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

Syk:IC50: 7.7 nM

In cells, GS-9973 also displays excellent specificity for Syk, and effectively inhibits BCR-mediated activation and proliferation of B-cells, as well as immune-complex-stimulated cytokine production in monocytes. In Caco-2 cell, GS-9973 exhibits good bidirectional permeability across cell monolayers.

In rat and dog, Entospletinib (1 mg/kg) by oral administration shows moderate to high bioavailability. In a rat collagen-induced arthritis model, Entospletinib (1-10 mg/kg) by oral administrations significantly suppress ankle inflammation. Entospletinib(ED50 =1.2 -3.9 mg/kg) exhibits disease-modifying activity such as pannus formation, cartilage damage, bone resorption, and periosteal bone formation.

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.