WO2019037482A1 - 具有血栓溶解活性的多肽 - Google Patents

具有血栓溶解活性的多肽 Download PDF

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WO2019037482A1
WO2019037482A1 PCT/CN2018/087099 CN2018087099W WO2019037482A1 WO 2019037482 A1 WO2019037482 A1 WO 2019037482A1 CN 2018087099 W CN2018087099 W CN 2018087099W WO 2019037482 A1 WO2019037482 A1 WO 2019037482A1
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polypeptide
ile
thr
asp
ala
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PCT/CN2018/087099
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French (fr)
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赵树民
鲍勇刚
石松传
周方
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北京博肽聚康生物技术有限公司
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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
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • the invention belongs to the field of biomedicine, and in particular relates to a polypeptide having thrombolytic activity.
  • cardiovascular and cerebrovascular diseases such as coronary heart disease has increased year by year.
  • cardiovascular and cerebrovascular diseases rank first in the cause of death.
  • cerebral thrombosis in the incidence of acute thrombotic diseases in the middle-aged and elderly population, morbidity and mortality, the current study found that cerebral thrombosis and other cardiovascular and cerebrovascular diseases have a trend of youth, has seriously threatened human health .
  • Thrombosis is the culprits of the onset of cardiovascular and cerebrovascular diseases. It refers to the pathology in which blood forms a embolus in the blood vessels (mostly small blood vessels) under certain conditions, causing partial or complete occlusion of blood vessels and blood supply disorders at corresponding sites. process.
  • Anticoagulant therapy and thrombolytic therapy are the most promising treatments for ischemic cardiovascular and cerebrovascular diseases.
  • the bioavailability, half-life and anticoagulant effects of heparin vary from person to person and the risk of postoperative bleeding is high.
  • Blockers The commonly used direct thrombin inhibitors are hirudin and recombinant hirudin, with a short half-life of 0.5-1 hour, mainly cleared by the kidneys, so patients with renal insufficiency should be used with caution. And the safe dose range of these two drugs is narrow and must be monitored in the laboratory.
  • the thrombolytic drugs used in clinical practice may cause fatal bleeding complications, and the dose is too small may affect the therapeutic effect of thrombolytic therapy, and the application thereof is greatly limited, so it is necessary to continuously develop new ones.
  • Anticoagulant drugs are used in clinical practice.
  • the Peptide drug research is on the rise, and peptide drugs mainly target the key proteins in the in vivo pathway to change the structure of the protein to treat diseases.
  • the Peptide Array Technology Platform is an advanced platform for high-throughput screening of peptide drugs.
  • the thrombolytically active polypeptide prepared by the present invention can be developed into a novel polypeptide drug having thrombolytic activity.
  • the present invention provides a group of polypeptides having thrombolytic activity.
  • the present invention provides a polypeptide having thrombolytic activity, the amino acid sequence of which is: Ile-Thr-Met-Ala-X1-X2, wherein X1 is selected from the group consisting of Ala, Ile, Asp; and X2 is selected from the group consisting of Gln, Lys, Ser.
  • amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, specifically: Ile-Thr-Met-Ala-Ala-Gln, Ile-Thr-Met- Ala-Ile-Lys or Ile-Thr-Met-Ala-Asp-Ser.
  • the present invention provides a polypeptide having thrombolytic activity, the amino acid sequence of which is: Glu-Asp-Ser-Arg-X3-X4, wherein X3 is selected from Gln, Ile, Tyr; X4 is selected From His, Thr, Gln.
  • the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, specifically Glu-Asp-Ser-Arg-Gln-His, Glu-Asp-Ser-Arg -Ile-Thr or Glu-Asp-Ser-Arg-Tyr-Gln.
  • the invention provides the use of the polypeptide in the manufacture of a medicament for the treatment and/or prevention of thrombosis.
  • composition comprising an active ingredient polypeptide and a pharmaceutically acceptable carrier.
  • compositions include, but are not limited to, injections, tablets, granules, capsules, oral solutions, pills, and the like.
  • the pharmaceutically acceptable carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrating agents, absorption enhancers, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field, and the like. Flavoring agents, sweeteners and the like can also be added.
  • the polypeptide of the present invention should be "effective amount" as an active ingredient, and the "effective amount” means a non-toxic, but sufficient amount of a drug or agent that provides a desired effect.
  • an "effective amount” of an ingredient refers to an amount of the ingredient that is effective to provide the desired effect when used in combination with other ingredients.
  • the “effective amount” will vary from subject to subject, depending on the age and general condition of the individual, the particular active drug, and the like. Thus, it is not always possible to refer to an accurate “effective amount”, however, a suitable “effective amount” in any individual case can be determined by one of ordinary skill in the art using routine experimental methods.
  • polypeptides of the present invention can be prepared by methods known to those skilled in the art, such as solid phase synthesis methods, and can be isolated and purified using separation and purification methods known in the art, such as high performance liquid chromatography.
  • results of the present invention show that the six polypeptides can significantly prolong the time of carotid thrombosis in rats, and can significantly shorten the length of thrombus in vitro and reduce the weight of thrombus, indicating that the polypeptide has good thrombolytic activity and can be used for A drug for thrombolysis is prepared.
  • Figure 1 is an image synthesized by screening a polypeptide chip
  • Figure 2 is a graph showing the in vitro thrombolysis of a polypeptide
  • Figure 3 is an HPLC chromatogram of the polypeptide Pep1;
  • Figure 4 is an HPLC chromatogram of the polypeptide Pep2
  • Figure 5 is an HPLC chromatogram of the polypeptide Pep3
  • Figure 6 is an HPLC chromatogram of the polypeptide Pep4
  • Figure 7 is an HPLC chromatogram of the polypeptide Pep5
  • Figure 8 is an HPLC chromatogram of the polypeptide Pep6
  • Figure 9 is a MS spectrum of the polypeptide Pep1;
  • Figure 10 is a MS spectrum of the polypeptide Pep2
  • Figure 11 is a MS spectrum of the polypeptide Pep3
  • Figure 12 is a MS spectrum of the polypeptide Pep4;
  • Figure 13 is a MS spectrum of the polypeptide Pep5;
  • Figure 14 is a MS map of the polypeptide Pep6.
  • X-X-X-X-X-X is Fmoc protected D-Gly, D-Ala, D-Val, D-Leu, D-Ile, D-Phe, D-Pro, D-Tyr, D-Ser, D-Thr, D-Trp, A molar mixture of D-Met, D-Glu, D-Gln, D-Asp, D-Asn, D-Lys, D-Arg, D-His, and the like.
  • the polypeptide chips synthesized in the above steps 1) to 4) are used for screening with a thrombolytic active polypeptide.
  • a selected group of peptides with significant thrombolytic activity Ile-Thr-Met-Ala-Ala-Gln, Ile-Thr-Met-Ala-Ile-Lys, Ile-Thr-Met-Ala-Asp-Ser, Glu -Asp-Ser-Arg-Gln-His, Glu-Asp-Ser-Arg-Ile-Thr, Glu-Asp-Ser-Arg-Tyr-Gln, the screening results, the data curve of thrombolytic active peptide is shown in Figure 2 (Note :Pep1 stands for Ile-Thr-Met-Ala-Ala-Gln; Pep2 stands for Ile-Thr-Met-Ala-Ile-Lys; Pep3 stands for Ile-Thr-Met-Ala-Asp-Ser; Pep4 stands for Glu-Asp-Ser -Arg-Gln-His; Pep5 represents Glu-Asp-Ser
  • the synthesis uses a solid phase synthesis method to synthesize a polypeptide from the C-terminus to the N-terminus.
  • the synthesis was synthesized by chemical synthesizer (AMS 586 Multiple Peptide Synthesiser, ABIMED, Germany), using Fmoc-protected amino acid as raw material, Fmoc-Rink linker resin as adhesion matrix, HOBT as condensing agent, DIC as activator, layer by layer. Synthetic polypeptide.
  • polypeptides Ile-Thr-Met-Ala-Ala-Gln Pep1
  • Ile-Thr-Met-Ala-Ile-Lys Pep2
  • Ile-Thr-Met-Ala-Asp-Ser Pep3
  • Glu-Asp- The HPLC profiles of Ser-Arg-Gln-His (Pep4), Glu-Asp-Ser-Arg-Ile-Thr (Pep5), Glu-Asp-Ser-Arg-Tyr-Gln (Pep6) are shown in Figure 3-8 (chromatography). The conditions are shown in Table 1), and the MS spectrum is shown in Figure 9-14.
  • HPLC chromatogram shows that each polypeptide has a characteristic peak composition
  • the molecular weight of the MS assay polypeptide is shown in Table 2.
  • mice 200 Wistar rats were randomly divided into 20 groups, 10 in each group, which were blank control group, positive control group, low, medium and high dose groups (the experimental peptide samples were 0.25, 0.5, 1.0 respectively). Mg/kg), the positive drug group was administered with urokinase 3000 U/kg, and the control group was given the same volume of normal saline. After 45 minutes of intravenous administration, the rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (1 ml/kg), and the rat carotid artery was damaged by electrical stimulation to form a thrombus, and the thrombus formation time was observed by a thrombometer. The experimental results are shown in Table 3.
  • ⁇ P ⁇ 0.05 was statistically different between the experimental group and the control group.
  • mice 200 Wistar rats were randomly divided into 20 groups, 10 in each group, which were blank control group, positive control group, low, medium and high dose groups (the experimental peptide samples were 0.25, 0.5, 1.0 respectively). Mg/kg), the positive drug group was administered with urokinase 3000 U/kg, and the control group was given the same volume of normal saline.
  • any polypeptide of Pep1-Pep6 add appropriate excipients to tablets (including controlled release tablets, matrix tablets, coated tablets, dispersible tablets, etc.), according to tablets (including controlled release tablets, matrix tablets, coated tablets) , dispersible tablets, etc.) process to prepare thrombolytic tablets.

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Abstract

本发明涉及一种溶栓多肽，其氨基酸序列为Ile-Thr-Met-Ala-Ala-Gln、Ile-Thr-Met-Ala-Ile-Lys、Ile-Thr-Met-Ala-Asp-Ser、Glu-Asp-Ser-Arg-Gln-His、Glu-Asp-Ser-Arg-Ile-Thr、Glu-Asp-Ser-Arg-Tyr-Gln，体内和体外溶栓实验结果表明所述多肽具有良好的血栓溶解活性，可以用于制备用于血栓溶解的药物。

Description

具有血栓溶解活性的多肽 技术领域
本发明属于生物医药领域，具体涉及具有血栓溶解活性的多肽。
背景技术
随着人类生活水平的不断提高，冠心病等心脑血管发病率逐年增高，目前心脑血管疾病在疾病死亡原因中高居榜首。特别是脑血栓在中老年人群中急性血栓性疾病的发病率、致残率和死亡率的特点，目前研究发现，脑血栓等心脑血管疾病具有年青化的趋势，已严重威胁着人类的健康。血栓形成是心脑血管疾病发病的罪魁，它是指在一定条件下，血液有形成分在血管内(多数为小血管)形成栓子，造成血管部分或完全堵塞、相应部位血供障碍的病理过程。
抗凝治疗和溶栓治疗做为目前缺血性心脑血管疾病最有前途的治疗方法。传统的抗凝药物主要有两类，分别是阻滞剂和肝素。肝素的生物利用度、半衰期和抗凝效果因人而异，术后出血风险较高。阻滞剂类目前常用的直接凝血酶抑制剂有水蛭素和重组水蛭素，半衰期短，为0.5-1小时，主要由肾脏清除，因此肾功能不全的患者慎用。并且这两种药物安全剂量范围狭窄，必须进行实验室监测。并且，目前临床上使用的溶栓药物，剂量过大可以导致致死性出血并发症，而剂量过小会影响溶栓治疗效果等特点，而使其应用受到很大限制，因此需要不断研发新的抗凝药物。
多肽药物研究方兴未艾，多肽药物主要针对体内通路上的关键蛋白做靶标结合来改变蛋白结构治疗疾病。多肽阵列技术平台是先进的高通量筛选多肽药物的平台。本发明制备的具有血栓溶解活性的多肽可以开发成新型具有溶栓活性的多肽药物。
发明内容
针对上述溶栓药物的缺陷，本发明提供一组具有溶栓活性的多肽。
根据本发明的一个方面，本发明提供一种具有溶栓活性的多肽，其氨基酸 序列为：Ile-Thr-Met-Ala-X1-X2，其中，X1选自Ala、Ile、Asp；X2选自Gln、Lys、Ser。
优选的，所述多肽氨基酸序列如SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：3所示，具体为：Ile-Thr-Met-Ala-Ala-Gln、Ile-Thr-Met-Ala-Ile-Lys或Ile-Thr-Met-Ala-Asp-Ser。
根据本发明的另一个方面，本发明提供一种具有溶栓活性的多肽，其氨基酸序列为：Glu-Asp-Ser-Arg-X3-X4，其中，X3选自Gln、Ile、Tyr；X4选自His、Thr、Gln。
优选的，所述多肽氨基酸序列如SEQ ID NO：4、SEQ ID NO：5或SEQ ID NO：6所示，具体为Glu-Asp-Ser-Arg-Gln-His、Glu-Asp-Ser-Arg-Ile-Thr或Glu-Asp-Ser-Arg-Tyr-Gln。
根据本发明的另一个方面，本发明提供所述多肽在制备治疗和/或预防血栓的药物中的应用。
根据本发明的另一个方面，本发明提供一种药物组合物，该药物组合物包括活性成分多肽和药学上可接受的载体。
所述药物组合物包括但不限于注射剂、片剂、颗粒剂、胶囊剂、口服液、丸剂等。
所述药学上可接受的载体包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等，必要时还可以加入香味剂、甜味剂等。
本发明所述多肽作为活性成分时应为“有效量”的，所述“有效量”是指无毒性，但足够量的提供所需的作用的药物或药剂。在本发明的药物组合物中，一种成分的“有效量”是指该成分在和其他成分联合应用时有效提供所需效应的量。“有效量”会因受试者的不同而不同，依据年龄和个体的一般情况，特定的活性药物等等。因此，不可能总是指精确的“有效量”，然而，任何个体病例中合适的“有效量”可以由本领域普通技术人员应用常规的实验方法来测定。
本发明所述多肽可以采用本领域技术人员已知的方法(例如固相合成方法)制备得到，以及可以采用本领域已知的分离纯化方法(例如高效液相色谱法)分离纯化。
本发明研究结果显示六种多肽均能明显延长大鼠颈动脉血栓形成的时间，且能够明显的缩短大鼠体外血栓的长度，减轻血栓的重量，表明多肽具有良好的血栓溶解活性，可以用于制备用于血栓溶解的药物。
附图说明
图1为筛选用多肽芯片合成完的图像；
图2为多肽体外溶栓实验曲线；
图3为多肽Pep1的HPLC图谱；
图4为多肽Pep2的HPLC图谱；
图5为多肽Pep3的HPLC图谱；
图6为多肽Pep4的HPLC图谱；
图7为多肽Pep5的HPLC图谱；
图8为多肽Pep6的HPLC图谱；
图9为多肽Pep1的MS图谱；
图10为多肽Pep2的MS图谱；
图11为多肽Pep3的MS图谱；
图12为多肽Pep4的MS图谱；
图13为多肽Pep5的MS图谱；
图14为多肽Pep6的MS图谱。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外，应理解，在阅读了本发明所记载的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本发明所限定的范围。
实施例1 溶栓活性多肽的筛选
1、设计筛选数据库
1)建立随机初级六肽数据库全库：X-X-X-X-X-X。其中X是Fmoc保护的D-Gly、D-Ala、D-Val、D-Leu、D-Ile、D-Phe、D-Pro、D-Tyr、D-Ser、D-Thr、 D-Trp、D-Met、D-Glu、D-Gln、D-Asp、D-Asn、D-Lys、D-Arg、D-His等摩尔量混合物。
2)建立二级六肽随机数据库：Ile-Thr-X-X-X-X和Glu-Asp-X-X-X-X。其中X是Fmoc保护的D-Gly、D-Ala、D-Val、D-Leu、D-Ile、D-Phe、D-Pro、D-Tyr、D-Ser、D-Thr、D-Trp、D-Met、D-Glu、D-Gln、D-Asp、D-Asn、D-Lys、D-Arg、D-His等摩尔量混合物。
3)建立三级六肽随机数据库：Ile-Thr-Met-Ala-X-X-X-X和Glu-Asp-Ser-Arg-X-X-X-X。其中X是Fmoc保护的D-Gly、D-Ala、D-Val、D-Leu、D-Ile、D-Phe、D-Pro、D-Tyr、D-Ser、D-Thr、D-Trp、D-Met、D-Glu、D-Gln、D-Asp、D-Asn、D-Lys、D-Arg、D-His等摩尔量混合物。
4)根据1)、2)、3)建立的多肽组合数据库。利用多肽阵列合成仪，使用特殊处理的纤维素膜最为基质，合成多肽芯片多肽芯片基质为FMOC-PEG-纤维素膜，多肽芯片大小是15cm*11cm，每张芯片承载198个多肽点，一组芯片361个多肽点需要两张芯片。采用原位合成，逐层合成来实现。合成参数为每个肽点氨基酸上样量为0.6ul，肽点直径为0.5cm，肽点间距为0.1cm，氨基酸浓度为0.25M，肽点合成重复一次。合成的筛选芯片如图1所示。
上述步骤1)-4)合成的多肽芯片用于具有溶栓活性多肽的筛查。
2、活性筛查
1)对芯片上每一个多肽复合物进行血栓溶解活性筛选，测试采取的测定方法是：将合成完的芯片用饱和氨气室温处理12h，然后将合成在芯片上的361个多肽用打孔器打下置于1.5mlEP管底部，每管加入30ul，pH值为8.0的50mM的Tris Buffer室温溶解2小时取液体20ul用于血栓溶解活性筛选测试。
2)配制1M的CaCl 2、50mM的Tris Buffer、浓度1mg/ml的羧肽酶抑制肽溶液、阳性对照肽CPI-2ki、Thrombomodulin、Trombin/CaCl 2mixture：860ul Tris buffer+100ulCaCl 2溶液+40uldd Tris溶解Trombin、130μg/ml的tPA、上述筛查得到的具有血栓溶解活性的多肽各1mg/ml用于体外血栓溶解测定实验。
3)取96孔板上用于实验，按下表从上到下加试剂：
Figure PCTCN2018087099-appb-000001
Figure PCTCN2018087099-appb-000002
取对板孔应量(如上表)的Plasma用前37摄氏度预热5min，然后加入对应量的tPA，再加入体系。即时开始测定420nm的OD值：前5min，每分钟测定1次，后续开始测定每5min测定1次共测定2h。根据测定数据绘制折线图。找出具有明显溶栓活性的多肽组(对应芯片上的一个多肽点，可能是多种多肽的混合物)
筛选出的一组具有明显血栓溶解活性的多肽，Ile-Thr-Met-Ala-Ala-Gln、Ile-Thr-Met-Ala-Ile-Lys、Ile-Thr-Met-Ala-Asp-Ser、Glu-Asp-Ser-Arg-Gln-His、Glu-Asp-Ser-Arg-Ile-Thr、Glu-Asp-Ser-Arg-Tyr-Gln其筛选结果，具有溶栓活性多肽数据曲线见图2(注：Pep1代表Ile-Thr-Met-Ala-Ala-Gln；Pep2代表Ile-Thr-Met-Ala-Ile-Lys；Pep3代表Ile-Thr-Met-Ala-Asp-Ser；Pep4代表Glu-Asp-Ser-Arg-Gln-His；Pep5代表Glu-Asp-Ser-Arg-Ile-Thr；Pep6代表Glu-Asp-Ser-Arg-Tyr-Gln)。
实施例2 多肽的合成与纯化
合成采用固相合成法从C-端到N-端合成多肽。合成采用化学合成仪(AMS 586 Multiple Peptide Synthesiser，ABIMED，Germany)合成，用Fmoc保护的氨基酸作为原料，以Fmoc-Rink linker resin树脂为附着基质，用HOBT作为缩合剂，DIC作为激活剂，逐层合成多肽。
合成过程中用2％乙酸酐的DMF溶液做侧链封闭试剂；用20％哌啶做Fmoc去除试剂，合成完毕经过TFA切割和侧链基团的去除操作。合成完毕的粗产物经离心收集，用R-HPLC(Waters 741)和C 18-column(Waters Delta-pak TM，40*100mm，15um，100埃)纯化得到纯度大于98％的多肽，冻干成粉末。
多肽Ile-Thr-Met-Ala-Ala-Gln(Pep1)、Ile-Thr-Met-Ala-Ile-Lys(Pep2)、 Ile-Thr-Met-Ala-Asp-Ser(Pep3)、Glu-Asp-Ser-Arg-Gln-His(Pep4)、Glu-Asp-Ser-Arg-Ile-Thr(Pep5)、Glu-Asp-Ser-Arg-Tyr-Gln(Pep6)其HPLC图谱见图3-8(色谱条件见表1)，MS图谱见图9-14。
表1 多肽HPLC色谱条件
Figure PCTCN2018087099-appb-000003
HPLC图谱显示各多肽分别有一个特征峰组成；
MS分析多肽的分子量见表2。
表2 多肽分子量
  理论Mr/g/mol MS测定Mr/g/mol
Ile-Thr-Met-Ala-Ala-Gln 633.77 634.05
Ile-Thr-Met-Ala-Ile-Lys 675.89 676.10
Ile-Thr-Met-Ala-Asp-Ser 636.72 637.00
Glu-Asp-Ser-Arg-Gln-His 770.76 770.90
Glu-Asp-Ser-Arg-Ile-Thr 719.76 720.00
Glu-Asp-Ser-Arg-Tyr-Gln 783.76 783.90
实施例3 多肽溶栓活性的动物实验
1体内血栓溶解实验
实验方法：选择Wistar大鼠200只，随机分成20组，每组10只，分别为空白对照组、阳性对照组、给药低、中、高剂量组(实验多肽样本分别是0.25、0.5、1.0mg/kg)，阳性药组给药尿激酶3000U/kg，对照组给同体积生理盐水。各组静脉给药45分钟后，腹腔注射3％戊巴比妥钠(1ml/kg)麻醉，电刺激损伤大鼠颈动脉以形成血栓，用血栓测定仪观察血栓形成时间。实验结果见表3。
表3 体内血栓溶解实验结果
Figure PCTCN2018087099-appb-000004
Figure PCTCN2018087099-appb-000005
※P＜0.05实验组与对照组有统计学差异
实验结果显示，给药低、中、高剂量组(实验多肽样本分别是0.25、0.5、1.0mg/kg)均能明显延长大鼠颈动脉血栓形成的时间，并且高剂量组抑制血栓形成的作用强于阳性对照药物尿激酶。表明实验多肽样本对电刺激大鼠颈动脉血栓形成具有明显抑制作用。
2体外血栓溶解实验
实验方法：选择Wistar大鼠200只，随机分成20组，每组10只，分别为空白对照组、阳性对照组、给药低、中、高剂量组(实验多肽样本分别是0.25、0.5、1.0mg/kg)，阳性药组给药尿激酶3000U/kg，对照组给同体积生理盐水。静脉给药10分钟后，腹腔注射3％戊巴比妥钠(1ml/kg)麻醉，沿腹中线切开腹壁，腹主动脉取血约1.8ml，注入硅化塑胶管中，将塑胶管两端对接紧密，放入体外血栓形成仪中，37℃恒温15分钟，取出塑胶管中血栓置于滤纸上，吸净表面血迹，测血栓长度、湿重、干重，并记录。实验结果见表4。
表4体外血栓溶解实验结果
Figure PCTCN2018087099-appb-000006
Figure PCTCN2018087099-appb-000007
※代表和空白对照做比较P＜0.05， ※※代表P＜0.01， ※※※代表P＜0.001
实验结果显示，低、中、高剂量组(实验多肽样本分别是0.25、0.5、1.0mg/kg)的多肽均能够明显的缩短大鼠体外血栓的长度，减轻血栓的重量，其中高剂量组(多肽样本量1.0mg/kg)作用优于阳性对照(尿激酶3000U/kg)。
实施例5
取Pep1-Pep6任意一种多肽10g，加入注射剂(包括冻干粉针剂和无菌分装干粉针剂)适当辅料，按注射剂(包括冻干粉针剂和无菌分装干粉针剂)工艺制备成溶栓注射剂。
实施例6
取Pep1-Pep6任意一种多肽10g，加入片剂(包括缓控释片、骨架片、包衣片、分散片等)适当辅料，按片剂(包括缓控释片、骨架片、包衣片、分散片等)工艺制备成溶栓片剂。
实施例7
取Pep1-Pep6任意一种多肽10g，加入胶囊剂适当辅料，按胶囊剂工艺制备成溶栓胶囊剂。
实施例8
取Pep1-Pep6任意一种多肽10g，加入乳剂(包括微乳、纳米乳等)适当辅料，按乳剂(包括微乳、纳米乳等)工艺制备成溶栓乳剂。
实施例9
取Pep1-Pep6任意一种多肽10g，加入颗粒剂适当辅料，按颗粒剂工艺制备成溶栓颗粒剂。
实施例10
取Pep1-Pep6任意一种多肽10g，加入缓释控释剂适当辅料，按缓释控释剂工艺制成溶栓缓释控释剂。
实施例11
取Pep1-Pep6任意一种多肽10g，加入口服液适当辅料，按口服液工艺制备成溶栓口服液。
实施例12
取Pep1-Pep6任意一种多肽10g，加入脂质体剂型适当辅料，按脂质体工艺制备成溶栓脂质体剂型。

Claims (10)

  1. 一种溶栓多肽，其特征在于，所述多肽的氨基酸序列为：
    Ile-Thr-Met-Ala-X1-X2，其中，X1选自Ala、Ile、Asp；X2选自Gln、Lys、Ser。
  2. 如权利要求1所述的多肽，其特征在于，所述多肽的氨基酸序列如SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：3所示。
  3. 如权利要求1或2所述多肽在制备治疗和/或预防血栓的药物中的应用。
  4. 一种药物组合物，其特征在于，所述药物组合物包括权利要求1或2所述的多肽和药学上可接受的载体。
  5. 如权利要求4所述的药物组合物，其特征在于，所述药物组合物包括注射剂、片剂、颗粒剂、胶囊剂、口服液或丸剂。
  6. 一种溶栓多肽，其特征在于，所述多肽的氨基酸序列为：
    Glu-Asp-Ser-Arg-X3-X4，其中，X3选自Gln、Ile、Tyr；X4选自His、Thr、Gln。
  7. 如权利要求6所述的多肽，其特征在于，所述多肽的氨基酸序列如SEQ ID NO：4、SEQ ID NO：5或SEQ ID NO：6所示。
  8. 如权利要求6或7所述多肽在制备治疗和/或预防血栓的药物中的应用。
  9. 一种药物组合物，其特征在于，所述药物组合物包括权利要求6或7所述的多肽和药学上可接受的载体。
  10. 如权利要求9所述的药物组合物，其特征在于，所述药物组合物包括注射剂、片剂、颗粒剂、胶囊剂、口服液或丸剂。
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CN1727008A (zh) * 2005-06-07 2006-02-01 西安交通大学 血栓诊断和/或治疗用含气干粉活性剂及其制备工艺
WO2006073119A1 (ja) * 2005-01-06 2006-07-13 Hiroshima-Ken ポリペプチドおよびその用途
CN101597322A (zh) * 2008-06-06 2009-12-09 首都医科大学 Tyr-Ile-Gly-Ser-Arg的17种类似物，其合成及在医学中的应用
CN107586316A (zh) * 2017-08-25 2018-01-16 北京博肽聚康生物技术有限公司 具有血栓溶解活性的多肽

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006073119A1 (ja) * 2005-01-06 2006-07-13 Hiroshima-Ken ポリペプチドおよびその用途
CN1727008A (zh) * 2005-06-07 2006-02-01 西安交通大学 血栓诊断和/或治疗用含气干粉活性剂及其制备工艺
CN101597322A (zh) * 2008-06-06 2009-12-09 首都医科大学 Tyr-Ile-Gly-Ser-Arg的17种类似物，其合成及在医学中的应用
CN107586316A (zh) * 2017-08-25 2018-01-16 北京博肽聚康生物技术有限公司 具有血栓溶解活性的多肽

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