Donafenib (Sorafenib D3)

BAY 43-9006 suppresses both wild-type and V599E mutant BRAF activity in vitro. In addition, BAY 43-9006 demonstrates significant activity against several receptor tyrosine kinases involved in neovascularization and tumor progression, including vascular endothelial growth factor receptor (VEGFR)-2, VEGFR-3, platelet-derived growth factor receptor β, Flt-3, and c-KIT. In cellular mechanistic assays, BAY 43-9006 demonstrates inhibition of the mitogen-activated protein kinase pathway in colon, pancreatic, and breast tumor cell lines expressing mutant KRAS or wild-type or mutant BRAF, whereas non–small-cell lung cancer cell lines expressing mutant KRAS are insensitive to inhibition of the mitogen-activated protein kinase pathway by BAY 43-9006. Potent inhibition of VEGFR-2, platelet-derived growth factor receptor β, and VEGFR-3 cellular receptor autophosphorylation is also observed for BAY 43-9006.^[1]

Tumor cell lines are plated at 2 × 10⁵ cells per well in 12-well tissue culture plates in DMEM growth media (10% heat-inactivated FCS) overnight. Cells are washed once with serum-free media and incubated in DMEM supplemented with 0.1% fatty acid-free BSA containing various concentrations of Donafenib (Sorafenib D3, BAY 43-9006) in 0.1% DMSO for 120 minutes to measure changes in basal pMEK 1/2, pERK 1/2, or pPKB. Cells are washed with cold PBS (PBS containing 0.1 mmol/L vanadate) and lysed in a 1% (v/v) Triton X-100 solution containing protease inhibitors. Lysates are clarified by centrifugation, subjected to SDS-PAGE, transferred to nitrocellulose membranes, blocked in TBS-BSA, and probed with anti-pMEK 1/2 (Ser²¹⁷/Ser²²¹; 1:1000), anti-MEK 1/2, anti-pERK 1/2 (Thr²⁰²/Tyr²⁰⁴; 1:1000), anti-ERK 1/2, anti-pPKB (Ser⁴⁷³; 1:1000), or anti-PKB primary antibodies. Blots are developed with horseradish peroxidase (HRP)-conjugated secondary antibodies and developed with Amersham ECL reagent on Amersham Hyperfilm.

Once daily oral dosing of BAY 43-9006 demonstrates broad-spectrum antitumor activity in colon, breast, and non–small-cell lung cancer xenograft models. Immunohistochemistry demonstrates a close association between inhibition of tumor growth and inhibition of the extracellular signal-regulated kinases (ERKs) 1/2 phosphorylation in two of three xenograft models examined, consistent with inhibition of the RAF/MEK/ERK pathway in some but not all models. Additional analyses of microvessel density and microvessel area in the same tumor sections using antimurine CD31 antibodies demonstrates significant inhibition of neovascularization in all three of the xenograft models.^[1]