CN104045693B - 一种关于per2蛋白激动剂多肽及其应用 - Google Patents
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Abstract
本发明涉及药物领域，具体涉及具有促进PER2蛋白表达，治疗治疗食道癌的多肽。其序列为AAMMANMEIKIC 是全新的序列，该多肽可治疗食道癌，提高在体内荷瘤小鼠生存率；抑制人食道癌细胞TE1 和ECA109的增殖；具有潜在的新药开发价值。
Description
技术领域
本发明涉及PER2蛋白激动剂多肽4及其应用，具体涉及具有促进PER2蛋白表达、治疗食道癌的多肽。
背景技术
食管癌的治疗上根据病灶的不同位置、疾病分期可以选择手术、放疗、化疗、生物靶向治疗方式。食管癌的手术常切除癌变组织与转移淋巴结，管状胃代替其功能。但是手术难以完全切除癌组织与亚临床病灶。其次就是放化疗，但是这两种治疗方式均对正常组织器官有明显副反应，进一步提高疗效并减少对正常组织的损伤是值得研究的课题。目前生物靶向治疗肿瘤是非常有前景的，靶向治疗是针对肿瘤发生过程中阻断某个或多个信号通路，达到治疗肿瘤的目的。
在探索肿瘤病因，寻找对肿瘤的治疗方法过程中，发现生物节律的紊乱与肿瘤的发生发展有很大关系。生物节律的紊乱与食管癌的发生发展有一定关系。在用现代时间生物学手段来探测肿瘤节律基因的变化已成为食管癌治疗的新兴研究课题。
生物节律基因PER2表达的PER2蛋白与正常光暗循环条件下均成自发性节律变化。PER2在PER系列基因中与肿瘤关系最为密切。研究表明，PER2蛋白在食管癌组织中低表达，而在癌旁组织中高表达，PER2可以看做是一个保护性基因，其表达可以上调抑癌基因p53，下调癌基因c-cmyc的表达。PER2的表达可以抑制瘤细胞的增殖、使永生化能力的丧失，同时可以通过阻滞瘤细胞的G2/M期，达到调控细胞周期增加放疗敏感性的目的。
PER2蛋白功能降低是食管癌发生的一个重要因素。因此，促进PER2蛋白表达，抑制食管癌发展，是治疗食管癌的新靶点。但是，尚未有开发成熟的PER2蛋白激动剂多肽的治疗食管癌的药物
本专利中的PER2蛋白激动剂多肽4已证明在食管癌中有效，具有在其他肿瘤模型中开发的前景。
发明内容
发明目的
本发明提供全新的序列，该序列PER2蛋白激动剂，对食管癌具有很好的疗效。
技术方案
PER2蛋白激动剂多肽4，其特征在于其序列为AAMMANMEIKIC。
一种药物组合物，其特征在于其包含如权利要求1所述多肽与一种以上药学上可接受的赋形剂、填充剂、粘合剂、润滑剂、崩解剂、或稳定剂。
所述的药物组合物，其特征在于，所述组合物为注射剂。
所述的多肽，其特征在于有效剂量为10mg/kg。
PER2蛋白激动剂多肽4在制备治疗食管癌药物中的应用。
有益效果
利用固相合成法化学合成PER2蛋白激动剂多肽4，该多肽具有全新的序列，该多肽可治疗食道癌，提高在体内荷瘤小鼠生存率；抑制人食道癌细胞TE1和ECA109的增殖；具有潜在的新药开发价值。
具体实施方式
本发明涉及多肽有吉尔生化(上海)合成。
实施例1
PER2蛋白激动剂多肽4人食道癌细胞TE1增殖的作用。
采用MTT比色法。将对数生长的TE1细胞，以1.0×105加入96孔培养板中，培养24h，实验孔、阳性药物对照孔分别加入不同浓度的实验药物PER2蛋白激动剂多肽4和阳性对照药物长春新碱；空白组加入相同体积的溶剂。每孔设五个复孔，培养48h，分别在0h、2h、8h、14h、20h、24h、36h、48h每孔加入MTT，作用4h后，加入DMSO，孵育30min，在酶标仪620nm处测定吸光度A值，按公式肿瘤细胞生长抑制率＝(1-实验组吸光值/对照组吸光值)×100％。计算出实验药物的IC50为6.40μM。当浓度为6.40μM时，对Eca109增殖抑制率为70.3％。
实施例2
PER2蛋白激动剂多肽4人食道癌细胞ECA109增殖的作用。
采用MTT比色法。将对数生长的ECA109细胞，以1.0×105加入96孔培养板中，培养24h，实验孔、阳性药物对照孔分别加入不同浓度的实验药物PER2蛋白激动剂多肽4和阳性对照药物长春新碱；空白组加入相同体积的溶剂。每孔设五个复孔，培养48h，分别在0h、2h、8h、14h、20h、24h、36h、48h每孔加入MTT，作用4h后，加入DMSO，孵育30min，在酶标仪620nm处测定吸光度A值，按公式肿瘤细胞生长抑制率＝(1-实验组吸光值/对照组吸光值)×100％。计算出实验药物的IC50为7.10μM。当浓度为7.10μM时，对Eca109增殖抑制率为60.8％。
实施例3
用肿瘤模型检测PER2蛋白激动剂多肽4的体内活力。
建立Eca109肿瘤模型，阳性对照药物长春新碱；空白组加入相同体积的溶剂，实验组设3个剂量：6、12、24mg/Kg。21天后，观察小鼠存活数量，计算存活率。结果显示，PER2蛋白激动剂多肽4可有效地保护小白鼠，提高荷瘤小鼠的生存率，生存率达到88.4％。
实施例4
PER2蛋白激动剂多肽对人食道癌细胞TE1裸鼠异种移植肿瘤生长抑制试验
取对数生长期的人食道癌细胞TE1细胞株，在无菌条件下制备成5×107/ml细胞悬液，以0.1ml接种于裸鼠右侧腋窝皮下。用游标卡尺测量裸鼠移植瘤直径，待肿瘤生长至100-200mm3后动物随机分组。使用测量瘤径的方法，动态观察被试多肽的抗肿瘤效果。肿瘤直径的测量次数为每2天1次，每次测量同时还需称量鼠重。给药方式均采用尾静脉注射。阴性对照组注射等量生理盐水，每天1次；顺铂组10mg/kg，每周给药1次；恩度组2.5mg/kg，每天给药1次；多肽高中低组分别以20mg/kg、10mg/kg、5mg/kg，每天给药1次。肿瘤体积计算公式：
TV＝0.52×a×b2
其中a、b分别表示长宽。根据测量的结果计算出相对肿瘤体积。抗肿瘤活性的评价指标为相对肿瘤增殖率T/C(％)，计算公式如下：
T/C(％)＝TRTV/CRTV×100％
TRTV：治疗组RTV；CRTV：阴性对照组RTV
表1多肽对人食道癌细胞ECA109裸鼠异种移植肿瘤生长的抑制作用
多肽对人食道癌细胞ECA109裸鼠移植瘤生长抑制试验结果表明，与阴性对照组相比，多肽20mg/kg组对人食道癌细胞ECA109移植瘤的生长具有极显著性的抑制作用，多肽10mg/kg组对人食道癌细胞ECA109移植瘤的生长具有显著性的抑制作用。与阳性对照顺铂相比，多肽对实验动物的体重无明显影响，未见明显的毒副反应。
SEQUENCE LISTING
<110> 苏州普罗达生物科技有限公司
<120> 一种关于PER2蛋白激动剂多肽及其应用
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 12
<212> PRT
<213> 人工序列
<400> 1
Ala Ala Met Met Ala Asn Met Glu Ile Lys Ile Cys
1 5 10
Claims (5)
1.PER2蛋白激动剂多肽，其特征在于其序列为AAMMANMEIKIC。
2.一种药物组合物，其特征在于其包含如权利要求1所述多肽与一种以上药学上可接受的填充剂、粘合剂、润滑剂、崩解剂、或稳定剂。
3.如权利要求2所述的药物组合物，其特征在于，所述组合物为注射剂。
4.如权利要求1所述的多肽，其特征在于有效剂量为10mg/kg。
5.如权利要求1所述的PER2蛋白激动剂多肽在制备治疗食道癌药物中的应用。
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