Combretastatin A-1 phosphate (OXi-4503) tetrasodium, a prodrug of Combretastatin A-1, is a tubulin-binding microtubule polymerization inhibitor targeting the colchicine-binding site. By inducing tubulin depolymerization, it inhibits the Wnt/β-catenin pathway and deactivates AKT, possessing both anti-tumor and anti-vascular properties.

Combretastatin A-1 phosphate effectively inhibits growth in a variety of tumor cell lines in vitro, including HepG2, SMMC-7721, Hepa 1-6, LM-3, Bel-7402, Huh7, BGC-803, MDA-MB-231, MCF-7, A375, NCI-1975, CT-26, HT-29, and A549 cells, with IC50 values ranging from 9.2 to 2247.0 nM after a treatment duration of 72 hours. At concentrations between 1 and 10 nM over 24 hours, it initiates apoptosis in HepG2 cells through the disruption of microtubules leading to AKT inactivation and subsequent removal of GSK-3β inhibition. Additionally, within 6 hours at concentrations of 1 to 50 nM, it decreases the mitochondrial membrane potential (MMP) and induces dose-dependent ROS accumulation in HepG2 cells. Western blot analysis confirms that at concentrations of 1, 5, and 10 nM, treated for 24 hours, there is a significant decrease in Mcl-1 expression level in HepG2 cells with unchanged Bcl-2 levels, a reduction in phospho-GSK 3β (Ser9) indicating GSK-3β activation, and lowered AKT phosphorylation at Ser473 without a change in total AKT protein levels.

Combretastatin A-1 phosphate administered intravenously (i.v.) at doses of 1-4 mg/kg every other day for four weeks significantly reduced tumor volume in a HepG2 subcutaneous xenograft model, with marked effects observed at 2 mg/kg or 4 mg/kg doses. Additionally, a dose of 2 mg/kg administered every other day for 21 days demonstrated enhanced apoptosis in an orthotopic hepatocellular carcinoma mouse model. This was tested using male athymic BALB/c nu/nu mice, ranging from 16-18 g and aged between 4-6 weeks, which were inoculated with HepG2 cells.