Ralimetinib targets p38 MAPK α and β isoforms. It selectively inhibits phosphorylation of MK2 at Thr334, without affecting the phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or c-Myc. This inhibition prevents p38 MAPK-mediated signaling, which plays a crucial role in tumor cell proliferation, angiogenesis, and metastasis.

LY2228820 inhibits p38α, as well as the level of phosphoMAPKAPK-2 (pMK2) in RAW 264.7 cells, with IC50 values of 7 nM and 34.3 nM, respectively. Furthermore, LY2228820 inhibits lipopolysaccharide (LPS)-induced TNFα formation in murine peritoneal macrophages, with IC50 of 5.2 nM. In multiple myeloma (MM) cells, including INA6, RPMI-8226, U266, and RPMI-Dox40, LY2228820 (200 nM–800 nM) significantly blocks p38MAPK signaling, as revealed by its inhibition on phosphorylation of HSP27, a downstream target of p38MAPK, without affecting the expression level of HSP27. LY2228820 (200 nM–400 nM) enhances bortezomib-induced cytotoxicity and apoptosis, but LY2228820 alone doesnt inhibit the growth of MM.1S cells. LY2228820 (200 nM–800 nM) also inhibits secretion of IL-6 and MIP-1α in long-term BM stromal cells (LT-BMSCs), BM mononuclear cells (BMMNCs), peripheral blood (PB) CD138+, CD138− or PB CD14+ cells. LY2228820 (400 nM–800 nM) also blocks osteoclastogenesis from CD14+ cells.

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Kinase 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: MTT assays and APO 2.7 staining are performed to assess cellular proliferation and induction of apoptosis, respectively. Viability is expressed as percent viable cells. Apoptosis in cells is evaluated by APO 2.7 staining. For detection of mitochondrial membrane protein 7A6 expressed in apoptotic cells, cells are incubated with APO 2.7 reagent for 20 min. Expression of APO 2.7 is determined using an EPICS XL flow cytometer.

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Ralimetinib inhibits p38α and p38β with IC50s of 5.3 and 3.2 nM, respectively. It selectively inhibits phosphorylation of MK2 (Thr334) without affecting other MAPK pathways. This leads to inhibition of pro-inflammatory cytokine production (TNFα, IL-1, IL-6, IL-8) and VEGF, ultimately inducing apoptosis and reducing tumor cell proliferation.

In LPS-induced mice, LY2228820 effectively inhibits the formation of TNFα with a threshold minimum 50% effective dose (TMED50) less than 1 mg/kg. In a rat model of collagen-inducedarthritis (CIA), LY2228820 displays potent effects on paw swelling, bone erosion, and cartilage destruction, with a threshold minimum 50% effective dose (TMED50)of 1.5 mg/kg.

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In vivo, Ralimetinib is an orally available p38 MAPK inhibitor with potential anti-inflammatory and antineoplastic activities. It has been evaluated in clinical trials for various cancers, including glioblastoma and metastatic breast cancer. Inhibition of the p38 MAPK pathway increases the antineoplastic activity of certain chemotherapeutic agents.

p38 MAPK α/β inhibition is measured using a biochemical kinase assay with purified enzymes and a peptide substrate. Ralimetinib is incubated with the enzyme and substrate; the incorporation of phosphate is measured using a radiometric or fluorescence-based method. IC50 values are calculated from dose-response curves. The assay confirms ATP-competitive inhibition.

In vitro cell-based assays evaluate the anti-inflammatory and anticancer effects of Ralimetinib. Cells are stimulated with LPS or other inflammatory stimuli in the presence of Ralimetinib; cytokine production (TNFα, IL-1, IL-6, IL-8) is measured by ELISA. Cancer cell proliferation and apoptosis are assessed using standard assays. MK2 phosphorylation is measured by Western blot.

Dissolved in 1% CMC/0.25% Tween 80 in water; 0–20 mg/kg; Administered via p.o.Lipopolysaccharide (LPS)-induced Balb/c mice

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In vivo efficacy is evaluated in animal models of cancer and inflammation. Tumor xenograft models are used to assess tumor growth inhibition. The compound is administered orally; tumor size is measured; biomarkers of p38 MAPK inhibition (e.g., MK2 phosphorylation) are assessed in tumor tissues. Pharmacokinetic parameters are evaluated.

Bioorg Med Chem Lett.2008 Jan 1;18(1):179-83;Br J Haematol.2008 May;141(5):598-606.

Lelimetinib has been used in clinical trials for the treatment of various cancers, including postmenopausal cancer, advanced cancer, adult glioblastoma, fallopian tube cancer, and metastatic breast cancer. See also: Lelimetinib mesylate (note moved to).

C24H29FN6

420.52566

420.243

862505-00-8

Ralimetinib dimesylate;862507-23-1

11539025

White to off-white powder

1.2±0.1 g/cm3

634.4±65.0 °C at 760 mmHg

337.5±34.3 °C

0.0±1.9 mmHg at 25°C

1.631

4.94

86.14

2

5

5

31

608

0

CC(C)(C)CN1C2=C(C=CC(=N2)C3=C(C4=CC=C(C=C4)F)N=C(C(C)(C)C)N3)NC1=N

5-(2-(tert-butyl)-5-(4-fluorophenyl)-1H-imidazol-4-yl)-3-neopentyl-3H-imidazo[4,5-b]pyridin-2-amine

XPPBBJCBDOEXDN-UHFFFAOYSA-N

LY2228820 LY2228820 LY 2228820 Ralimetinib.

LY2228820 LY-2228820 LY 2228820

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)

DMSO: ~100 mg/mL (~237.8 mM; with ultrasonication)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.94 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween-80 + 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 DMSO stock solution (25.0 mg/mL) to 400 μL of PEG300 and mix well; then add 50 μL of Tween-80 and mix well; finally add 450 μL of physiological saline and 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 (5.94 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 DMSO stock solution (25.0 mg/mL) to 900 μL of corn oil and mix well.

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