CN114848620B - Tlr2抑制剂联合folfox在治疗结直肠癌中的应用 - Google Patents
Tlr2抑制剂联合folfox在治疗结直肠癌中的应用 Download PDFInfo
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Abstract
本发明提供一种TLR2抑制剂联合FOLFOX在治疗结直肠癌中的应用。本发明一方面提供了TLR2抑制剂在制备提高结直肠癌对FOLFOX敏感性药物中的应用。所述TLR2抑制剂可联合FOLFOX,同时用于治疗结直肠癌。研究表明TLR2激活了treg细胞导致CD8+T细胞杀伤肿瘤作用减低,进而治疗不敏感,而TLR2抑制剂可抑制这一现象的发生。本发明的另一方面提供了一种用于治疗结直肠癌的药物组合物,所述药物组合物中含有治疗有效量的TLR2抑制剂和FOLFOX。本发明技术方案中TLR2抑制剂可以显著降低结直肠癌患者对FOLFOX的不敏感性,TLR2抑制剂联合FOLFOX可以提高FOLFOX对患者的治疗作用。
Description
技术领域
本发明涉及医疗药物领域,特别是涉及TLR2抑制剂联合FOLFOX在治疗结直肠癌中的应用。
背景技术
结直肠癌(Colorectal cancer,CRC)是最常见的消化道恶性肿瘤之一,其发病率和死亡率在全球范围内居恶性肿瘤第二位。约20%的CRC患者伴有远处转移,对于不可手术切除的晚期CRC患者,治疗方案包括化疗、放疗、靶向治疗和免疫治疗等。目前,根据CRC的美国国立综合癌症网络(National Comprehensive Cancer Network,NCCN)指南指出,以5-氟尿嘧啶和奥沙利铂为基础的化疗方案(FOLFOX)仍是进展期以及转移性CRC的主要治疗手段。然而,只有约一半的CRC患者对FOLFOX方案敏感,其余患者则表现为FOLFOX方案不敏感并伴发有害的、甚至危及生命的副作用,极大的影响晚期CRC治疗效果。因此,深入研究关于增加FOLFOX敏感的分子机制并寻找潜在的增敏靶点,对于改善 CRC患者的预后具有重要的意义。
TLR是一种模式识别受体,可对外源性危险信号(通常是病毒或细菌的产物) 作出反应,并触发固有免疫应答。大量研究表明TLR活化可促进获得性免疫应答。TLR活化可以启动树突状细胞(DC)的成熟与活化。免疫系统参与控制肿瘤的发生和发展已被充分证明。
关于TLR与结直肠癌的关系,目前尚未有明确的研究结论。
发明内容
鉴于以上所述现有技术的缺点,本发明的目的在于提供TLR2抑制剂联合 FOLFOX在治疗结直肠癌中的应用,用于解决现有技术中患者对FOLFOX不敏感的问题。
本发明通过对FOLFOX不同敏感性的CRC患者的研究结果分析发现TLR2 在调控FOLFOX敏感性中起到作用,解决了对FOLFOX不敏感的CRC患者由于体内高水平的TLR2表达,抑制了体内的抗肿瘤免疫过程,免疫细胞无法杀伤肿瘤细胞,造成FOLFOX的疗效不佳的问题。
为了解决上述问题,本发明的一方面提供了TLR2抑制剂在制备提高结直肠癌对FOLFOX敏感性药物中的应用。所述TLR2抑制剂可联合FOLFOX,同时用于治疗结直肠癌。研究表明TLR2激活了treg细胞导致CD8+T细胞杀伤肿瘤作用减低,进而治疗不敏感,而TLR2抑制剂可抑制这一现象的发生。其中 FOLFOX是指5-氟尿嘧啶和奥沙利铂为基础的化疗方案。
进一步地,所述TLR2抑制剂可以为现有已知的任一可以降低TLR2表达的化合物,例如3-((2-羟基-3-甲氧基亚苄基)氨基)-2-甲基苯甲酸,即C29,分子式 C16H15NO4。
本发明的另一方面提供了一种可提高结直肠癌对FOLFOX敏感性药物,所述药物含有治疗有效量的TLR2抑制剂。
进一步地,所述药物中还含有人体可接受的载体。这类载体包括(但并不限于):盐水、缓冲液、葡萄糖、水、甘油、乙醇、粉剂、及其组合。
所述药物或者药物组合物的形式无特殊限制,可以为固体、液体、凝胶、半流质、气雾等各种物质形式。
进一步地,所述TLR2抑制剂可以为现有已知的任一可以降低TLR2表达的化合物,例如3-((2-羟基-3-甲氧基亚苄基)氨基)-2-甲基苯甲酸,即C29,分子式 C16H15NO4。
本发明的另一方面提供了一种用于治疗结直肠癌的药物组合物,所述药物组合物中含有治疗有效量的TLR2抑制剂和FOLFOX。
进一步地,所述TLR2抑制剂可以为现有已知的任一可以降低TLR2表达的化合物,
例如3-((2-羟基-3-甲氧基亚苄基)氨基)-2-甲基苯甲酸,即C29,分子式C16H15NO4。
本发明的组合物可以被制成针剂形式,例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。诸如片剂和胶囊之类的药物组合物,可通过常规方法进行制备。药物组合物如针剂、溶液、片剂和胶囊宜在无菌条件下制造。
本发明的药物、制剂或者药物组合物可通过常规的方式施用于所需的对象 (如人和非人哺乳动物)。代表性的施用方式包括(但并不限于):口服或注射(包括静脉注射、静脉滴注、肌肉注射或皮下注射等中的一种或多种)等中的一种或多种给药途径。使用药物组合物时,是将安全有效量的药物施用于哺乳动物。当然,具体剂量和方法还应考虑给药途径、病人健康状况等因素,这些都是熟练医师技能范围之内的。
如上所述,本发明的TLR2抑制剂联合FOLFOX在治疗结直肠癌中的应用,具有以下有益效果:TLR2抑制剂可以显著降低结直肠癌患者对FOLFOX的不敏感性,TLR2抑制剂联合FOLFOX可以提高FOLFOX对患者的治疗作用。
附图说明
图1显示为两组患者在接受FOLFOX化疗后病理标本及测序热图。
图2显示为敏感组与不敏感组测序差异分子信号通路分析。
图3显示为敏感组与不敏感组TLR2,CD8和treg表达情况。
图4显示为结直肠癌中TLR2表达与CD8+T细胞和treg细胞浸润相关性。
图5A显示为两组小鼠体内肿瘤照片。
图5B显示为小鼠体内肿瘤大小曲线图。
图5C显示为小鼠体内肿瘤重量直方图。
图5D显示为小鼠体内肿瘤组织免疫组化结果图。
具体实施方式
本发明的研究结果显示,与对FOLFOX化疗方案敏感的患者相比,在对 FOLFOX化疗方案不敏感的患者中,TOLL样受体信号通路显著激活,其中TLR2 与抗肿瘤免疫抑制显著相关。在免疫正常的小鼠中,TLR2抑制剂(例如C29, CAS 363600-92-4)能够增强FOLFOX化疗对CRC的作用。基于此本发明首次提出了一种基于TLR2抑制剂和FOLFOX药物治疗结直肠癌联合协同治疗新策略,这将有助于提高结直肠癌化学药物治疗水平,与此同时增加C29在肿瘤治疗中的适应范围。
实验方法:
1.RNA提取和转录组测序
22例分离肿瘤组织样本,加入Trizol裂解液。按照试剂盒说明书提取总RNA,用Bioanalyzer2200检测RNA含量和纯度。选择RNA完整性(RIN)为8.0以上的样本参与后续cDNA文库的构建,使用TruSeqTMRNA Sample进行cDNA文库的定量分析,采用凝胶电泳和Bioanalyzer 2200进行分析。基于IlliminaHiSeqTM 2000双端测序技术构建22个样本,转录组数据库、相关cDNA文库构建及后续深入数据分析均在上海烈兵生物技术有限公司完成。
2.GO分析
根据NCBI的关键功能分类——基因本体论(Gene Ontology),应用GO分析对差异表达基因的主要功能进行分析。一般采用Fisher精确检验和χ2检验对 GO类进行分类,通过计算错误发现率(FDR)对p值进行校正,FDR越小,对p 值的判断误差越小。FDR定义为FDR=1Nk/T,其中Nk为Fisher检验p值小于χ2检验p值的个数。我们计算了所有差异基因的GOs的p值。显著GO项定义为P值<0.05和FDR<0.05。对于GO项冗余的处理,我们采用了从叶到根的每条路径中只选取一个项的过滤策略。
3.通路分析
同样,根据KEGG、Biocarta和Reactome的分析,也采用了Pathway分析的方法来找出差异基因的重要通路。然而,我们仍然采用Fisher精确检验和χ2检验来选择显著性途径,显著性阈值由P-value和FDR定义。P<0.05和FDR<0.05。浓缩的计算方法与上面的公式相似。KEGG数据库包括代谢、膜转运、信号转导、细胞周期通路以及它们之间相互作用的信息。我们选择的基因可能涉及两个或更多的信号通路。由于同一基因可能存在于不同的路径中,不同路径之间的重叠会突出关键基因。我们选择了富集生物学通路中的基因,并使用Cytoscape 进行通路的图示。
4.免疫荧光检测
细胞用4%多聚甲醛固定15min,PBS洗涤后,0.1%Triton X-100在室温下渗透5min,PBS洗涤后,5%BSA在室温下封闭1小时。过夜之间主要抗体被用来培养细胞在4摄氏度,包括FOXP3(CST,12653),CD8(ABCAM AB237709)和 TLR2(ABCAM AB209216)。二抗选用Alexa Fluor 488和Alexa Fluor 555二抗 (Abcam)。将细胞在37℃孵育1小时。用二胺基苯基吲哚(DAPI,Santa Cruz,USA)复染细胞核。使用激光扫描共聚焦显微镜(Zeiss,LSM510)观察结果。
5.balb/c皮下成瘤实验
将呈稳定生长的CT26细胞制备成无血清悬液,取4-6周龄雄性裸鼠小鼠,分别于小鼠右下腹注射肿瘤细胞。FOLFOX和C29通过尾静脉/腹腔注射给药。 C29的给药浓度为1mg/kg,奥沙利铂6mg/kg两小时后,予90mg/kg叶酸+50mg/kg 5-FU。种植肿瘤后第四天给药并用游标卡尺测量瘤体直径,每隔3天给药并用游标卡尺测量瘤体直径,连续观察4次。
以下通过特定的具体实施例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。须知,下列实施例中未具体注明的工艺设备或装置均采用本领域内的常规设备或装置。此外应理解,本发明中提到的一个或多个方法步骤并不排斥在所述组合步骤前后还可以存在其他方法步骤或在这些明确提到的步骤之间还可以插入其他方法步骤,除非另有说明;还应理解,本发明中提到的一个或多个设备/装置之间的组合连接关系并不排斥在所述组合设备/装置前后还可以存在其他设备/装置或在这些明确提到的两个设备/装置之间还可以插入其他设备/装置,除非另有说明。而且,除非另有说明,各方法步骤的编号仅为鉴别各方法步骤的便利工具,而非为限制各方法步骤的排列次序或限定本发明可实施的范围,其相对关系的改变或调整,在无实质变更技术内容的情况下,当亦视为本发明可实施的范畴。
实施例1对FOLFOX敏感和不敏感的患者进行测序分析
(1)实验对象:收集2017年1月至2019年1月在我院胃肠外科就诊的患者。22例CRC患者因TNM晚期而术前接受新辅助治疗。22例病理类型为腺癌的CRC患者签署知情同意书并获得上海交通大学医学院附属瑞金医院伦理委员会批准。收集22例患者的一般资料、新辅助治疗前后肿瘤相关指标、肿瘤回归分级(tumor regression grade,TRG)等相关资料,所有患者术前均接受2-4疗程的 mFOLFOX新辅助治疗。因此,本研究共纳入22例患者。新辅助治疗后按TRG 进行分组。TRG是指原发病灶在放化疗干预后的相对回归程度,是衡量肿瘤新辅助治疗的重要评价依据之一。患者有显著的回归,纳入新辅助治疗敏感组。 13例TRG评级为2~3的患者纳入新辅助治疗不敏感组。9例TRG评级为0~1 的患者纳入新辅助治疗敏感组。
(2)检测步骤:进行转库组测序和HE染色。
(3)检测结果:与不敏感组相比,敏感组肿瘤病理学明显退缩(图1中B)。测序结果显示一致性良好(图1中A)。敏感组和不敏感组的FOLFOX新辅助化疗前后的MRI典型图像(图1中C)。
实施例2对实施例1中的测序数据进行分析
(1)实验对象:两组测序数据。
(2)检测步骤:通过基因本体论(GO)分析和KEGG通路数据库进行通路分析来对FOLFOX在CRC中差异敏感的相关的潜在生物学过程。
(3)检测结果:对FOLFOX敏感组和不敏感组的分析显示,局部炎症反应、免疫反应和TOLL样受体信号通路显著富集(图2),说明在不敏感组中存在 TOLL样受体信号通路激活和抗肿瘤免疫抑制的情况。
实施例3免疫荧光染色比较两组间免疫细胞分布差异
(1)实验对象:两组患者肿瘤组织。
(2)检测步骤:免疫荧光染色TLR2,CD8和FOXP3。
(3)检测结果:比较两组间荧光分布差异,结果显示,不敏感组TLR2和 FOXP3阳性细胞显著高于敏感组,不敏感组CD8阳性细胞显著低于敏感组(图 3)。
实施例4分析结直肠癌中TLR2表达与CD8+T细胞和treg细胞浸润相关性
(1)实验对象:TIMER2数据库结直肠癌患者数据。
(2)检测步骤:运用数据库分析结直肠癌中TLR2与CD8+T细胞和treg 细胞浸润相关性。
(3)检测结果:TLR2表达与treg细胞显著正相关(Y=58.70*X+1.602) (Y=TLR2,X=treg),与CD8+T细胞显著负相关(Y=-19.70*X+2.520)(Y=TLR2, X=CD8+T)(图4)。
实施例5体内实验证实免疫正常小鼠中TLR2抑制剂C29对FOLFOX治疗的增敏作用
(1)实验对象:鼠结肠癌细胞CT26细胞和balb/c小鼠。
(2)检测步骤:用CT26构建balb/c小鼠进行皮下成瘤实验,建立肿瘤模型后第四天,进行FOLFOX治疗(静脉或腹腔给药,奥沙利铂6mg/kg两小时后,予90mg/kg叶酸+50mg/kg5-FU),同时施用或不施用TLR2激动剂(HSP70, 10mg/kg)及TLR2抑制剂(C29,1mg/kg),隔三天给药一次,共四次后评估疗效。
分别设计如下四组分组(生理盐水对照,FOLFOX,FOLFOX+C29, FOLFOX+C29+HSP70)治疗过程中每周计量小鼠肿瘤体积,治疗结束后取出小鼠肿瘤,称量肿瘤。
(3)检测结果:FOLFOX对于CT26形成的肿瘤有治疗效果,FOLFOX联合C29治疗效果最佳,肿瘤相比较于单独使用FOLFOX组肿瘤体积明显减小84%(FOLFOX组0.953±0.071cm3对比FOLFOX+C29组0.155±0.089cm3, P<0.0001)、肿瘤重量明细减少约84%(FOLFOX组1.068±0.079g对比 FOLFOX+C29组0.170±0.098g,P<0.0001),而重组蛋白HSP70可以抵消TLR2 对FOLFOX的增敏作用(图5A-C)。免疫组织化学染色法(图5D)证实在免疫正常的小鼠中,C29可以增强FOLFOX化疗对肿瘤增殖(Ki-67)的抑制,并减少肿瘤组织中的CD8+T细胞浸润,增加Treg(FOXP3)的细胞浸润。
结论与分析
本发明的研究结果显示,通过转录组测序实验,以NCCN肿瘤退缩分级(tumorregression grading,TRG)为标准,系统比较分析了9例FOLFOX敏感和13例FOLFOX不敏感CRC患者之间基因的差异表达(9例TRG 0~1分,列入新辅助治疗敏感组;13例TRG 2~3分,列入新辅助治疗不敏感组),发现在对FOLFOX化疗方案敏感组的患者肿瘤退缩和病理学缓解均优于不敏感组人群,并取两组患者肿瘤组织进行转录组测序(图1)。而敏感组与不敏感组测序差异分子信号通路分析显示主要富集炎症反应、中性粒细胞趋化,免疫反应以及TOLL样受体信号通路(图2),取两组患者标本切片进行免疫荧光实验检测 TLR2和主要免疫细胞,结果显示在FOLFOX不敏感组中TLR2和FOXP3(treg) 表达量高于敏感组,CD8表达量低于敏感组(图3)。进一步发现在结直肠癌中, TLR2表达与treg细胞显著正相关,与CD8+显著负相关(图4)。提示不敏感组中是由于TLR2激活了treg细胞导致CD8+T细胞杀伤肿瘤作用减低,进而治疗不敏感。体内实验结果表明,在免疫正常的小鼠中,C29能够增强FOLFOX化疗对CRC的作用(图5A-C)。
以上的实施例是为了说明本发明公开的实施方案,并不能理解为对本发明的限制。此外,本文所列出的各种修改以及发明中方法、组合物的变化,在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述,但应当理解,本发明不应仅限于这些具体实施例。事实上,各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。
Claims (4)
1.一种TLR2抑制剂与FOLFOX药物联合在制备提高结直肠癌对FOLFOX敏感性的药物中的应用,所述TLR2抑制剂为C29。
2.根据权利要求1所述的应用,其特征在于:所述药物具有至少以下任一作用:
a.降低TLR2的表达;
b.降低TLR2对treg细胞的激活作用;
c.增强CD8+T对肿瘤细胞的杀伤作用。
3.根据权利要求1所述的应用,其特征在于:所述药物中还含有人体可接受的载体。
4.一种用于治疗结直肠癌的药物组合物,其特征在于:所述药物组合物中含有治疗有效量的TLR2抑制剂和FOLFOX疗法的药物,所述TLR2抑制剂为C29。
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