Entospletinib (GS-9973) (IC50= 7.7 nM)is a specific Syk inhibitor, which is orally bioavailable.

Syk:IC50: 7.7 nM

在细胞内，GS-9973对Syk展现出极高的特异性，并有效抑制BCR介导的B细胞激活与增殖，同时抑制免疫复合物刺激的单核细胞中的细胞因子产生。在Caco-2细胞中，GS-9973显示出良好的双向跨细胞单层的渗透性。

在大鼠和狗的实验中，通过口服给予Entospletinib (1 mg/kg) 显示出中等至高的生物利用度。在大鼠胶原诱导的关节炎模型中，Entospletinib (1-10 mg/kg) 通过口服给药明显抑制踝部炎症。Entospletinib(ED50=1.2-3.9 mg/kg) 表现出改变疾病进程的活性，如滑膜炎形成、软骨损伤、骨吸收及骨膜下骨形成等。

In the determination of full-length baculovirus expressed Syk kinase activity, the reaction system was 25 μL containing 25 mM Tris-HCl, pH 7.5, 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2, 0.5 μM Promega PTK Biotin Peptide Substrate 1, 0.01% casein, 1, 0.01% Triton X-100, 0.25% Glycerol, and 40 mM ATP (Km for ATP). After incubation for 60 minutes at room temperature, the reaction was stopped by adding 30 mM EDTA (30 μL of SA-APC and 4 nM PT-66 antibody). After measuring the plate, the IC50 value of the test compound was calculated using a 4-parameter linear regression algorithm.

In MV-4-11 cells, the functional effect of the compound on the cellular Flt3 activity was determined by inhibition of cell proliferation. Cells were diluted in 96 well flat bottom tissue culture plates in RPMI medium containing 10% FBS and compound dilutions were added and incubated at 37°C for 72 hours. Aramazol (10%) was added to the cells and the cells were incubated for another 12-18 hours at 37°C. Finally, the inhibition of the relative cell number was measured at 570/600 nm in the spectrophotometer.

In the rat collagen-induced arthritis (CIA) model, Entospletinib (10 mg/kg), which is dissolved in hydrogenated castor oil,ethanol or physiological saline, is taken by oral.