Osimertinib (AZD9291)

AZD9291 shows significantly more potent inhibition of proliferation in mutant EGFR cell lines compared to wild-type in vitro. ^[2]

AZD9291(5mg/kg p.o.) causes profound regression of tumors across EGFRm+ (PC9) and EGFRm+/T790M (H1975) tumor models with profound inhibition of EGFR phosphorylation and key downstream signaling pathways such as AKT and ERK in vivo. ^[2]

• EGFR cellular phosphorylation assay

  Cells are seeded (10000 cells/well) in growth medium in Corning black, clear- bottomed 384-well plates and incubated at 37°C with 5% CO₂ overnight. Cells are acoustically dosed using an Echo 555, with compounds serially diluted in 100% DMSO. Plates are incubated for a further 2h, then following aspiration of medium, 40μL lx lysis buffer is added to each well. Greiner black high bind 384-well plates are coated with capture antibody and then blocked with 3% BSA. Following removal of block, 15μL of lysate are transferred to the Greiner black high bind 384-well plates and incubated for 2 hours. Following aspiration and washing of the plates with PBS, 20μL, of detection antibody were added and incubated for 2 hours. Following aspiration and washing of the plates with PBS, 20μL of QuantaBlu fluorogenic peroxidase substrate are added and incubated for 1 hour. 20μL QuantaBlu stop solution are added to plates and fluorescence read on an Envision plate reader using Excitation 352nm wavelength and emission 460nm wavelength. The data obtained with each compound is exported into a suitable software package to perform curve fitting analysis. From this data an IC50 value is determined by calculation of the concentration of compound that is required to give a 50% effect.

• Cell lines

  PC9 cells

• Concentrations

  10, 50, & 100 nM

• Incubation Time

  24 h

• Method

  Cells were treated with increasing concentrations of osimertinib for 24h.

• Animal Models

  Mice bearing PC9 and H1975 xenograft tumors

• Dosages

  ~5 mg/kg

• Administration

  p.o.