WO2018121457A1 - 一种多肽在预防或治疗心肌缺血再灌注损伤相关疾病中的药物应用 - Google Patents

一种多肽在预防或治疗心肌缺血再灌注损伤相关疾病中的药物应用 Download PDF

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WO2018121457A1
WO2018121457A1 PCT/CN2017/118161 CN2017118161W WO2018121457A1 WO 2018121457 A1 WO2018121457 A1 WO 2018121457A1 CN 2017118161 W CN2017118161 W CN 2017118161W WO 2018121457 A1 WO2018121457 A1 WO 2018121457A1
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reperfusion injury
myocardial ischemia
polypeptide
myocardial
preventing
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彭礼飞
罗辉
邵正
司徒永立
邓莉
何庆丰
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广东医科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

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  • the present invention relates to the field of biomedicine, and in particular to the use of a polypeptide for preventing or treating diseases associated with myocardial ischemia-reperfusion injury.
  • MI myocardial infarction
  • Reperfusion Restoring ischemic myocardial blood flow (reperfusion) is the most effective measure for the treatment of myocardial ischemia, but reperfusion itself can lead to complex pathophysiological reactions, aggravate myocardial damage, and cause myocardial ischemia reperfusion injury (myocardial ischemia reperfusion injury, MIRI).
  • the problem to be solved by the present invention is to provide a polypeptide drug for anti-myocardial ischemia-reperfusion injury to reduce or avoid myocardial ischemia-reperfusion injury.
  • the present invention provides a pharmaceutical application of the complete amino acid sequence of the polypeptide AdKI4 or a mutant thereof against diseases associated with myocardial ischemia-reperfusion injury.
  • the complete amino acid sequence of AdKI4 is: SEQ ID NO. 1, or a mutant of AdKI4, such as a mutant formed after deletion of one or more amino acid residues at the amino terminus or carboxy terminus of the entire amino acid, such as amino acid sequence: SEQ ID NO .2.
  • the invention proves that AdKI4 can significantly reduce myocardial infarct size after myocardial ischemia-reperfusion and effectively reduce myocardial ischemia-reperfusion injury by pharmacological experiments. Therefore, the polypeptide can be used for preventing or treating myocardial ischemia-reperfusion injury-related diseases.
  • myocardial infarction, coronary heart disease, etc. has a wide range of clinical applications.
  • Figure 2 shows the effect of AdKI4 on myocardial infarct size in SD rats with myocardial ischemia-reperfusion injury (NBT staining, gray-white area is myocardial infarction area).
  • A. Control group sham operation group
  • B. Low dose group 1.0 mg/kg. BW
  • C. Medium dose group 2.0 mg/kg. BW
  • D. High dose group (4.0 mg/kg. BW) ).
  • BW is the weight.
  • the present inventors isolated a polypeptide-AdKI4 in the early stage of Ancylostoma duodenale.
  • the inventors used the SD rat model of myocardial ischemia-reperfusion injury to first confirm that AdKI4 can significantly reduce myocardial infarct size after myocardial ischemia-reperfusion in a dose-dependent manner, indicating that AdKI4 is resistant to ischemia-reperfusion.
  • Myocardial injury has a good protective effect. Therefore, AdKI4 can be used as a drug to prevent or treat diseases related to myocardial ischemia-reperfusion injury.
  • AdKI4 Recombinant AdKI4
  • SPF SD rats were purchased from the Guangdong Medical Laboratory Animal Center [Experimental Animal Card: SCXK (Yue) 2013-0002; SYXK (Guangdong) 2012] -0122]; Tetranitroblue tetrazolium chloride (NBT) is a product of Sigma.
  • AdKI4 The preparation method of recombinant AdKI4 can be found in: "Prokaryotic expression, purification and activity study of Kunitz-type serine protease inhibitor IsKuI-1" (Cui Hongzhen, Shao Zheng, Deng Li et al; Chinese Journal of Biotechnology, 2014, Vol. 34, No. 12 Period, pp. 30-35).
  • the pET32a-sumo/AdKI4 prokaryotic expression plasmid was constructed and transferred into Escherichia coli BL21 (DE3), and the sequence was confirmed by sequencing. The expression was induced by IPTG, and the expressed product was purified by nickel affinity chromatography.
  • the protein, the fusion protein is cleaved by SUMO protease, and a recombinant polypeptide having a purity greater than 95% is obtained by chromatography, that is, a recombinant polypeptide of interest, recombinant AdKI4 (rAdKI4), is obtained.
  • AdKI4 The complete amino acid sequence of AdKI4 was obtained as follows: SEQ ID NO. 1, or a mutant of AdKI4, such as SEQ ID NO. 2, which deleted 7 amino acid residues at the amino terminus of SEQ ID NO. 1, and deleted 3 amino acids at the carboxy terminus. The sequence of the residue.
  • I/R myocardial ischemia-reperfusion
  • LAD anterior descending artery
  • AdKI4 has a significant protective effect on myocardial ischemia-reperfusion injury in a dose-dependent manner, and can be used as a drug to prevent or treat myocardial ischemia-reperfusion injury-related diseases.

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Abstract

提供一种多肽在预防或治疗心肌缺血再灌注损伤相关疾病中的应用。该多肽能显著减少心肌缺血再灌注损伤中的心肌梗死面积，可作为预防或治疗心肌缺血再灌注损伤相关疾病的药物。

Description

一种多肽在预防或治疗心肌缺血再灌注损伤相关疾病中的药物应用 技术领域
本发明涉及生物医药领域，具体地说，本发明涉及一种多肽在预防或治疗心肌缺血再灌注损伤相关疾病中的应用。
背景技术
心肌缺血引起的心脏病，即缺血性心脏病，亦称冠心病，临床常见且危害严重，其中心肌梗死(myocardial infarction，MI)是心肌缺血心脏病中危害最大、最致命的一种，致死率很高。通过恢复缺血心肌血流(再灌注)是治疗心肌缺血最有效的措施，但是再灌注本身可导致复杂的病理生理反应，加重心肌损伤，引起心肌缺血再灌注损伤(myocardial ischemia reperfusion injury，MIRI)。目前，临床上应用抗氧化治疗、腺苷受体激动剂、钙拈抗剂、Na +/H +、Na +/Ca 2+离子交换抑制剂、缺血预适应、后适应处理、他汀类、β受体阻滞剂、抗血小板聚集剂等探索性地治疗MIRI，但临床疗效并不能令人满意[徐盟.心肌缺血再灌注损伤的主要机制与相关药物治疗的研究进展.实用药物与临床.2014；17(8)：1052-1055；彭军，李元建.第十九章抗心肌缺血药//苏定冯，陈丰原主编.心血管药理学(第4版).北京：人民卫生出版社.2011年，P401-417]。目前，国外正在进行应用环孢素A(Cyclosporine-A)、美托洛尔(metoprolol)、阿昔单抗(abciximab)和艾塞那肽(Exenatide)等药物治疗MIRI的临床II期研究[
Figure PCTCN2017118161-appb-000001
B，et al.Evolving therapies for myocardial ischemia/reperfusion injury.JAm Coll Cardiol.2015；65(14)：1454-1471]。虽然目前用于防治MIRI药物虽有一定疗效，但却十分有限。
发明内容
本发明要解决的问题是提供一种抗心肌缺血再灌注损伤的多肽药物的应用，以减少或避免引起心肌缺血再灌注损伤。
为减少或避免引起心肌缺血再灌注损伤，本发明提供了一种多肽AdKI4的完整氨基酸序列或其突变体在抗心肌缺血再灌注损伤相关疾病中的药物应用。AdKI4的完整氨基酸序列为：SEQ ID NO.1，或AdKI4的突变体，如在完整氨基酸的氨基端或羧基端缺失一个或多个氨基酸残基后形成的突变体，如氨基酸序列：SEQ ID NO.2。
本发明通过药理实验证实了AdKI4能明显减少心肌缺血再灌注后的心肌梗死面积，有效减轻心肌缺血再灌注损伤，因此，该多肽可应用于预防或治疗心肌缺血再灌注损伤相关疾病，如心肌梗死、冠心病等，有广泛的临床应用价值。
附图说明
以下附图仅旨在于对本发明做示意性说明和解释，并不限定本发明的范围。其中，
图1显示的是AdKI4对心肌缺血再灌注损伤心肌梗死率的影响(n＝8)。
图2显示的是心脏切片示AdKI4对SD大鼠心肌缺血再灌注损伤心肌梗死面积的影响(NBT染色，灰白色区域为心肌梗死区)。A.对照组(假手术组)；B.低剂量组(1.0mg/kg.BW)；C.中剂量组(2.0mg/kg.BW)；D.高剂量组(4.0mg/kg.BW)。BW为体重。
具体实施方式
为了对本发明的技术特征、目的和效果有更加清楚的理解，现对照附图说明本发明的具体实施方式。以下具体实施例仅用于说明本发明而不用于限制本发明的范围。
本发明人前期在十二指肠钩口线虫(Ancylostoma duodenale)中分离到一种多肽-AdKI4。发明人通过系列探索研究，利用SD大鼠心肌缺血再灌注损伤模型，首先明确了AdKI4能明显减少心肌缺血再灌注后的心肌梗死面积，并呈剂量依赖关系，表明AdKI4对缺血再灌注心肌损伤具有很好保护作用，因此，AdKI4可作为药物应用于预防或治疗心肌缺血再灌注损伤相关疾病。
实施例rAdKI4对心肌缺血再灌注损伤的保护作用
1实验材料
重组AdKI4(rAdKI4)由发明人在实验室重组制备，纯度大于95％；SPF级SD大鼠购自广东省医学实验动物中心[实验动物证：SCXK(粤)2013-0002；SYXK(粤)2012-0122]；氯化硝基四氮唑蓝(Tetranitroblue tetrazolium chloride，NBT)为Sigma公司产品。
重组AdKI4的制备方法可参见：《Kunitz型丝氨酸蛋白酶抑制剂IsKuI-1的原核表达、纯化及活性研究》，(崔宏娣，邵正，邓莉等；中国生物工程杂志，2014年第34卷第12期，第30～35页)。具体来说，通过构建pET32a-sumo/AdKI4原核表达质粒，转入到大肠埃希菌BL21(DE3)中，经测序证实序列正确；用IPTG诱导表达，表达产物经镍亲和层析纯化获得融合蛋白，经SUMO蛋白酶切割融合蛋白，并通过层析制备获得纯度大于95％重组多肽，即获得重组的目的多肽-重组AdKI4(rAdKI4)。
实验获得AdKI4的完整氨基酸序列为：SEQ ID NO.1，或AdKI4的突变体，如SEQ ID NO.2，是在SEQ ID NO.1氨基端缺失7个氨基酸残基、羧基端缺失3个氨基酸残基的序列。
2实验方法
2.1动物分组：体重180～250g的SPF级SD大鼠40只，随机分为5组，每组8只，分别为：空白对照组(假手术组)、模型组、rAdKI4高剂量组(4.0mg/kg.BW)、中剂量组(2.0mg/kg.BW)、低剂量组(1.0mg/kg.BW)。BW为体重(body weight，BW)。
2.2构建模型及给药：
(1)SD大鼠心肌缺血再灌注(I/R)模型构建：异氟烷呼吸麻醉机麻醉大鼠，仰卧固定，37℃维持体温，气管插管，连接小动物呼吸机辅助呼吸(吸呼比＝1∶1，呼吸频率＝80次/min，潮气量＝4.0m1)，经胸骨左缘第2～3肋间打开胸腔，用开睑器轻轻拉开开口，暴露心脏，剪开心包膜，找到左心耳和肺动脉圆锥，在左心耳根部下方2mm处以4-0线进针，穿过心肌表层在肺动脉圆锥处稍下方出针，中间放一已消毒1cm长的棉线，结扎冠状动脉前降支 (LAD)，合上并排除胸腔空气，缺血30min，显微剪剪断结扎线，再灌注24h；空白对照组(假手术组)只穿线不结扎。动物缺血前及缺血后记录心电图。
(2)给药：药物组于缺血前2min内尾静脉注射给药，空白对照组和模型组于缺血前2min内尾静脉注射给予等量生理盐水。
2.3心肌梗死面积测量：再灌注24h后，异氟烷呼吸麻醉实验大鼠，取出心脏后，生理盐水冲洗干净，于-20℃冰冻30min，于心脏结扎处向下横切片(2mm/片)，37℃下，氯化硝基四氮唑蓝液(NBT)染色30min，扫描仪扫描并计算梗死面积。梗死率＝心肌梗死区面积/心肌面积
2.4统计方法：用SPSS 16.0软件包进行统计分析，数据用均数±标准差
Figure PCTCN2017118161-appb-000002
表示。3组之间比较用单因素方差分析(ANOVA)，两两比较用q检验，以P＜0.05为差异有统计学意义。
3实验结果
AdKI4能显著减少心肌缺血再灌注损伤心肌梗死面积(图2)。与模型组比较，AdKI4各组均显著减少心肌梗死率(P＜0.05)，且高、中、低剂量组减少心肌梗死率呈明显剂量依赖关系(P＜0.05)(表1，图1)。
表1 重组AdKI4对SD大鼠心肌梗死率的影响(n＝8)
Figure PCTCN2017118161-appb-000003
注： *与模型组比较，P＜0.05； #高、中、低剂量组比较，P＜0.05
4结论：AdKI4对心肌缺血再灌注损伤具有显著保护作用，且呈剂量依赖关系，可作为药物应用于预防或治疗心肌缺血再灌注损伤相关疾病。
以上所述仅为本发明示意性的具体实施方式，并非用以限定本发明的范围。任何本领域的技术人员，在不脱离本发明的构思和原则的前提下所作的等同变化、修改与结合，均应属于本发明保护的范围。

Claims (2)

  1. 一种多肽在预防或治疗心肌缺血再灌注损伤相关疾病中的药物应用，该多肽氨基酸序列为：SEQ ID NO.1或SEQ ID NO.2。
  2. 如权利要求1所述的应用，其中所述心肌缺血再灌注损伤相关疾病为心肌梗死或冠心病。
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CN113702645A (zh) * 2021-08-30 2021-11-26 复旦大学附属中山医院 Sirt4在心血管疾病治疗中的用途
CN114249837A (zh) * 2021-12-28 2022-03-29 徐州医科大学 一种多肽、及其制备方法和应用

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CN113702645A (zh) * 2021-08-30 2021-11-26 复旦大学附属中山医院 Sirt4在心血管疾病治疗中的用途
CN114249837A (zh) * 2021-12-28 2022-03-29 徐州医科大学 一种多肽、及其制备方法和应用
CN114249837B (zh) * 2021-12-28 2023-10-20 徐州医科大学 一种多肽、及其制备方法和应用

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