CN116763766A - 聚乙二醇修饰的姜黄素在制备plk1激酶抑制剂中的应用 - Google Patents
聚乙二醇修饰的姜黄素在制备plk1激酶抑制剂中的应用 Download PDFInfo
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Abstract
本发明公开了一种聚乙二醇修饰的姜黄素在制备PLK1激酶抑制剂中的应用。本发明聚乙二醇修饰的姜黄素能够高效抑制PLK1的表达,可以用于制备PLK1激酶抑制剂;同时PLK1激酶抑制剂可以用于制备预防和/或治疗因PLK1激酶活性失调导致和/或与之相关的疾病的药物中的用途,包括细胞增殖性疾病、癌症、病毒感染、自身免疫性和神经变性疾病等。
Description
技术领域
本发明涉及医药技术领域,具体涉及一种聚乙二醇修饰的姜黄素在制备PLK1激酶抑制剂中的应用。
背景技术
Polo样激酶(Poloike kinases,PLKs)是一类结构高度保守的丝氨酸/苏氨酸蛋白激酶,各亚型的结构具有很高的相似性。在人类PLK家族的所有成员中,对PLK1的研究是最为透彻的。PLK1主要参与调节中心体成熟、纺体形成以及染色体分离等过程,对细胞有丝分裂过程起重要的调控作用。亚细胞区域定位实验表明,PLK1在不同时期定位于中心体、赤道板、着丝粒及胞质分裂处。在GO期到S期之间,PLK1的表达量和活性停留在较低水平,从G2期开始上升,于M期达到顶峰。动物实验表明抑制小鼠PLK1功能会干扰纺锤体组装,影响有丝分裂检查点的激活,进而延长分裂停滞时间,最终引起增殖细胞的凋亡。在某些人类细胞株实验中,敲除PLK1的基因也会诱导细胞周期停滞。因此,PLK1对于正常细胞的增殖具有不可或缺的作用。
但是,过高的PLK1表达也会产生一些因PLK1激酶活性失调导致和/或与之相关的疾病,如细胞增殖性疾病、癌症、病毒感染、自身免疫性和神经变性疾病等。PLK1在正常组织中胎盘、脾、卵巢、睾丸等生长增殖较快的细胞外中表达。PLK1的活化可诱导NIH3T3成纤维细胞的恶性转化,将其移植入裸鼠体内可诱导肿瘤形成。多项研究表明,PLK1与肿瘤的发生发展亦有重要的联系。PLK1在结直肠癌,胃癌,肺癌及乳腺癌等多种恶性肿瘤中异常高表达,其过表达也是肿瘤不良预后的标志之一。因此,PLK1在肿瘤诊断和治疗中是广受关注的靶点。
结直肠癌(Colorectal cancer,CRC)是我国第二高发的恶性肿瘤。CRC晚期患者需接受根治性手术和辅助治疗。目前,超过30%的患者治疗后仍会发生复发转移,发生复发转移的CRC患者5年生存率仅约13%。由于CRC的致癌靶点及其确切作用和机制尚未被充分认知,部分患者对现有药物具有原发和继发耐药性,且不同的靶向药物上尚存在不同程度的毒副作用,CRC现有临床干预效果十分有限。因此,急需寻找毒副作用小、能够有效治疗CRC的新药物及新疗法。许多临床实验已经表明靶向PLK1激酶的抑制剂成为CRC的有效治疗工具。迄今为止,已经开发了几种PLK1激酶抑制剂用于癌症治疗的临床试验。Volasertib是其中一种PLK1抑制剂,已被证明可以改善铂耐药癌症的临床疗效。
姜黄素(Curcumin,Cur)是一种多酚植物成分,从草本植物姜黄中提取,对正常组织的毒性很低。本发明基于姜黄素开发了一种新型的PLK1抑制剂,并公布了其在制备预防或治疗因PLK1激酶活性失调导致和/或与之相关的疾病的药物中的用途,如癌症(结直肠癌、胃癌和肺癌等)、细胞增殖性疾病(骨髓增生异常综合症)、病毒感染、自身免疫性和神经变性疾病。这种PLK1激酶抑制剂聚乙二醇修饰的姜黄素是一种两亲性分子,其在水溶液中自组织成纳米颗粒,并能在细胞内酸性条件下快速释放出姜黄素。与大多数通过纳米技术包装PLK1激酶抑制剂在体内实现对病灶组织的靶向不同,我们首次报道了一种原药不具备PLK1抑制效应,化学修饰后却能特异性抑制PLK1激酶的纳米剂型。该PLK1激酶抑制剂可以抑制PLK1基因的转录,抑制PLK1蛋白的表达,造成细胞的生长抑制或凋亡。此外,其还克服了小分子激酶抑制剂的体内应用时靶向难题,可以通过纳米剂型特定的EPR效应靶向灶组织。
发明内容
针对现有技术存在的不足,本发明的目的是提供聚乙二醇修饰的姜黄素在制备PLK1激酶抑制剂中的应用。本发明的PLK1激酶抑制剂可以用于制备预防和/或治疗因PLK1激酶活性失调导致和/或与之相关的疾病的药物,包括细胞增殖性疾病、癌症、病毒感染、自身免疫性和神经变性疾病等。
为实现上述目的,本发明采取的技术方案如下:
一种聚乙二醇修饰的姜黄素在制备PLK1激酶抑制剂中的应用,所述聚乙二醇修饰的姜黄素的结构式如下:
其中5≤n≤100,n为正整数。
优选的,所述PLK1激酶抑制剂包括聚乙二醇修饰的姜黄素的前药和/或聚乙二醇修饰的姜黄素的纳米制剂。
优选的,所述PLK1激酶抑制剂还包括至少一种药学上可接受的载体。
优选的,所述PLK1激酶抑制剂为口服制剂或注射剂。
上述的应用制备的PLK1激酶抑制剂在制备预防和/或治疗因PLK1激酶活性失调导致和/或与之相关的疾病的药物中的用途。
优选的,所述因PLK1激酶活性失调导致和/或与之相关的疾病包括细胞增殖性疾病、癌症、病毒感染、自身免疫性和神经变性疾病。
进一步优选的,所述细胞增殖性疾病包括骨髓增生异常综合症。
进一步优选的,所述预防和/或治疗癌症的药物单独给药或联合治疗。
更优选的,所述联合治疗包括放疗、化疗和手术治疗。
进一步优选的,所述癌症包括结直肠癌、胃癌、肺癌和乳腺癌。
本发明中的姜黄素的分子量为368,其结构式如下式所示:
姜黄素(CAS:458-37-7)是植物提取物,几乎不溶于水,具有抗炎、抗氧化等药理作用。据报道,在pH=7.2的磷酸盐缓冲液(0.1M,37℃)中培养时,约90%的姜黄素分子在30min内会分解。(Y.-J.Wang,M.-H.Pan,A.-L.Cheng,L.-I.Lin,Y.-S.Ho,C.-Y.Hsieh,J.-K.Lin,Stability of curcumin in buffer solutions and characterization of itsdegradation products,Journal of Pharmaceutical and Biomedical Analysis 15(12)(1997)1867-1876.)。姜黄素在生理环境中较低的溶解度和生物利用度显著限制了其临床应用。增殖实验表明姜黄素可抑制CRC细胞的增殖,并呈剂量依赖效应。人源性CRC异种移植(patient-derivedxenografts,PDX)中姜黄素可抑制肿瘤体内生长。本发明将聚乙二醇修饰的姜黄素制剂作为一种新的PLK1激酶抑制剂。
与现有技术相比,本发明的有益效果为:
(1)本发明的聚乙二醇修饰的姜黄素能够高效抑制PLK1的表达,可以用于制备PLK1激酶抑制剂;同时PLK1激酶抑制剂可以用于制备预防和/或治疗因PLK1激酶活性失调导致和/或与之相关的疾病的药物中的用途,包括细胞增殖性疾病、癌症、病毒感染、自身免疫性和神经变性疾病等。
(2)本发明将一种新的姜黄素制剂(聚乙二醇修饰的姜黄素)作为靶向治疗CRC的药物,来预防(治疗)和/或靶向CRC的发生或进展。细胞实验表明,聚乙二醇修饰的姜黄素可显著抑制CRC细胞的增殖。使用CRC患者来源的肿瘤PDX模型进行的体内实验表明,在PLK1高表达的PDX中,这种新的姜黄素制剂通过更显著的下调PLK1表现出更好的抗肿瘤作用,且没有明显的全身毒性。
(3)本发明所提供的新PLK1激酶抑制剂为CRC等肿瘤的预防和治疗提供了新的方向和方法。
附图说明
图1为MTT法检测新姜黄素制剂对结直肠癌HCT116细胞活力的影响;
图2为MTT法检测新姜黄素制剂对胃癌细胞HGC和AGS细胞活力的影响;
图3为流式细胞术检测新姜黄素制剂对CRC细胞周期的影响;
图4为RNA-seq检测新姜黄素制剂在基因水平对PLK1的影响;
图5为蛋白质免疫印迹法检测新姜黄素制剂在蛋白水平对PLK1的影响;
图6为新姜黄素制剂对PDX动物模型的影响;
图7为显微镜下PDX动物模型的脏器病理切片图。
具体实施方式
为更好地说明本发明的目的、技术方案和优点,下面将结合具体实施例对本发明作进一步说明。本领域技术人员应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。
实施例中所用的试验方法如无特殊说明,均为常规方法;所用的材料、试剂等,如无特殊说明,均可从商业途径得到。实施例中所用的姜黄素购买自上海麦克林生化科技股份有限公司。
实施例中所用的新姜黄素制剂(聚乙二醇修饰的姜黄素的前药)属于自制,结构式如下:
实施例1:
细胞实验评价姜黄素和新姜黄素制剂对CRC细胞增殖能力的影响
(1)细胞培养
结直肠癌HCT116细胞用含10%胎牛血清(Invitrogen,USA)的RPMI-1640(Gibco,USA)培养基培养,置于含5% CO2的37℃恒温细胞培养箱中培养。
(2)MTT法检测新姜黄素制剂对CRC细胞活性的影响:
用MTT法检测HCT116的细胞活性。
MTT法检测细胞活性的具体步骤为:
1)将培养至对数生长期的HCT116细胞接种于96孔板,每孔100μL培养基中含有5×103个细胞,培养一天。分别设置空白组、对照组和药物处理组(新姜黄素制剂和姜黄素),其中空白组不含细胞只有培养基,对照组含有细胞不加药物,药物处理组含有2.5、5、10、15和20μg/mL的不同浓度的新姜黄素制剂或姜黄素。
2)第二天,向每孔加入200μL培养基或含有不同药物浓度的培养基。细胞在培养箱中继续培养48h后,每孔加入MTT溶液避光孵育。
用全波长多功能酶标仪在波长570nm处测定吸光度值。
用GraphPad Prism 8.0.1软件计算IC50值。
MTT法检测细胞活力结果如图1所示,姜黄素及其新制剂对HCT116的IC50值分别为12.58±1.89μg/mL和9.37±1.37μg/mL。
实施例2:
细胞实验评价姜黄素和新姜黄素制剂对胃癌HGC和AGS细胞增殖能力的影响
(1)细胞培养
胃癌HGC和AGS细胞用含10%胎牛血清(Invitrogen,USA)的RPMI-1640(Gibco,USA)培养基培养,置于含5% CO2的37℃恒温细胞培养箱中培养。
(2)MTT法检测新姜黄素制剂对HGC和AGS细胞活性的影响:
MTT法检测细胞活性的具体步骤为:
1)将培养至对数生长期的HGC和AGS细胞接种于96孔板,每孔100μL培养基中含有5×103个细胞,培养一天。分别设置空白组、对照组和药物处理组(新姜黄素制剂和姜黄素),其中空白组不含细胞只有培养基,对照组含有细胞不加药物,药物处理组含有1.25、2.5、3.75、5.0和7.5μg/mL的不同浓度的姜黄素及其新制剂。
2)第二天,向每孔加入200μL培养基或含有不同药物浓度的培养基。细胞在培养箱中继续培养48h后,每孔加入MTT溶液避光孵育。
用全波长多功能酶标仪在波长570nm处测定吸光度值。
用GraphPad Prism 8.0.1软件计算IC50值。
MTT法检测细胞活力结果如图2所示,姜黄素及其新制剂对HGC细胞的IC50值分别为3.28±0.21μg/mL和2.57±0.27μg/mL,对AGS细胞的IC50值分别为4.73±0.56μg/mL和2.90±0.10μg/mL。
实施例3:
姜黄素及其新制剂阻滞HCT116细胞周期
流式细胞术检测姜黄素及其新制剂阻滞HCT116细胞周期。
将贴壁HCT116细胞用无血清培养基培养48h,获得G0/G1期同步细胞。同样,用胸苷(Sigma,德国)处理贴壁细胞24h,获得S期细胞。用nocodazole(MCE,USA)处理HCT116细胞18h,获得G2/M期细胞。不同阶段的同步细胞用含药物的新鲜营养物以10μg/mL等剂量姜黄素培养。处理24h后,收集不同时间点的细胞样本,用流式细胞仪进行细胞周期检测。
如图3所示,姜黄素及其新制剂可以不同程度地阻滞HCT116细胞于G0/G1,S和G2/M期。
实施例4:
RNA-seq检测姜黄素和新姜黄素制剂在基因水平对PLK1的影响
利用RNA测序(RNA Sequencing,RNA-seq)技术深入分析姜黄素和新姜黄素制剂在基因水平对PLK1的影响。将HCT116细胞用含姜黄素或新姜黄素制剂的培养基培养24h后,用Trizol法提取细胞总RNA。RNA-seq由Novagene股份有限公司在Illumina平台上进行检测。采用DEseq法分析样品间的差异表达基因(DEGs)。以FDR值<0.05和|log2 Fold Change|≥1为阈值识别差异表达基因(DEGs)。每个样本有三个生物学重复。
如图4所示,与没有药物处理的对照组相比,姜黄素原药没能抑制PLK1的表达,然而新姜黄素制剂却能极显著地特异性靶向抑制HCT细胞中PLK1的表达。
实施例5:
蛋白质免疫印迹法检测姜黄素和新姜黄素制剂在蛋白水平对PLK1的影响
具体步骤如下:空白对照或10μg/mL的姜黄素及其新制剂处理HCT116细胞24h后,吸去培养基,加入蛋白裂解液,置于冰上裂解,裂解液转移至1.5mL的EP管中,超声仪4℃破碎10min,12000rpm离心10min,吸取上清蛋白样品依次进行SDS-PAGE凝胶电泳、转膜、封闭、一抗(MEK、PLK1和GAPDH)4℃孵育过夜、二抗室温孵育1h、化学发光显影。
如图5所示,姜黄素不能在蛋白水平特异性抑制HCT细胞中PLK1的表达,新姜黄素制剂可以在蛋白水平特异性抑制HCT细胞中PLK1的表达,这表明新姜黄素制剂可以作为PLK1的靶向抑制剂。
实施例6:
动物实验评价姜黄素和新姜黄素制剂对PDX动物模型的影响
利用PLK1高低表达的PDX动物模型验证新姜黄素制剂的抗肿瘤效果。
具体如下:
实验使用的雌性BALB/c裸鼠(SPF级)购自中国广东药康股份有限公司。肿瘤组织(PDXlow:IIA期;PDXhigh:IIIB期)取自中山大学附属第六医院未经术前化疗的晚期结直肠癌患者。将新鲜切除的不同患者PLK1低表达和PLK1高表达的肿瘤组织接种于血供丰富的BALB/c裸鼠右前腋窝,构建PLK1高/低表达的PDX模型,分别为PDXlow(IIA期)和PDXhigh(IIIB期)模型。接种后,待肿瘤生长大小至50mm3左右后,将裸鼠进行随机分组,分别设对照组(生理盐水)、姜黄素处理组(35mg/kg)及新姜黄素制剂处理组(35mg/kg),给药方式为隔日腹腔注射,连续治疗20天。
每隔2日测量肿瘤大小。肿瘤体积计算公式为:V=a2b/2(a为肿瘤短直径,b为肿瘤长直径)。
治疗结束后安乐死裸鼠,解剖肿瘤。动物实验所有操作均严格按照动物实验保护准则进行。
PDX动物模型结果如图6所示,PLK1高表达(PLK1high)的肿瘤比PLK1低表达(PLK1low)的肿瘤生长更快(图6)。在体内,Cur和mCur可以抑制肿瘤生长。mCur表现出比Cur更好的抗肿瘤活性。值得注意的是,对于PLK1high的肿瘤,mCur表现出明显强于Cur的抗癌作用。如图6所示,mCur组的肿瘤大小和肿瘤重量均明显小于Cur组。与体外实验证据一致,PLK1的表达在体内被mCur特异性下调。这些结果表明,PLK1在体内对mCur的抗肿瘤活性起着至关重要的作用,mCur是一种有效的靶向治疗CRC的PLK1抑制剂。
实施例7:
动物实验评估姜黄素和新姜黄素制剂的体内脏器毒性
取实施例6中实验后的PDX动物模型,分离肿瘤后,摘取心脏、肺脏、肝脏和肾脏,用体积分数为10%的甲醛固定后,石蜡包埋,切片,HE染色,显微镜下观察脏器组织的形态学变化。
肾脏和肝脏病理切片结果如图7所示,新姜黄素制剂对裸鼠的心、肺、肝和肾均无病理毒性。
综上所述,体外细胞实验结果显示,姜黄素及其新制剂对HCT116的IC50值分别为12.58±1.89μg/mL和9.37±1.37μg/mL。姜黄素及其新制剂对HGC细胞的IC50值分别为3.28±0.21μg/mL和2.57±0.27μg/mL,对AGS细胞的IC50值分别为4.73±0.56μg/mL和2.90±0.10μg/mL。同时,新姜黄素制剂可以有效抑制HCT细胞于G0/G1,S和G2/M期。此外,RNA-seq和蛋白质免疫印迹检测表明新姜黄素制剂可以有效抑制PLK1的表达。PDX动物模型结果表明新姜黄素制剂可特异性抑制PLK1高表达的PDX模型的肿瘤生长,对裸鼠的心、肺、肝和肾均无病理毒性。因此,新姜黄素制剂可作为靶向治疗CRC的PLK1抑制剂,为CRC等肿瘤的预防和治疗提供了新的治疗方案。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (10)
1.聚乙二醇修饰的姜黄素在制备PLK1激酶抑制剂中的应用,其特征在于,所述聚乙二醇修饰的姜黄素的结构式如下:
其中5≤n≤100,n为正整数。
2.根据权利要求1所述的应用,其特征在于,所述PLK1激酶抑制剂包括聚乙二醇修饰的姜黄素的前药和/或聚乙二醇修饰的姜黄素的纳米制剂。
3.根据权利要求1所述的应用,其特征在于,所述PLK1激酶抑制剂还包括至少一种药学上可接受的载体。
4.根据权利要求1所述的应用,其特征在于,所述PLK1激酶抑制剂为口服制剂或注射剂。
5.权利要求1-4任一项所述的应用制备的PLK1激酶抑制剂在制备预防和/或治疗因PLK1激酶活性失调导致和/或与之相关的疾病的药物中的用途。
6.根据权利要求5所述的用途,其特征在于,所述因PLK1激酶活性失调导致和/或与之相关的疾病包括细胞增殖性疾病、癌症、病毒感染、自身免疫性和神经变性疾病。
7.根据权利要求6所述的用途,其特征在于,所述细胞增殖性疾病包括骨髓增生异常综合症。
8.根据权利要求6所述的用途,其特征在于,所述预防和/或治疗癌症的药物单独给药或联合治疗。
9.根据权利要求8所述的用途,其特征在于,所述联合治疗包括放疗、化疗和手术治疗。
10.根据权利要求6所述的用途,其特征在于,所述癌症包括结直肠癌、胃癌、肺癌和乳腺癌。
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