Desmethyl Erlotinib (CP-473420)

EGFR

379, 41

Male 5-week-old BALB-nu/nu with HPAC

Phase 1 Study of Erlotinib Prior to Surgery in Patients With Head and Neck Cancer is ongoing.

50 mg/kg

2 nM[1], 2 nM[1], 2 nM[1], 2 nM[1], 2 nM[1], 2 nM[1]

At doses of 100 mg/kg, Erlotinib HCl completely prevents EGF-induced autophosphorylation of EGFR in human HN5 tumors growing as xenografts in athymic mice and of the hepatic EGFR of the treated mice.[2] Erlotinib HCl (100 mg/Kg) inhibits H460a and A549 tumor models with 71 and 93% inhibition rate.[6]

[2], Kinase assays, 96-well plates are coated by incubation overnight at 37 C with 100 uL per well of 0.25 mg/mL PGT in PBS. Excess PGT is removed by aspiration, and the plate is washed 3 times with washing buffer (0.1% Tween 20 in PBS). The kinase reaction is performed in 50 uL of 50 mM HEPES (pH 7.3), containing 125 mM sodium chloride, 24 mM magnesium chloride, 0.1 mM sodium orthovanadate, 20 uM ATP, 1.6 ug/mL EGF, and 15 ng of EGFR, affinity purified from A431 cell membranes. Erlotinib HCl in DMSO is added to give a final DMSO concentration of 2.5%. Phosphorylation is initiated by addition of ATP and proceeded for 8 minutes at room temperature, with constant shaking. The kinase reaction is terminated by aspiration of the reaction mixture and is washed 4 times with washing buffer. Phosphorylated PGT is measured by 25 minutes of incubation with 50 uL per well HRP-conjugated PY54 antiphosphotyrosine antibody, diluted to 0.2 ug/mL in blocking buffer (3% BSA and 0.05% Tween 20 in PBS). Antibody is removed by aspiration, and the plate is washed 4 times with washing buffer. The colonmetric signal is developed by addition of TMB Microwell Peroxidase Substrate, 50uL per well, and stopped by the addition of 0.09 M sulfuric acid, 50 uL per well. Phosphotyrosine is estimated by measurement of absorbance at 450 nm. The signal for controls is typically 0.6-1.2 absorbance units, with essentially no back ground in wells without AlP, EGFR, or PGT and is proportional to the time of incubation for 10 minutes.

DMSO 11 mg/mL, Water <1 mg/mL, Ethanol 5 mg/mL

2-(4-(3-ethynylphenylamino)-7-(2-methoxyethoxy)quinazolin-6-yloxy)ethanol