Ralimetinib dimesylate (LY2228820 dimesylate) is the dimesylate salt form of LY2228820, a tri-substituted imidazole derivative and orally available, p38 mitogen-activated protein kinase (MAPK) inhibitor with potential anti-inflammatory and antineoplastic activities.

p38α:7 nM

Ralimetinib抑制p38α及RAW 264.7细胞中的phosphoMAPKAPK-2 (pMK2)水平，其IC50值分别为7 nM和34.3 nM。此外，Ralimetinib还能抑制小鼠腹膜巨噬细胞中脂多糖（LPS）诱导的TNFα形成，IC50为5.2 nM。[1] 在多发性骨髓瘤（MM）细胞，Ralimetinib（200 nM–800 nM）显著阻断p38MAPK信号传导，通过抑制HSP27的磷酸化来显示，HSP27是p38MAPK的下游靶标，而不影响HSP27的表达水平。Ralimetinib（200 nM–400 nM）能增强波替佐米诱导的细胞毒性和凋亡，但单独使用Ralimetinib并不抑制MM.1S细胞的生长。Ralimetinib（200 nM–800 nM）还能抑制长期骨髓基质细胞（LT-BMSCs）、骨髓单核细胞（BMMNCs）、外周血（PB）CD138+、CD138- 或PB CD14+细胞中的IL-6和MIP-1α的分泌。Ralimetinib（400 nM–800 nM）还能阻止CD14+细胞的成骨细胞生成。[2]

在LPS诱导的小鼠模型中，Ralimetinib有效抑制了TNFα的生成，其阈值最小50%有效剂量(TMED50)低于1 mg/kg。在大鼠胶原诱导关节炎(CIA)模型中，Ralimetinib展现出对爪肿胀、骨侵蚀和软骨破坏的强大效果，其阈值最小50%有效剂量(TMED50)为1.5 mg/kg。[1]

Inhibition of p38α: 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.

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.(Only for Reference)