CN110664818B - 一种治疗肺癌的药物 - Google Patents
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Abstract
本发明公开了一种治疗肺癌的药物，该药物是由NG25和曲美替尼按质量比4:1组成；所述的肺癌是CLU基因功能缺失或下调的肺癌。进一步地，所述的肺癌是CLU基因功能缺失或下调的、由KRASG12D癌基因驱动的肺癌。体外实验发现，CLU敲低的肺癌细胞对NG25更为敏感，与对照相比，NG25可显著抑制肺癌细胞生长，而当联合Trametinib共同处理细胞后，发现对肺癌细胞的抑制更为显著。同样，在动物模型中，TAK1抑制剂NG25可抑制CLU敲低的细胞移植瘤和CLU敲除原位肺癌的生长，而且联合Trametinib共同治疗能使肿瘤显著萎缩，达到治疗该类型肺癌的目的。
Description
技术领域
本发明属于生物医药领域，具体涉及一种治疗肺癌的药物，特别涉及TAK1抑制剂NG25联合曲美替尼(Trametinib)对伴有CLU基因功能缺失的KRASG12D癌基因驱动的肺癌的治疗药物。
背景技术
目前已知肺癌的发生主要与致癌基因的改变有关。KRAS是肺癌常见的驱动基因突变，在10-30％肺癌病人中呈现该基因的突变。到目前为止，临床上缺乏针对该种肺癌有效的靶向治疗药物或方法。
尽管肺癌靶向治疗已取得一定的效果，但仍有相当一部分病人服用了针对肿瘤中突变的癌基因的抑制剂后，没有表现出应有的治疗效果。其中原因很多，但抑癌基因的作用值得关注。深入研究也表明这些抑癌基因对肺癌的发展及对药物的敏感性有重要影响。因此，阐明肺癌中抑癌基因的作用机制将有助于为由癌基因和抑癌基因突变引起的肺癌的治疗提供有效的治疗策略。
目前，肺癌抑癌基因的发现非常有限，除P53基因、RB基因和CDKN2A基因在肺癌中钝化的频率非常高外，也发现如SMAD2、SMAD4、TGFBR2、PTEN等一批频率非常低的基因。另外，还有一些基因的低表达也发现对肺癌的发生发展有影响，如CLU基因。
CLU基因位于八号染色体，含有9个外显子，其蛋白Clusterin在组织中广泛分布，并参与多个细胞进程，包括：组织分化、脂质运输、细胞增殖、凋亡等过程。Clusterin表达受生长因子调控。另外，原癌基因H-Ras、c-Myc和N-Myc抑制CLU表达。因此有人认为：抑制CLU的表达是癌基因依赖的细胞转化所需要的。前列腺癌的研究发现小鼠敲除CLU，会诱发前列腺上皮肉瘤(PIN)生成，促进细胞增殖和NF-kB信号增强，并且可促进肿瘤的恶性发展。而神经胶质瘤中也发现CLU对肿瘤起负调控作用。根据Oncomine数据库，发现CLU在大部分原发瘤中mRNA水平下调，并与癌症的恶性阶段和程度呈反比列，包括前列腺癌、乳腺癌、肺癌。这暗示着CLU功能的缺失对肺癌的发生发展起重要作用。但针对该类型肺癌，甚至还同时携带KRAS突变的肺癌目前的治疗缺乏有效的治疗。
发明内容
基于目前针对CLU缺失的肺癌缺乏指引性和有效的治疗手段，本发明的目的在于提供一种治疗肺癌的组合物。本发明通过构建了CLU敲低的稳定细胞、CLU敲低的小鼠移植瘤以及CLU敲除同时伴有KRASG12D小鼠原位肺癌的动物模型最大限度的模拟CLU缺失的肺癌，然后利用这些模型评价TAK1抑制剂对此类肺癌的治疗效果，并且联合Trametinib对同时携带KRAS突变的此类肺癌进行治疗。
本发明的目的通过下述技术方案实现：
一种治疗肺癌的药物，由NG25(N-[4-[(4-乙基-1-哌嗪基)甲基]-3-(三氟甲基)苯基]-4-甲基-3-(1H-吡咯并[2,3-B]吡啶-4-基氧基)苯甲酰胺)和曲美替尼组成；
优选地，所述的药物中，NG25与曲美替尼的质量比为4:1；
优选地，所述的肺癌是CLU基因功能缺失或下调的肺癌；
优选地，所述的肺癌是CLU基因功能缺失或下调的、由KRASG12D癌基因驱动的肺癌；
进一步地，所述的肺癌包括原位癌和移植癌；
所述的药物还可以含有一种以上药学上可以接受的载体；所述的载体优选为缓释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、吸附载体、表面活性剂或润滑剂等；
所述的药物可以进一步制成注射剂、片剂、粒剂或胶囊等多种形式，各种剂型的药物可以按照药学领域的常规方法制备。
本发明发现CLU功能的缺失可促进TAK1通路的激活，从而激活其下游NFKB信号，促进细胞生长；而抑制TAK1信号的激活，可抑制Clusterin表达下调而引起的肺癌细胞的生长。这些研究提示TAK1信号是Clusterin下调所依赖的肺癌治疗的潜在靶点。
N-[4-[(4-乙基-1-哌嗪基)甲基]-3-(三氟甲基)苯基]-4-甲基-3-(1H-吡咯并[2,3-B]吡啶-4-基氧基)苯甲酰胺(NG25 trihydrochloride)，简称NG25，是TAK1和MAP4K2的双重抑制剂。NG25可选择性与TAK1和MAP4K2结合抑制其活性，从而抑制其下游如IKK、p38和JNK蛋白的磷酸化的激活。
本发明相对于现有技术具有如下的优点及效果：
本申请通过大量创造性劳动发现：TAK1信号通路在CLU功能缺失的KRAS突变的肺癌生长中起重要作用，抑制TAK1信号可有效阻止此类肺癌细胞生长，联合MEK抑制剂Trametinib可更有效地抑制肺癌生长。在体外实验中发现，CLU敲低的肺癌细胞对NG25更为敏感，与对照相比，NG25可显著抑制肺癌细胞生长，而当联合Trametinib共同处理细胞后，发现对肺癌细胞的抑制更为显著。同样，在动物模型中，TAK1抑制剂NG25可抑制CLU敲低的细胞移植瘤和CLU敲除原位肺癌的生长，而且联合Trametinib共同治疗能使肿瘤显著萎缩，达到治疗该类型肺癌的目的。
附图说明
图1为CLU敲低的肺癌细胞Hop62对NG25敏感性的评价图；其中，A为Hop62细胞CLU敲低效果的蛋白免疫印迹评价图；B为对照组Hop62-shGFP细胞对NG25的半抑制浓度(IC50)；C为CLU敲低细胞Hop62-shCLU对NG25的半抑制浓度(IC50)；D为NG25处理Hop62-shGFP和Hop62-shCLU细胞的平板克隆结果，右边为细胞克隆数的统计图；E为NG25对细胞克隆抑制程度图。
图2为NG25和Trametinib对细胞生长抑制的协同评价图；其中，A为CCK8实验评价NG25和Trametinib单独和协同处理的细胞生长影响的结果；B为平板克隆评价所示药物及协同处理对克隆形成影响的结果，右边图为细胞克隆数的统计。
图3为药物在裸鼠皮下移植瘤中对肿瘤的生长抑制的协同作用评价图；其中，A为所示药物及协同治疗两周过程中肿瘤大小变化的记录结果；B为药物治疗两周后取出的肿瘤大小的对比图，右边为肿瘤重量的统计图。
图4为LSL-KRASG12D肺癌转基因小鼠模型中评价所示药物及协同治疗对肺癌原发瘤的抑制效果图；其中，A为小鼠治疗前后的CT图；B为药物治疗两周后肿瘤大小变化的定量图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
以下实施例中NG25是TAK1的抑制剂，Trametinib是MEK的抑制剂
实施例1
CLU敲低的肺癌细胞对TAK1抑制剂NG25更敏感
一、实验方法
(1)Hop62细胞构建CLU稳定敲低的细胞系构建：首先将表达载体pLKO.1-shCLU(Sigma，TRCN0000078611，序列：CCGGGCTAAAGTCCTACCAGTGGAACTCGAGTTCCACTGGTAGGACTTTAGCTTTTTG)与病毒骨架载体psPAX和PM2.G共转293T细胞进行慢病毒包装，pLKO.1-shGFP(Sigma，SHC004)载体为对照。用包装好的shCLU和shGFP的病毒分别感染Hop62细胞，然后用2ug/mL的Puromycin进筛选稳定敲低CLU的细胞株。用免疫印迹的方法评价细胞株的敲低效果。
(2)通过CCK8的方法检测Hop62-shCLU细胞对NG25的敏感性：分别在96孔板中加入Hop62-shGFP和Hop62-shCLU细胞(800cell/孔)，然后分别用0、0.01625、0.3125、0.625、1.25、2.5、5和10μM浓度的NG25处理细胞6天，用CCK8的方法检测细胞活性，最后通过GraphPad软件计算药物对细胞的半抑制浓度(IC50)
(3)平板克隆形成实验评价CLU敲低细胞对NG25的敏感性：在6孔板中分别种800/孔的Hop62-shGFP和Hop62-shCLU细胞，用不同浓度的NG25分别处理。细胞培养7天后进行固定和结晶紫染色，并进行细胞克隆数的统计和变化分析。
二、实验结果及分析
(1)通过免疫印迹方法，显示Hop62-shCLU稳定细胞系中的CLU表达与对照Hop62-shGFP相比显著地被下调(图1A)。
(2)通过CCK8细胞活性检测发现，Hop62-shGFP细胞对NG25的半抑制浓度比Hop62-shCLU细胞高(图1B和图1C)，表明CLU敲低后Hop62细胞对药物NG25更为敏感。
(3)平板克隆形成能力的评价中，发现在浓度2.5μM的NG25处理下，Hop62-shCLU细胞平板克隆的能力与对照细胞相比显著降低(图1D)。从药物影响的程度分析，对照细胞shGFP克隆下降8.9％，而Hop62-shCLU细胞克隆数下降36.2％(图1E)。这些结果同样表明Hop62-shCLU细胞对NG25更敏感。
实施例2
NG25协同Trametinib可显著抑制CLU下调的肺癌细胞的生长
一、实验方法
(1)在96孔板中接种Hop62-shCLU细胞，每孔800cells，然后分别用DMSO(对照)、NG25(1.25μM)、Trametinib(Tram,25nM)和NG25联合Tram(1.25μM+25nM)四种方式处理细胞。每天用CCK8检测细胞的生长活性，6天后绘制细胞的生长曲线图。
(2)平板克隆形成评价NG25联合Tram对细胞生长的影响：6孔板中接种400cell/孔，分别用DMSO(对照)、NG25(1.25μM)、Tram(25nM)和NG25联合Tram(1.25μM+25nM)四种方式处理细胞。培养7天后，细胞固定、染色、统计分析。
二、实验结果及分析
(1)细胞生长检测中，与DMSO组相比较，在该浓度处理下NG25对细胞生长有轻微抑制，Tram对细胞有明显的抑制效果，而当两种药联合处理细胞，细胞的生长受到非常显著地抑制，其效果明显好于两种单药的处理(图2A)。
(2)平板克隆形成实验中同样发现，单药NG25和Tram对Hop62-shCLU细胞的克隆形成能力有明显地抑制效果。而当两种药联合作用使，细胞的克隆形成能力被极大地抑制(图2B)。
实施例3
NG25联合Trametinib可有效抑制CLU下调的肺癌细胞皮下移植瘤的生长
(1)将CLU敲低的肺癌细胞Hop62-shCLU重悬于BD Matrigel基质胶中(按照每个移植瘤需要3×106细胞计算，将细胞重悬于100μL BD Matrigel基质胶中)。将上述细胞和基质混合物接种到裸鼠的左右侧腰背部皮下各100μL。
(2)裸鼠接种6天左右肿瘤固定后，将裸鼠进行分组，将Hop62-shCLU分成4组，分别为空白组(不治疗)、NG25治疗组(腹腔注射，4mg/kg/Day)、Tram治疗组(灌胃，1mg/kg/Day)和NG25与Tram联合治疗组(两种药物同时给药)。Hop62-shGFP为细胞对照组。
(3)治疗开始每两天测量肿瘤大小，按公式：体积＝长×宽2/2计数大小。治疗2周后处死裸鼠，解剖肿瘤。
二、实验结果及分析
与shGFP对照相比，Hop62-shCLU肿瘤的生长速度更快，肿瘤体积更大。与不治疗的肿瘤相比，NG25和Tram单药对Hop62-shCLU肿瘤的生长有抑制作用，而当NG25和Tram联合治疗裸鼠时，可发现肿瘤的生长受到非常显著地抑制(图3A)，并且肿瘤的体积和肿瘤显著缩小(图3B)。
实施例4
NG25联合Trametinib能有效治疗原位瘤
移植瘤治疗模型在评价药物作用可方便的观察和测量肿瘤生长的大小变化且体系单一，能清晰的说明问题。但缺点是不能模拟原发肺癌复杂的癌变、肿瘤形成过程，及肿瘤和基质细胞的相互作用。因此，对预测药物在病人体内的治疗能力较差。为此，本发明进一步利用LSL-KRASG12D转基因小鼠的肺癌原位瘤模型评价NG25联合Tram的治疗效果。
一、实验方法
(1)8周左右的LSL-KRASG12D的小鼠用pSECC-sgCLU的病毒(病毒中含有Cre、Cas9和sgCLU，序列：GGAGATTCAGAACGCCGTCC)滴鼻，让病毒从气管进入小鼠肺部，从而将肺上皮细胞中的KRASG12D激活的同时敲掉细胞中的CLU基因，模拟CLU下调的情况下，KRAS突变诱导的肺癌的发生。由于CLU敲除，半年后小鼠更快得肺癌。
(2)计算机断层扫描(Computer Tomography,CT)记录肿瘤的大小及严重程度。
(3)将得肺癌的小鼠分成NG25治疗组(腹腔注射，4mg/kg/Day)、Tram治疗组(灌胃，1mg/kg/Day)和NG25与Tram联合治疗组(两种药物同时给药)三个组，分别进行12天的给药治疗。治疗后CT检测记录肿瘤大小变化情况。
二、实验结果及分析
经过12天的治疗，用CT扫描不同治疗组小鼠肺部肿瘤情况。结果发现，NG25单药治疗使肺部肿瘤面积轻微缩小(比用前缩小大约38.5％)，单药Tram使肿瘤缩小大约46.5％，而NG25联合Tram对肿瘤有非常显著的抑制效果，肿瘤缩小大约76.4％(图4A和图4B)。原位瘤模型的治疗效果同样说明NG25联合Tram对CLU下调的肺癌具有显著的治疗效果。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (4)
1.一种治疗肺癌的药物，其特征在于是由NG25和曲美替尼按质量比4:1组成；
所述NG25和曲美替尼的结构式如下所示：
所述的肺癌是CLU基因功能缺失或下调的、由KRASG12D癌基因驱动的肺癌，包括原位癌和移植癌。
2.根据权利要求1所述的药物，其特征在于：所述的药物还含有一种以上的载体。
3.根据权利要求2所述的药物，其特征在于：所述的载体为缓释剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、吸附载体、表面活性剂或润滑剂。
4.根据权利要求1所述的药物，其特征在于：所述的药物是注射剂、片剂、颗粒剂或胶囊剂。
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