CN116492471A - 一种治疗braf v600e突变晚期结直肠癌的组合药物 - Google Patents
一种治疗braf v600e突变晚期结直肠癌的组合药物 Download PDFInfo
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Abstract
本申请提出了一种治疗BRAF V600E突变晚期结直肠癌的组合药物,涉及生物医药技术领域。其有效成分由以下物质组成:Encorafenib、Cetuximab和MOGAT3的小分子抑制剂。采用MOGAT3的小分子抑制剂和康奈非尼(Encorafenib)以及西妥昔单抗(Cetuximab)进行联合用药能够有效促进双靶药物实现靶向治疗的效果,同时协同增效,逆转了耐药,为提供耐药的药物以及临床用药提供了可靠的选择。
Description
技术领域
本申请涉及生物医药技术领域,具体而言,涉及一种治疗BRAF V600E突变晚期结直肠癌的组合药物。
背景技术
BRAF V600E突变晚期结直肠癌患者的生存时间有1年,靶向BRAF(编码RAF家族丝氨酸/苏氨酸蛋白激酶)及EGFR(Epidermal Growth Factor Receptor)的联合治疗是最新的标准治疗方案,但治疗后肿瘤在4.3个月就会出现耐药及进展,当前治疗上述疾病的药物多为分子靶向药物,其价格昂贵,副作用大,并且不久就迅速出现耐药,目前亟待更深入地研究耐药机制,寻找逆转策略延长患者的生存。
发明内容
本申请的目的在于提供一种治疗BRAF V600E突变晚期结直肠癌的组合药物,此药物具有能延缓分子靶向药迅速耐药的特点。
本申请解决其技术问题是采用以下技术方案来实现的。
本申请实施例提供一种治疗BRAF V600E突变晚期结直肠癌的组合药物,其有效成分由以下物质组成:Encorafenib、Cetuximab和MOGAT3的小分子抑制剂。
本申请研究人员在BRAF V600E突变结直肠癌PDX模型上动态检测了肿瘤对BRAF和EGFR双靶向药物从敏感到耐药过程中的信号通路的变化,发现MOGAT3调控的脂质代谢可以促进耐药,MOGAT3的小分子抑制剂PF-06471553可以下调脂代谢并逆转耐药。另外,研究发现在双靶耐药PDX肿瘤中脂质合成异常激活,其中,单酰基甘油酰基转移酶MOGAT3的表达也显著增高,MAPK信号通路重新激活,在敲除MOGAT3后,耐药细胞中的脂质的累积显著减少,细胞也重新对双靶敏感,MOGAT3的小分子抑制剂PF-06471553体内也能够逆转耐药。因此采用MOGAT3的小分子抑制剂和康奈非尼(Encorafenib)以及西妥昔单抗(Cetuximab)进行联合用药能够有效促进双靶药物实现靶向治疗的效果,同时协同增效,逆转了耐药,这为提供耐药的药物,临床用药提供了可靠的新选择。
相对于现有技术,本申请的实施例至少具有如下优点或有益效果:
本申请提供一种治疗BRAF V600E突变晚期结直肠癌的组合药物,此药物具有能延缓分子靶向药迅速耐药的特点。采用MOGAT3的小分子抑制剂和康奈非尼(Encorafenib)以及西妥昔单抗(Cetuximab)进行联合用药能够有效促进双靶药物实现靶向治疗的效果,同时协同增效,逆转了耐药,为提供耐药的药物以及临床用药提供了可靠的选择。
附图说明
为了更清楚地说明本申请实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本申请的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为本申请实施例1筛选试验中KEGG富集图;
图2为本申请实施例1筛选试验中QPCR验证代谢通路相关基因结果图;
图3为本申请实施例1筛选试验中免疫组化染色结果图;
图4为本申请实施例1筛选试验中免疫组化定量结果图;
图5为本申请实施例1筛选试验中代谢通路分类图;
图6为本申请实施例1筛选试验中甘油三酯含量对照图;
图7为本申请实施例1筛选试验中染色结果图;
图8为本申请实施例1筛选试验中WB试验结果图;
图9为本申请实施例1筛选试验中代谢组学试验结果图;
图10为本申请实施例3体外试验中染色结果图;
图11为本申请实施例3体外试验中细胞活力测试结果图;
图12为本申请实施例3体外试验中细胞凋亡水平图;
图13为本申请实施例3体外试验中细胞凋亡速率图;
图14为本申请实施例4体内试验中肿瘤体积大小对比图;
图15为本申请实施例4体内试验中肿瘤重量对比图;
图16为本申请实施例4体内试验中血液中脂质水平含量检测图;
图17为本申请实施例4体内试验中肿瘤生长数据图;
图18为本申请实施例4体内试验中小鼠体重变化对比图;
图19为本申请实施例4体内试验中切片染色结果图;
图20为本申请实施例4体内试验中免疫组化定量结果图。
具体实施方式
为使本申请实施例的目的、技术方案和优点更加清楚,下面将对本申请实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互组合。下面将参考具体实施例来详细说明本申请。
本申请实施例提供一种治疗BRAF V600E突变晚期结直肠癌的组合药物,其由以下成分组成:Encorafenib、Cetuximab和MOGAT3的小分子抑制剂。
此药物具有能延缓分子靶向药迅速耐药的特点。采用MOGAT3的小分子抑制剂和康奈非尼(Encorafenib)以及西妥昔单抗(Cetuximab)进行联合用药能够有效促进双靶药物实现靶向治疗的效果,同时协同增效,逆转了耐药,为提供耐药的药物以及临床用药提供了可靠的选择。
在本申请的一些实施例中,上述MOGAT3的小分子抑制剂为PF-06471553。
在本申请研究中,具体采用了PF-06471553进行试验验证,其中PF-06471553和Encorafenib以及Cetuximab联合使用组的表现出可以显著抑制耐药细胞脂质增加,降低耐药细胞中的脂滴,下调脂代谢;同时评估细胞增殖活力中,发现双靶联合PF-06471553可以显著抑制耐药细胞的增殖,使耐药细胞恢复敏感性,逆转耐药;而且采用流式细胞术评估细胞凋亡水平,发现双靶联合PF-06471553(Pf)可以明显促进耐药细胞的凋亡,达到协同增效的作用。
在本申请的一些实施例中,上述组合药物中Encorafenib、Cetuximab和PF-06471553的摩尔浓度比为1:1:10。
采用以上比例组合的药物,具有很好的耐药特性和促进双靶药物实现靶向治疗的协同效果。
以下结合实施例对本申请的特征和性能作进一步的详细描述。
实施例1
本实施例筛选了与BRAF V600E突变晚期结直肠癌双靶耐药肿瘤调控相关代谢物质。
1、试验材料和试验方法(下同)
人体标本试验材料:肿瘤标本采集自浙江大学附属邵逸夫医院结直肠癌手术切除患者。切除的肿瘤组织标本用液氮快速冷冻后保存在-80℃。
采用脂质代谢组学试验测定双靶耐药细胞和敏感细胞脂质含量,LC-MS非靶向代谢组学由美吉生物(中国上海)测定。
细胞活力及凋亡测定:使用CCK8试剂盒(Dojindo,Japan)测试细胞活力。取对数期生长的细胞,胰酶消化后离心、重悬,使用细胞计数仪准确计数,于96孔板中每孔接种5×103个细胞/200微升完全培养基,24小时后待细胞完全贴壁,吸弃培养基,每孔添加100微升含有指定试剂的完全培养基,再培养72小时,培养结束后,吸弃培养基,每孔添加新配置的10%V/V的水溶性WST-8染料,培养环境下孵育0.5-2小时,然后使用酶标仪检测450nm处吸光度,即OD值,并计算细胞活率。使用凋亡检测试剂盒(BD,美国),检测使用BDFACSARIALL流式细胞仪检测,设门排除细胞碎片和黏连细胞团,记录10000个细胞事件。结果使用Flowjo(Version X)软件进行分析。
Nile Red、IHC、TUNEL染色、甘油三酯检测:Nile red Staining Solution试剂盒,TUNEL细胞凋亡检测试剂盒(APPLYGEN,中国);Ki67抗体Cat No.28074-1-AP(PROTEINTECH中国),MOGAT3抗体DF9099(Affnity中国),采用Avidin Biotin Comlex(ABC)免疫组化试剂盒(北京中衫金桥生物科技有限公司)进行免疫组化法检测。外周血标本3000-4000r,离心10min,取上清,上机检测TG(全自动生化分析仪LW C400,深圳蓝韵医疗器械)。组织标本的包埋与切片制作由邵逸夫医院病理科协作完成。
2、试验结果
1)转录组学测序分析
对BRAF V600E突变晚期结直肠癌双靶耐药肿瘤与敏感肿瘤进行了转录组学测序分析,如图1所示,KEGG富集提示BRAF V600E突变晚期结直肠癌双靶耐药肿瘤与敏感肿瘤中代谢通路变化明显。
2)QPCR验证代谢通路相关基因
通过QPCR验证了代谢通路相关基因,如图2所示,发现MOGAT3在耐药组织里RNA水平上调最为明显。
3)免疫组化
对BRAF V600E突变晚期结直肠癌双靶耐药肿瘤与敏感肿瘤进行免疫组化分析,如图3和图4所示,耐药组织MOGAT3蛋白水平显著上升。
4)代谢通路分析
如图5所示,脂代谢通路变化在代谢改变中最明显。如图6所示,耐药组与敏感组相比甘油三酯明显上升。
5)切片染色
如图7所示,尼罗红染色提示耐药瘤组织中脂质含量增多。
6)WB试验
如图8所示,WB结果提示BRAF V600E突变的Encorafenib+Cetuximab耐药细胞RKOEC-R中MOGAT3蛋白明显上升。
7)代谢组学试验
如图9所示,代谢组学试验测定双靶耐药细胞(RKO EC-R)比敏感细胞(RKO)甘油三酯含量显著上升。
综合以上试验结果,研究人员发现脂质代谢在肿瘤发生和进展中起关键作用,但关于脂质如何引起肿瘤耐药的研究还需要进一步的探索。因此,为了深入地了解脂质重塑在肿瘤耐药中产生作用的分子机制,本研究进一步对双靶耐药PDX肿瘤中异常增高的代谢物质单酰基甘油酰基转移酶MOGAT3进行了探究。
实施例2
本实施例提供一种治疗BRAF V600E突变晚期结直肠癌的组合药物,其含有以下组分:2μM的Encorafenib、2μM的Cetuximab和20μM的MOGAT3小分子抑制剂PF-06471553,其余成分为生理盐水。
实施例3
本实施例对实施例2提供的组合药物进行了体外试验验证。
细胞培养:人结肠癌细胞系RKO购自中国科学院上海细胞库。用含10%胎牛血清的DMEM培养液(RKO)培养细胞。
1)细胞处理后,尼罗红染色试验
将双靶耐药细胞系接种12孔板并分成对照组(Vehicle)、单独PF-06471553(Pf)组(20μM)、Encorafenib+Cetuximab组(2μM的Encorafenib和2μM的Cetuximab)和实施例2中的药物处理组,每组均加药处理48小时,通过尼罗红染色评估脂质合成情况,如图10所示,双靶联合PF-06471553(Pf)可以显著抑制耐药细胞的脂质增加,降低耐药细胞中的脂滴。
2)细胞CCK8试验
将双靶耐药细胞系接种96孔板并分成对照组(Vehicle)、单独PF-06471553(Pf)组(20μM)、Encorafenib+Cetuximab组(2μM,Encorafenib和Cetuximab的摩尔浓度比为1:1)和实施例2中的药物处理组,每组均加药处理96小时,通过CCK8评估细胞增殖活力,如图11所示,双靶联合PF-06471553(Pf)可以显著抑制耐药细胞的增殖,使耐药细胞恢复敏感性。
3)细胞处理后,流式细胞术评估
将双靶耐药细胞系接种96孔板并分成对照组(Vehicle)、单独PF-06471553(Pf)组(20μM)、Encorafenib+Cetuximab组(2μM,Encorafenib和Cetuximab的摩尔浓度比为1:1)和实施例2中的药物处理组,每组均加药处理48小时,药物处理48小时后使用流式细胞术评估细胞凋亡水平如图12所示,双靶联合PF-06471553(Pf)可以明显促进耐药细胞的凋亡(图13)。
总的来说,考虑到PF-06471553或Encorafenib+Cetuximab单独处理在BRAF-V600E耐药细胞系中的作用有限,而在联合PF-06471553与Encorafenib+Cetuximab治疗后显著提高了Encorafenib+Cetuximab的效用。这些数据表明PF-06471553和Encorafenib+Cetuximab联合用药协同增强Encorafenib+Cetuximab的细胞杀伤作用并逆转耐药细胞对于双靶药的抗药性。
实施例4
本实施例为了进一步探讨在体内环境采用实施例2中的药物是否可以增加耐药肿瘤对双靶药的敏感性,构建了病人来源的双靶耐药肿瘤小鼠异种皮下移植肿瘤模型PDXs,用于评估PF-06471553联合Encorafenib+Cetuximab的组合效应。
1、小鼠PDX模型构建
取BRAF V600E突变结直肠癌患者的手术肿瘤标本,将肿瘤碎片接种到4周龄雌性裸鼠腹股沟皮下。当肿瘤大小达到200mm3时,作为后续给药疗效的控制时间点,小鼠被随机分配到一个队列和药物或载体每天给药。四组小鼠分别接受以下四个组别中的治疗方式:
对照组,生理盐水,与试验组(Pf组、Enc+Cet组和Enc+Cet+Pf组)等剂量灌胃每天;
Pf组,单独PF-06471553,20mg/kg(按小鼠体重计算,下同),灌胃每天一次;
Enc+Cet组,Encorafenib+Cetuximab组,按照Encorafenib:Cetuximab为1:1的摩尔浓度比组合,20mg/kg,灌胃每天一次;
Enc+Cet+Pf组,采用实施例2中的药物,即双靶药联合PF-06471553治疗,20mg/kg,灌胃每天一次。
治疗3周后处死小鼠,计数肿瘤大小并进行分析。
2、试验结果
1)小鼠肿瘤大小和重量
如图14和图15所示,双靶药联合PF-06471553(Pf)共处理后显著抑制肿瘤的生长,表现为肿瘤体积和重量的减小。
2)小鼠血液中脂质水平含量
在最后一次测量肿瘤数据后,眼眶外周血取血检测甘油三酯含量,结果如图16所示,外周血甘油三酯检测发现双靶药联合PF-06471553(Pf)共处理后TG水平显著降低。
3)小鼠体内移植的肿瘤生长数据
如图17所示,相较于对照组,单独使用PF-06471553(Pf)治疗组的肿瘤几乎没有受到抑制;而双靶药物处理组并没有表现出抑制肿瘤生长的趋势;联合使用双靶药与PF-06471553(Pf)显著抑制肿瘤生长,在四个组中肿瘤生长抑制程度最强。如图18所示,我们发现与其他治疗方法相比小鼠对于联合治疗的耐受性良好,联合组的体重减少与其他组间的差异可以忽略不计。
4)肿瘤中脂质含量
我们还使用了切片染色显示肿瘤内脂质的含量,IHC以及IF分析各组肿瘤内的蛋白表达。如图19和图20所示,Ki67和TUNEL染色,发现双靶药联合PF-06471553处理的肿瘤组织中的增殖减少,凋亡增加,有效缓解了双靶药的耐药问题,与对照组或仅双靶药处理组相比,PF-06471553(Pf)联合双靶药处理组明显降低了MOGAT3的蛋白表达水平,减少了Ki67增殖指标表达的同时增加了凋亡TUNEL。
综上,体内试验结果证实PF-06471553(Pf)与双靶药联合使用在体内同样能达到逆转耐药状态,这一结果与体外试验结果吻合。
综上所述,本申请实施例深入探索脂质代谢促进双靶药物耐药的具体机制,PDX动物模型上双靶药物联合MOGAT3的小分子抑制剂能发挥了协同增效作用,逆转耐药。因此提供一种双靶药物联合MOGAT3的小分子抑制剂的组合药物能够为治疗BRAF V600E突变晚期结直肠癌提供了一种新的用药选择。
以上所描述的实施例是本申请一部分实施例,而不是全部的实施例。本申请的实施例的详细描述并非旨在限制要求保护的本申请的范围,而是仅仅表示本申请的选定实施例。基于本申请中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本申请保护的范围。
Claims (3)
1.一种治疗BRAF V600E突变晚期结直肠癌的组合药物,其特征在于,其有效成分由以下物质组成:Encorafenib、Cetuximab和MOGAT3的小分子抑制剂。
2.根据权利要求1所述的BRAF V600E突变晚期结直肠癌的组合药物,其特征在于,所述MOGAT3的小分子抑制剂为PF-06471553。
3.根据权利要求2所述的BRAF V600E突变晚期结直肠癌的组合药物,其特征在于,组合药物中Encorafenib、Cetuximab和PF-06471553的摩尔浓度比为1:1:10。
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