Entospletinib (GS-9973) is a Syk inhibitor (IC50=7.7 nM) with selective, oral activity. Entospletinib has potential value in the treatment of a variety of hematological malignancies and immune-related diseases.

Ramos cells:2 nM (EC50), HUVEC:> 1000 nM (EC50), MV4-11 cells:327 nM (EC50), Bone marrow cells:582 nM, Syk:7.7 nM, TF-1 cells:453 nM (EC50)

METHODS: NALM-6 and SEM cell lines were treated with Entospletinib (1, 5, and 10 μM) for 72 hours, and the expression levels of target proteins were detected using Western Blot.
RESULTS: The expressions of key proteins including pAKT, pERK, pGSK3β, p53 and BCL-6 were significantly affected by Entospletinib. [1]

METHODS: To investigate the anti-inflammatory activity of Entospletinib, Entospletinib (100 mg/kg) was administered orally twice daily to K/BxN serum-induced arthritis mice.
RESULTS: Entospletinib significantly reduced macroscopic manifestations of joint inflammation, including joint swelling and clinical scores. [2]
METHODS: To study the anti-inflammatory activity of Entospletinib, Entospletinib (10 mg/kg) was administered orally to a rat CIA model.
RESULTS: Entospletinib showed significant anti-inflammatory effect. [3]

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.