R406 (free base)

SYK

470, 45

Female C57BL/6 mice challenged intravenously with 1% ovalbumin (OVA) in saline (10 mg/kg) containing 1% Evans blue dye, female Balb/c mice with the anticollagen antibody-induced arthritis, and female C57BL/6 mice with arthritis induced by intraperitoneal injection of pooled sera from adult K/BxN mice

A number of studies examining the pharmacokinetics and safety of the prodrug formulation of R406 have been completed. Presently, the compound remains active in Phase I clinical evaluation to assess its bioavailability.

ca.10 mg/kg/day

41 nM [1], 41 nM [1], 41 nM [1], 41 nM [1], 41 nM [1], 41 nM [1]

R406 has shown efficacy in a number of animal models of immune disorders. Oral administration of R406 in mice with immune complex-mediated inflammation significantly inhibits the cutaneous reverse passive Arthus reaction by approximately 72% and 86% at 1 mg/kg and 5 mg/kg, respectively, compared with the control. R406 treatment at 10 mg/kg significantly reduces inflammation and swelling, decreases the progressive arthritis to a lower level in the passive anticollagen antibody-challenged mice, and delays the onset and reduces paw thickening and clinical arthritis by approximately 50% in the K/BxN serum transfer mice model. [1]

In-vitro Fluorescence Polarization Kinase Assays, 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 uL containing 5 uM HS1 peptide substrate and 4 uM 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 uL 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 in duplicate and curve-fitting is performed by non-linear regression analysis using Prism GraphPad Software.

DMSO 29 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL

6-(5-fluoro-2-(3, 4, 5-trimethoxyphenylamino)pyrimidin-4-ylamino)-2, 2-dimethyl-2H-pyrido[3, 2-b][1, 4]oxazin-3(4H)-one