Fostamatinib

Fostamatinib (R788) is a small-molecule inhibitor and an oral prodrug of R406, a Syk/FLT3-targeted inhibitor (IC50 = 41 nM) that also inhibits Lyn (IC50 = 63 nM) and Lck (IC50 = 37 nM), featuring oral activity and cell permeability, used for the treatment of immune thrombocytopenia.

Syk/FLT3:30 nM (Ki), Lck:37 nM, Syk:41 nM, Lyn:63 nM

Methods: In in vitro experiments, monocytes isolated from healthy controls were co-incubated with R406 (the active metabolite of fostamatinib, 2 µM) for 24 hours, and erythrophagocytosis and TNF-α production were measured.
Results: R406 significantly reduced monocyte phagocytosis of IgG-coated erythrocytes (from 58.8% to 5.6%) and strongly inhibited TNF-α production induced by heme or erythrocytes from AIHA patients. [1]
Methods: HK, K30, and TE1 cells were treated with 10 μM Fostamatinib for 24–48 hours, and assays including CCK-8, Transwell, scratch wound healing, and Western blot were performed.
Results: Fostamatinib significantly inhibited cell proliferation, invasion, migration, and LYN protein expression. [2]

Methods: In Louvain rats, a single oral dose of Fostamatinib (R788) at 10 mg/kg or 20 mg/kg was administered, plasma concentrations of the active metabolite R406 were measured, and pharmacokinetic parameters including AUC0-16 hrs, Cmax, and half-life were calculated; the presence of the prodrug in plasma was also monitored.
Results: At 10 mg/kg and 20 mg/kg doses, the AUC0-16 hrs of R406 were 10618 ng·h/mL and 30650 ng·h/mL, respectively; Cmax values were 2600 ng/mL and 6500 ng/mL (both reached at 1 hour post-dose), with half-lives of 4.2 hours for both doses; the prodrug R788 was not detected in plasma, indicating complete conversion to R406. [3]

Fluorescence polarization kinase assay and Ki determination: The fluorescence polarization reactions are performed. For Ki determination, duplicate 200-μL reactions are set up at eight different ATP concentrations from 200 μM (2-fold serial dilutions) in the presence of either DMSO or R788 at 125, 62.5, 31.25, 15.5, or 7.8 nM. At different time points, 20 μL of each reaction is removed and quenched to stop the reaction. For each concentration of R788, the rate of reaction at each concentration of ATP is determined and plotted against the ATP concentration to determine the apparent Km and Vmax (maximal rate). Finally the apparent Km (or apparent Ki/Vmax) is plotted against the inhibitor concentration to determine the Ki.

Cultured human mast cells (CHMC) are derived from cord blood CD34+ progenitor cells and grown, primed, and stimulated and shown in supplemental data. Before stimulation, cells are incubated with R788 or DMSO for 30 minutes. Cells are then stimulated with either 0.25 to 2 mg/mL anti-IgE or anti-IgG or 2 μM ionomycin. For tryptase measurement, ∼1500 cells per well are stimulated for 30 min in modified Tyrode's buffer. For LTC4 and cytokine production, 100,000 cells per well are stimulated for 1 or 7 hours, respectively. Tryptase activity is measured by luminescence readout of a peptide substrate, and LTC4 and cytokines are measured using Luminex multiplex technology. (Only for Reference)

H2O: < 1 mg/mL (insoluble or slightly soluble)

Ethanol: < 1 mg/mL (insoluble or slightly soluble)

DMSO: 127.5 mg/mL (219.65 mM), Sonication is recommended.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 4 mg/mL (6.89 mM), Sonication is recommended.