Erlotinib hydrochloride (NSC 718781) is an EGFR inhibitor (IC50: 2 nM). It is used for the treatment of non-small cell lung cancer.

EGFR:2 nM (cell free)

Erlotinib 是一种直接作用于人类EGFR酪氨酸激酶的抑制剂（IC50为2nM），能够显著降低完整肿瘤细胞中EGFR的自磷酸化作用（IC50为20nM）。同时，Erlotinib 对重组的EGFR细胞内（激酶）域也是一种有效抑制剂（IC50为1nM）。DiFi细胞的增殖受到Erlotinib 的强力抑制，在为期8天的增殖试验中，IC50达到100nM[1]。

Erlotinib（20 mg/kg，口服）显著减轻顺铂(CP)所引起的大鼠体重(BW)损失，与CP+载体(V)组相比(P<0.05)。Erlotinib 治疗明显改善了CP-N（正常对照组，NC）大鼠的肾功能。与CP+V大鼠相比，CP+E（Erlotinib ）组大鼠的血清肌酐(s-Cr)水平、血尿素氮(BUN)、尿N-乙酰-β-D-葡萄糖胺酶(NAG)指数显著降低（P<0.05），且尿量(UV)及肌酐清除率(Ccr)显著提高（P<0.05）[2]。Erlotinib 在小鼠体内显著抑制人头颈癌HN5肿瘤异种移植物的生长，ED50值为9 mg/kg [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].