Erlotinib (CP358774) is a first-generation EGFR inhibitor that inhibits the EGFR 19 Del and L858R mutations. Erlotinib has antitumor activity and is used for the treatment of EGFR-mutated non-small-cell lung cancers. Erlotinib administration results in the development of the EGFR C797S resistance mutation.

EGFR:2 nM (cell free)

方法：人非小细胞肺癌细胞 PC9 用 Erlotinib (0.0002-2 µM) 处理 72 h，检测细胞生存曲线。
结果：Erlotinib 抑制 PC9细胞生长，IC50 为 30 nM。[1]
方法：人头颈癌细胞 HN5 用 Erlotinib (20-1000 nM) 处理 1 h，随后用 EGF (50 ng/mL) 刺激 5 min，使用 Western Blot 检测靶点蛋白表达水平。
结果：Erlotinib 抑制人头颈部肿瘤细胞中 EGFR 自磷酸化。[2]

方法：为测试体内抗肿瘤活性，将 Erlotinib (2.9-92 mg/kg，sterile+pyrogen-free 10% DMSO+0.85% sodium chloride+0.1% Pluronic P105) 灌胃给药给携带人头颈癌肿瘤 HN5 或人鳞状细胞癌肿瘤 A431 的 CD-1 nu/nu 小鼠，每天一次，持续二十天。
结果：Erlotinib 抑制 HN5 或 A431 的肿瘤生长。[3]

96-well plates are coated by incubation overnight at 37 °C with 100 μL 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 μL of 50 mM HEPES (pH 7.3), containing 125 mM sodium chloride, 24 mM magnesium chloride, 0.1 mM sodium orthovanadate, 20 μM ATP, 1.6 μg/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 μL per well HRP-conjugated PY54 anti-phosphotyrosine antibody, diluted to 0.2 μg/mL in blocking buffer (3% BSA and 0.05% Tween 20 in PBS). The antibody is removed by aspiration, and the plate is washed 4 times with washing buffer. The colorimetric signal is developed by addition of TMB Microwell Peroxidase Substrate, 50μL per well, and stopped by the addition of 0.09 M sulfuric acid, 50 μL 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 background in wells without AlP, EGFR, or PGT and is proportional to the time of incubation for 10 minutes [1].

Exponentially growing cells are seeded in 96-well plastic plates and exposed to serial dilutions of erlotinib (30 nM-20 μM), pemetrexed, or the combination at a constant concentration ratio of 4:1 in triplicates for 72 h. Cell viability is assayed by cell count and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Growth inhibition is expressed as the percentage of surviving cells in drug-treated versus PBS-treated control cells (which is considered as 100% viability). The IC50 value is the concentration resulting in 50% cell growth inhibition by a 72-h exposure to the drug(s) compared with untreated control cells and is calculated by the CalcuSyn software [4].

Six-week-old male SD rats weighing 180 to 210 g are used. Cisplatin (CP) is freshly prepared in saline at a concentration of 1 mg/mL and then injected intraperitoneally in SD rats (n=28) at a dose of 7 mg/kg on day 0. To investigate the effect of Erlotinib, 28 CP-N rats are divided into two groups. Separate groups (n=14) each of animals are administered with either Erlotinib (20 mg/kg) (CP+E, n=14) or vehicle (CP+V, n=14) daily by oral gavage from the day -1 (24 hours prior to the CP injection) to day 3. Vehicle-treated groups receive an equivalent volume of saline. Five male SD rats at the age of 6 weeks are used as a normal control group (NC, n=5). The NC rats are given an equivalent volume of saline daily by oral gavage from the day -1 to day 3. At day 4 (96 hours after CP injection), each rat is anesthetized and sacrificed by exsanguination after the cardiac puncture; blood is collected by cardiac puncture and kidneys are collected. Renal tissue is divided; separate portions are snap-frozen in liquid nitrogen or fixed in 2% paraformaldehyde/phosphate-buffered saline (PBS) for later use. All surgery is performed under diethyl ether gas anesthesia, and all efforts are made to minimize suffering [2].