Binding assays

The kinase reaction was performed in50 μl of 50 mM HEPES(pH 7.3), containing 125 mM NaCl, 24 mM MgCl2, 0.1 mM sodium orthovanadate, 20 μM ATP, 1.6 μg/ml EGF, and 15 ng of EGFR. The compound in DMSO was added to give a final DMSO concentration of 2.5%. Phosphorylation was initiated by addition of ATP and proceeded for 8 min at room temperature with constant shaking. The kinase reaction was terminated by aspiration of the reaction mixture and was washed 4 times with wash buffer. Phosphorylated POT was measured by 25 min of incubation with 50 μl per well HRP-conjugated PY54 antiphosphotyrosine antibody, diluted to 0.2 μg/mI in blocking buffer (3% BSA and 0.05% Tween 20 in PBS). Removed antibody by aspiration and washed the plate 4 times with wash buffer. The colonmetric signal was developed by addition of TMB Microwell Peroxidase Substrate, 50 μl per well, and stopped by the addition of 0.09 M sulfuric acid, 50 μ1 per well. Phosphotyrosine is estimated by measurement of absorbance at 450 nm.

Cell lines

Human NSCLC cell lines H322, A549, H1650, and H1975 cells.

Preparation method

The solubility of this compound in DMSO is >19.7mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

2 μmol/L, 24h

Applications

Erlotinib alone induced G1-phase arrest in ~80% H322 cells. Erlotinib (2 μM) significantly inhibited growth of AsPC-1 and BxPC-3 pancreatic cells. Erlotinib potently inhibited EGFR activation in HNS human head and neck tumor cells, DiFi human colon cancer cells and MDA MB-468 human breast cancer cells. Erlotinib (1 μM) induced apoptosis in DiFi human colon cancer cells.

Animal models

H460a and A549 tumor models

Dosage form

100 mg/kg

Application

Erlotinib (100 mg/kg) exhibited antitumor activity at the MTD in both the H460a and A549 tumor models.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1]. Moyer J D, Barbacci E G, Iwata K K, et al. Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase[J]. Cancer research, 1997, 57(21): 4838-4848.

[2]. Li T, Ling Y H, Goldman I D, et al. Schedule-dependent cytotoxic synergism of pemetrexed and erlotinib in human non–small cell lung cancer cells[J]. Clinical Cancer Research, 2007, 13(11): 3413-3422.

[3]. Ali S, Banerjee S, Ahmad A, et al. Apoptosis-inducing effect of erlotinib is potentiated by 3, 3′-diindolylmethane in vitro and in vivo using an orthotopic model of pancreatic cancer[J]. Molecular cancer therapeutics, 2008, 7(6): 1708-1719.

[4]. Higgins B, Kolinsky K, Smith M, et al. Antitumor activity of erlotinib (OSI-774, Tarceva) alone or in combination in human non-small cell lung cancer tumor xenograft models[J]. Anti-cancer drugs, 2004, 15(5): 503-512.