WO2017161621A1 - 抗血小板溶栓素在制备治疗血栓性血小板减少性紫癜的药物中的应用 - Google Patents

抗血小板溶栓素在制备治疗血栓性血小板减少性紫癜的药物中的应用 Download PDF

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WO2017161621A1
WO2017161621A1 PCT/CN2016/079973 CN2016079973W WO2017161621A1 WO 2017161621 A1 WO2017161621 A1 WO 2017161621A1 CN 2016079973 W CN2016079973 W CN 2016079973W WO 2017161621 A1 WO2017161621 A1 WO 2017161621A1
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platelet
ttp
thrombocytopenic purpura
thrombotic thrombocytopenic
aggregation
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PCT/CN2016/079973
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English (en)
French (fr)
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李小羿
戴向荣
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兆科药业（合肥）有限公司
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Priority to NZ747528A priority Critical patent/NZ747528A/en
Priority to KR1020187030356A priority patent/KR102302688B1/ko
Priority to US16/095,607 priority patent/US20190125843A1/en
Priority to AU2016399395A priority patent/AU2016399395B2/en
Priority to EP16894974.1A priority patent/EP3434278B1/en
Priority to ES16894974T priority patent/ES2895105T3/es
Publication of WO2017161621A1 publication Critical patent/WO2017161621A1/zh
Priority to AU2018101584A priority patent/AU2018101584A4/en

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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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4806—Hydrolases (3) acting on peptide bonds (3.4) from animals other than mammals, e.g. snakes
    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • 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
    • 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
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • 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
    • A61P7/06—Antianaemics

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  • the invention relates to the field of medicine, in particular to the application of an anti-platelet thrombolysin in the preparation of a medicament for treating thrombotic thrombocytopenic purpura (TTP).
  • TTP thrombotic thrombocytopenic purpura
  • Thrombotic Thrombocytopenic Purpura is a severe diffuse thrombotic microangiopathy with microvascular hemolytic anemia, reduced consumption of platelet aggregation, and organ damage caused by microthrombus (eg kidney, The central nervous system, etc. is characterized. Its pathogenesis is closely related to a vWF proteolytic enzyme (vascular hemophilic factor lyase, ADAMTS13). The role of the enzyme is to specifically lyse von Willebrand Factor (vWF) in the blood to ensure normal hemostasis.
  • vWF vascular hemophilic factor lyase
  • vWF aggregates abundantly on vascular endothelial cells, becoming a plurality of oversized multimers, which in turn can bind to the GPIb receptor on the platelet surface in the bloodstream. It mediates the adhesion and aggregation of platelets on endothelial cells, which eventually leads to thrombosis and further TTP.
  • TTP can be divided into hereditary TTP and acquired TTP.
  • the difference in pathogenesis between the two is that the cause of the decrease in ADAMTS13 enzyme activity is different: hereditary patients are caused by genetic defects due to insufficient congenital expression of this enzyme.
  • the reason for the decrease in enzyme activity is that the patient can produce an autoantibody against ADAMTS13, which causes the enzyme to be inactivated.
  • the most effective treatment for TTP is fresh plasma infusion and plasmapheresis, but its drawback is that it depends on objective conditions and has a high recurrence rate and mortality rate in patients with acquired TTP.
  • anti-ADAMTS13 autoantibodies can be inhibited by immunosuppressive drugs or gamma spheres, but such treatments have a slower onset of action, and the effects vary from patient to patient.
  • the method of supplementing the ADAMTS13 protein is only effective in patients with congenital TTP, because the supplemental ADAMTS13 protein is rapidly inhibited by autoantibodies in patients with acquired TTP. 70%-80% of patients with TTP are acquired, so the clinical application of this method is very limited.
  • the technical problem to be solved by the present invention is to provide an anti-platelet thrombolysin for the preparation of a medicament for treating thrombotic thrombocytopenic purpura.
  • the present invention finds that anti-platelet thrombolysis can be in the blood of TTP patients. Restores platelet, red blood cell and hemoglobin levels, while lowering the level of lactate dehydrogenase, effectively inhibiting thrombocytopenia and cytolytic hemolytic anemia, inhibiting platelet aggregation, thereby preventing thrombosis and improving TTP symptoms. .
  • the antiplatelet thrombolysin (APT) of the present invention is a proteolytic enzyme isolated from the snake venom of Agkistrodon acutus, consisting of two peptide chains of ⁇ chain and ⁇ chain, and the amino acid sequence of the ⁇ chain is SEQ. As shown in ID NO. 1, the ⁇ -chain amino acid sequence is shown in SEQ ID NO. 2, which is disclosed in Patent 201310228219.2.
  • thrombotic thrombocytopenic purpura is congenital or acquired thrombotic thrombocytopenic purpura.
  • the medicament for treating thrombotic thrombocytopenic purpura provided by the present invention is a chemical medicine or a biological preparation.
  • the medicament for treating thrombotic thrombocytopenic purpura provided by the present invention is an oral preparation or an injection.
  • the oral preparation for treating thrombotic thrombocytopenic purpura is a tablet, capsule, pill, granule, pill, microcapsule or pellet.
  • the mass-volume concentration of the anti-platelet thrombopoietin in the oral preparation or injection for treating thrombotic thrombocytopenic purpura is 1.8 to 10 ⁇ g/mL.
  • the dose of the anti-platelet lysin in the oral preparation or injection for treating thrombotic thrombocytopenic purpura is 0.03 to 3 mg/kg.
  • the invention provides an anti-platelet thrombolysis agent for use in the preparation of a medicament for treating thrombotic thrombocytopenic purpura.
  • Anti-platelet thrombolysis can prevent and treat the main symptoms in the acquired TTP ⁇ model, restore platelet, red blood cell and hemoglobin levels, while reducing lactate dehydrogenase levels, effectively inhibiting thrombocytopenia and cytolytic hemolytic anemia, Thereby achieving the purpose of treating TTP. It can be seen that the use of the anti-platelet thrombolysin provided by the present invention is different from the use disclosed in the prior art, and its mechanism of action is also superior to the existing means for treating TTP, and can supplement their deficiencies.
  • Figure 1 shows the inhibitory effect of different concentrations of anti-platelet thrombolysis on platelet aggregation induced by normal human plasma Ristotecin: the green curve indicates the platelet aggregation and time curve of the blank control group; the purple curve indicates the addition of 1.5ug/mL anti-platelet thrombolysis Platelet aggregation and time curve; blue curve indicates platelet aggregation and time curve after adding 1.8ug/mL anti-platelet thrombolysis; black curve indicates platelet aggregation and time after adding 2.1ug/mL anti-platelet thrombolysis curve.
  • Figure 2 shows the effect of anti-platelet thrombolysis on plasma ADAMTS13 activity in normal and TTP patients
  • Figure 3 shows the anti-ADAMTS13 antibody 3H9 in different groups of ADAMTS13 antigen concentration and The effect of activity on the establishment of a partial response acquired TTP model:
  • Figure 3A shows the control group (the legend is a solid circle), the prevention group (the legend is a hollow square) and the treatment group (the legend is a solid triangle) after 3H9 injection.
  • Figure 3B shows the concentration of ADAMTS13 antigen in the control group (the legend is a solid circle), the prevention group (the legend is a hollow square) and the treatment group (the legend is a solid triangle) after injection of 3H9.
  • the curve of change shows the concentration of ADAMTS13 antigen in the control group (the legend is a solid circle), the prevention group (the legend is a hollow square) and the treatment group (the legend is a solid triangle) after injection of 3H9.
  • Figure 4 shows the preventive and therapeutic effects of anti-platelet thrombolysis on various indicators of thrombocytopenia and cytolytic hemolytic anemia:
  • Figure 4A shows the control group (the legend is a solid circle), the prevention group (the legend is a hollow square) And the treatment group (the legend is a solid triangle) curve of platelet count with time;
  • Figure 4B shows the combination of the control group (the legend is a solid circle), the prevention group (the legend is a hollow square) and the treatment group (the legend is a solid triangle) Curve of globin concentration over time;
  • Figure 4C shows the change of red blood cell count over time in the control group (the legend is a solid circle), the prevention group (the legend is a hollow square), and the treatment group (the legend is a solid triangle);
  • 4D represents the change in lactate dehydrogenase levels over time in the control group (the legend is a solid circle), the prevention group (the legend is a hollow square), and the treatment group (the legend is a solid triangle).
  • the invention provides the application of anti-platelet thrombolysin in the preparation of a medicament for treating thrombotic thrombocytopenic purpura, and those skilled in the art can learn from the contents of the present invention and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • the reagents used in the present invention are all commercially available, and are commercially available.
  • Example 1 Antiplatelet thrombolysis induced by rospirin in normal humans and TTP patients in vitro Inhibition effect of platelet aggregation
  • PDP was prepared by taking 3.2% sodium citrate anticoagulated whole blood, 800 prm, and centrifuging for 10 min. The remaining blood was centrifuged at 3000 ppm for 10 min to prepare platelet-poor plasma (PPP). The PRP platelet concentration was adjusted to about 300 ⁇ 109/L with PPP, and the PPP was adjusted to zero. After the final concentration of 10 umol/L of ADP was added to the PRP, the change in transmittance within 5 min was observed, and the maximum transmittance was used as the PRP. Maximum aggregation rate (PAGm).
  • PAGm Maximum aggregation rate
  • the plasma to be tested was mixed with a fluorescent buffer (5 mmol/L Bis-Tris, 25 mmoI/L CaCl2, 0.005% Tween 20, pH 6.0) at a concentration of 1:25, and normal human mixed plasma was used as a standard. Dilute with 1:12.5, 1:25, 1:50, 1:100, 1:200, 1:400 with the above buffer as a standard curve.
  • the above-mentioned different concentrations of normal human mixed plasma and the sample to be tested are added together to a 96-well half-white plate, 50 ul per well, and then a certain concentration (2 umol/L) of the fluorescently labeled substrate FRETS-VWF73 is 50 ul per well, and the above 96-well white plate is added.
  • Enzyme digestion reaction with blood test to be tested After 5 minutes of reaction, the sample was placed in a microplate reader for a reaction temperature of 4 degrees, the fluorescence excitation/emission wavelength was 485/530 nm, the frequency was read every 2 minutes, and the reading was terminated for 60 minutes. The entire operation was no more than 2 hours. Enter the data of each hole at each time point Line curve fitting, the sample reaction curve equation is obtained, and the slope of the sample to be tested is substituted into the above logarithmic linear regression equation, and the activity of ADAMTS13 in the normal mixed plasma at 1:25 concentration is taken as 100%, and the test is obtained. Sample ADAMTS13 activity (%).
  • anti-platelet thrombolysis inhibits the platelet aggregation induced by Ristocetin in normal human plasma at a final concentration of 1.8ug/mL, and the inhibition rate is 93%. From the characteristics of aggregation curve and dose-effect relationship, anti-platelet thrombolysis mainly inhibits platelet aggregation.
  • Antiplatelet thrombolysis inhibits Ristocastin-induced platelet aggregation in patients with TTP. From the results of three TTP samples, anti-platelet thrombolysis at 1.8 ug/mL is equally effective in inhibiting platelet aggregation in TTP patients (see table below). The lower side shows the inhibitory effect of anti-platelet thrombolysis (1.8ug/mL) on platelet aggregation in different populations.
  • the trials were divided into three groups of 4 animals each.
  • the first group was a positive control. From the 0th day of the test, the anti-ADAMTS13 antibody 3H9 was injected from the femoral vein at a dose of 600 ug/kg every 48 hours until the end of the test on the 9th day to establish a TTP model.
  • the second group was the prevention group, and the same dose of 3H9 was injected on the first day and the third day, and 0.3 mg/kg of anti-platelet thrombolysis was subcutaneously injected on the first day and the fifth day to observe the prevention of TTP. effect.
  • the third group was the treatment group.
  • the anti-ADAMTS13 antibody 3H9 was injected from the femoral vein at a dose of 600 ug/kg every 48 hours until the end of the test on the 9th day to establish a TTP model.
  • 0.3 mg/kg of anti-platelet thrombolysis was subcutaneously injected to observe the effect of treating TTP.
  • Blood samples were taken daily and blood parameters were measured, including: whole blood count, lactate dehydrogenase level, haptoglobin level, split red blood cell count, etc., while ADAMTS13 antigen concentration and activity parameters were determined using a dedicated ELISA assay.
  • anti-platelet thrombolysis prevented the reduction of platelet count and inhibited the occurrence of symptoms such as erythrocytosis, hemolytic anemia, and abnormal lactate dehydrogenase levels. There was also no reduction in red blood cell count and hemoglobin concentration in this group of animals (Fig. 4).
  • anti-platelet thrombolysis can prevent and treat the main symptoms in the acquired TTP ⁇ model, restore platelet, red blood cell and hemoglobin levels, while reducing lactate dehydrogenase levels, effectively inhibiting thrombocytopenia and lytic hemolysis Anemia, in order to achieve the purpose of treating TTP.
  • an anti-platelet thrombopoietin according to the present invention for the preparation of a medicament for the treatment of thrombotic thrombocytopenic purpura has been described by way of example, and it will be apparent to those skilled in the art that The use of the anti-platelet thrombolysin described herein in the preparation of a medicament for the treatment of thrombotic thrombocytopenic purpura is modified or combined as appropriate to achieve the techniques of the present invention. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the spirit, scope and content of the invention.

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Abstract

一种抗血小板溶栓素在制备治疗血栓性血小板减少性紫癜(Thrombotic Thrombocytopenic Purpura，TTP)的药物中的应用。TTP的主要发病机制为血管性血友病因子裂解酶，即ADAMTS13的先天表达缺陷或其被自身抗体所抑制，从而导致血管性血友病因子(vWF)在体内大量聚集，成为为多个超大的多聚体，其介导病理性血小板聚集并形成血栓，最终导致TTP。而该抗血小板溶栓素限制了血小板的黏附和聚集，恢复血小板、红细胞和血红蛋白水平，同时降低乳酸脱氢酶水平，有效的抑制了血小板减少症和裂细胞性溶血性贫血，从而达到治疗TTP的目的。

Description

抗血小板溶栓素在制备治疗血栓性血小板减少性紫癜的药物中的应用
本申请要求于2016年3月23日提交中国专利局、申请号为201610180263.4、发明名称为“抗血小板溶栓素在制备治疗血栓性血小板减少性紫癜的药物中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及医药领域，尤其涉及一种抗血小板溶栓素在制备治疗血栓性血小板减少性紫癜(Thrombotic Thrombocytopenic Purpura，TTP)的药物中的应用。
背景技术
血栓性血小板减少性紫癜(Thrombotic Thrombocytopenic Purpura，TTP)是一种严重的弥散性血栓性微血管病，以微血管病性溶血性贫血、血小板聚集消耗性减少，以及微血栓形成造成器官损害(如肾脏、中枢神经系统等)为特征。其发病机制与一种vWF蛋白裂解酶(血管性血友病因子裂解酶，ADAMTS13)密切相关。该酶的作用在于特异性地裂解血液中的血管性血友病因子(vWF)，以保证人体正常的止血作用。在TTP的病程中，由于此酶的活性降低，vWF在血管内皮细胞上大量聚集，成为为多个超大的多聚体，该多聚体又可与血流中血小板表面的GPIb受体结合，介导血小板在内皮细胞上的黏附及聚集，最终导致血栓形成及进一步的TTP发生。
根据病因可将TTP分为遗传性TTP和获得性TTP，两者发病机理的差异在于引起ADAMTS13酶活性降低的起因不同：遗传性的患者是由于这种酶先天性表达不足，是基因缺陷引起的；而在获得性的患者中，造成酶活性降低的原因在于患者体内可产生一种针对ADAMTS13的自身抗体，这种抗体造成了酶的灭活。
对于以上病因，目前最有效的治疗TTP方法为新鲜血浆输注和血浆置换,但其缺陷在于依赖客观条件，并且对获得性TTP患者具有很高的复发率和死亡率。对于获得性TTP患者，可用免疫抑制药物或丙球等抑制抗ADAMTS13自身抗体的产生，但这类治疗起效较慢，效果因患者而异、并不确切。而补充ADAMTS13蛋白的方法只对于先天性TTP患者有效，因为对于获得性TTP患者而言，补充的ADAMTS13蛋白会很快被自身抗体所抑制。TTP患者中有70％-80％属于获得性，因此这种方法的临床应用非常局限。
发明内容
有鉴于此，本发明要解决的技术问题在于提供一种抗血小板溶栓素在制备治疗血栓性血小板减少性紫癜的药物中的应用，本发明发现，抗血小板溶栓素在TTP患者血液中能够恢复血小板、红细胞和血红蛋白水平，同时降低乳酸脱氢酶水平，有效的抑制了血小板减少症和裂细胞性溶血性贫血，起到抑制血小板聚集的作用，从而达到预防血栓形成，改善TTP症状的目的。
本发明所述抗血小板溶栓素(Antiplatelet thrombolysin，APT)是从尖吻蝮蛇蛇毒中分离出来的一种蛋白水解酶，由α链、β链两条肽链组成，α链氨基酸序列如SEQ ID NO.1所示，β链氨基酸序列如SEQ ID NO.2所示，其在专利201310228219.2中公开。
作为优选，血栓性血小板减少性紫癜为先天性或获得性血栓性血小板减少性紫癜。
作为优选，本发明提供的治疗血栓性血小板减少性紫癜的药物为化学药物或生物制剂。
作为优选，治疗血栓性血小板减少性紫癜的药物，包括抗血小板溶栓素与药学上可接受的辅料。
优选的，本发明提供的治疗血栓性血小板减少性紫癜的药物为口服制剂或注射剂。
更优选的，治疗血栓性血小板减少性紫癜的口服制剂为片剂、胶囊剂、丸剂、颗粒剂、滴丸剂、微囊剂或微丸剂。
更优选的，治疗血栓性血小板减少性紫癜的口服制剂或注射剂中抗血小板溶栓素的质量-体积浓度为1.8-10μg/mL。
更优选的，治疗血栓性血小板减少性紫癜的口服制剂或注射剂中抗血小板溶栓素的剂量为0.03-3mg/kg。
本发明提供了一种抗血小板溶栓素在制备治疗血栓性血小板减少性紫癜的药物中的应用。抗血小板溶栓素可以预防并治疗获得性TTP狒狒模型中的主要病症，恢复血小板、红细胞和血红蛋白水平，同时降低乳酸脱氢酶水平，有效的抑制了血小板减少症和裂细胞性溶血性贫血，从而达到治疗TTP的目的。可见，本发明提供的抗血小板溶栓素的用途，与现有技术中公布的用途不相同，其作用机理也优于现有的治疗TTP的手段，且可以补充它们的不足。实验显示，经抗血小板溶栓素处理后，TTP患者的富含血小板血浆(PRP)样本中的血小板聚集抑制率较空白对照组有了显著提高，表明抗血小板溶栓素对TTP患者有着明显的抑制血小板聚集，防止血栓形成和血小板减少的作用，具有良好的临床应用前景。
附图说明
图1示不同浓度抗血小板溶栓素对正常人血浆Ristocetin诱导血小板聚集抑制效果：绿色曲线表示空白对照组的血小板聚集情况与时间曲线；紫色曲线表示加入1.5ug/mL抗血小板溶栓素后的血小板聚集情况与时间曲线；蓝色曲线表示加入1.8ug/mL抗血小板溶栓素后的血小板聚集情况与时间曲线；黑色曲线表示加入2.1ug/mL抗血小板溶栓素后的血小板聚集情况与时间曲线。
图2示抗血小板溶栓素对正常人和TTP患者血浆ADAMTS13活性的影响；
图3示抗ADAMTS13抗体3H9在不同组别中对ADAMTS13抗原浓度及 活性的影响，用以部分反应获得性TTP模型的建立情况：图3A表示注射3H9后，对照组(图例为实心圆形)、预防组(图例为空心方块)及治疗组(图例为实心三角)中ADAMTS13酶活性随时间的变化曲线；图3B表示注射3H9后，对照组(图例为实心圆形)、预防组(图例为空心方块)及治疗组(图例为实心三角)中ADAMTS13抗原浓度随时间的变化曲线。
图4示抗血小板溶栓素对血小板减少症和裂细胞性溶血性贫血的各项指标的预防和治疗效果：图4A表示对照组(图例为实心圆形)、预防组(图例为空心方块)及治疗组(图例为实心三角)中血小板计数随时间的变化曲线；图4B表示对照组(图例为实心圆形)、预防组(图例为空心方块)及治疗组(图例为实心三角)中结合珠蛋白浓度随时间的变化曲线；图4C表示对照组(图例为实心圆形)、预防组(图例为空心方块)及治疗组(图例为实心三角)中裂红细胞计数随时间的变化曲线；图4D表示对照组(图例为实心圆形)、预防组(图例为空心方块)及治疗组(图例为实心三角)中乳酸脱氢酶水平随时间的变化曲线。
具体实施方式
本发明提供了抗血小板溶栓素在制备治疗血栓性血小板减少性紫癜的药物中的应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明采用的试剂皆为普通市售品，皆可于市场购得。
下面结合实施例，进一步阐述本发明：
实施例1 抗血小板溶栓素在体外对正常人及TTP患者瑞斯妥霉素诱导的 血小板聚集的抑制效果
1.样本采集
正常人血样3例采自健康鲜血员。TTP患者3例采自急诊TTP患者，3例患者均有临床上典型的“TTP三联征”，实验室检测其ADAMTS13活性均低于10％。上述样本均取外周静脉血，枸橼酸三钠抗凝。
2.实验方法
(1)血小板聚集抑制试验(光学比浊法)：
取3.2％枸橼酸钠抗凝全血，800prm，离心10min，制备PRP，其余血液以3000prm，离心10min制备贫血小板血浆(PPP)。将PRP血小板浓度以PPP调整为300×109/L左右，以PPP调零，观察PRP中加入终浓度10umol/L的ADP后，5min内透光度的变化，以最大透光度，作为该PRP的最大聚集率(PAGm)。
Figure PCTCN2016079973-appb-000001
(2)血浆ADAMTS13活性检测试验(化学荧光FRET法)
待测血浆按1:25浓度与荧光缓冲液(5mmol/L Bis-Tris,25mmoI/L CaCl2,0.005％Tween 20,pH 6.0)混匀，同时将正常人混合血浆作为标准品。按1:12.5、1:25、1:50、1:100、1:200、1:400与上述缓冲液进行稀释，作为标准曲线。将上述不同浓度的正常人混合血浆及待测标本一同加入96孔半量白板，每孔50ul，再将一定浓度(2umol/L)的荧光标记底物FRETS-VWF73每孔50ul,加入上述96孔白板与待测血楽进行酶切反应。反应5分钟后放入酶标仪进行检测，设置反应温度为4度，荧光激发/发射波长为485/530nm，频率为每2分读数一次，60分钟终止读数，整个操作过程不超过2小时。将各孔各时间点数据进 行曲线拟合，得到样本反应曲线方程，将待测样本的斜率分别代入以上对数直线回归方程，以1:25浓度正常混合血浆的ADAMTS13活性作为100％,与之相比即可得到待测样本ADAMTS13活性(％)。
3.实验结果
(1)血小板聚集抑制试验：
抗血小板溶栓素对正常人血浆Ristocetin诱导的血小板聚集抑制终浓度1.8ug/mL即可达抑制率93％，由聚集曲线及量效关系的特征来看，抗血小板溶栓素主要抑制血小板聚集的“第一相”，即通过对“outside-in”信号途径的阻断从而抑制了血小板的活化以及进一步的聚集反应，其抑制聚集的效果十分有效(图1)。
抗血小板溶栓素对TTP患者血浆Ristocetin诱导的血小板聚集抑制由三例TTP样本检测结果来看，1.8ug/mL浓度的抗血小板溶栓素对TTP患者血小板聚集的抑制同样有效(见下表)，下表示抗血小板溶栓素(1.8ug/mL)对不同人群血小板聚集的抑制效果。
Figure PCTCN2016079973-appb-000002
(2)血浆ADAMTS13活性检测试验
两种不同浓度的抗血小板溶栓素(5ug/mL、1.8ug/mL)对正常人或TTP患者血浆中的ADAMTS13活性均无明显影响(图2)。
4.结论
(1)抗血小板溶栓素对正常人或TTP患者血浆Ristocetin诱导的血小 板聚集均有明显抑制作用，该批次(20130603)产品1.8ug/mL(终浓度)即可达到最大抑制率90％以上，对TTP患者血小板聚集的抑制与正常人无差异。
(2)正常治疗浓度的抗血小板溶栓素对正常人血浆中的ADAMTS13活性无明显影响，说明其不会对正常的ADAMTS13裂解vWF行为产生抑制。
实施例2 抗血小板溶栓素用于获得性TTP狒狒模型的有效性实验
1.实验设计和方法
试验分三组，每组4只动物。第一组为阳性对照，从试验第0天开始，每48小时由股静脉注射剂量为为600ug/kg的抗ADAMTS13抗体3H9，直至试验第9天结束，用以建立TTP模型。第2组为预防组，在第1天和第3天注射相同剂量的3H9，并在第1天与第5天皮下注射0.3mg/kg的抗血小板溶栓素，用以观察其预防TTP的作用。而第3组为治疗组，从试验第0天开始，每48小时由股静脉注射剂量为为600ug/kg的抗ADAMTS13抗体3H9，直至试验第9天结束，用以建立TTP模型。其中，在第5到第11天，每天由皮下注射0.3mg/kg的抗血小板溶栓素，用以观察其治疗TTP的作用。
每天抽取血样并进行血液学参数的测量，其包括：全血细胞计数，乳酸脱氢酶水平，结合珠蛋白水平，裂红细胞计数等，而ADAMTS13抗原浓度及活性参数用专用的ELISA试验测定。
2.实验结果
(1)TTP模型的建立：
每隔48小时的抗ADAMTS13抗体3H9的注射导致了ADAMTS13酶活性的显著降低，并同时引起ADAMTS13抗原浓度的降低(图3)。这直接导致了动物体内出现血小板减少和溶血性贫血，表现为血小板计数和结合珠蛋白计数减少，并伴有裂红细胞计数和乳酸脱氢酶水平升高(图4)。这些都是TTP的典型症状，也是该模型建立成功的指标。
(2)预防组中溶栓素的有效性：
相对于对照组，预防组中，抗血小板溶栓素防止了血小板计数的减少，抑制了裂红细胞症，溶血性贫血和乳酸脱氢酶水平异常等症状的出现。该组动物也没有出现红细胞计数和血红蛋白浓度的减少(图4)。
(3)治疗组中溶栓素的有效性：
治疗组中，在试验第5天，动物出现了TTP症状，各项指标与对照组一致，提示模型建立良好。第5天开始注射抗血小板溶栓素后，血小板计数有了明显增加，并在第9天恢复初始水平，而在第11天升高至比初始水平的180％。裂细胞性溶血性贫血在第5天后得到了有效的治疗，裂红细胞计数也显著降低，而结合珠蛋白浓度则从第10天开始趋于正常化(图4)。
3.结论
实验证实，抗血小板溶栓素可以预防并治疗获得性TTP狒狒模型中的主要病症，恢复血小板、红细胞和血红蛋白水平，同时降低乳酸脱氢酶水平，有效的抑制了血小板减少症和裂细胞性溶血性贫血，从而达到治疗TTP的目的。
本发明提出的一种抗血小板溶栓素在制备治疗血栓性血小板减少性紫癜的药物中的应用已通过实施例进行了描述，相关技术人员明显能在不脱离本发明内容、精神和范围内对本文所述的抗血小板溶栓素在制备治疗血栓性血小板减少性紫癜的药物中的应用进行改动或适当变更与组合，来实现本发明技术。特别需要指出的是，所有相类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明的精神、范围和内容中。
Figure PCTCN2016079973-appb-000003
Figure PCTCN2016079973-appb-000004
Figure PCTCN2016079973-appb-000005

Claims (8)

  1. 一种抗血小板溶栓素在制备治疗血栓性血小板减少性紫癜的药物中的应用，所述抗血小板溶栓素由α链、β链两条肽链组成，其中，α链氨基酸序列如SEQ ID NO.1所示，β链氨基酸序列如SEQ ID NO.2所示。
  2. 根据权利要求1所述的应用，其特征在于，所述血栓性血小板减少性紫癜为先天性或获得性血栓性血小板减少性紫癜。
  3. 根据权利要求1所述的应用，其特征在于，所述药物为化学药物或生物制剂。
  4. 根据权利要求1所述的应用，其特征在于，所述药物包括抗血小板溶栓素与药学上可接受的辅料。
  5. 根据权利要求1所述的应用，其特征在于，所述药物为口服制剂或注射剂。
  6. 根据权利要求5所述的应用，其特征在于，所述口服制剂为片剂、胶囊剂、丸剂、颗粒剂、滴丸剂、微囊剂或微丸剂。
  7. 根据权利要求5所述的应用，其特征在于，所述口服制剂或注射剂中抗血小板溶栓素的质量-体积浓度为1.8-10μg/mL。
  8. 根据权利要求5所述的应用，其特征在于，所述口服制剂或注射剂中抗血小板溶栓素的剂量为0.03-3mg/kg。
PCT/CN2016/079973 2016-03-23 2016-04-22 抗血小板溶栓素在制备治疗血栓性血小板减少性紫癜的药物中的应用 WO2017161621A1 (zh)

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