Neflamapimod (VX-745) , a specific and effective inhibitor of p38α(IC50=10 nM), is 22-fold greater specificity against p38β and no inhibition activity to p38γ.

p38α:10 nM, p38β:220 nM

Neflamapimod 抑制p38α和p38β MAPK，其IC50分别为10 nM和220 nM，但对p38γ MAPK及其他大量激酶不产生抑制作用。在人类外周血单核细胞（PBMC）检测中，Neflamapimod对IL-1β和TNFα的IC50分别为56 nM和52 nM。Neflamapimod阻断由IL-1和TNFα诱导的IL-6及IL-8的产生，以及由LPS和IL-1β介导的COX-2合成。[1-3] Neflamapimod（60 nM-20 μM）抑制骨髓基质细胞（BMSCs）中IL-6和VEGF的分泌，但不影响其存活率。Neflamapimod还抑制TNF-α在BMSCs中引发的IL-6分泌。Neflamapimod通过抑制多发性骨髓瘤（MM）细胞在BMSCs黏附后引起的IL-6分泌和MM细胞增殖，表明Neflamapimod能抑制骨髓微环境中的旁分泌多发性骨髓瘤（MM）细胞生长，并克服细胞黏附相关的化合物抗性。[4]

Neflamapimod 对由佐剂引发的大鼠关节炎(AA)具有有效性，ED50为5 mg/kg。在AA大鼠中，Neflamapimod的组织学评分显示93%的骨吸收抑制和56%的炎症抑制。在经典的软骨诱导性关节炎模型中，Neflamapimod表现出剂量响应性的严重程度下降。[1-3] 在II型胶原诱导的关节炎(CIA)小鼠模型中，与对照组小鼠相比，Neflamapimod (2.5, 5, 和 10 mg/kg) 分别改善了27%、31%和44%的炎症评分。此外，组织学评分显示Neflamapimod在保护骨骼和软骨侵蚀方面有32-39%的效果。[5]

Spectrophotometric coupled-enzyme assay: The IC50 for the inhibition of p38α and p38β homologs are obtained by a spectrophotometric coupled-enzyme assay. A fixed concentration of enzyme (15 nM of p38α or p38β) is incubated with VX-745 in DMSO for 10 min. at 30 °C in 0.1 M HEPES buffer, pH 7.5, containing 10% glycerol, 10 mM MgCl2, 2.5 mM phosphoenolpyruvate, 200 μM NADH, 150 μg/mL pyruvate kinase, 50 μg/mL lactate dehydrogenase, and 200 μM EGF receptor peptide (KRELVEPLTPSGEAPNQALLR). The reaction is initiated with 100 μM and 70 μM ATP for p38α and p38β assays, respectively. The decrease of absorbance at 340 nm is monitored to follow the rate of the reaction. IC50 is evaluated from the rate data as a function of the inhibitor concentration.

BMSCs (5 × 104 cells/well) or MM cells (3 × 104 cells/well) are incubated in 96-well culture plates in the presence or absence of VX-745 for 48 hours at 37 °C. DNA synthesis is measured by [3H]-thymidine ([3H]TdR) uptake. Cells are pulsed with [3H]TdR (0.5 μCi/well [.0185 MBq]) during the last 8 hours of 48-hour cultures. Growth inhibition of both MM cells and BMSCs by VX-745 is also assessed by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye absorbance.(Only for Reference)