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家族抑制剂,抑制EGFRwt,EGFRL858R,EGFRL858R\u002FT790M 和 HER2的 IC50 分别为0.5 nM,0.4 nM,10 nM 和 14 nM。",{"key":243,"value":244},"相关类别","信号通路\n>>\n自噬\n>>\n自噬\n信号通路\n>>\nJAK\u002FSTAT信号通路\n>>\nEGFR\n信号通路\n>>\n蛋白酪氨酸激酶\n>>\nEGFR\n研究领域\n>>\n癌症",{"key":246,"value":247},"靶点","EGFR:0.5 nM (IC50)\nHER2:14 nM (IC50)\nEGFRL858R:0.4 nM (IC50)\nEGFRL858R\u002FT790M:10 nM (IC50)",{"key":249,"value":250},"体外研究","在无细胞体外激酶测定中,Afatinib(BIBW2992)dimaleate显示出对抗EGFR和HER2的野生型和突变形式的有效活性,类似于吉非替尼对L858R EGFR的效力,但对抗吉非替尼抗性的活性约高100倍。 L858R-T790M EGFR双突变体,IC50为10 nM。此外,BIBW2992与拉帕替尼和卡那替尼相比具有抗HER2的体外效力,IC50为14nM。该评估中最敏感的激酶是lyn,IC50为736 nM [1]。 Afatinib是这些ErbB家族受体的不可逆抑制剂。食管鳞状细胞癌(ESCC)细胞系对Afatinib敏感,IC50浓度在较低的微摩尔范围内(孵育48小时：HKESC-1 = 78 nM,HKESC-2 = 115 nM,KYSE510 =3.182μM,SLMT-1 =4.625μM,EC-1 =1.489μM;孵育72小时：HKESC-1 = 2 nM,HKESC-2 = 2 nM,KYSE510 =1.090μM,SLMT-1 =1.161μM,EC-1 = 109 nM)最大生长抑制超过95％。 Afatinib可以剂量和时间依赖的方式强烈诱导HKESC-2和EC-1中的G0\u002FG1细胞周期停滞[2]。",{"key":252,"value":253},"体内研究","一旦治疗开始,阿法替尼(15mg\u002Fkg)强烈抑制HKESC-2肿瘤的生长。车辆的平均肿瘤大小和终点处理分别为348±24 mm3和108±36 mm3,两者之间存在显着差异。显然,在Afatinib给药结束后,肿瘤大小不会在短时间内反弹。在整个治疗过程中体重没有快速变化表明Afatinib的毒性很小,这种药物耐受性良好[2]。",{"key":255,"value":256},"激酶实验","感染后72小时，用HEPEX（20mM HEPES pH 7.4,100mM NaCl，10mMβ-甘油磷酸盐，10mM对硝基苯基磷酸盐，30mM NaF，从Sf9生物质中提取EGFR激酶结构域-GST融合蛋白， 5 mM EDTA，5％甘油，1％Triton X-100,1 mM Na3VO4,0.1％SDS，0.5μg\u002F mL胃蛋白酶抑制剂A，抑肽酶20 KIU \u002F mL，Leupeptin2μg\u002F mL，苯甲脒1 mM，2.5μg\u002F mL 3,4-二氯异香豆素，2.5μg\u002F mL反式环氧琥珀酰基-L-亮氨酰-L-酰氨基丁烷和0.002％PMSF）并用于测定IC 50值。每个100μL酶反应在50％Me2SO中含有10μLAfatinib（BIBW2992），20μL底物溶液（200 mM HEPES pH 7.4,50 mM Mg-acetate，2.5 mg \u002F mL poly（EY），5μg\u002F mL bio -pEY）和20μL酶制剂。通过加入50μL在10mM MgCl 2中制备的100μMATP溶液开始酶促反应。测定在室温下进行30分钟，并通过加入50μL终止溶液（在20mM HEPES pH 7.4中的250mM EDTA）终止。将100μL转移至链霉抗生物素蛋白包被的微量滴定板，在室温下孵育60分钟后，用200μL洗涤溶液（50mM Tris，0.05％Tween20）洗涤板。向孔中加入100μL等份的HRPO标记的抗PY抗体（PY20H Anti-Ptyr：HRP）250ng \u002F mL。孵育60分钟后，将板用200μL洗涤溶液洗涤三次。然后用100μLTMB过氧化物酶溶液（A：B = 1：1）显色样品。 10分钟后停止反应。将板转移至ELISA读数器并在OD 450nm下测量消光。所有数据点一式三份执行[1]。",{"key":258,"value":259},"细胞实验","将人ESCC细胞系EC-1，HKESC1和HKESC2，SLMT1和KYSE510在含有10％胎牛血清（FBS）的RPMI中培养。使用MTT通过比色测定评估细胞毒性。将肿瘤细胞在各孔培养基中的48孔板（每孔3000-8000个细胞）中培养。在细胞铺板后24小时加入完全培养基中的阿法替尼，并在37℃，5％CO 2下培养48和72小时。细胞生长抑制表示为用酶标仪在570nm处测量的对照培养物的吸光度百分比，并且通过GraphPad PRISM计算50％的最大生长抑制（IC 50）。在每个实验中，对于每种药物浓度（n = 3）进行一式三份孔，并且在三个独立实验中重复测定[2]。",{"key":261,"value":262},"动物实验","小鼠[2]使用体重约16-20克的六周龄雌性无胸腺裸鼠（nu \u002F nu）。通过将HKESC-2（重悬于50μLHBSS缓冲液中的6×104细胞）皮下接种到裸鼠的两侧，建立ESCC异种移植物。当肿瘤大小达到4-6mm直径时，它们在治疗（15mg \u002F kg）或载体对照组中随机化。用于治疗的阿法替尼通过在给药前溶解在0.5％甲基纤维素中来制备。通过口服强饲法将药物或媒介物以5天的时间表给予小鼠，加上2天，连续两周。通过用卡尺监测肿瘤大小的变化来评估药物功效。用公式肿瘤体积=（宽度2×长度）\u002F 2计算肿瘤体积。",{"key":264,"value":265},"参考文献","[1].\nLi D, et al. BIBW2992, an irreversible EGFR\u002FHER2 inhibitor highly effective in preclinical lung cancer models. Oncogene. 2008 Aug 7;27(34):4702-11.\n[2].\nWong CH, et al. Preclinical evaluation of afatinib (BIBW2992) in esophageal squamous cell carcinoma (ESCC). Am J Cancer Res. 2015 Nov 15;5(12):3588-99.\n[3].\nWang XK, et al. Afatinib circumvents multidrug resistance via dually inhibiting ATP binding cassette subfamily G member 2 in vitro and in vivo. Oncotarget. 2014 Dec 15;5(23):11971-85.\n[4].\nYoshioka T, et al. Antitumor activity of pan-HER inhibitors in HER2-positive gastric cancer. 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