Description

Cancer

Description

R406 is a potent spleen tyrosine kinase (Syk) inhibitor with EC50 and K_i of 56 nM and 30 nM, respectively.

Targets

IC₅₀

In Vitro

R406 is a potent inhibitor of immunoglobulin E (IgE)- and IgG-mediated activation of Fc receptor signaling. R406 inhibits the anti-IgE-induced production and release of LTC4 and cytokines and chemokines, including TNFα, IL-8, and GM-CSF. R406 inhibits phosphorylation of Syk substrate linker for activation of T cells in mast cells and B-cell linker protein/SLP65 in B cells. R406 binds to the ATP binding pocket of Syk and inhibits its kinase activity as an ATP-competitive inhibitor with K_i of 30 nM. R406 blocks Syk-dependent FcR-mediated activation of monocytes/macrophages and neutrophils and Bcr-mediated activation of B lymphocytes. ^[1] R406 significantly induces chronic lymphocytic leukemia (CLL) cell apoptosis in nurselike cells cocultures and blocks CCL3 and CCL4 secretion by CLL cells in response to B-cell antigen receptor (Bcr) triggering. ^[2] R406 is a potent inhibitor of platelet signaling and functions initiated by FcγRIIA cross-linking by specific antibodies or by sera from HIT patients. ^[3]

In Vivo

R406 reduces cutaneous reverse passive Arthus reaction by approximately 86% at 5 mg/kg in prophylactic treated mice. R406 also shows efficacy in inhibiting paw inflammation in antibody-induced arthritis mouse models. ^[1] R406 does not adversely affect macrophage or neutrophil function in innate immune responses and has minimal functional immunotoxicity notwithstanding its lymphocytopenic effect. ^[4]

Clinical Trials

R406’s prodrug, Fostamatinib, is completed in Phase I clinical trials in subjects with rheumatoid arthritis or renal impairment.

Features

Lead drug candidate for rheumatoid arthritis

In Vitro Fluorescence Polarization Kinase Assay

R406 is serially diluted in DMSO and then diluted to 1% DMSO in kinase buffer (20 mM HEPES, pH 7.4, 5 mM MgCl₂, 2 mM MnCl₂, 1 mM DTT, 0.1 mg/mL acetylated BGG). ATP and substrate in kinase buffer are added at room temperature, resulting in a final DMSO concentration on 0.2%. The kinase reactions are performed in a final volume of 20?mL containing 5?mM HS1 peptide substrate, 4?mM ATP and started by addition of 0.125 ng of Syk in kinase buffer. The reaction is allowed to proceed for 40 minutes at room temperature. The reaction is stopped by the addition of 20?mL of PTK quench mix containing EDTA/anti-phosphotyrosine antibody (1X final)/fluorescent phosphopeptide tracer (0.5X final) diluted in FP Dilution Buffer. The plate is incubated for 30 minutes in the dark at room temperature and then read on a Polarion fluorescence polarization plate reader. Data are converted to amount of phosphopeptide present using a calibration curve generated by competition with the phosphopeptide competitor provided in the Tyrosine Kinase Assay Kit. For IC50 determination, R406 is tested at eleven concentrations and curve-fitting is performed by non-linear regression analysis.

Ki Determination

For K_i 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 R406 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 R406, the rate of reaction at each concentration of ATP is determined and plotted against the ATP concentration to determine the apparent K_m and V_max (maximal rate). Finally the apparent K_m (or apparent K_m/V_max) is plotted against the inhibitor concentration to determine the K_i.

Animal Models

Arthritis is induced in C57BL/6 mice by intraperitoneal injection of 150 μL of pooled sera from adult K/BxN mice.

Formulation

35% TPGS, 60% PEG 400, and 5% propylene glycol

Doses

1 or 5 mg/kg

Administration

Administered orally