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Purity: ≥98%
Ralimetinib dimysylate (also known as LY-2228820; LY2228820) is a novel, potent and selective ATP-competitive inhibitor of p38 MAPK with potential anti-inflammatory activity. In a cell-free assay, it inhibits p38 MAPK with an IC50 of 7 nM and does not affect p38 MAPK activation. In cell-based assays, LY2228820 potently and selectively inhibited phosphorylation of MK2 (Thr334) in anisomycin-stimulated HeLa cells (at 9.8 nmol/L by Western blot analysis) and anisomycin-induced mouse RAW264.7 macrophages (IC(50) = 35.3 nmol/L) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or c-Myc ≤ 10 μmol/L.
Targets |
p38β MAPK (IC50 = 3.2 nM); p38α MAPK (IC50 = 5.8 nM)
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ln Vitro |
Ralimetinib dimesylate inhibits p38α and the quantity of phosphoMAPKAPK-2 (pMK2) in RAW 264.7 cells with IC50 values of 7 nM and 34.3 nM, respectively. Additionally, Ralimetinib dimesylate inhibits lipopolysaccharide (LPS)-induced TNFα synthesis in murine peritoneal macrophages with an IC50 of 5.2 nM[1]. Ralimetinib dimesylate (LY2228820) (200 nM-800 nM) significantly inhibits p38MAPK signaling in multiple myeloma (MM) cells, including INA6, RPMI-8226, U266 and RPMI-Dox40, as shown by its inhibition on phosphorylation of HSP27, a downstream target of p38MAPK, without changing the expression level of HSP27. Ralimetinib dimesylate (200 nM–400 nM) increases bortezomib-induced cytotoxicity and apoptosis, but Ralimetinib dimesylate alone does not inhibit the growth of MM.1S cells. Additionally, ralimetinib dimesylate (200 nM-800 nM) inhibits the release of IL-6 and MIP-1 from long-term BM stromal cells (LT-BMSCs), BM mononuclear cells (BMMNCs), and peripheral blood (PB) CD138+, CD138-, or PB CD14+ cells. Additionally, osteoclastogenesis from CD14+ cells is prevented by ralimetinib dimesylate (400 nM–800 nM)[2].
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ln Vivo |
Ralimetinib dimesylate inhibits the production of TNFα in LPS-induced mice with a threshold minimum 50% effective dose (TMED50) of less than 1 mg/kg. Ralimetinib dimesylate exhibits strong effects on paw swelling, bone deterioration, and cartilage destruction in a rat model of collagen-induced arthritis (CIA), with a threshold minimum 50% effective dose (TMED50) of 1.5 mg/kg[1]. In mice implanted with B16-F10 melanoma, ralimetinib dimesylate inhibits tumor phospho-MK2 in a dose-dependent manner (TED50=1.95 mg/kg, TED70=11.17 mg/kg). Mouse in vivo TED50=1.01 mg/kg (compound exposure approximately 100 nM) and human ex vivo IC50=0.12 μM with either mouse or human PBMC[3] are the values for ralimetinib dimesylate's ability to inhibit MK2 phosphorylation.
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Enzyme Assay |
Inhibition of p38α is determined using recombinant human p38α in a standard filter binding protocol using ATP[γ-33P] and EGFR 21-mer peptide as substrate. Functional inhibition of TNFα in murine peritoneal macrophages is determined using LPS stimulation in the presence of LY2228820. To assess p38α activity in cells more directly, RAW 264.7 cells are treated with LY2228820 and then stimulated with anisomycin. The level of p38α activity is detected using a phosphoMAPKAPK-2 (pMK2) (Thr 334) antibody which reacts with a residue specifically phosphorylated by p38α.
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Cell Assay |
Recombinant human p38α is used in a standard filter binding protocol with ATP[γ-33P] and the EGFR 21-mer peptide as substrate to measure the inhibition of p38. Using LPS stimulation and Ralimetinib, the functional inhibition of TNFα in murine peritoneal macrophages is assessed. RAW 264.7 cells are given ralimetinib treatment before being stimulated with anisomycin in order to more accurately measure p38α activity in the cells. A phosphoMAPKAPK-2 (pMK2) (Thr 334) antibody that reacts with a residue that has been specifically phosphorylated by p38 is used to measure the level of p38α activity.
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Animal Protocol |
Murine B16-F10 melanoma cells are cultured in Dulbecco's Modified Eagle Medium with l-glutamine, high glucose, and 10% FBS (GIBCO 11965-092). B16-F10 cells (2×106) are implanted into the rear flank of C57/bl6 mice, and when the tumors grow to a size of about 200 mm3, Ralimetinib dimesylate in 1% carboxymethylcellulose/0.25% Tween 80 is given orally. Tumors are removed, homogenized, and lysed two hours after the dose for Western blot analysis. Chemiluminescent detection is used to measure MK2 phosphorylation (p-Thr334), which is normalized to total glyceraldehyde-3-phosphate dehydrogenase. In xenograft models, the 50% or 70% threshold effective dose (TED50 and TED70, respectively) is calculated to approximate the effective dose ranges where significant target inhibition is seen. The TED50 or TED70 is the dose at which a statistically significant effect is obtained and there is at least 50% or 70% inhibition, in comparison to the vehicle control, respectively.
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References |
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Molecular Formula |
C24H29FN6.2CH4O3S
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Molecular Weight |
612.74
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Elemental Analysis |
C, 50.97; H, 6.09; F, 3.10; N, 13.72; O, 15.67; S, 10.46
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CAS # |
862507-23-1
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Related CAS # |
Ralimetinib;862505-00-8
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Appearance |
Solid powder
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SMILES |
CC(C)(C)CN1C2=C(C=CC(=N2)C3=C(N=C(N3)C(C)(C)C)C4=CC=C(C=C4)F)N=C1N.CS(=O)(=O)O.CS(=O)(=O)O
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InChi Key |
NARMJPIBAXVUIE-UHFFFAOYSA-N
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InChi Code |
InChI=1S/C24H29FN6.2CH4O3S/c1-23(2,3)13-31-20-17(28-22(31)26)12-11-16(27-20)19-18(14-7-9-15(25)10-8-14)29-21(30-19)24(4,5)6;2*1-5(2,3)4/h7-12H,13H2,1-6H3,(H2,26,28)(H,29,30);2*1H3,(H,2,3,4)
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Chemical Name |
5-[2-tert-butyl-4-(4-fluorophenyl)-1H-imidazol-5-yl]-3-(2,2-dimethylpropyl)imidazo[4,5-b]pyridin-2-amine;methanesulfonic acid
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Synonyms |
LY-2228820; LY 2228820; Ralimetinib; LY2228820; LY2228820 dimesylate; Ralimetinib dimesylate
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HS Tariff Code |
2934.99.9001
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Storage |
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. |
Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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Solubility (In Vitro) |
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Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.08 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (4.08 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (4.08 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: Saline: 30mg/mL |
Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
1 mM | 1.6320 mL | 8.1601 mL | 16.3201 mL | |
5 mM | 0.3264 mL | 1.6320 mL | 3.2640 mL | |
10 mM | 0.1632 mL | 0.8160 mL | 1.6320 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration: mg/mL;
Method for preparing DMSO stock solution: mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation::Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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