Roxadustat [808118-40-3]

Roxadustat (FG-4592) is an orally bioavailable, hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI), with potential anti-anemic activity.

FG-4592 (25 mg/kg) enhances mitophagy and attenuates retinal histopathologic damage in the photoreceptor layer after RD in an RD animal model[1].

Kinase assays: Potencies (IC50 values) are determined by assays of active kinase domains cloned and expressed in baculovirus using the FastBacbaculovirus expression system or obtained commercially. For tyrosine kinase assays, a biotinylated peptide substrate containing a single tyrosine is used with 1 mM ATP, anEu-cryptate–labeled anti-phosphotyrosine antibody (PT66), and Strepavidin-APC in a homogeneous time-resolved fluorescence assay. Serine/threonine kinases are assayed using 5 μM ATP, [33P]ATP, and a biotinylated peptide substrate with peptide capture and incorporation of 33P determined using a SA-Flashplate. Linifanib is assayed at multiple concentrations prepared by serial dilution of a DMSO stock solution of Linifanib. The concentration resulting in 50% inhibition of activity is calculated using nonlinear regression analysis of the concentration response data.

The left eye of each rat is retro-orbitally injected with 25 mg/kg FG-4592 every 2 days, and an equal volume of 20% DMSO diluted with 0.9% NaCl is administrated with retro-orbital injection and served as the control. The retinae (attached, 1 day, 3 days, 5 days, and 7 days after RD) are homogenized and lysed with buffer containing 50 mM hydroxymethyl(Tris)-aminomethane(HCl), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and a protease inhibitor tablet. Samples are run on 8% to 12% 2-hydroxyethyl (Bis)-hydroxymethyl (Tris) gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (0.2-mm pores). After blocking with 3% nonfat dried milk, the membranes are incubated overnight with primary antibody HIF-1α, LC3, BNIP3, autophagy-related gene 5 (Atg5), and β-actin. The blotted membranes are then incubated for 60 minutes at room temperature with a horseradish peroxidase (HRP)-labeled secondary antibody. Immunoreactive bands are visualized by enhanced chemiluminescence (ECL) and detected with an Amersham Imager 600. A minimum of three rats are used for each condition.

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

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

DMSO: 240 mg/mL (681.16 mM), Sonication is recommended.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 5 mg/mL (14.19 mM), Sonication is recommended.