| Protocol | In Vitro | In vitro activity: GS-9973 shows good bidirectional permeability across Caco-2 cell monolayers in vitro. In cells, GS-9973 also shows excellent selectivity for Syk, and potently inhibits BCR-mediated activation and proliferation of B-cells as well as immune-complex-stimulated cytokine production in monocytes. The combination of idelalisib and GS-9973 synergistically inhibits CLL cell viability and further disrupts chemokine signaling. Kinase Assay: Full-length baculovirus-expressed Syk kinase activity is measured in a Lance-based assay format in a final volume of 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 biotinylated peptide substrate 1, 0.01% casein, 0.01% Triton X-100, 0.25% glycerol, and 40 mM ATP (Km for ATP) incubated at room temperature for 60 min. Reactions are stopped with the addition of 30 mM EDTA containing 30 μL of SA-APC and 4 nM PT-66 antibody and the plates measured on a Perkin-Elmer Envision. IC50 values for test compounds are determined using a four-parameter linear regression algorithm. Cell Assay: Functional impact on cellular Flt3 activity is determined by measuring compound inhibition of MV-4-11 cell proliferation. A total of 104 cells are diluted in RPMI medium containing 10% FBS in 96-well flat-bottomed tissue culture plates and incubated with compound dilutions for 72 h at 37 °C. Alamar blue (10%) is added to the cells, which were incubated for an additional 12-18 h at 37 °C, and inhibition of the relative cell numbers is determined by spectrophotometer readings at 570/600 nm. |
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| In Vivo | GS-9973 (1 mg/kg p.o.) shows moderate to high bioavailability in rat and dog. In a rat collagen-induced arthritis model, GS-9973 (1-10 mg/kg p.o.) significantly inhibits ankle inflammation. Moreover, GS-9973 also shows disease-modifying activity in multiple histological measurements, including inhibition of pannus formation, cartilage damage, bone resorption, and peritosteal bone formation with ED50 ranging from 1.2 to 3.9 mg/kg. |
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| Animal model | Rat Collagen-Induced Arthritis (CIA) Model |
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| Formulation | Dissolved in Cremophor/ethanol/saline |
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| Dosages | 10 mg/kg; oral administration |
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These protocols are for reference only. InvivoChem does not
independently validate these methods.
Preparing Stock Solutions
| Solvent volume to be added |
Mass (the weight of a compound) |
| Mother liquor concentration |
1mg | 5mg | 10mg | 20mg |
| 1mM | 2.4304 mL | 12.1518 mL | 24.3037 mL | 48.6074 mL |
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| 5mM | 0.4861 mL | 2.4304 mL | 4.8607 mL | 9.7215 mL |
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| 10mM | 0.2430 mL | 1.2152 mL | 2.4304 mL | 4.8607 mL |
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| 20mM | 0.1215 mL | 0.6076 mL | 1.2152 mL | 2.4304 mL |
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Quality Control Documentation
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