Osimertinib mesylate (AZD-9291 mesylate) is an EGFR third-generation inhibitor that inhibits the T790M resistance mutation produced by second-generation EGFR inhibitors with irreversible and oral activity. Osimertinib mesylate has antitumor activity for the treatment of EGFR-mutated non-small-cell lung cancer.

EGFR (L858R/T790M):1 nM (cell free), EGFR (L858R):12 nM (cell free)

方法: 人非小细胞肺癌细胞 PC-9 (exon 19del)、H3255 (L858R)、PC-9ER (exon 19del+T790M) 和 H1975 (L858R+T790M) 用 Osimertinib mesylate (0.0001-10 µmol/L) 处理 72 h，使用 MTS 方法检测细胞生长抑制情况。
结果: Osimertinib 剂量依赖诱导 PC-9、H3255、PC-9ER 和 H1975 细胞生长，IC50 分别为 41、26、41 和 31 nM。[1]
方法: EGFR 突变的人非小细胞肺癌细胞 PC-9、H1975、H1650 和 H3255 用 Osimertinib mesylate (0.1-1000 nM) 处理 6 h，使用 Western Blot 方法检测靶点蛋白表达水平。
结果: Osimertinib 抑制 EGFR 突变肿瘤细胞的 pEGFR(Y1068)、pERK(P-p44/42)、pAKT(S473)。[2]
方法: 人非小细胞肺癌细胞 NCI-H1975 用 Osimertinib mesylate (10-100 nM) 处理 24 h，然后用 2-20 Gy 的剂量照射，使用 Flow Cytometry 方法分析细胞周期情况。
结果: 联合治疗组 G2/M 和 S 期细胞比例呈剂量依赖性降低。Osimertinib 减少照射后 G2/M 期细胞周期停滞。[3]

方法: 为检测体内抗肿瘤活性，将 Osimertinib mesylate (5-10 mg/kg) 口服给药给携带人肺癌肿瘤 H1975、PC9 和 A431 的 SCID 小鼠，每天一次，持续七天。
结果: Osimertinib 治疗显著抑制 H1975、PC9 和 A431 肿瘤的生长，表明在体内具有抗肿瘤活性。[4]
方法: 为检测体内抗肿瘤活性，将 Osimertinib mesylate (6 mg/kg) 口服给药给用 PC-9/ffluc 细胞构建软脑膜癌病 (LMC) 模型的 SHO-SCID 小鼠，每天一次，持续五十天。
结果: Osimertinib 治疗明显延缓了 LMC 的发展。第三代 EGFR-TKI Osimertinib 可能是由 EGFR 突变型肿瘤引起的第一代或第二代 EGFR-TKI 耐药性 LMC 的有效治疗方法。[5]

Kinase assays were performed as per the EMD Millipore profiling service protocol using peptide or protein substrates in a filter-binding radioactive ATP transferase assay for protein [1].

PC-9 cells were seeded into T75 flasks (5 × 10^5 cells/flask) in RPMI growth media and incubated at 37°C, 5% CO2. The following day the media was replaced with media supplemented with a concentration of EGFR inhibitor equal to the EC50 concentration predetermined in PC-9 cells. Media changes were carried out every 2-3 days and resistant clones allowed to grow to 80% confluency prior to the cells being trypsinised and reseeded at the original seeding density in media containing twice the concentration of EGFR inhibitor. Dose escalations were continued until a final concentration of 1.5μM gefitinib, 1.5μM afatinib, 1.5μM WZ4002 or 160nM AZD9291 were achieved [1].

The generation of EGFRL858R and EGFRL858R+T790M mice (male and female) was previously described Doxycycline was administered by feeding mice (aprox 3 week old) with doxycycline-impregnated food pellets (625 ppm) and treated for about 3 months during which time tumors developed. Afatinib and AZD9291 were suspended in 1% Polysorbate 80 and administered via oral gavage once daily at the doses of 7.5 mg/kg and 5 mg/kg, respectively. Mice were imaged weekly at the Vanderbilt University Institute of Imaging Science. For immunoblot analysis, mice were treated for eight hours with drug as described before dissection and flash freezing of the lungs. Lungs were pulverized in liquid nitrogen before lysis as described above [1].