CN114207437A - 筛选抗癌剂的方法和用于治疗胰腺癌的激酶抑制剂的组合药物 - Google Patents
筛选抗癌剂的方法和用于治疗胰腺癌的激酶抑制剂的组合药物 Download PDFInfo
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Abstract
本发明涉及一种筛选抗癌剂的方法，使具有下述特征的果蝇摄取被检物质，将其生存率与未摄取被检物质的果蝇的生存率进行比较，从而筛选抗癌剂，所述特征为：a)变异型Ras85D的表达、b)p53基因缺失或表达被抑制、c)细胞周期蛋白E基因的过量表达、和d)Med基因缺失或表达被抑制。本发明另外涉及一种用于治疗胰腺癌的至少2种激酶抑制剂的组合药物、以及用于该组合药物的激酶抑制剂。
Description
技术领域
本发明涉及筛选针对胰腺癌的抗癌剂的方法、用于治疗胰腺癌的至少2种激酶抑制剂的组合药物、和该组合药物中使用的激酶抑制剂。
背景技术
胰腺癌是最难治疗的癌症之一。目前胰腺癌占癌症死亡的第4位，但是预期未来10年将升至第2位，担心其会成为巨大的社会问题。
胰腺癌的90％以上为发生于胰管的胰导管癌，具有即使发病也无自觉症状的特征。因此患者会错过接受诊断的机会，接受诊断时癌细胞大多已经转移到了其它器官。转移会带来严重的预后不良，因此胰腺癌患者的生存率处于各癌症种类中的最低水平，胰腺癌的预防、治疗法的开发一直是极其重要的课题。
但是，已知胰腺癌显示出极高的药物治疗抵抗性，新药的开发非常困难。为数不多的得到批准的代谢拮抗剂吉西他滨、EGFR抑制药厄洛替尼等也被指出效果不充分、毒性令人担忧等问题。
胰腺癌中频繁发生4种基因变异，即，KRAS基因变异所致的KRAS活化、TP53基因变异所致的TP53失活、CDKN2A基因变异所致的CDKN2A失活和SMAD4基因变异所致的SMAD4失活，已知预后最差的胰腺癌患者具有全部上述4种基因变异(非专利文献1)。因此，具有上述4种基因变异的模型动物和使用其的治疗法的研究在胰腺癌治疗方法的开发中具有重要的意义。但是，迄今为止，尚未制作模拟这4个基因异常的动物模型，成为研究的重大障碍。
近年来，对使用果蝇(Drosophila)作为模型动物这一点产生了浓厚兴趣。果蝇与哺乳类之间具有高度遗传保守性(例如，人类疾病中的异常基因中，75％以上也存在于蝇类中)，具有与哺乳类在功能上对应的体内结构(上皮结构、主要器官等)，遗传学分析工具较丰富(能够获得几乎全基因的siRNA敲除系统、变异体)，能够极迅速且廉价地繁育(10天内能够产生下一代，繁育费用仅为小鼠的约1/1000)，其作为模型动物具有有益的特质。
本发明人们报道了：以表达甲状腺髓样癌相关受体酪氨酸激酶、即RET的变异型的果蝇(ptc>dRetM955T)为模型动物，来探索甲状腺髓样癌相关激酶且筛选以该激酶为靶点的抗癌剂(专利文献1、非专利文献2～4)。ptc>dRetM955T是利用了能够在果蝇内强制表达外源基因的二元系统-gal4-UAS系统和ptc启动子、使RET变异体在局限于幼虫的翅原基的上皮细胞内进行表达的方式改造的果蝇，显示出发生肿瘤样病变、所有个体在未形成成熟个体前全部死亡的性质。
如此，以果蝇为模型动物来开发癌症治疗法是一种有效的手段，但是ptc>dRetM955T为甲状腺髓样癌的模型动物，因此为了探索胰腺癌的治疗法，需要重新制作胰腺癌的模型动物。
现有技术文献
专利文献：
专利文献1：日本特表2019-528279
非专利文献
非专利文献1：Qian et al.,JAMA Oncol.2018；4(3):e173420.
非专利文献2：Sonoshita et al.,Curr.Top.Dev.Biol.2017；121:287-309.
非专利文献3：Sonoshita et al.,Nat.Chem.Biol.2018；14(3):291-298.
非专利文献4：Ung,Sonoshita et al.,PLoS Comput.Biol.2019；15(4):e1006878.
发明内容
发明要解决的课题
在人胰腺癌中确认到的4种变异基因中，CDKN2A基因在果蝇中不存在。因此，不能如甲状腺髓样癌的果蝇模型那样，通过将在人胰腺癌中确认到的基因变异直接在果蝇中再现来制作胰腺癌的果蝇模型。本发明的目的在于，研发可成为胰腺癌的模型动物的果蝇，探索使用其的胰腺癌的新型治疗手段的方法，以及提供用于治疗胰腺癌的药物。
用于解决课题的方案
本发明人们研发了具有与4种人胰腺癌相关基因变异相当的特征的果蝇，通过使用其进行筛选，发现多种激酶抑制剂的组合对治疗胰腺癌有效，从而完成了以下的发明。
(1)一种筛选抗癌剂的方法，其包括：
使具有下述a)～d)的特征的果蝇摄取被检物质的步骤，
a)表达序列号1的氨基酸序列的第12位的甘氨酸被置换为天冬氨酸、缬氨酸或半胱氨酸的变异型Ras85D、
b)p53基因的缺失或表达抑制、
c)细胞周期蛋白E(Cyclin E)基因的过量表达、以及
d)Med基因的缺失或表达抑制；
测定摄取了被检物质的果蝇的生存率的步骤；以及，
当摄取了被检物质的果蝇的生存率比未摄取被检物质的果蝇的生存率高时，将该被检物质作为抗癌剂的候选物质筛选出来的步骤。
(2)根据(1)所述的方法，其中，变异型Ras85D是序列号1的氨基酸序列的第12位的甘氨酸被置换为天冬氨酸的蛋白质，p53基因的表达抑制通过导入抑制p53基因的表达的核酸来进行，细胞周期蛋白E的过量表达通过导入编码细胞周期蛋白E的核酸来进行，以及，Med基因的表达抑制通过导入抑制Med基因表达的核酸来进行。
(3)根据(1)所述的方法，其中，果蝇是导入了编码序列号1的氨基酸序列的第12位的甘氨酸被置换为天冬氨酸的变异型Ras85D的核酸、针对p53基因的敲除核酸、编码细胞周期蛋白E基因的核酸、以及针对Med基因的敲除核酸的果蝇。
(4)一种筛选抗癌剂的方法，其包括：
在含有被检物质的饲料上饲养由导入了下述a')～d')的果蝇和导入了gal4基因的果蝇交配而产的卵的步骤，
a')在UAS序列的下游具有编码序列号1的氨基酸序列的第12位的甘氨酸被置换为天冬氨酸的变异型Ras85D的碱基序列的核酸、
b')在UAS序列的下游具有编码针对p53基因的shRNA的碱基序列的核酸、
c')在UAS序列的下游具有编码细胞周期蛋白E基因的碱基序列的核酸、以及
d')在UAS序列的下游具有编码针对Med基因的shRNA的碱基序列的核酸；
测定在含有被检物质的饲料上饲养的果蝇的生存率的步骤；以及，
在含有被检物质的饲料上饲养的果蝇的生存率比在不含被检物质的饲料上饲养的果蝇的生存率高时，将该被检物质作为抗癌剂的候选物质筛选出来的步骤。
(5)根据(1)～(4)中任一项所述的方法，其中，通过调节果蝇的饲养温度，来控制未摄取被检物质的果蝇或在不含被检物质的饲料上饲养的果蝇的生存率。
(6)一种果蝇，其具有下述a)～d)的特征：
a)表达序列号1的氨基酸序列的第12位的甘氨酸被置换为天冬氨酸、缬氨酸或半胱氨酸的变异型Ras85D、
b)p53基因的缺失或表达抑制、
c)细胞周期蛋白E基因的过量表达、以及
d)Med基因的缺失或表达抑制。
(7)根据(6)所述的果蝇，其导入了下述a')～d')：
a')在UAS序列的下游具有编码序列号1的氨基酸序列的第12位的甘氨酸被置换为天冬氨酸的变异型Ras85D的碱基序列的核酸、
b')在UAS序列的下游具有编码针对p53基因的shRNA的碱基序列的核酸、
c')在UAS序列的下游具有编码细胞周期蛋白E基因的碱基序列的核酸、以及
d')在UAS序列的下游具有编码针对Med基因的shRNA的碱基序列的核酸。
(8)一种组合药物，其为用于治疗胰腺癌的、选自由MEK抑制剂、FRK抑制剂、WEE抑制剂、AURK抑制剂和ROCK抑制剂组成的组中的至少2种激酶抑制剂的组合药物。
(9)一种组合药物，其为用于治疗胰腺癌的、MEK抑制剂与选自由FRK抑制剂、WEE抑制剂、AURK抑制剂和ROCK抑制剂组成的组中的至少1种激酶抑制剂的组合药物。
(10)根据(8)或(9)所述的组合药物，其中，MEK抑制剂为曲美替尼。
(11)根据(8)～(10)中任一项所述的组合药物，其中，FRK抑制剂为AD80，WEE抑制剂为阿达色替，AURK抑制剂为阿利色昔或BI-831266。
(12)一种激酶抑制剂，其用于在胰腺癌的治疗中与MEK抑制剂组合，且选自由FRK抑制剂、WEE抑制剂、AURK抑制剂和ROCK抑制剂组成的组。
(13)根据(12)所述的激酶抑制剂，其中，MEK抑制剂为曲美替尼。
(14)根据(12)或(13)所述的激酶抑制剂，其中，FRK抑制剂为AD80，WEE抑制剂为阿达色替，AURK抑制剂为阿利色昔或BI-831266。
(15)一种MEK抑制剂，其用于在胰腺癌的治疗中与选自由FRK抑制剂、WEE抑制剂、AURK抑制剂和ROCK抑制剂组成的组中的至少1种激酶抑制剂组合。
(16)根据(15)所述的MEK抑制剂，其为曲美替尼。
(17)根据(15)或(16)所述的MEK抑制剂，其中，FRK抑制剂为AD80，WEE抑制剂为阿达色替，AURK抑制剂为阿利色昔(Alisertib)或BI-831266。
发明的效果
根据本发明，能够制作具有与4种人胰腺癌相关基因变异相当的特征的果蝇，通过使用该果蝇作为胰腺癌模型动物，能够高效且低成本地探索可对胰腺癌发挥治疗效果的物质。另外，可以提供特定激酶抑制剂的2种以上的组合作为胰腺癌的治疗药。
附图说明
图1为用荧光显微镜观察对照蝇(ptc>GFP)、表达在胰腺癌细胞中频繁出现的变异型Ras85D的果蝇(1-hit蝇)和模拟在胰腺癌细胞中频繁出现的4种基因变异的果蝇(4-hit蝇)的幼虫的翅原基而得的图像。
图2为示出向4-hit蝇中导入激酶基因的杂合性变异而制作激酶杂合性变异4-hit蝇的简要步骤的图。
图3为示出具有MEK、FRK、WEE、ROCK或AURK的杂合性变异的4-hit蝇的生存率的图。
图4为示出使之摄取MEK抑制剂(曲美替尼)、FRK抑制剂(AD80)、WEE抑制剂(MK-1775)、ROCK抑制剂(Y-27632)和AURK抑制剂(BI-831266)中的一种、或使其摄取MEK抑制剂与此外的4种激酶抑制剂中的任一种的组合时的4-hit蝇(non-GFP)的生存率的图。*p<0.01。
图5为示出在作为第1化合物的MEK抑制剂(曲美替尼)与作为第2化合物的FRK抑制剂(AD80)、WEE抑制剂(MK-1775)或AURK抑制剂(阿利色昔、BI-831266)的组合的存在下培养的人胰腺癌细胞(MIAPaCa-2细胞)的相对细胞数的图。*：与无第2化合物(0nM)相比显著减少(p<0.05)。#：与无曲美替尼(0nM)相比显著减少(p<0.05)。
图6为示出在22～29℃饲养时4-hit蝇(non-GFP)的生存率的图。
图7A为示出分别单独给药或组合给药MEK抑制剂(曲美替尼)和AURK抑制剂(BI-831266)的荷瘤小鼠的肿瘤体积的经时变化的图。*p<0.05、**p<0.01(均为给药第21天的曼-惠特尼U检验(Mann-Whitney U test))。NS：not significant(无显著差异)。误差棒表示8只的标准偏差。
图7B为示出分别单独给药或组合给药MEK抑制剂(曲美替尼)和AURK抑制剂(BI-831266)的荷瘤小鼠的、给药第21天的肿瘤体积相对于给药开始时的肿瘤体积的变化率的瀑布图。1根条棒表示1只小鼠的肿瘤体积变化率。
具体实施方式
模拟人胰腺癌的果蝇
本发明的第1方式涉及一种果蝇，其具有下述a)～d)的特征：
a)表达序列号1的氨基酸序列的第12位的甘氨酸被置换为天冬氨酸、缬氨酸或半胱氨酸的变异型Ras85D、
b)p53基因的缺失或表达抑制、
c)细胞周期蛋白E基因的过量表达、以及
d)Med基因的缺失或表达抑制。
Ras85D蛋白为人KRAS在果蝇中的直系同源物，其氨基酸序列(序列号1)以登录号NP_476699.1登录于NCBI中，编码其的碱基序列以登录号NM_057351.5登录于NCBI中。
人KRAS基因外显子2的密码子12中的变异已知为引起KRAS的活化的癌诱导性变异，在胰腺癌细胞中可频繁观察到。变异型Ras85D为向Ras85D中导入了与人KRAS的癌诱导性变异相当的变异而成的，具体而言为序列号1所示的氨基酸序列的第12位的甘氨酸被置换为天冬氨酸、缬氨酸或半胱氨酸者，作为活化KRAS起作用。一般认为，果蝇中的编码变异型Ras85D的基因的强制表达能够在果蝇中模拟与引起人活化KRAS时相同的情况。
果蝇的p53为人TP53的直系同源物，编码其的碱基序列以登录号NM_206545.2登录于NCBI中。已知对于人类而言，TP53基因的变异所引起的p53功能下降会促进癌症的发生和进展，在胰腺癌细胞中也频繁观察到p53变异。可认为，果蝇中的p53基因的功能性表达的抑制、例如p53基因缺失或表达抑制能够在果蝇中模拟与人失活p53所导致的情况相同的情况。
细胞周期蛋白依赖性激酶抑制剂2A(cyclin-dependent kinase inhibitor 2A，CDKN2A)也被称为P16，是参与细胞周期调节的蛋白。已知CDKN2A基因变异所致的CDKN2A失活与各种癌症的发生有关，在胰腺癌细胞中也可频繁观察到。果蝇中不存在人CDKN2A的直系同源物，因此本发明中使用果蝇细胞周期蛋白E的过量表达来代替CDKN2A基因变异。已知的是，细胞周期蛋白E是控制细胞周期的G1期的进展和进入S期的因子，与细胞周期蛋白依赖性激酶2(CDK2)结合而使其活化。据报道，果蝇中的细胞周期蛋白E的过量表达可在功能上代替CDKN2A失活，在果蝇中模拟与CDKN2A失活所导致的情况相同的情况(Datar etal.EMBO J.19:4543,2000)。编码果蝇的细胞周期蛋白E的碱基序列以登录号NP_476959.1登录于NCBI中。
Med(Mothers against decapentaplegic，母源抗皮肤生长因子)为人SMAD4在果蝇中的直系同源物，编码其的碱基序列以登录号NM_079871.4登录于NCBI中。在人类中，SMAD4参与对细胞增殖进行抑制性控制的TGF-β信号转导。SMAD4基因的变异所致的SMAD4失活据说与癌症发生有关，在胰腺癌细胞中可频繁观察到。可认为，果蝇中的Med基因的功能性表达的抑制、例如Med基因的缺失或表达抑制能够在果蝇中模仿与人失活SMAD4所导致的情况相同的情况。
本发明中，对基因导入变异、基因的缺失和表达抑制可以使用该技术领域中通常使用的分子生物方法来进行。
在优选的实施方式中，果蝇中，变异型Ras85D的第12位的甘氨酸被置换成天冬氨酸(表示为RasG12D)，通过导入抑制p53基因表达的核酸而进行p53基因的表达抑制，通过导入编码细胞周期蛋白E的核酸而进行细胞周期蛋白E的过量表达，以及通过导入抑制Med基因表达的核酸而进行Med基因的表达抑制。
作为本发明中可利用的抑制基因表达的核酸，可列举反义核酸、siRNA之类的敲除核酸(反义核酸、siRNA、作为其前体的shRNA、或编码shRNA的shDNA等)。敲除核酸可以以靶基因的碱基序列为基础以对靶基因特异性且脱靶效应低的方式来适宜设计。在更优选的实施方式中，果蝇导入了编码RasG12D的核酸、针对p53基因的敲除核酸、编码细胞周期蛋白E基因的核酸、以及针对Med基因的敲除核酸。可以以这4种核酸均存在于1个控制序列下的方式构成，或者可以以这4种核酸分别存在于彼此不同的控制序列下的方式构成。
上述4种核酸优选随着能够控制它们的表达时机、程度的控制系统一起整合于果蝇的基因组。为了实现这一点，典型情况下利用gal4-UAS系统。gal4-UAS系统为组合了酵母的转录活化因子gal4和作为其靶序列的UAS(Upstream activating sequence，上游激活序列)的、诱导基因的强制表达的二元系统。例如，通过使基因组中整合有在UAS的下游配置了上述4种核酸的构建物的果蝇与表达gal4蛋白的果蝇(gal4驱动系统)交配而得到的F1果蝇为，能够按照gal4蛋白的表达模式控制述4种核酸的表达的果蝇。gal4-UAS系统会依赖于温度而改变gal4蛋白的转录能力，因此可以通过调节果蝇的饲养温度来控制gal4蛋白的活性、控制上述4种核酸的表达水平。另外，该F1果蝇中的上述4种核酸的表达程度也可能根据具有控制gal4表达的启动子/增强子区域的活性的细胞种类/组织、这些部位的启动子/增强子区域的活性强度而不同。因此，也可以通过在用于制作F1的亲代的gal4驱动系统中适宜地设定控制gal4表达的启动子/增强子区域来控制上述4种核酸的表达水平，这些均在本领域技术人员可实施的范围内。
果蝇基因组中的整合上述4种核酸的位置没有特别限定，优选整合于作为常染色体的第二染色体或第三染色体。
在进一步优选的实施方式中，果蝇中导入了下述a')～d')：
a')在UAS序列的下游具有编码序列号1的氨基酸序列的第12位的甘氨酸被置换为天冬氨酸的变异型Ras85D的碱基序列的核酸、
b')在UAS序列的下游具有编码针对p53基因的shRNA的碱基序列的核酸、
c')在UAS序列的下游具有编码细胞周期蛋白E基因的碱基序列的核酸、以及
d')在UAS序列的下游具有编码针对Med基因的shRNA的碱基序列的核酸。
具有a)～d)的特征的果蝇的制作可以参照非专利文献2～4中记载的ptc>dRetM955T的制作而进行。制作步骤的一例的概要如以下(i)～(iii)所示，但是制作方法和果蝇不受下述例子限定。
(i)使用以处于UAS的控制下的方式重组有编码变异型Ras85D的碱基序列和编码针对p53基因的shRNA的碱基序列的果蝇用表达载体来改造果蝇基因组，制作转化蝇1。
(ii)使用以在UAS的控制下重组有细胞周期蛋白E基因的碱基序列和编码针对Med基因的shRNA的碱基序列的果蝇用表达载体来改造果蝇基因组，制作转化蝇2。
(iii)使转化蝇1和2交配而制作转化蝇3，进而使其与gal4驱动系统交配，制作具有a)～d)的特征的模型果蝇。
具有a)～d)的特征的果蝇会产生细胞的异常增殖、迁移能力亢进的肿瘤细胞。可认为这些现象是重现人胰腺癌的性状的结果，因此具有a)～d)的特征的果蝇能够作为人胰腺癌的模型来使用。另外，通过调查使该果蝇与下述杂合性变异果蝇交配而得到的F1的性状、生存率，还能够探索对具有a)～d)的特征的果蝇所呈现的人胰腺癌样性状有影响的基因，所述杂合性变异果蝇是向任意基因的一个等位基因中导入了缺失等变异、从而使该基因的功能下降的果蝇。
抗癌剂的筛选方法
作为另一方式，本发明提供一种筛选抗癌剂的方法，其包括：使上述人胰腺癌模拟果蝇、即具有上述a)～d)的特征的果蝇摄取被检物质的步骤；测定摄取了被检物质的果蝇的生存率的步骤；以及，在摄取了被检物质的果蝇的生存率比未摄取被检物质的果蝇的生存率高时，将该被检物质作为抗癌剂的候选物质筛选出来的步骤。
抗癌剂的筛选方法包括使上述人胰腺癌模拟果蝇、即具有上述a)～d)的特征的果蝇摄取被检物质的步骤。该步骤典型情况下可通过使具有a)～d)的特征的果蝇摄取含有被检物质的饲料来进行。该饲料除了含有适量的被检物质以外可以使用通常制备果蝇用饲料的方法和材料来制备。被检物质的量、与其它材料的混合方法可根据被检物质的物性、所期待的有效浓度等来适宜调节。另外，摄取饲料期间可以是果蝇的幼虫期、即果蝇从卵中孵化出来至蛹化的时期。本步骤中，只要为了得到期望的生存率而如后述那样调节饲养温度、且以饲料不干燥的程度维持湿度，则可以在饲养作为实验动物的果蝇时通常使用的条件下饲养果蝇。
本步骤的优选例之一为将具有上述a)～d)的特征的果蝇的卵放置在含有被检物质的饲料上，使孵化出的幼虫连同饲料一起摄取被检物质的步骤。另外，另一优选例为将具有上述a)～d)的特征的果蝇的幼虫放置在含有被检物质的饲料上，使其连同饲料一起摄取被检物质的步骤。
抗癌剂的筛选方法包括：测定摄取了被检物质的果蝇的生存率的步骤；以及，在摄取了被检物质的果蝇的生存率比未摄取被检物质的果蝇的生存率高时，将该被检物质作为抗癌剂的候选物质筛选出来的步骤。生存率可以通过将从蛹中羽化的个体数除以蛹的总数来计算。
具有上述a)～d)的特征的果蝇中会产生细胞的异常增殖、迁移能力亢进的肿瘤细胞，生存率会下降。另外，利用gal4-UAS系统时，若饲养温度升高到16～29℃的范围内，则该蝇的幼虫不会从蛹中羽化，死亡概率上升，例如实施例3和5中记载的Ser-gal4；UAS-RasG12D,UAS-p53shRNA,UAS-CycE,UAS-MedshRNA 4-hit蝇(non-GFP)时，25℃以上时生存率基本上为0％。因此，摄取被检物质而饲养的果蝇的生存率比未摄取被检物质且在相同条件下饲养的果蝇的生存率高时，例如未摄取被检物质而饲养的果蝇的生存率为0％、而摄取被检物质而饲养的果蝇的生存率超过0％时，推测该物质具有抑制具有a)～d)的特征的果蝇所呈现的人胰腺癌样性状的表达的作用，因此可以将该物质作为胰腺癌的抗癌剂的候选物质筛选出来。
利用gal4-UAS系统的具有上述a)～d)的特征的果蝇的情况下，可以通过调节饲养温度来控制上述4种核酸的表达水平，从而控制其生存率。例如，对于实施例3和5中记载的Ser-gal4；UAS-RasG12D,UAS-p53shRNA,UAS-CycE,UAS-MedshRNA 4-hit蝇(non-GFP)而言，若在25℃以上饲养，则其生存率基本上为0％，若在22～24℃饲养，则生存率可上升到10～20％左右。在22～24℃的饲养温度下实施本筛选方法而筛选出的被检物质与在25℃以上的饲养温度下实施本筛选方法而筛选出的被检物质相比，可期待具有比较温和的抗癌作用。
本筛选方法的优选实施方式包括：
在含有被检物质的饲料上饲养由导入了下述a')～d')的果蝇和导入了gal4基因的果蝇交配而产的卵的步骤，
a')在UAS序列的下游具有编码序列号1的氨基酸序列的第12位的甘氨酸被置换为天冬氨酸的变异型Ras85D的碱基序列的核酸、
b')在UAS序列的下游具有编码针对p53基因的shRNA的碱基序列的核酸、
c')在UAS序列的下游具有编码细胞周期蛋白E基因的碱基序列的核酸、以及
d')在UAS序列的下游具有编码针对Med基因的shRNA的碱基序列的核酸；
测定在含有被检物质的饲料上饲养的果蝇的生存率的步骤；以及，
在含有被检物质的饲料上饲养的果蝇的生存率比在不含被检物质的饲料上饲养的果蝇的生存率高时，将该被检物质作为抗癌剂的候选物质筛选出来的步骤。
组合药物
本发明的另一方式涉及一种用于治疗胰腺癌的、选自由MEK抑制剂、FRK抑制剂、WEE抑制剂、AURK抑制剂和ROCK抑制剂组成的组中的至少2种激酶抑制剂的组合药物。
MEK(MAPK(丝裂原活化蛋白激酶)/ERK(细胞外信号控制激酶)激酶)是构成MAPK级联的蛋白激酶之一。MAPK级联形成了控制细胞增殖、生长、分化和细胞凋亡之类的多种细胞过程的复杂的信号转导网络。通过抑制MEK，可使细胞增殖停止、诱导细胞凋亡，因此MEK作为抗癌剂的靶分子受到关注，已报道了各种MEK抑制剂。
作为本发明中使用的MEK抑制剂的例子，可列举曲美替尼(Trametinib)、考比替尼(Cobimetinib)、贝美替尼(Binimetinib)、舍美替尼(Selumetinib)、匹马司替(pimasertib)、莫达美替尼(Mirdametinib)、瑞法替尼(Refametinib)、PD184352、PD98059、BIX02189、BIX02188、TAK-733、AZD8330、PD318088、Myricetin、BI-847325、GDC-0623、Ro5126766、PD198306、RO4987655、HI TOPK 032等。
FRK(Fyn相关激酶(Fyn related kinase),也被称为RAK)是属于SRC亚家族成员的、存在于核内的非受体型酪氨酸激酶，已知在乳腺癌细胞、肾癌细胞中表达上升。
作为本发明中使用的FRK抑制剂的例子，可列举AD80、RAF709等。
AURORA激酶(AURK)是控制细胞分裂的丝氨酸/苏氨酸激酶，已知与细胞分裂期的中心体分离、双极纺锤体形成有关。存在AURK-A、AURK-B、AURK-C这3种亚型，它们在C末端激酶结构域具有高同源性。AURK在多种癌细胞中高表达，因此作为抗癌剂的靶分子受到关注，已报道了各种AURK抑制剂。
作为本发明中使用的AURK抑制剂的例子，可列举阿利色昔(Alisertib，AURK-A抑制剂)、BI-831266(AURK-B抑制剂)、巴拉塞替(Barasertib、AURK-B抑制剂)、陶扎色替(Tozasertib，AURK-A选择性高的通用AURK抑制剂)、达鲁舍替(Danusertib，通用AURK抑制剂)、CCT137690(通用AURK抑制剂)CCT129202(AURK-A选择性高的通用AURK抑制剂)、CCT241736(AURK-A,B抑制剂)、SNS-314(通用AURK抑制剂)、橙皮甙(Hesperadin，AURK-B抑制剂)、MK-5108(AURK-A抑制剂)、MLN8054(AURK-A抑制剂)、ZM 447439(AURK-A,B抑制剂)、PF03814735(AURK-A,B抑制剂)、AT9283(AURK-A,B抑制剂)、GSK1070916(AURK-B,C抑制剂)、PHA-680632(AURK-A选择性高的通用AURK抑制剂)、逆转素(Reversine，通用AURK抑制剂)、CYC116(AURK-A,B抑制剂)、ENMD-2076(AURK-A抑制剂)、TAK-901(AURK-A,B抑制剂)、AMG900(通用AURK抑制剂)、MK-8745(AURK-A抑制剂)、JNJ-7706621(AURK-A,B抑制剂)、SCH-1473759(AURK-A,B抑制剂)、艾雷舍替(Ilorasertib，AURK-B,C选择性高的通用AURK抑制剂)、TCS7010(AURK-A抑制剂)、LY3295668(AURK-A抑制剂)、BI-811283(AURK-B抑制剂)、西奥罗尼(Chiauranib，AURK-B抑制剂)、NMI-900(AURK-B,C抑制剂)等。
WEE激酶是介导CDK-1/细胞周期蛋白B复合体的由磷酸化引起的失活，对细胞分裂进行负调控的核内酪氨酸激酶，存在WEE1(WEE1A)、WEE2(WEE1B)和Myt1(PKMYT1)这3种。WEE1在有丝分裂的G2/M检查点承担着重要的作用，具有与MYT1一起使细胞进入G2-M阻滞(arrest)的功能。癌细胞中的WEE1抑制使G1检查点失活，引起染色体不稳定性，最终诱导分裂期细胞死亡(mitotic catastrophe)，因此WEE激酶、特别是WEE1作为抗癌剂的靶分子受到关注。
作为本发明中使用的WEE抑制剂的例子，可列举阿达色替(Adavosertib，也被称为MK-1775或AZD1775)、PD0166285、PD407824等。
ROCK(Rho-associated protein kinase，Rho相关蛋白激酶)是控制细胞骨架的丝氨酸/苏氨酸激酶，存在ROCK1和ROCK2这2种亚型。ROCK是低分子GTPaseRhoA的下游靶标，对多种底物进行磷酸化，与细胞运动、细胞极性、细胞粘附、细胞分裂、细胞凋亡、转录控制等多种生命功能相关，因此作为包括抗癌剂在内的各种药物的靶分子受到关注，已报道了各种ROCK抑制剂。
作为本发明中使用的ROCK抑制剂的例子，可列举Y-27632(ROCK1抑制剂)、法舒地尔(Fasudil，ROCK2抑制剂)、氮杂吲哚1(Azaindole 1，通用ROCK抑制剂)、AT13148(通用ROCK抑制剂)、硫酸阿扎那韦(Thiazovivin)、奈他地尔(Netarsudil)、瑞舒地尔(Ripasudil)、色满酮1(Chroman1)、GSK429286A、RKI-1447、GSK269962A、Y-39983、KD025、SAR407899、BDP5290、SB-772077B、H-1152、LX7101、SR-3677、Y-33075、CMPD101、SLx-2119等。
本方式的组合药物为包含以上说明的激酶抑制剂中的至少2种、即选自由MEK抑制剂、FRK抑制剂、WEE抑制剂、AURK抑制剂和ROCK抑制剂组成的组中的至少2种激酶抑制剂的组合的药物。“至少2种激酶抑制剂的组合”包括2种激酶抑制剂的组合、3种激酶抑制剂的组合、4种激酶抑制剂的组合、全部5种激酶抑制剂的组合。该术语不包括同种激酶抑制剂中的至少2种的组合，例如不包括属于MEK抑制剂的激酶抑制剂中的2种以上的组合。因此，例如“2种激酶抑制剂的组合”是指，MEK抑制剂与FRK抑制剂、MEK抑制剂与WEE抑制剂、MEK抑制剂与AURK抑制剂、MEK抑制剂与ROCK抑制剂、FRK抑制剂与WEE抑制剂、FRK抑制剂与AURK抑制剂、FRK抑制剂与ROCK抑制剂、WEE抑制剂与AURK抑制剂、WEE抑制剂与ROCK抑制剂、以及AURK抑制剂与ROCK抑制剂的各组合。
另外，构成组合的各抑制剂不限于1种化合物。例如MEK抑制剂与FRK抑制剂的组合不限于1种MEK抑制剂与1种FRK抑制剂的组合，而是包括1种MEK抑制剂与2种以上FRK抑制剂的组合、2种以上MEK抑制剂与1种FRK抑制剂的组合、以及2种以上MEK抑制剂与2种以上FRK抑制剂的组合。
本方式的组合药物可包括由上述5种激酶抑制剂中的2种以上激酶抑制剂构成的任意组合。一实施方式中，组合为选自由下述物质组成的组中的至少2种激酶抑制剂的组合，上述物质为：作为MEK抑制剂的曲美替尼、作为FRK抑制剂的AD80、作为WEE抑制剂的阿达色替以及作为AURK抑制剂的阿利色昔和BI-831266。另外，组合可以为MEK抑制剂与选自其它4种激酶抑制剂中的至少1种激酶抑制剂的组合。另一实施方式中，组合是作为MEK抑制剂的曲美替尼与选自由作为FRK抑制剂的AD80、作为WEE抑制剂的阿达色替以及作为AURK抑制剂的阿利色昔和BI-831266组成的组中的至少1种激酶抑制剂的组合。
组合药物中所含的2种以上激酶抑制剂可以一起或分别地、同时或逐次地给药于有此需求、即希望治疗胰腺癌的对象。组合药物可以为同时含有2种以上激酶抑制剂的制剂形态，也可以为将各激酶抑制剂的单个制剂组合的形态。组合药物为各制剂的组合时，各制剂的给药顺序和给药时期没有特别限制，可以同时给药，也可以隔开时间而在不同的时间或不同的日子给药。
意图用于上述组合药物的各激酶抑制剂也为本发明的另一方式。
上述组合药物可用于治疗胰腺癌。这里术语“治疗”包括以疾病的治愈或暂时性缓解为目的的、医学上允许的全部类型的治疗性干预。即，胰腺癌的治疗包括包含使胰腺癌的进展延迟或停止、使病变萎缩或消失、防止复发等在内的各种目的的医学上允许的干预。
上述组合药物可给药于罹患了胰腺癌的对象，例如包括小鼠、大鼠、仓鼠、豚鼠在内的啮齿类；包括人、黑猩猩、罗猴在内的灵长类；包括猪、牛、山羊、马、绵羊在内的家畜；包括狗、猫在内的观赏动物等等哺乳动物。优选的对象为人。
作为所治疗的胰腺癌，可列举外分泌瘤、例如浸润性胰管癌、胰腺腺泡细胞癌、导管内乳头状粘液瘤等以及内分泌肿瘤、例如神经内分泌瘤等。
关于组合药物中的各激酶抑制剂的量，只要是在与构成组合的其它激酶抑制剂组合时能治疗胰腺癌的量即可，通常与单独使用时的量相等或更少。以与单独使用时的量相等的量包含各激酶抑制剂的组合药物可以对胰腺癌发挥更强的效果，可在更短时间内或对于单独使用时无效的对象治疗胰腺癌。另外，以比单独使用时的量更少的量包含各激酶抑制剂的组合药物具有能够在维持对胰腺癌的效果的同时减少各激酶抑制剂的量的优点。
上述组合药物和用于组合药物的激酶抑制剂分别可以以包含激酶抑制剂以外的药物、缓冲剂、抗氧化剂、保存剂、蛋白质、亲水性聚合物、氨基酸、螯合剂、非离子表面活性剂、赋形剂、稳定化剂、载体等药学上可接受的成分的药物组合物形态来使用。药学上可接受的成分对于本领域技术人员而言是熟知的，本领域技术人员可以在通常可实施的范围内根据制剂形态从例如第十七次修订日本药典等标准所记载的成分中适宜选择、使用。
药物组合物含有有效量的各激酶抑制剂。这里有效量是指如上述那样在与构成组合的其它激酶抑制剂组合时能够治疗癌症的量。有效量可根据所组合的各激酶抑制剂的种类、比例、用法、对象的年龄、疾病的状态等条件来适宜决定。
药物组合物的剂形是任意的，作为优选例，可列举口服剂(片剂、胶囊剂、散剂、颗粒剂、细粒剂、丸剂、悬浮剂、乳剂、液剂、糖浆剂等)和非口服制剂(注射剂、点滴剂、经肠剂、经皮剂等)形态。另外，药物组合物的给药途径没有特别限制，在非口服制剂时，例如可列举血管内给药(优选为静脉内给药)、腹腔内给药、肠内给药、皮下给药、对靶标位置的局部给药等。优选的实施方式中，药物组合物通过口服给药或静脉内给药来进行给药。
癌症的治疗方法
作为另一方式，本发明还提供一种胰腺癌的治疗方法，其包括：将有效量的选自由MEK抑制剂、FRK抑制剂、WEE抑制剂、AURK抑制剂和ROCK抑制剂组成的组中的至少2种激酶抑制剂的组合一起或分别地、同时或逐次地给药于有此必要的对象。
通过以下的实施例进一步详细说明本发明，但是本发明不受这些限定。
实施例
实施例1人胰腺癌果蝇模型的制作
利用常规方法从果蝇w-(布鲁明顿果蝇库存中心，Bloomington DrosophilaStock Center)提取基因组DNA。以该基因组DNA为模板，使用以扩增Ras85D基因的方式设计的底物DNA进行PCR，得到包含Ras85D基因的碱基序列的DNA片段。另外，设计合成了用于将编码Ras85D的氨基酸序列的第12位的甘氨酸的密码子置换为编码天冬氨酸的密码子的位点特异性变异导入用引物DNA，以上述DNA片段为模板进行PCR而制作编码Ras85D变异体(RasG12D)的DNA片段。将该DNA片段插入到作为果蝇敲除用载体的pWALIUM载体(哈佛医学院，Harvard Medical School)中，由此制作pWALIUM.UAS-RasG12D载体。将其显微注射到果蝇y1w67c23；P{CaryP}attP2中，通过同源重组制作在3号染色体L区插入有编码RasG12D的DNA的UAS-RasG12D蝇。
另外，与编码RasG12D的DNA片段一起，将包含p53基因敲除序列(将有义链TGCTGAAGCAATAACCACCGA(序列号2)、发夹环TAGTTATATTCAAGCATA(序列号6)和反义链TCGGTGGTTATTGCTTCAGCA(序列号3)连接而成的序列)的DNA片段插入到pWALIUM载体中，由此制作pWALIUM.UAS-RasG12D,UAS-p53shRNA载体。将其同样地显微注射到果蝇y1w67c23；P{CaryP}attP2中，从而制作在3号染色体L区域中插入有编码RasG12D的DNA和针对p53基因的shRNA的UAS-RasG12D,UAS-p53shRNA蝇。
进而，以果蝇w-的基因组DNA为模板，使用以扩增细胞周期蛋白E(CycE)基因方式设计的引物DNA进行PCR，得到包含CycE基因的碱基序列的DNA片段。将该DNA片段与包含Med基因敲除序列(将有义链TTCAGTGCGATGAACATTGCT(序列号4)、发夹环TAGTTATATTCAAGCATA(序列号6)和反义链AGCAATGTTCATCGCACTGAA(序列号5)连接而成的序列)的DNA片段一起导入到pWALIUM载体中，由此制作pWALIUM.UAS-CycE,UAS-MedshRNA载体。将其显微注射到果蝇PBac{yellow[+]-attP-9A}VK00027中，通过同源重组制作在3号染色体R区域中插入了编码CycE基因的DNA和针对Med基因的shRNA的UAS-CycE,UAS-MedshRNA蝇。
接着，使UAS-RasG12D,UAS-p53shRNA蝇和UAS-CycE,UAS-MedshRNA蝇在25℃下交配3天，由此制作UAS-RasG12D,UAS-p53shRNA,UAS-CycE,UAS-MedshRNA蝇。
使UAS-RasG12D蝇和UAS-RasG12D,UAS-p53shRNA,UAS-CycE,UAS-MedshRNA蝇分别与ptc-gal4,UAS-GFP蝇(ptc>GFP蝇，Bloomington Drosophila Stock Center)在25℃下交配3天，由此制作ptc>GFP；UAS-RasG12D蝇(记作1-hit蝇(ptc>GFP))和ptc>GFP；UAS-RasG12D,UAS-p53shRNA,UAS-CycE,UAS-MedshRNA蝇(记作4-hit蝇(ptc>GFP))。这些蝇中，在ptc启动子控制下在局限于翅原基的上皮细胞中诱导导入基因的表达。
将用荧光显微镜观察1-hit蝇(ptc>GFP)、4-hit蝇(ptc>GFP)和作为对照的ptc>GFP蝇的3龄幼虫的翅原基的图像示于图1。对照中，在约10个细胞宽度的单层上皮细胞中观察到GFP的表达(从左侧起第2个图和中间的图)。1-hit蝇(ptc>GFP)中，在更宽的宽度观察到GFP表达细胞(从右侧起第2个图。该倾向在4-hit蝇(ptc>GFP)中更为显著(从右侧起第1个图)，另外确认出现了脱离原区域的迁移能力亢进的肿瘤细胞(图中箭头所示)。
另外，在进行用于制作1-hit蝇(ptc>GFP)和4-hit蝇(ptc>GFP)的交配时，交配后的亲代蝇在饲料上产卵2天，每1瓶得到20～50个卵。将其在16℃饲养25天后，将羽化的个体数除以蛹的总数，从而计算生存率。对照即ptc>GFP蝇的生存率为100％，与此相对地，1-hit蝇(ptc>GFP)的生存率为55％，另外4-hit蝇(ptc>GFP)的生存率为0％，即，致死。
由这些结果可确认，由于RasG12D的表达，翅原基上皮细胞的增殖亢进，在表达RasG12D的基础上诱导p53的敲除、CycE的表达亢进和Med基因的敲除时，翅原基上皮细胞的增殖进一步亢进，另外迁移能力也亢进，表明4-hit蝇可能成为人胰腺癌果蝇模型。
实施例2对4-hit蝇的生存率造成影响的激酶基因的探索
按照Sonoshita等(Curr.Top.Dev.Biol.,2017,121,287-309)的方法如图2所示那样进行交配，探索对4-hit蝇的生存率造成影响的激酶基因。具体而言，使实施例1中制作的UAS-RasG12D,UAS-p53shRNA,UAS-CycE,UAS-MedshRNA蝇与SM5tubgal80-TM6B平衡子蝇(Dr.RossCagan,Icahn School of Medicine at Mount Sinai,NY,USA)进行交配，由此制作UAS-RasG12D,UAS-p53shRNA,UAS-CycE,UAS-MedshRNA/SM5tubgal80-TM6B蝇。接着，使其与Ser>GFP蝇(Bloomington Drosophila Stock Center)进行交配，由此制作Ser>GFP；UAS-RasG12D,UAS-p53shRNA,UAS-CycE,UAS-MedshRNA/SM5tubgal80-TM6B蝇。
另外，从布鲁明顿果蝇库存中心(Bloomington Drosophila Stock Center，美国)中获得蝇总激酶组中的激酶的杂合性变异系统中可获得的220个系统，使这些在27℃下分别与Ser>GFP；UAS-RasG12D,UAS-p53shRNA,UAS-CycE,UAS-MedshRNA/SM5tubgal80-TM6B蝇交配3天，得到激酶杂合性变异4-hit蝇的卵。作为对照，使w-蝇与Ser>GFP；UAS-RasG12D,UAS-p53shRNA,UAS-CycE,UAS-MedshRNA/SM5tubgal80-TM6B蝇交配，得到激酶野生型4-hit蝇的卵。将这些蝇在27℃下饲养13天后，将羽化的个体数除以蛹的总数，由此计算生存率。
蝇激酶基因的总括检索的结果是，发现RAS通路因子MEK、SRC家族激酶FRK、细胞分裂控制因子组WEE和AURORA、以及细胞骨架控制因子ROCK各自的杂合性变异抑制4-hit蝇的致死性(图3)。将这些杂合性变异蝇的生存率示于图3。这表明，MEK抑制剂、FRK抑制剂、WEE抑制剂、AURK抑制剂和ROCK抑制剂的应用可能对提升4-hit蝇的生存率有效。
实施例3激酶抑制剂对4-hit蝇的生存率的药效评价
将曲美替尼(MEK抑制药)、阿达色替(也称为MK-1775，WEE1抑制剂)、AD80(FRK抑制剂)、Y-27632(ROCK抑制剂)、阿利色昔(AURKA抑制剂，以上购自MedChem Express)、BI-831266(AURKB抑制剂，勃林格殷格翰(Boehringer Ingelheim)惠赠)溶解于DMSO(SIGMA)，在-20℃保存。
将琼脂(和光)、啤酒酵母(MPBio)、酵母提取物(西格玛奥德里奇)、bacto蛋白胨(BD)、蔗糖(和光)、葡萄糖(和光)、MgCl2(和光)、CaCl2(和光)、丙酸(和光)、防霉剂(10％甲基-4-羟基苯甲酸的95％乙醇液；和光)溶解于超纯水，由此制作标准饲料。将其于50℃保温并添加溶解于DMSO的激酶抑制剂(曲美替尼单独、或曲美替尼与其它激酶抑制剂的组合)并混合，冷却，由此制备含药饲料。各化合物在饲料中的最终浓度设为曲美替尼1μM、阿达色替100μM、AD80 50μM、Y-27632 10μM、BI-831266 100μM。
使实施例1中制作的UAS-RasG12D,UAS-p53shRNA,UAS-CycE,UAS-MedshRNA蝇与Ser-gal4蝇(Bloomington Drosophila Stock Center)交配后，使亲代蝇在标准饲料或含药饲料上产卵2天，每1瓶得到20～50个Ser-gal4；UAS-RasG12D,UAS-p53shRNA,UAS-CycE,UAS-MedshRNA 4-hit蝇(non-GFP)的卵。将其在25℃下饲养13天后，将羽化的个体数除以蛹的总数，由此计算在各饲料上饲养的4-hit蝇(non-GFP)的生存率。
曲美替尼单独的情况下，使4-hit蝇(non-GFP)的生存率提高20％。另外，其它激酶抑制剂单独的情况下不改变4-hit蝇(non-GFP)的生存率，但是与曲美替尼组合时，与曲美替尼单独时相比提高生存率，因此表明了曲美替尼与其它激酶抑制剂的协同效应(图4)。
实施例4使用人胰腺癌细胞的激酶抑制剂的药效评价
由国立研究开发法人理化学研究所生物资源研究中心购买人胰腺癌细胞株(MIAPaCa-2)，使用在杜尔贝科改良伊戈尔培养基(nakalai tesque)中加入了10％胎牛血清(gibco)和1％青霉素-链霉素(nakalai tesque)而成的培养基，在5％CO2存在下在37℃下进行培养。
分别制备曲美替尼(仅DMSO、1μM、300μM)、阿达色替(300μM、1mM)、AD80(30μM、3mM)、阿利色昔(30μM、1mM)、BI-831266(3μM、3mM)，用培养基稀释100倍作为药剂液。
将MIAPaCa-2细胞以1,000个细胞/孔播种到96孔板中(100μL培养基中)。培养1天后，将曲美替尼单独或曲美替尼与其它激酶抑制剂的组合药剂液以10μL添加到96孔板的细胞中(DMSO的最终浓度0.2％)。各组合分别以5个孔进行试验。药剂液添加后、在5％CO2存在下在37℃培养72小时后，使用MTS测试(CellTiter96(注册商标)，Promega)测定细胞生存率。生存率以相对于对照(仅溶剂)的比例来表示。
曲美替尼单独的情况下，用量依赖性地降低细胞生存率。若将曲美替尼与阿达色替、AD80、阿利色昔、BI-831266分别组合，则任一组合的情况下与曲美替尼单独时相比细胞生存率均大幅降低，因此表明存在曲美替尼与其它激酶抑制剂的协同效应(图5)。
实施例5 4-hit蝇的致死性的温度依赖性的评价
将实施例3中制作的4-hit蝇(non-GFP)在22、24、25、27、29℃的各温度下饲养，结果生存率随着温度上升而下降，在25℃以上致死(图6)。
实施例6使用荷瘤小鼠的激酶抑制剂的药效评价
对BALB/c-nu/nu免疫缺陷裸小鼠(6周龄，无特定病原体饲养)实施麻醉，向其皮下移植5×106个MIA PaCa-2细胞。在计测肿瘤体积(长径×短径×短径/2)的情况下继续饲养，在肿瘤达到100mm3的时间点分为4组(n＝8)。向各组每周5天口服给药溶剂(5％DMSO)、曲美替尼(1mg/kg体重/天)、BI-831266(10mg/kg体重/天)、或曲美替尼(1mg/kg体重/天)和BI-831266(10mg/kg体重/天)，经时测定肿瘤体积。
在曲美替尼与BI-831266的组合给药组(T+B组)的情况下，与曲美替尼单独给药组(T组)和BI-831266单独给药组(B组)相比，肿瘤体积平均值的增加受到显著抑制(图7A)。以各小鼠来观察时，组合给药组的情况下，观察到多例实现了部分缓解(肿瘤体积缩小30％以上)或完全缓解(肿瘤消失)的病例，与此相对地，溶剂给药组和曲美替尼单独给药组的情况下未观察到部分缓解或完全缓解，BI-831266给药组的情况下仅发现1例完全缓解(图7B)。由这些结果可确认，曲美替尼与BI-831266的组合对移植到小鼠中的人胰腺癌细胞显示出协同的细胞增殖抑制效果，并且该效果的个体差异较小。
序列表自由文本
序列号1Ras85D的氨基酸序列
序列号2编码以p53基因为靶的shRNA的有义链的DNA的碱基序列
序列号3编码以p53基因为靶的shRNA的反义链的DNA的碱基序列
序列号4编码以Med基因为靶的shRNA的有义链的DNA的碱基序列序列号5编码以Med基因为靶的shRNA的反义链的DNA的碱基序列序列号6编码shRNA发夹环的DNA的碱基序列
Claims (17)
1.一种筛选抗癌剂的方法，其包括：
使具有下述a)～d)的特征的果蝇摄取被检物质的步骤，
a)表达序列号1的氨基酸序列的第12位的甘氨酸被置换为天冬氨酸、缬氨酸或半胱氨酸的变异型Ras85D、
b)p53基因的缺失或表达抑制、
c)细胞周期蛋白E基因的过量表达、以及
d)Med基因的缺失或表达抑制；
测定摄取了被检物质的果蝇的生存率的步骤；以及，
当摄取了被检物质的果蝇的生存率比未摄取被检物质的果蝇的生存率高时，将该被检物质作为抗癌剂的候选物质筛选出来的步骤。
2.根据权利要求1所述的方法，其中，
变异型Ras85D是序列号1的氨基酸序列的第12位的甘氨酸被置换为天冬氨酸的蛋白质，
p53基因的表达抑制通过导入抑制p53基因的表达的核酸来进行，
细胞周期蛋白E的过量表达通过导入编码细胞周期蛋白E的核酸来进行，并且，
Med基因的表达抑制通过导入抑制Med基因表达的核酸来进行。
3.根据权利要求1所述的方法，其中，果蝇是导入了编码序列号1的氨基酸序列的第12位的甘氨酸被置换为天冬氨酸的变异型Ras85D的核酸、针对p53基因的敲除核酸、编码细胞周期蛋白E基因的核酸、以及针对Med基因的敲除核酸的果蝇。
4.一种筛选抗癌剂的方法，其包括：
在含有被检物质的饲料上饲养由导入了下述a')～d')的果蝇和导入了gal4基因的果蝇交配而产的卵的步骤，
a')在UAS序列的下游具有编码序列号1的氨基酸序列的第12位的甘氨酸被置换为天冬氨酸的变异型Ras85D的碱基序列的核酸、
b')在UAS序列的下游具有编码针对p53基因的shRNA的碱基序列的核酸、c')在UAS序列的下游具有编码细胞周期蛋白E基因的碱基序列的核酸、以及
d')在UAS序列的下游具有编码针对Med基因的shRNA的碱基序列的核酸；
测定在含有被检物质的饲料上饲养的果蝇的生存率的步骤；以及，
在含有被检物质的饲料上饲养的果蝇的生存率比在不含被检物质的饲料上饲养的果蝇的生存率高时，将该被检物质作为抗癌剂的候选物质筛选出来的步骤。
5.根据权利要求1～4中任一项所述的方法，其中，通过调节果蝇的饲养温度，来控制未摄取被检物质的果蝇或在不含被检物质的饲料上饲养的果蝇的生存率。
6.一种果蝇，其具有下述a)～d)的特征：
a)表达序列号1的氨基酸序列的第12位的甘氨酸被置换为天冬氨酸、缬氨酸或半胱氨酸的变异型Ras85D、
b)p53基因的缺失或表达抑制、
c)细胞周期蛋白E基因的过量表达、以及
d)Med基因的缺失或表达抑制。
7.根据权利要求6所述的果蝇，其导入了下述a')～d')：
a')在UAS序列的下游具有编码序列号1的氨基酸序列的第12位的甘氨酸被置换为天冬氨酸的变异型Ras85D的碱基序列的核酸、
b')在UAS序列的下游具有编码针对p53基因的shRNA的碱基序列的核酸、c')在UAS序列的下游具有编码细胞周期蛋白E基因的碱基序列的核酸、以及
d')在UAS序列的下游具有编码针对Med基因的shRNA的碱基序列的核酸。
8.一种组合药物，其为用于治疗胰腺癌的、选自由MEK抑制剂、FRK抑制剂、WEE抑制剂、AURK抑制剂和ROCK抑制剂组成的组中的至少2种激酶抑制剂的组合药物。
9.一种组合药物，其为用于治疗胰腺癌的、MEK抑制剂与选自由FRK抑制剂、WEE抑制剂、AURK抑制剂和ROCK抑制剂组成的组中的至少1种激酶抑制剂的组合药物。
10.根据权利要求8或9所述的组合药物，其中，MEK抑制剂为曲美替尼。
11.根据权利要求8～10中任一项所述的组合药物，其中，FRK抑制剂为AD80，WEE抑制剂为阿达色替，AURK抑制剂为阿利色昔或BI-831266。
12.一种激酶抑制剂，其用于在胰腺癌的治疗中与MEK抑制剂组合，且选自由FRK抑制剂、WEE抑制剂、AURK抑制剂和ROCK抑制剂组成的组。
13.根据权利要求12所述的激酶抑制剂，其中，MEK抑制剂为曲美替尼。
14.根据权利要求12或13所述的激酶抑制剂，其中，FRK抑制剂为AD80，WEE抑制剂为阿达色替，AURK抑制剂为阿利色昔或BI-831266。
15.一种MEK抑制剂，其用于在胰腺癌的治疗中与选自由FRK抑制剂、WEE抑制剂、AURK抑制剂和ROCK抑制剂组成的组中的至少1种激酶抑制剂组合。
16.根据权利要求15所述的MEK抑制剂，其为曲美替尼。
17.根据权利要求15或16所述的MEK抑制剂，其中，FRK抑制剂为AD80，WEE抑制剂为阿达色替，AURK抑制剂为阿利色昔或BI-831266。
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| Publication number | Publication date |
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| JPWO2021172582A1 (zh) | 2021-09-02 |
| WO2021172582A1 (ja) | 2021-09-02 |
| AU2021226119B2 (en) | 2024-04-18 |
| CA3151403A1 (en) | 2021-09-02 |
| KR20220032607A (ko) | 2022-03-15 |
| EP4115907A1 (en) | 2023-01-11 |
| AU2021226119A1 (en) | 2022-03-24 |
| US20220387630A1 (en) | 2022-12-08 |
| CN114207437B (zh) | 2024-05-28 |
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