailable small-molecule inhibitor of MEK1 and MEK2 (MEK1/2) with potential antineoplastic activity. Pimasertib selectively binds to and inhibits the activity of MEK1/2, preventing the activation of MEK1/2-dependent effector proteins and transcription factors, which may result in the inhibition of growth factor-mediated cell signaling and tumor cell proliferation. MEK1/2 (MAP2K1/K2) are dual-specificity threonine/tyrosine kinases that play key roles in the activation of the RAS/RAF/MEK/ERK pathway and are often upregulated in a variety of tumor cell types.

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We investigate cytotoxicity and mechanism of action of AS703026, a novel, selective, orally bioavailable MEK1/2 inhibitor, in human multiple myeloma (MM). AS703026, more potently (9-10 fold) than AZD6244, inhibited growth and survival of MM cells and cytokine-induced osteoclast differentiation. Inhibition of proliferation induced by AS703026 was mediated by G0-G1 cell cycle arrest and was accompanied by reduction of c-maf oncogene expression. AS703026 further induced apoptosis via caspase 3 and PARP cleavage in MM cells, both in the presence or absence of bone marrow stromal cells (BMSCs). Importantly, AS703026 sensitized MM cells to a broad spectrum of conventional (dexamethasone, melphalan), novel or emerging (lenalidomide, perifosine, bortezomib, rapamycin) anti-MM therapies. Significant tumor growth reduction in AS703026- vs. vehicle-treated mice bearing H929 MM xenograft tumors correlated with downregulated pERK1/2, induced PARP cleavage, and decreased microvessels in vivo. Moreover, AS703026 (<200 nM) was cytotoxic against the majority of tumor cells tested from patients with relapsed and refractory MM (84%), regardless of mutational status of RAS and BRAF genes. Importantly, BMSC-induced viability of MM patient cells was similarly blocked within the same dose range. Our results therefore support clinical evaluation of AS703026, alone or in combination with other anti-MM agents, to improve patient outcome.[1]

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Background: Although inhibitors of the proto-oncogene BRAF have shown excellent antitumor activity against malignant melanoma, their efficacy is limited by the development of acquired drug resistance, a process in which reactivation of MAP kinase (MEK) is known to play an important role. In this study, we evaluated the efficacy of AS703026, a new MEK inhibitor, in BRAF inhibitor-resistant melanoma cell lines. Methods: Two melanoma cells lines, RPMI-7951 and SK-MEL5, harboring an activating mutation of BRAF (V600E) were treated with the BRAF inhibitor PLX4032 to select a BRAF inhibitor-resistant cell line for further study. Cell viability assay was determined with MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay and trypan blue exclusion method; apoptosis assay was performed by annexin-V staining. Knockdown of BRAF was investigated by small interfering RNA. Results: RPMI-7951 cells exhibited an increased sensitivity to combined treatment with PLX4032 and AS703026 compared to either drug alone. Consistent with this, the combination of PLX4032 and AS703026 significantly induced apoptosis, whereas each drug used alone did not, as demonstrated by a flow cytometric analysis of annexin-V/propidium iodide-stained cells and Western blot analysis of cleaved caspase-3. Notably, immunoblot analyses also showed a depletion of phosphorylated-ERK with combined drug treatment. In addition, AS703026 synergized with small interfering RNA-mediated downregulation of BRAF to produce results similar to those of combined treatment with PLX4032 and AS703026. Conclusions: Our results suggest that combined treatment with AS703026 and a BRAF inhibitor overcomes the resistance to BRAF inhibitors in malignant melanoma cells harboring a mutant form of BRAF.[2]

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Epidermal growth factor receptor (EGFR) monoclonal antibodies (mAb) are used widely to treat metastatic colorectal cancer (mCRC) patients, but it is now clear that patients harboring K-ras mutation are resistant to EGFR mAbs such as cetuximab (Erbitux) and panitumumab (Vectibix). For this reason, current recommendations for patient care involve diagnosing the K-ras mutational status of patients prior to EGFR mAb therapy. In this study, we investigated the ability of two MEK inhibitors currently in clinical trials, AS703026 and AZD6244, to address the challenge posed by the resistance of K-ras mutated colorectal cancers to EGFR mAb. AS703026 and AZD6244 were tested in various cell-based assays and tumor xenograft studies, focusing on isogenic human colorectal tumor cell lines that expressed only WT or mutant K-Ras (D-WT or D-MUT). The EGFR mAb cetuximab inhibited the Ras-ERK pathway and proliferation of D-WT cells in vitro and in vivo, but it did not inhibit proliferation of D-MUT cells in either setting. In contrast, AS703026 and AZD6244 effectively inhibited the growth of D-MUT cells in vitro and in vivo by specific inhibition of the key MEK downstream target kinase ERK. Inhibition of MEK by AS703026 or AZD6244 also suppressed cetuximab-resistant colorectal cancer cells attributed to K-ras mutation both in vitro and in vivo. Our findings offer proof-of-concept for the use of MEK inhibitors as an effective therapy in K-ras mutated CRC.[3]

C15H15FIN3O3

431.20

431.014

C, 41.78; H, 3.51; F, 4.41; I, 29.43; N, 9.74; O, 11.13

1236699-92-5

1236361-78-6 (HCl); 1236699-92-5;

44187362

Light yellow to khaki solid powder

1.8±0.1 g/cm3

623.2±55.0 °C at 760 mmHg

330.7±31.5 °C

0.0±1.9 mmHg at 25°C

1.684

3.05

4

6

6

23

391

1

FC1=C(C=CC(I)=C1)NC2=CN=CC=C2C(NC[C@@H](CO)O)=O

VIUAUNHCRHHYNE-JTQLQIEISA-N

InChI=1S/C15H15FIN3O3/c16-12-5-9(17)1-2-13(12)20-14-7-18-4-3-11(14)15(23)19-6-10(22)8-21/h1-5,7,10,20-22H,6,8H2,(H,19,23)/t10-/m0/s1

N-[(2S)-2,3-dihydroxypropyl]-3-(2-fluoro-4-iodoanilino)pyridine-4-carboxamide

MSC 1936369B; SAR 245509; AS-703026; SAR245509; SAR-245509; AS703026; AS 703026

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.80 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 (5.80 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 (5.80 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: 0.5% CMC+0.25% Tween 80: 30mg/mL

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