WO2020087856A1 - 抗血小板溶栓素在制备治疗贫血药物中的应用 - Google Patents

抗血小板溶栓素在制备治疗贫血药物中的应用 Download PDF

Info

Publication number
WO2020087856A1
WO2020087856A1 PCT/CN2019/080448 CN2019080448W WO2020087856A1 WO 2020087856 A1 WO2020087856 A1 WO 2020087856A1 CN 2019080448 W CN2019080448 W CN 2019080448W WO 2020087856 A1 WO2020087856 A1 WO 2020087856A1
Authority
WO
WIPO (PCT)
Prior art keywords
anemia
amino acid
platelet
chain
acid sequence
Prior art date
Application number
PCT/CN2019/080448
Other languages
English (en)
French (fr)
Inventor
楚在玹
李小羿
戴向荣
Original Assignee
兆科药业（合肥）有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 兆科药业（合肥）有限公司 filed Critical 兆科药业（合肥）有限公司
Priority to EP19878870.5A priority Critical patent/EP3875109A4/en
Priority to JP2021523006A priority patent/JP7189342B2/ja
Priority to KR1020217016085A priority patent/KR102669871B1/ko
Priority to CA3118237A priority patent/CA3118237C/en
Priority to BR112021008313-8A priority patent/BR112021008313A2/pt
Priority to US17/289,743 priority patent/US20220370572A1/en
Publication of WO2020087856A1 publication Critical patent/WO2020087856A1/zh

Links

Images

Classifications

    • 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
    • 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
    • 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

Definitions

  • the invention relates to the field of medicine, in particular to the application of an antiplatelet thrombolytic agent in the preparation of anemia treatment drugs.
  • Anemia is a common clinical symptom. It refers to a state in which the total amount of red blood cells in the human blood decreases below the normal value. It can be caused by a variety of system diseases. Due to the complexity of measuring red blood cell volume, hemoglobin Hb concentration is often used in clinical practice.
  • anemia has different classifications. According to the speed of anemia, it is divided into acute and chronic anemia; according to the morphology of red blood cells, it is divided into large cell anemia, normal cell anemia and small cell hypochromic anemia; according to the concentration of hemoglobin, it is divided into mild, moderate, severe and extremely severe anemia; The hyperplasia of the system is divided into proliferative anemia including hemolytic anemia, iron deficiency anemia, megaloblastic anemia, etc. and hypoplastic anemia such as aplastic anemia.
  • Hematopoietic cells, bone marrow hematopoietic microenvironment and abnormal hematopoietic raw materials affect erythropoiesis production anemia, such as aplastic anemia, pure red blood cell aplastic anemia, congenital erythropoiesis anemia, malignant clonal anemia of hematopoietic system ;
  • Anemia caused by abnormal hematopoietic microenvironment including anemia caused by the damage of bone marrow stromal and stromal cells, anemia caused by abnormal levels of hematopoietic regulatory factors, hyperlymphocyte function and other autoimmune hemolytic anemias such as sickle cell anemia and hematopoiesis Anemia due to increased apoptosis;
  • Anemia caused by insufficient hematopoietic raw materials or utilization disorders including anemia due to folic acid or vitamin B12 deficiency or utilization disorders, iron deficiency, and iron utilization disorders
  • Red blood cells destroy excessive anemia, including anemia caused by abnormal red blood cells and abnormal surrounding environment of red blood cells;
  • the etiology of anemia the degree to which the ability of the blood to carry itch drops, the degree to which the blood volume decreases, the rate of occurrence of anemia, and the compensation and tolerance of the blood, circulation, and respiratory systems all affect the clinical manifestations of anemia.
  • the severity of anemia depends on the speed of anemia, the degree of anemia and the body's ability to compensate.
  • Sickle cell anemia also known as sickle cell anemia, sickle cell anemia, sickleemia, sickle cell, disease, SCD
  • Sickle cell anemia is a potentially human lethal genetic disease, an autosomal inherited hemoglobin disease, and the clinical manifestation is chronic hemolytic anemia , Prone to infection and recurrent pain, resulting in chronic ischemia leading to organ tissue damage.
  • Sickle-changed red blood cells are stiff and poorly deformable. Hemolysis can occur due to the destruction of vascular mechanisms and phagocytosis by the mononuclear macrophage system. It is also easy to block capillaries and cause local hypoxia and inflammation.
  • Sickle cell anemia is also a serious disease that threatens the health of mother and child, and can cause a fetal mortality rate of 5% and a maternal mortality rate of 4.62%. Because the 6th amino acid glutamic acid in the ⁇ -peptide chain is replaced by valine, it constitutes sickle hemoglobin (HbS), replacing normal Hb (HbA). Normal adult hemoglobin is a tetramer composed of two ⁇ chains and two ⁇ chains combined with each other. The ⁇ chain and ⁇ chain are composed of 141 and 146 amino acids connected in sequence.
  • red blood cells Even if the cells are placed under aerobic conditions, the red blood cells remain sickle-shaped. Sickle-changed red blood cells can also increase blood viscosity, slow blood flow, and poor deformability. It is easy to block capillaries and cause local hypoxia and inflammation to cause pain crisis in the corresponding parts, which mostly occur in muscles, bones, joints of limbs, Chest and abdomen, especially joints and chest and abdomen are common.
  • hydroxyurea can reduce the number of pain crises and the need for blood transfusions, and it can also reduce the frequency of chest syndrome.
  • This disease is mainly caused by hypoxia that causes red blood cell sickle to block capillaries and cause pain crisis. It is appropriate to use alternative treatments and vasodilator drugs.
  • Folic acid supplementation can reduce the increased cysteine levels and improve the function of vascular endothelium.
  • bone marrow transplants and fetal liver hematopoietic stem cell transplants to save patients and improve quality of life.
  • Using an indirect flow blood cell separator to separate the red blood cells of the patient and input the blood cells to the donor's red blood cells to obtain better results.
  • anti-platelet thrombolysin can significantly reduce platelet-neutrophil aggregation in anemia mice, prolong the survival time of anemia mice, and is expected to be applied to the treatment of anemia.
  • the object of the present invention is to provide a new application of antiplatelet thrombolytics.
  • the antiplatelet thrombolysin (Antiplatelet thrombolysin, APT, Anfibatide) is a C-type lectin-like protein isolated from the snake venom of Agkistrodon acutus, which is composed of two peptide chains of ⁇ chain and ⁇ chain.
  • Amino acid sequence The alpha chain amino acid sequence is shown in SEQ ID NO.1, and the beta chain amino acid sequence is shown in SEQ ID NO.2; or the anti-platelet thrombolysin is substituted, deleted, or added with an alpha chain amino acid sequence Or a few amino acids derived from the amino acid sequence of SEQ ID NO. 1 has at least 95% identity, and its ⁇ chain amino acid sequence is substituted, deleted, or added one or a few amino acids derived from SEQ ID NO. 2 The amino acid sequence has at least 95% identity and has anemia-improving activity.
  • the anemia is sickle cell anemia.
  • the medicine of the present invention is a chemical medicine or a biological agent.
  • the medicine includes antiplatelet thrombolysin and pharmaceutically acceptable excipients.
  • the medicine for treating anemia provided by the present invention is an external preparation or an injection.
  • the external preparation is tincture, ointment, cream, lotion, rinse, liniment or gel.
  • antiplatelet thrombolytic agent provided by the present invention in the preparation of a medicament for improving anemia is not the same as the application published in the prior art, and it can also supplement the deficiency of the existing medicinal treatment.
  • antiplatelet thrombolysin can significantly reduce the aggregation of platelet-neutrophils in sickle cell anemia model induced by TNF- ⁇ , and prolong the survival time of TNF- ⁇ -induced sickle cell anemia in vivo.
  • Figure 1 shows the role of neutrophils in sickle cell anemia mouse model
  • Figure 2 is a platelet-neutrophil interaction between sickle cell anemia mouse model
  • Figure 3 is a representative image of in vivo microscopic imaging. From left to right are the BSA group, IgG group, anti-platelet thrombolytic group, antiPDI group, antiPDI + anti-platelet thrombolytic group. The large and small arrows show blood flow and Neutrophil rolling direction;
  • Figure 4 shows the survival time of sickle cell anemia mice after administration.
  • the present invention provides an application of anti-platelet thrombolysin in the preparation of a medicament for treating anemia.
  • Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve.
  • all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the methods and applications of the present invention have been described through preferred embodiments, and it is obvious that relevant personnel can modify or appropriately modify and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention. Apply the technology of the present invention.
  • the reagents used in the present invention are all ordinary commercial products, and are all commercially available.
  • Example 1 Antiplatelet thrombolysin reduces TNF- ⁇ -induced sickle cell anemia mouse model platelet-neutrophil aggregation
  • platelet-selective receptors mediate the binding and adhesion of activated platelets and neutrophils, neutrophil aggregation, and accelerate tissue damage; on the other hand, platelet adhesion to neutrophils promotes platelet aggregation and mediates vascular occlusion
  • the neutrophil cell membrane can release the occurrence of tetraenoic acid.
  • thromboxane and prostaglandin produced by it have a significant effect on regulating the diameter and permeability of blood vessels, and can also cause Inflammation and pain.
  • Sickle cell anemia Sickle red blood cells in the blood are stiff and poorly deformable, and easily block capillaries to cause local hypoxia and inflammation.
  • the clinical manifestations are chronic hemolytic anemia, easy to interfere and recurrent pain, and ischemia leads to damage to organ tissues .
  • the current treatment of sickle cell anemia is to prevent hypoxia, dehydration, and infection to relieve symptoms, reduce complications of organ damage, promote hematopoiesis, and prolong life.
  • Platelets (2x10 7 cells / ml) and neutrophils (1x10 6 cells / ml) of sickle cell anemia model mice were labeled with DyLight488-anti-CD42C and Alexa Fluor647-anti-Ly-6G antibodies, human neutrophils and Platelets were labeled with Alexa Fluor 488-anti-CD41 and FITC-anti-L selectin antibodies, neutrophils were induced with 20ng / ml TNF- ⁇ for 5 minutes, and platelets were compared with control IgG (10 ⁇ g / ml) and anti PDI antibody (protein II) Sulfur bond isomerase inhibitor, 10 ⁇ g / ml), BSA (0.2 ⁇ g / ml), anti-platelet thrombolysin (0.2 ⁇ g / ml) and anti-platelet thrombolysin + antiPDI pre-incubated at room temperature for 30min, and then with 0.025U / ml thrombin was incubated at 37
  • the ordinate is relative to the cell-to-cell interaction of the control group
  • the abscissa is the experimental group from left to right: control IgG group, anti PDI group, BSA group, anfibatide group, IgG + BSA group, anti PDI + anfibatide group.
  • the ordinate in Figure 2 is the interaction between platelets and neutrophils relative to the control group, and the abscissa is the experimental group from left to right: control IgG group, anti PDI group, BSA group, anfibatide group, IgG + BSA group, anti PDI + anfibatide group.
  • the experimental data was statistically analyzed by ANOVA and Tukey ’s test.
  • n 4, * P ⁇ 0.05, ** P ⁇ 0.01, or *** P ⁇ 0.001
  • the administration group included: IgG control (1.5 ⁇ g / g), antiPDI (BD34, 1.5 ⁇ g / g) , BSA (25ng / g), anfibatide (25ng / g), antiPDI (1.5 ⁇ g / g) + anfibatide (25ng / g).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Hematology (AREA)
  • Diabetes (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Zoology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)
  • Peptides Or Proteins (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

提供了抗血小板溶栓素在制备治疗贫血药物中的应用。

Description

抗血小板溶栓素在制备治疗贫血药物中的应用
本申请要求于2018年10月29日提交中国专利局、申请号为201811267000.2、发明名称为“抗血小板溶栓素在制备治疗贫血药物中的应用”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贫血药物中的应用。
背景技术
贫血是常见的临床症状，是指人体血液中红细胞总量减少至正常值以下的一种状态，可由多种系统的疾病引起，由于红细胞容量测定较复杂，临床上常以血红蛋白Hb浓度来代替。
基于不同的临床特点，贫血有不同的分类。按贫血进展速度分急、慢性贫血；按红细胞形态分大细胞性贫血、正常细胞性贫血和小细胞低色素性贫血；按血红蛋白浓度分轻度、中度、重度和极重度贫血；按骨髓红系增生情况分增生性贫血包括溶血性贫血、缺铁性贫血、巨幼细胞贫血等和增生低下性贫血如再生障碍性贫血。
临床上根据其发病机制及病因主要有:
1)造血细胞、骨髓造血微环境和造血原料异常影响红细胞生产的红细胞生成减少性贫血，如再生障碍性贫血、纯红细胞再生障碍贫血、先天性红细胞生成异常性贫血、造血系统恶性克隆性贫血等；
2)造血微环境异常所致贫血，包括骨髓基质和基质细胞受损所致贫血、 造血调节因子水平异常所致贫血、淋巴细胞功能亢进等所致自身免疫性溶血性贫血如镰刀细胞贫血及造血细胞凋亡亢进所致贫血；
3)造血原料不足或利用障碍所致贫血，包括叶酸或维生素B12缺乏或利用障碍所致贫血、缺铁和铁利用障碍所致贫血；
4)红细胞破坏过多性贫血，包括红细胞自身异常以及红细胞周围环境异常等所致贫血；
5)失血性贫血等。
贫血的病因、血液携痒能力下降的程度、血容量下降的程度、发生贫血的速度和血液、循环、呼吸等系统的代偿和耐受能力均会影响贫血的临床表现。贫血症状的轻重取决于贫血的速度、贫血的程度和机体的代偿能力。
镰状细胞贫血(又名镰刀型细胞贫血，镰状细胞性贫血，sicklemia，Sickle Cell Disease,SCD)是一种潜在的人类致死遗传病，为常染色体遗传血红蛋白病，临床表现为慢性溶血性贫血、易感染和再发性疼痛以致慢性局部缺血导致器官组织损伤。镰变的红细胞僵硬，变形性差，可受血管机制破坏和单核巨噬系统吞噬而发生溶血，也易堵塞毛细血管引起局部缺氧和炎症反应。
镰状细胞贫血也是严重危害母子健康的疾病，可使胎儿死亡率达5％，孕妇死亡率达4.62％。因β-肽链第6位氨基酸谷氨酸被缬氨酸所代替，构成镰状血红蛋白(HbS)，取代了正常Hb(HbA)。正常成人血红蛋白是由两条α链和两条β链相互结合成的四聚体，α链和β链分别由141和146个氨基酸顺序连结构成。镰状细胞贫血患者因β链第6位氨基酸谷氨酸被缬氨酸所代替，形成了异常的血红蛋白S(hemoglobin S，HbS)，取代了正常血红蛋白(HbA)，在氧分压下降时HbS分子间相互作用，成为溶解度很低的螺旋形多聚体，使红细胞扭曲成 镰状细胞即发生镰变。红细胞内HbS浓度、脱氧程度、酸中毒、红细胞脱水程度等许多因素与红细胞镰变有关。红细胞镰变的初期是可逆的，给予氧可逆转镰变过程。但当镰变已严重损害红细胞膜后，镰变就变为不可逆，即使将这种细胞置于有氧条件下，红细胞仍保持镰状。镰变的红细胞还可使血液黏滞性增加，血流缓慢，加之变形性差，易堵塞毛细血管引起局部缺氧和炎症反应导致相应部位产生疼痛危象，多发生于肌肉、骨骼、四肢关节、胸腹部，尤以关节和胸腹部为常见。
镰状细胞贫血患者血液脱氧合时，导致细胞失水变形，此外，红细胞与毛细血管内皮细胞黏连性增加，尤其是脱落到血液中的血管内皮细胞对黏附过程的促进作用，镰刀形细胞大大增多，这种细胞极脆易破损造成血红蛋白低水平，严重的后果是某些器官的毛细血管被异形细胞堵塞，加剧了血管阻塞及溶血反应，这是许多镰状细胞贫血患者死亡的主要原因。
在严重病例中，羟基脲可以减少疼痛危象和需要输血的次数，还能降低胸部综合征的发作频率。本病主要是因缺氧使红细胞镰变阻碍毛细血管而造成疼痛危象，宜用替代治疗和血管扩张药物。叶酸的补充能降低增高的半胱氨酸水平，改善血管内皮的功能。也有进行骨髓移植和胎肝造血干细胞移植以拯救患者和提高生存质量。用间接流动血细胞分离器分离出病人的红细胞同时输入供血者去甘油的红细胞获得较好疗效。也有主张部分换血同时静滴5％葡萄糖来降低血液粘稠度。小剂量双香豆素治疗可以减少病人血栓形成前状态的发生。本病尚无明确患病个体遗传基因的构成变化，故病因治疗无意义，目前常规治疗在于预防缺氧、脱水、感染以缓解症状，减少器官损伤并发症，促进造血和延长生命。
发明内容
本专利发明人意外发现，抗血小板溶栓素能够显著减少贫血小鼠血小板-中性粒细胞聚集，延长贫血小鼠的存活时间，有望应用于贫血的治疗。
有鉴于此，本发明的目的在于提供一种抗血小板溶栓素的新应用。本发明所述抗血小板溶栓素(Antiplatelet thrombolysin，APT，Anfibatide)是从尖吻蝮蛇蛇毒中分离出来的一种C型凝集素类似蛋白，由α链、β链两条肽链组成，其氨基酸序列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；或所述抗血小板溶栓素为其α链氨基酸序列经过取代、缺失、或添加一个或几个氨基酸衍生的与SEQ ID NO.1的氨基酸序列至少有95％的一致性，且其β链氨基酸序列经过取代、缺失、或添加一个或几个氨基酸衍生的与SEQ ID NO.2的氨基酸序列至少有95％的一致性，且具有改善贫血的活性。
作为优选，所述贫血为镰状细胞贫血症。
作为优选，本发明所述药物为化学药物或生物制剂。
作为优选，所述药物包括抗血小板溶栓素与药学上可接受的辅料。
优选的，本发明提供的治疗贫血的药物为外用制剂或注射剂。
更优选的，所述外用制剂为酊剂、软膏剂、乳膏剂、洗剂、冲洗剂、搽剂或凝胶剂。
本发明提供的抗血小板溶栓素在制备改善贫症的药物中的应用，与现有技术中公布的用途不相同，亦可以补充现有药物治疗的不足。经体内外实验证实，抗血小板溶栓素能显著减少TNF-α诱导的镰状细胞贫血模型血小板-中性粒细 胞聚集，延长TNF-α诱导镰状细胞贫血活体的存活时间。
附图说明
图1为镰刀细胞贫血小鼠模型中性粒细胞细胞间作用；
图2为镰刀细胞贫血小鼠模型血小板-中性粒细胞间作用；
图3为活体显微成像代表图，从左到右依次是BSA组、IgG组、抗血小板溶栓素组、antiPDI组、antiPDI+抗血小板溶栓素组，大箭头和小箭头分别显示血流和中性粒细胞滚动方向；
图4示镰刀细胞贫血小鼠给药后存活时间。
具体实施方式
本发明提供了一种抗血小板溶栓素在制备治疗贫血药物中的应用，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 抗血小板溶栓素减少TNF-α诱导镰刀细胞贫血小鼠模型血小板-中性粒细胞聚集作用
中性粒细胞(neutrophil)在血液的非特异性细胞免疫系统中起着十分重要的作用。活化中性粒细胞通过趋化作用到达病变部位，其携带的杀菌物质在局部会大量释放，通过各自的破坏作用，导致组织损伤，进一步导致器官障碍，且中性粒细胞侵入一个器官之后，会诱导其他的重要器官中中性粒细胞的聚集，从而导致多器官损伤。同时，血小板选择受体介导活化血小板与中性粒细胞的结合与黏附，中性粒细胞聚集，加速组织损伤；另一方面，血小板与中性粒细胞黏附，促进血小板聚集，介导血管闭塞，同时中性粒细胞的细胞膜能释放出发生四烯酸，在酶的作用下，由它再进一步生成的血栓素和前列腺素等对调节血管口径和通透性有明显的作用，还能引起炎症反应和疼痛。镰刀细胞贫血血液中镰变的红细胞僵硬，变形性差，易堵塞毛细血管引起局部缺氧和炎症反应，其临床表现为慢性溶血性贫血、易干扰和再发性疼痛，局部缺血导致器官组织损伤。目前镰刀细胞贫血常规治疗在于预防缺氧、脱水、感染以缓解症状，减少器官损伤并发症，促进造血和延长生命。
通过将Berkeley小鼠骨髓移植到6周龄的经过致死剂量辐照的WT小鼠体内，产生嵌合对照和Berkeley小鼠，移植3个月后通过PCR和电泳分析确认嵌合体，这些嵌合Berkeley小鼠即为镰刀细胞贫血小鼠。
取镰刀细胞贫血模型小鼠血小板(2x10 7个/ml)和中性粒细胞(1x10 6个/ml)分别用DyLight488-抗CD42C和Alexa Fluor647-抗Ly-6G抗体标记，人中性粒细胞和血小板分别用Alexa Fluor 488-抗CD41和FITC-抗L选择素抗体标记，中性粒细胞用20ng/ml TNF-α诱导5min，血小板分别与对照IgG(10μg/ml)、 anti PDI抗体(蛋白二硫键异构酶抑制剂，10μg/ml)、BSA(0.2μg/ml)、抗血小板溶栓素(0.2μg/ml)及抗血小板溶栓素+antiPDI室温预孵30min，后与0.025U/ml凝血酶于37℃孵育5min，再与50uM PPACK孵育，1000rpm搅拌混合血小板及中性粒细胞，5min后固定并通过流式细胞仪进行分析。
在病理发生时，血小板与中性粒细胞黏附，促进血小板聚集，介导血管闭塞，加重病理生理反应。试验结果显示，0.2μg/ml的抗血小板溶栓素可降低镰刀细胞贫血模型小鼠中性粒细胞间的聚集作用(图1)，也可减少血小板与中性粒细胞间的结合作用(图2)，从而起到抑制血管损伤的作用。
图1中纵坐标为相对于对照组细胞与细胞间作用，横坐标为实验组从左至右依次为：对照IgG组、anti PDI组、BSA组、anfibatide组、IgG+BSA组，anti PDI+anfibatide组。
图2中纵坐标为相对于对照组血小板与中性粒细胞间作用，横坐标为实验组从左至右依次为：对照IgG组、anti PDI组、BSA组、anfibatide组、IgG+BSA组，anti PDI+anfibatide组。
实验数据经ANOVA和Tukey’s检验统计分析。
n＝4，*P<0.05,**P<0.01,or***P<0.001
实施例2 抗血小板溶栓素对镰刀型细胞贫血小鼠的治疗作用
通过将Berkeley小鼠骨髓移植到6周龄的经过致死剂量辐照的WT小鼠体内，产生嵌合对照和Berkeley小鼠，移植3个月后通过PCR和电泳分析确认嵌合体，这些嵌合Berkeley小鼠即为镰刀细胞贫血小鼠。取造模成功小鼠，通过腹腔注射500ng TNF-α诱导严炎症，诱导3h后采用单盲给药，给药组包 括：IgG对照(1.5μg/g)、antiPDI(BD34,1.5μg/g)、BSA(25ng/g)、anfibatide(25ng/g)、antiPDI(1.5μg/g)+anfibatide(25ng/g)。
给药后通过活体显微镜观察，研究期间记录每只小鼠的存活时间，记录时间从注射TNFα开始，并在小鼠死亡或注射TNF-α6h结束。使用GraphPad Prism7软件分析数据。通过Student's t-检验和ANOVA进行统计学分析。n＝8/组，*P<0.05，**P<0.01，***P<0.001，****P<0.0001。
结果显示，活体显微镜观察，相比较BSA对照组，25ng/g抗血小板溶栓素消除SCD小鼠提高血管壁上血小板与中性粒细胞的黏附(图3)，图中分别用DyLight488-抗CD42C和Alexa Fluor647-抗Ly-6G抗体标记血小板和中性粒细胞，大箭头指示血流方向，小箭头指示中性粒细胞滚动方向。源于TNF-α刺激及活体显微镜外科手术可引起急性血管栓塞，致使大多数SCD小鼠死亡，实验观察期为小鼠死亡或TNFα给药6h。
与各对照组相比，BSA与抗血小板溶栓素组比较P＝0.0249，BSA+IgG与BSA+抗PDI组比较P＝0.028，BSA+IgG与抗血小板溶栓素anti-PDI联用组比较P＝0.0165，抗血小板溶栓素(Anfibatide)治疗小鼠50％存活时间为5.0h，为在各给药组中小鼠存活时间最长(见图4)，显示出抗血小板溶栓素对镰刀细胞贫血活体的贫血治疗改善作用。

Claims (6)

  1. 抗血小板溶栓素在制备治疗贫血药物中的应用，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；或所述抗血小板溶栓素为其α链氨基酸序列经过取代、缺失、或添加一个或几个氨基酸衍生的与SEQ ID NO.1的氨基酸序列至少有95％的一致性，且其β链氨基酸序列经过取代、缺失、或添加一个或几个氨基酸衍生的与SEQ ID NO.2的氨基酸序列至少有95％的一致性，且具有改善贫血的活性。
  2. 根据权利要求1所述的应用，其特征在于，所述贫血为镰状细胞贫血症。
  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。
PCT/CN2019/080448 2018-10-29 2019-03-29 抗血小板溶栓素在制备治疗贫血药物中的应用 WO2020087856A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
EP19878870.5A EP3875109A4 (en) 2018-10-29 2019-03-29 APPLICATION OF ANTITHROMBOCYTE THROMBOLYSIN IN THE PREPARATION OF MEDICATIONS FOR THE TREATMENT OF ANEMIA
JP2021523006A JP7189342B2 (ja) 2018-10-29 2019-03-29 貧血治療薬の製造における抗血小板トロンボリシンの使用
KR1020217016085A KR102669871B1 (ko) 2018-10-29 2019-03-29 빈혈 치료 약물 제조에서 항혈소판 트롬볼리신의 응용
CA3118237A CA3118237C (en) 2018-10-29 2019-03-29 Application of anti-platelet thrombolysin in preparing drug for treating anemia
BR112021008313-8A BR112021008313A2 (pt) 2018-10-29 2019-03-29 Aplicação de trombolisina antiplaquetária na preparação de fármaco para tratar anemia
US17/289,743 US20220370572A1 (en) 2018-10-29 2019-03-29 Application of anti-platelet thrombolysin in preparing drug for treating anemia

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811267000.2 2018-10-29
CN201811267000.2A CN109464659B (zh) 2018-10-29 2018-10-29 抗血小板溶栓素在制备治疗贫血药物中的应用

Publications (1)

Publication Number Publication Date
WO2020087856A1 true WO2020087856A1 (zh) 2020-05-07

Family

ID=65666166

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/080448 WO2020087856A1 (zh) 2018-10-29 2019-03-29 抗血小板溶栓素在制备治疗贫血药物中的应用

Country Status (8)

Country Link
US (1) US20220370572A1 (zh)
EP (1) EP3875109A4 (zh)
JP (1) JP7189342B2 (zh)
KR (1) KR102669871B1 (zh)
CN (1) CN109464659B (zh)
BR (1) BR112021008313A2 (zh)
CA (1) CA3118237C (zh)
WO (1) WO2020087856A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109464659B (zh) * 2018-10-29 2021-10-15 兆科药业（合肥）有限公司 抗血小板溶栓素在制备治疗贫血药物中的应用
CN109260464B (zh) * 2018-10-29 2021-10-15 兆科药业（合肥）有限公司 抗血小板溶栓素的新用途

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101838323A (zh) * 2010-02-03 2010-09-22 兆科药业（合肥）有限公司 一种抗血小板溶栓素及其制备方法
CN105833255A (zh) * 2016-03-23 2016-08-10 兆科药业（合肥）有限公司 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
CN109464659A (zh) * 2018-10-29 2019-03-15 兆科药业（合肥）有限公司 抗血小板溶栓素在制备治疗贫血药物中的应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101838323A (zh) * 2010-02-03 2010-09-22 兆科药业（合肥）有限公司 一种抗血小板溶栓素及其制备方法
CN105833255A (zh) * 2016-03-23 2016-08-10 兆科药业（合肥）有限公司 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
CN109464659A (zh) * 2018-10-29 2019-03-15 兆科药业（合肥）有限公司 抗血小板溶栓素在制备治疗贫血药物中的应用

Also Published As

Publication number Publication date
BR112021008313A2 (pt) 2021-10-13
CA3118237C (en) 2023-10-17
JP7189342B2 (ja) 2022-12-13
JP2022512825A (ja) 2022-02-07
EP3875109A4 (en) 2022-08-10
KR20210113167A (ko) 2021-09-15
CN109464659A (zh) 2019-03-15
CA3118237A1 (en) 2020-05-07
CN109464659B (zh) 2021-10-15
EP3875109A1 (en) 2021-09-08
US20220370572A1 (en) 2022-11-24
KR102669871B1 (ko) 2024-05-29

Similar Documents

Publication Publication Date Title
JP6581208B2 (ja) 創傷治癒の方法
Williams et al. Healing responses of skin and muscle in critical illness
WO2020087856A1 (zh) 抗血小板溶栓素在制备治疗贫血药物中的应用
MX2008014648A (es) Metodos para tratar la anemia aguda por perdida de sangre con un sustituto de la sangre con hemoglobina reticulada.
BR112016009753B1 (pt) Métodos in vitro de cultura de amostras de células estromais mesenquimais e para a preparação de células estromais mesenquimais
CN109414542A (zh) 管理需要输血的患者群体中的不良事件的方法
Cherednikov et al. Innovative Endoscopic Technologies in the Complex Treatment of Patients with Unstable Stopped Gastroduodenal Bleeding
WO2009000150A1 (fr) Utilisation de la sanchinoside dans le traitement de la septicémie
Rathnayaka et al. Thrombotic microangiopathy, hemolytic uremic syndrome and thrombotic thrombocytopenic purpura: Rare manifestations of Russell’s viper (Daboia russelii) envenoming in Sri Lanka
LUTTGENS Thrombotic thrombocytopenic purpura with extensive hemorrhagic gangrene of the skin and subcutaneous tissue: Report of a case
CN110898069A (zh) 1,25-二羟基维生素d3在预防和治疗ams中的药物用途
JP2023518085A (ja) 自家生理学的液体から、強化された抗炎症性／抗異化性作用物質を作製するための方法
JP2009191015A (ja) 創傷治癒促進剤
Garvin et al. A comparison of ductal management techniques in an in situ canine pancreas model
Wang et al. Irisin Ameliorates Hypoxia/Reoxygenation-Induced Inflammation and Apoptosis in PC12 Cells by Inhibiting TLR4/MYD88 Signaling Pathway.
RU2758536C1 (ru) Способ снижения воспалительной гиперактивации нейтрофилов
KR102678583B1 (ko) Hapln1을 포함하는 혈관질환 예방 또는 치료용 조성물
US20230340414A1 (en) Method for obtaining platelet-derived mitochondria and use thereof
D'Angelo et al. SEVERE HYPOPROTHROMBINÆMIA AFTER PROPYLTHIOURACIL THERAPY
JP2008214274A (ja) すい臓β細胞インスリン分泌能促進剤および体内組織細胞グルコース取り込み能促進剤
US20020114846A1 (en) Amniotic apoptosis modulating substances
Smith Randomized, double-blinded pilot study: Nitrite Infusion in Islet Cell Transplantation: Test of Clinical Efficacy and Determination of Cytoprotective Mechanisms
Wang et al. Treatment of pyogenic liver abscess by surgical incision and drainage combined with platelet-rich plasma: A case report
Sayfutdinova et al. Evaluation of the effect of a new blood substitute on biochemical parameters in experimental toxic hepatitis
CA2542449C (en) Combined preparation for treating sepsis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19878870

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021523006

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 3118237

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112021008313

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 2019878870

Country of ref document: EP

Effective date: 20210531

REG Reference to national code

Ref country code: BR

Ref legal event code: B01E

Ref document number: 112021008313

Country of ref document: BR

Free format text: COM BASE NA PORTARIA 405 DE 21/12/2020, SOLICITA-SE QUE SEJA APRESENTADO, EM ATE 60 (SESSENTA) DIAS, NOVO CONTEUDO DE LISTAGEM DE SEQUENCIA POIS O CONTEUDO APRESENTADO NA PETICAO NO 870210057505 DE 25/06/2021 NAO ESTA EM LINGUA VERNACULA

ENP Entry into the national phase

Ref document number: 112021008313

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20210429