WO2005074979A1 - Utilisation de la prourokinase pour le traitement du syndrome pulmonaire thrombo-embolique ou de l'occlusion de l'artere centrale de la retine - Google Patents

Utilisation de la prourokinase pour le traitement du syndrome pulmonaire thrombo-embolique ou de l'occlusion de l'artere centrale de la retine Download PDF

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WO2005074979A1
WO2005074979A1 PCT/CN2005/000141 CN2005000141W WO2005074979A1 WO 2005074979 A1 WO2005074979 A1 WO 2005074979A1 CN 2005000141 W CN2005000141 W CN 2005000141W WO 2005074979 A1 WO2005074979 A1 WO 2005074979A1
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urokinase
pro
prourokinase
cells
blood
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PCT/CN2005/000141
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French (fr)
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Yan Jiang
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Shanghai Tasly Pharmaceutical Co., Ltd.
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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)
    • A61K38/49—Urokinase; Tissue plasminogen activator
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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

Definitions

  • the present invention relates to the use of urokinase for treating pulmonary embolism or retinal artery embolism. Background technique
  • Thrombotic diseases including acute myocardial infarction (AMI), cerebral infarction, pulmonary embolism, deep vein thrombosis of the lower limbs, retinal arteries, and venous thrombosis, are a class of diseases that seriously endanger the lives and health of people around the world.
  • AMI acute myocardial infarction
  • cerebral infarction cerebral infarction
  • pulmonary embolism deep vein thrombosis of the lower limbs
  • retinal arteries retinal arteries
  • venous thrombosis a class of diseases that seriously endanger the lives and health of people around the world.
  • AMI acute myocardial infarction
  • pulmonary embolism pulmonary embolism
  • deep vein thrombosis of the lower limbs deep vein thrombosis of the lower limbs
  • retinal arteries retinal arteries
  • venous thrombosis a class of diseases that seriously endanger the lives and health of people around the world.
  • thrombosis which blocks local blood flow or sheds emboli to block downstream blood flow (thromboembolism).
  • a thrombus is a small block formed by the bloodstream on the surface of the inner wall of the cardiovascular system where it is peeled off or repaired.
  • a thrombus consists of insoluble fibrin, deposited platelets, accumulated white blood cells, and trapped red blood cells.
  • Blood test formation is a multifactorial change process involving many genetic and environmental factors that interact with each other. Clinically, patients with thrombotic qualities are common.
  • the main treatments for thrombotic diseases include artificial mechanical methods such as balloon catheterization and surgical embolectomy and antithrombotic medications (thrombolysis and anticoagulation) and supportive therapies.
  • Urokinase Prourokinase, Pro-uk
  • Pro-uk is a glycoprotein
  • the native urokinase Asn 3 ° 2 is a glycosyl site.
  • the oligosaccharide chain at the glycosylation site As n 3 ° 2 can affect the half-life and activity of Pro-uk in the blood. Compared with non-glycosylated Pro-uk expressed by E.
  • Urokinase has a molecular weight of 54 kDa and is composed of 411 amino acid residues. The amino acid sequence can be found in Entrez Protein Database BAA01919. Pro-uk contains 12 pairs of disulfide bonds and has three protein domains (Reference 3: Homes WE, et al. Cloning and expression of the gene for pro-urokinase in Escherichia coli.
  • the epidermal growth factor (EGF-like) domain consisting of amino acid residues 5-49, is highly homologous to epidermal growth factor, is rich in cysteine, and is found in amino acid residues 11 and 19, 13 and There is a pair of disulfide bonds between 31, 33 and 42, each of which has an unclear function.
  • the kringle domain It consists of amino acid positions 50-136, has a finger-shaped ring, and has three pairs of disulfide bonds. Its positions are between residues 50-130, 71 and 113, 102, and 126, respectively. It is speculated that this region may be involved in protein-protein interactions, and its specific function is unclear.
  • Serine protease domain located at the carboxyl terminus of Pro-uk, of which His 2 . 4 , Asp 255 , and Ser 356 constitute the enzyme's active center (Flohe L, et al. Single-chain urokinase-type plasminogen activator: New hopes for clot-specific lysis. Eur Heart, 1985; 11: 851), Asn 3 ° 2 is a glycosylation site, and there are 5 pairs of disulfide bonds in this region.
  • Pro-uk belongs to the family of serine proteases and is a thrombolytic agent. It can lyse the inactive plasminogen to convert it into active plasmin (Plasmin), so as to achieve the effect of dissolving thrombus.
  • the thrombolytic effect of Pro-uk is thrombus-specific.
  • One explanation is that a competitive inhibitor of Pro-uk exists in plasma, but when fibrin is not present, Pro-uk Binding to inhibitors but not showing activity. When fibrin binds to urokinase-plasminogen activator (u-PA), the inhibitor is removed and Pro-uk activity is restored, which in turn activates fibrin.
  • the combined plasminogen produces a specific thrombolytic effect.
  • the second explanation is that the pigs present in plasma mainly exist in the form of Glu-plg, whose amino terminus is Glu. Glu-plg and certain amino acids at the carboxyl terminus of fibrin, such as After Lys binds, the configuration changes, u-PA can specifically activate plg of this configuration change, and produce specific thrombolytic effect.
  • Thromb Haemost, 1985; 54: 893 named it single-chain urokinase-type fiber 1 J (single-chain urokinase-type plasminogen activator (scu-PA)) per lysate activation.
  • scu-PA single-chain urokinase-type fiber 1 J
  • scu-PA single-chain urokinase-type plasminogen activator
  • Pulmonary thromboembolism also known as pulmonary thromboembolism, refers to the detachment of thrombi formed in the venous system or the right heart and clogging the pulmonary artery. Pulmonary thrombosis can also constitute pulmonary thromboembolism. Pulmonary embolism has a high incidence and is the most common cause of death in lung diseases. More than 50,000 people die of PTE each year in the United States. According to foreign autopsy reports, the incidence of PTE is as high as 10% to 25%. At present, in addition to general supportive and symptomatic treatment for pulmonary embolism, thrombolytic therapy can also be adopted.
  • Thrombolytic therapy can accelerate the dissolution of fibrin in the blood vessel cavity, eliminate or reduce thrombus, restore blocked blood flow as soon as possible, and correct blood. Disturbances in kinetics reduce mortality.
  • Thrombolytic drugs commonly used in clinical practice are: urokinase, streptokinase, and tissue-type plasminogen activator. There is no report on the use of prourokinase for pulmonary embolism.
  • the central retinal artery and its branches belong to peripheral arteries. Except for the retinal ciliary artery, it is the only blood vessel supplying nutrients to the inner layer of the retina. Impaired blood supply can cause retinal ischemia and hypoxia, which seriously damages visual function. Therefore, the central retinal artery Insufficient blood supply is a serious blinding disease, the incidence of which increases with age, and is often accompanied by other systemic vascular diseases. The common cause of this disease is thrombosis of the central retinal artery, and the obstruction site is mainly the retinal central artery at or above the retinal plate.
  • urokinase can be used to treat pulmonary embolism and retinal arterial embolism.
  • the urokinase can be natural human urokinase or recombinant human urokinase.
  • Natural urokinase is present in urine, plasma, tissue and cell culture fluids, and can be obtained from suitable tissues using techniques well known in the art. This separation process is generally performed by first preparing a cell-free crude tissue extract and several exogenous proteins, and then further purifying urokinase using, for example, column chromatography and / or other conventional techniques well known in the art. Methods known to those skilled in the art can also be used to utilize genetic recombination technology in prokaryotic cells such as E. coli or eukaryotic cells such as yeast cells, mammalian cells (mouse IC9 cells, CH0 cells, Namalwa cells, insect cells, myeloma cells).
  • urokinase can also be obtained from transgenic animals such as transgenic mice.
  • the urokinase is then purified from it. This isolation process is generally performed by first preparing a cell-free crude tissue extract and several foreign proteins, which are then further purified using, for example, column chromatography and / or other conventional techniques known in the art Prourokinase.
  • Urokinase can be used in individuals diagnosed with pulmonary embolism and retinal arterial embolism to treat pulmonary embolism and retinal arterial embolism. Prourokinase can be administered alone or in admixture with a pharmaceutically acceptable carrier or excipient.
  • urokinase can be prepared as an injectable form either as a liquid solution or a suspension.
  • the active ingredient is usually mixed with a carrier containing a pharmaceutically acceptable excipient which is compatible with the active ingredient.
  • Suitable carriers are, for example, water, salt, glucose and glycerol, and combinations thereof.
  • the carrier may contain minor amounts of adjuvant materials such as wetting agents or solubilizing agents or pH buffering agents.
  • composition or formulation to be administered will, in any event, contain urokinase in an amount sufficient to dissolve the thrombus.
  • the injectable composition is preferably administered intravenously.
  • Injectable formulations should contain an effective amount of the active ingredient in a carrier, and those skilled in the art will readily determine the exact amount stated.
  • a typical range of active ingredients may be from about 1% to about 95% (W / W) by weight of the composition, or even higher or lower if appropriate. Dosage depends on factors such as the age, weight and health of the recipient. Those of ordinary skill in the art will usually routinely establish a dose response curve to easily determine other effective doses. detailed description
  • Example 1 Dissolving effect of human urokinase on golden hamster pulmonary embolism
  • Bovine thrombin is produced by the Technology Company of the Institute of Hematology, Chinese Academy of Medical Sciences. White lyophilized powder, 400 IU per bottle, dissolved in physiological saline and diluted to the required concentration.
  • Heparin sodium a product of China Huixing Biochemical Reagent Co., Ltd. (Shanghai). lg / bottle, potency 150U / mg.
  • Sodium pentobarbital product of SERVA Company, Shanghai Xingzhi Chemical Plant], batch number 921019.
  • Other reagents are commercially available.
  • JC-1000 PC medical multi-probe gamma counter manufactured by Xi'an Kaipu Electromechanical Co., Ltd.
  • SJ-8900 Y pollution detector a product of Sanjia Instrument Co., Ltd.
  • BSJ-160 part of the collector is produced by Shanghai Huxi Instrument Factory.
  • WZ-50C2 microinfusion pump product of Zhejiang University Medical Instrument Co., Ltd.
  • the Iodogen method was adopted, that is, 20 ⁇ g of Iodogen was dissolved in chloroform, and then flattened in a 1.5 ml conical plastic tube. After being blown with nitrogen, 100 l of 0.05 mol / L phosphate buffer solution (pH 7.4) and 5 mg of human fiber After the proteinogen and 0.5mCiNa 125 I were shaken at room temperature for 30 minutes, 50 ⁇ 1 of a 4% sodium iodide (containing 5 mg human fibrinogen) carrier solution was added to terminate the reaction.
  • the reaction compound was added to a pre-treated Sephadex G-150 gel column to remove free radioactive iodine, the column volume was 1.2X 12cm, and the eluent was 0.05mol / L phosphate buffer solution (pH7.4), and the flow rate was 0.1 ml / min. Collect the eluate separately, collect 1 tube every 200 ⁇ 1, monitor the protein elution with a UV monitor, and keep the radioactive peak eluate with the same chromatographic behavior as the unlabeled fibrinogen. Store at 20 ° C until use.
  • Thrombolysis experiment on golden hamster pulmonary embolism model Six healthy volunteers each took 5 ml of venous blood, each was anticoagulated with 3.8% sodium citrate (the ratio of whole blood to anticoagulant was 9: 1), and centrifuged at 3000 rpm for 10 min to prepare platelet-poor plasma (platelet count ⁇ 5 X lOVml), platelet-poor plasma from 6 volunteers.
  • Golden hamsters were anesthetized by intraperitoneal injection of 3% barbituric sodium (60mg / kg), exposed the right external jugular vein, and a polyethylene catheter filled with heparinized saline was inserted at the proximal end to the vein of the head and arm for 125 injections 1-fibrin labeled plasma clot and administration.
  • the prepared 125 1-fibrin-labeled plasma clot was injected into the body with 1.5 ml of physiological saline, and the anterior wall of the chest of the animal was detected with a Y detector to confirm that a 125 1-plasma clot of the pulmonary artery had been caused.
  • the animals were injected intraperitoneally with potassium iodide (5mg) to saturate the iodine uptake capacity of the thyroid gland.
  • the animals were divided into 4 groups of 10 animals each.
  • the administration was started 10 minutes after the embolus was injected into the body to form a pulmonary embolism, and 10% of the total intravenous bolus (volume 0.1 ml, bolus time lmin) was used, and the remaining 90% was intravenously infused at a constant rate (volume 0.9ml, Infusion time lh).
  • the control group was given physiological saline, and the three doses of the test drug were given recombinant human urokinase (pro-uk) for injection, the doses were 1, 3, 10 X 10 4 IU / kg.
  • the total volume administered to each animal in the above groups was 1 ml. Animals were sacrificed at 120 min.
  • the total amount of radiation injected into the emboli was taken at the same time as the thyroid, liver, kidney, urine, blood, muscle and other tissues were measured for radioactivity, and the recovery rate was calculated. Use the dissolution rate over 50% as the effective standard to determine the effectiveness of each dose group.
  • the average radioactivity recovery rate of each experimental group was 101.6 ⁇ 10.8%.
  • the total radioactivity of the plug was 0.22 ⁇ 0.31%, indicating that the experimental method is reliable.
  • the radioactivity remaining in the lungs of the animals in the control group was mainly distributed in the hilar, and the percentage of thrombolysis was 15. 4 ⁇ 3.5%, indicating that the thrombus had some autolysis.
  • the low, medium, and high doses of pro-uk in the three dose groups were significantly reduced in the residual radioactivity in the lungs, and were mainly distributed at the edge of the lung lobe.
  • the percentage of thrombolysis was 19. 7 ⁇ 6.9% ( P> 0. 05), 42.9 ⁇ 15. 2% (P ⁇ 0. 001), 65. 5 ⁇ 11. 5% (P) 0. 001), the thrombolytic efficiency was 0%, 30%, respectively , 80%.
  • Test substance Recombinant human urokinase (PI-Uk) for injection provided by Tianjin Tasly Pharmaceutical Co., Ltd., Institute of Biotechnology, Academy of Military Medical Sciences, white lyophilized powder, 20 X 10 4 IU / bottle. Immediately before use, it is dissolved in water for injection and diluted with physiological saline to the required concentration for intravenous administration to animals.
  • PI-Uk Recombinant human urokinase
  • Tetrachlorotetraiodo-fluorescein sodium salt (Tiger Red), a product of Shanghai No. 3 Reagent Factory.
  • a 4% solution was prepared with physiological saline and filtered through a filtration membrane ( ⁇ 0.22 ⁇ m, Millipore SA, France), and stored at 4 ° C until use. '
  • Wistar rats weighing 298.3 ⁇ 12.3 (280 ⁇ 330) g, were anesthetized intraperitoneally with 20% urethane lg / kg, and the right side was fixed on the operating table, and the left and upper eyelids were sutured to make the eyelids Zhang Ye.
  • 6/0 non-invasive sutures were used to fix the traction, so that the eyeballs slightly protruded out of the orbit.
  • an SQ-III thrombosis test was performed Device (cold light source, ⁇ 560nm, ⁇ ⁇ 60nm, light intensity lW / cm 2 ) irradiates the central artery of the retinal membrane with a light beam of a specific wavelength for 15 minutes, and tetrasodium tetrachlorotetrafluorescein disodium in the blood vessel can absorb a large amount of energy and cause energy level transitions The energy is transferred to oxygen molecules, so that the oxygen in the blood is converted into oxygen free radicals (single-line oxygen). Oxygen free radicals re-oxidize and destroy vascular endothelial cells, damage endothelial cells and / or expose the basement membrane.
  • Oxygen free radicals re-oxidize and destroy vascular endothelial cells, damage endothelial cells and / or expose the basement membrane.
  • Platelet adhesion and aggregation thereby activating the endogenous and exogenous coagulation system.
  • oxygen free radicals can damage the platelet membrane.
  • the damaged platelets can release a series of procoagulant factors and provide a phospholipid surface for coagulation, which can greatly accelerate Formation of blood clots. After thrombosis of the central retinal artery, the blood flow in the retina of the retinal fundus dominates.
  • Animals were randomly divided into 4 groups based on body weight, with 10 in each group. After thrombosis formed and stabilized for 45 min, the drug was started through the right femoral vein, and 10% of the total intravenous bolus was used (volume 0.2 ml, bolus time lmin), and the remaining 90% was intravenously infused at a constant rate (volume 0.8 ml , Infusion time lh).
  • the sham operation group and the model control group were given normal saline, and the three test drug groups were given pro-uk, that is, the amounts were 2.5, 5.0, and 10. OX 10 4 IU / kg. 0ml ⁇
  • the total volume of each animal in each of the above groups was 2.0ml.
  • All dose data are expressed in ⁇ SD.
  • the dose data were compared using a paired t-test to compare the significance of the means before and after administration, and the unpaired t-test was used to compare the significance of the means between different groups.
  • the count data were statistically processed using the exact Fisher test.
  • the model control group did not recanalize the central retinal artery in 10 animals within 120 min. Intravenous administration of pro-uk 2. 5, 5. 0, 10. OX 10 4 IU / kg can obviously dissolve thrombi, and the embolized blood vessels are 5/10 (50%), 7/10 (70%), 9/10 (90%) Recanalization occurred, and the recanalization times were 34.6 ⁇ 33.4, 38.8 ⁇ 28.6, 11.5 ⁇ 7. 0min.

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Description

尿激酶原用于治疗肺栓塞或视网膜动脉栓塞的用凃 技术领域
本发明涉及尿激酶原用于治疗肺栓塞或视网膜动脉栓塞的用途。 背景技术
血栓性疾病， 包括急性心肌梗塞（AMI)、 脑梗塞、 肺栓塞， 下肢深静脉 血栓形成、 视网膜动、静脉栓塞形成等， 是一类严重危及世界各国人民生命 及健康的疾病。 在美国， 35〜50岁男性死亡者中， 35%是由 AMI所致。 全球 十大致死性疾病当中，冠心病（心绞痛、心肌梗塞及心源性猝死）排第一位， 每年死于该病 720万人，第三位是脑血管疾病，每年死亡人数达 460万。心、 脑血管疾病目前仍呈上升趋势。 全世界每年约有 1300万人患有各类血栓性 疾病， 需要使用溶栓药物治疗。 近 10年心血管病发病率一直上升， 特别是 冠心病在我国已成为内科常见病、 多发病， 并有由城市向农村发展， 患者趋 于年轻化的势头。
本病特征为血栓形成， 阻塞局部血流或脱落成栓子堵塞下游血流 (血栓 栓塞）。 血栓是血流在心血管系统血管内面剥落处或修补处的表面所形成的 小块， 在可变的流体依赖型中， 血栓由不溶性纤维蛋白，沉积的血小板，积聚 的白细胞和陷入的红细胞组成。血检形成是一种涉及许多彼此相互作用的遗 传和环境因素的多因素变化的过程， 在临床上常见到血栓形成素质的患者。
血栓性疾病的主要治疗方法包括：人工机械方法如球囊导管术和外科栓 子切除术和抗栓药物治疗（溶栓治疗和抗凝治疗） 以及支持疗法。
尿激酶原（Prourokinase, Pro- uk)是一种糖蛋白， 天然尿激酶原 Asn3°2 为糖基位点。糖基化位点 Asn 3°2上的寡糖链可影响 Pro- uk在血液中的半衰期 和活性。天然的或由哺乳动物细胞表达的糖基化的 Pro-uk，和大肠杆菌表达 的非糖基化的 Pro-uk相比， 二者对纤维蛋白的溶解都是血栓特异的， 但由 于糖基化的 Pro- uk被纤溶酶活化的能力比非糖基化 Pro- uk弱， 因此其催化 活性比非糖基化 Pro- uk弱 (文献 1 : Panne 11 R, et al. The relative and variable stabilities of glycosylated and non-glycosylated pro- urokinase in plasma. Thromb Haemost, 1997 ; Suppl : 504) , 但在血浆 中稳定性好 (文献 2: Lenich C, et al. The influence of glycosylation on the catalytic and fibrinolytic properties of pro-UK. Thromb Haemost. 1992 ; 68 : 539)。
尿激酶原分子量 54kDa，由 411个氨基酸残基组成，氨基酸序列见 Entrez Protein Database BAA01919。 Pro- uk含 12对二硫键， 具有三个蛋白结构域 (文献 3： Homes WE, et al. Cloning and expression of the gene for pro-urokinase in Escherichia coli. Biotechnology, 1985 ; 3 : 923)： (1) 表皮生长因子类 (EGF- like)结构域， 由第 5-49位氨基酸残基组成， 与表皮 生长因子高度同源， 富含半胱氨酸， 在氨基酸残基 11和 19、 13和 31、 33 和 42 间各有一对二硫键， 该结构域功能不清。 （2) kringle 结构域。 由第 50-136位氨基酸组成，呈指形环状，有三对二硫键，其位置分别在第 50-130、 71和 113、 102和 126残基间。 推测该区可能参与蛋白与蛋白之间的相互作 用， 具体功能尚不清楚。 （3)丝氨酸蛋白酶结构域： 位于 Pro- uk羧基端， 其 中 His2。4， Asp255,与 Ser356构成该酶活性中心（Flohe L, et al. single-chain urokinase— type plasminogen activator : New hopes for clot-specific lysis. Eur Heart, 1985 ; 11 : 851 )， Asn3°2为糖基化位点， 该区内共有 5对 二硫键。
Pro- uk属丝氨酸蛋白酶家族，是一溶栓剂，它通过将没有活性的纤溶酶 原裂解， 转变成有活性的纤溶酶 (Plasmin) , 从而达到溶解血栓的效果。 Pro-uk的溶栓作用是血栓特异性的，其机制主要有两种解释：一种解释是血 浆中存在着一种 Pro- uk的竞争性抑制因子， 但纤维蛋白不存在时， Pro-uk 与抑制因子结合而不能表现活性， 当纤维蛋白与尿激酶纤溶酶原激活物 (urokinase-plasminogen activator, u- PA)结合时， 抑制因子被除去， Pro-uk 活性恢复， 进而激活与纤维蛋白结合的纤溶酶原， 产生特异性溶栓作用； 第 二种解释是血浆中存在的 pig主要是以氨基末端为 Glu的 Glu-plg形式存在， Glu-plg与纤维蛋白羧基端的某些氨基酸如 Lys结合后，构型发生变化， u - PA 能特异性地激活这种构型变化的 plg， 产生特异性溶栓作用。
1973 年 Bernik (文献 4： Bernik MB. Increased plasminogen activaor (urokinase) in tissue culture after fiber deposition. J Clin Invest, 1973 ; 52 : 823)首先在组织培养液中发现尿激酶原， 1979年 Husain 等 (文献 5： Husain SS, et al. Isolation of plaminogen activators useful as therapeutic and diagnostic agents (single-chain, high-fibrin affinity urokinase) . US Patent No. 4381346 (filed 1979 ; issued 1983)， 文献 6: Husain SS, et al. Purification of a new, high molecular form of urokinase from urine. Thromb Haemost, 1981 ; 46 : 11 ) (2'3)从尿中纯化 出尿激酶原。 1981年 Wun等 （文献 7: Wun TC, et al. A proenzyme form of urokinase. J Biol Chem, 1982 ; 257 : 7262)证明它是尿激酶的前体， 所以 称它为尿激酶原 （prourokinase), 1985年国际血栓形成和止血委员会 （文 献 8: Report of the Subcommittee of Fibrinolysis, San Diego, CA， USA, July 13， 1985. Thromb Haemost, 1985 ; 54 : 893 )将其命名为单链尿激酶型 纤溶酉每原激活齐1 J ( single-chain urokinase- type plasminogen activator (scu-PA ) )。继 Husain之以后，人们又先后从人胎肾细胞（文献 9: Nolan C， et al. Plasminogen activator from himan embryonic kidney cell culture : Evidence of a proactivator. Biochim Biophys Acta, 1977 ; 496 : 384)、 恶性胶质瘤细胞 （文献 10： Nielsen LS, et al. Purification of zymogen to plasminogen activator from human glioblastoma cells by affinity chromatographywith monoclonal antibody. Biochemistry, 1982 ; 21 : 6410)、肾腺癌细胞培养液（文献 11 : Stump DC, et al. Purification and char act er i zat i on of single chain urokinase— type plasinogen activator from human cell cultures. J Biol Chem, 1986 ; 261 : 1274) 和人血浆中 (文献 12： Wun TC, et al. Isolation and characterization of urokinase from human plasma. J Biol Chem, 1982 ; 257 : 3276 ) 纯化获得了 Pro- uk。 但由于天然材料中 Pro- uk含量甚微， 难以大量制备， 于是人们开始采用基 因工程来大量生产重组 ProUK。 1985年 Homes 等首先在大肠杆菌中获得了表 达 (文献 13： Homes WE, et al. Cloning and expression of the gene for pro- urokinase in Escherichia coli. Biotechnology, 1985 ; 3 : 923 ) ( 13) 禾口纯化 (文献 14= Winkler ME, et al. Purification and characterization of recombinant single-chain urokinase produced in Escherichea coli. Biochemistry, 1986 ; 25 : 4041 ) ""。 1986年开始由德国的 Grunenthal 公司 生产， 称为 saruplase。 大肠杆菌表达的 Pro-uk， 由于产品呈不溶性的包含 体， 需要繁琐的变性和复性， 从而导致产品的不均一性和产量较低， 于是人 们开始了用动物细胞来表达糖基化 Pro- uk的研究， 包括小鼠 IC9细胞（文 献 15： Nolli ML, et al. Production and charaterisation of human recombinant single chain urokinase— "type plasminogen activator from mouse cells. Fibnrinolysis, 1989 : 3 : 101)， CH0细胞 (文献 16: Nelles L, et al. Characterization of recombinant human single chain urokinase— type plasminogen activator mutants produced by site-specific mutagenesis of Lysine. J Biol Chem, 1987 ; 262 : 5682； 文献 17: 李风知等. 人尿激酶原全长 cDNA在中国仓鼠卵巢细胞中的稳定表 达. 生物工程学报， 1991 ; 7 : 114), Namalwa细胞 (文献 18: Satoh M, et al. Stable production of recombinant pro- urokinase by human lymphoblastoid Namalwa KJM-l cells： Host-cell dependency of the espressed- protein stability. Cytotechnology, 1993 ; 13 : 79； 文献 19: Satoh M， et al. Efficient expression of pro— urokinase by human lymphoblastoid Namalwa KJM-l cells using Moloney retroviral promoter. Cytotechnology, 1996 ; 18 : 167), 昆虫细胞 （文献 20: 徐杨等. 人尿激酶 原 cDNA在昆虫杆状病毒真核表达系统中的高效表达. 生物化学杂志， 1993 ; 6 : 709 )， 骨髓瘤细胞 （文献 21 : Weaver WD, et al. New recombinant glycosylated prourokinase for treatment of patients with acute myocardial infarction. J Am Coll Cardiol, 1994 ; 24 : 1242 ) 等， 都已 获得了很高的表达。
继 Van de Warf F等首次报道了 proUK用于心梗病人的治疗（文献 22Van de Warf F， et al. Coronary thrombolysis with human single-chain, urokinase- type plasminogen activaor (pro- urokinase) in patients with acute myocardial infarction. Ann Inter Med, 1986； 104 : 345 ) 之后， 人们采用天然的、 基因重组非糖基化的和基因重组糖基化 的 pro- uk治疗心梗病人达数千人（文献 23: 俞炜源等， 尿剂酶原的性质、 结构、 功能及其药代动力学和临床应用效果）。
del Zoppo G. J.等对 20 名脑血栓病人向脑血管直接输入 6mg pr0UK (pr0lyse)，并静脉输入肝素（分高低两组）， 结果高肝素组血管再通率 高达 81. 8%，低肝素组 40. 0% (文献 24： del Zoppo G. J.， et al. The prolyse in acute cerebral thromboembolism trial (PR0ACT)： Results of 6 mg dose tier. Stroke, 1996， 27 : 164)。 Furlan A等公开了 pro- uk (prolyse)对于 发病 6小时之内的脑梗塞的溶栓作用， 121位使用 pro-uk的患者，血管再通 率为 66% (文献 25： Fur lan A et al, Intra- arterial prourokinase for acute ischemic stroke. The PR0ACT II study: a randomized controlled trial. Prolyse in Acute Cerebral Thromboembolism. JAMA. 1999 Dec 1 ; 282 (21) : 2003-11. )。 Moia，- M等对 Pro- uk在治疗下肢深静脉血栓 （deep vein thrombosis, DVT)方面进行了初步研究.，研究证实了 Pro- uk对于 DVT的安全、有效性（文 献 26 : Moia, M et al, A pilot study of pro- urokinase in the treatment of deep vein thrombosis. Thromb Haemost, 1994, 72 : 430)。 研究采用开 放的、非对照研究， 24 h内给 15位患者分别输注 pro- UK 120 mg (800. 000IU (5 mg) /h)和普通肝素， 其中 11条下肢的血液循环都有不同程度的改善。
肺栓塞 （pulmonary thromboembolism, PTE)又称肺血栓性栓塞， 是指 静脉系统或右心内形成的血栓脱落堵塞于肺动脉，肺动脉内血栓形成也可构 成肺血栓性栓塞。 肺栓塞发病率高， 是肺部疾病中最常见的死亡原因。 在美 国每年死于 PTE者超过 50000。 根据国外尸解报告的资料， PTE的发生率高 达 10%〜25%。 目前，肺栓塞的治疗除一般的支持疗法和对症治疗外，还可采 取溶栓治疗， 溶栓治疗可加速溶解血管腔内的纤维蛋白， 消除或缩小血栓， 尽快恢复阻塞的血流， 纠正血液动力学紊乱， 降低病死率。 目前临床上常用 的溶栓药物有： 尿激酶、 链激酶和组织型纤溶酶原激活剂。 关于尿激酶原用 于肺栓塞尚无报道。
视网膜中央动脉及其分支属于末梢动脉， 除了视网膜睫状动脉以外， 它 是供应视网膜内层营养的唯一血管， 血液供给障碍都可导致视网膜缺血缺 氧， 严重损害视功能， 因此， 视网膜中央动脉供血不足是一种严重的致盲性 疾病， 发病率随着年龄的增长而升高， 常伴发于其它全身性血管疾病。 该病 常见的原因为视网膜中央动脉血栓形成，阻塞部位以筛板处或筛板以上的视 网膜中央动脉为主。 急性视网膜动脉梗塞（CRA0)传统的治疗方法常采用前 房穿剌术、 视觉按摩术、 碳酸酐酶抑制剂等， 但几乎不能改变其疾病的自发 进程。 目前国内外有采用溶栓剂治疗 CRA0的文献报道， 但尚无 Pro-uk用于 治疗 CRA0的报道。 发明内容
发明人发现尿激酶原能用于治疗肺栓塞和治疗视网膜动脉栓塞。所述尿 激酶原可以是天然人尿激酶原或者重组人尿激酶原。
天然尿激酶原存在于尿液、血浆、 组织和细胞培养液中， 可以利用本领 域公知的技术从合适组织中。一般完成这一分离过程首先是制备无细胞成分 的粗组织提取物和数种外源蛋白， 然后利用如柱层析和 /或本领域熟知的其 它常规技术进一步纯化尿激酶原。 也可以利用本领域技术人员熟知的方法利用基因重组技术在原核细胞 如大肠杆菌或真核细胞如酵母细胞、 哺乳动物细胞 （小鼠 IC9细胞、 CH0细 胞、 Namalwa细胞、 昆虫细胞、 骨髓瘤细胞） 中表达尿激酶原， 还可以由转 基因动物如转基因小鼠获得表达。然后从中纯化出尿激酶原，一般完成这一 分离过程首先是制备无细胞成分的粗组织提取物和数种外源蛋白，然后利用 如柱层析和 /或本领域熟知的其它常规技术进一步纯化尿激酶原。
可将尿激酶原用于被诊断为肺栓塞和视网膜动脉栓塞的个体， 以治疗肺 栓塞和视网膜动脉栓塞。尿激酶原可以单独或与药用可接受载体或赋形剂混 合施用。
一般情况下，可将尿激酶原制备成为或者是液体溶液或者是悬浮液状的 可注射形式。如上所述， 活性成分通常与含有药用可接受并与活性成分相容 的赋形剂的载体相混合。 适宜的载体例如有： 水、 盐、 葡萄糖和甘油等及其 组合。 此外， 如果需要的话， 载体可含有小量的辅剂物质如湿润剂或者增溶 剂或 PH缓冲剂。
本领域的技术人员已知或者将容易了解到制备这类剂型的实际方法。将 被施用的组合物或配方在任何情况下都含有数量足以溶解血栓的尿激酶原。
可注射的组合物最好是通过静脉途径结药。可注射配方应在载体中含有 有效量的活性成分， 而本领域的技术人员很容易确定所说的确切含量。活性 成分的典型范围可以是组合物重量的约 1 %-约 95% (W/W)，或者如果合适的 话甚至可以更高或更低。用量有赖于诸如接受治疗者的年龄、体重和健康状 况的这些因素。本领域的普通技术人员通常常规试验建立剂量反应曲线很容 易确定其它的有效剂量。 具体实施方式
为了更好地理解本发明的实质下面将用尿激酶原的药理试验及结果来 说明其用途。 实施例仅用于说明目的， 并非意在以任何方式限制本发明的范 围。
实施例 1、 人尿激酶原对金黄地鼠肺栓塞的溶解作用
实验材料
受试物：
注射用重组人尿激酶原 (pro- uk)天津天士力制药股份有限公司、军事医 学科学院生物工程研究所提供， 白色冻干粉， 20 X 104IU/瓶。 临用前以注射 用水溶解， 并用生理盐水稀释至所需浓度供动物静脉给药用。
人纤维蛋白原 （fibrinogen, FG)标准品， Sigma公司产品， 含量 68%。 1，3，4，6-四氯 - 3α- 6α二苯基甘脲 (Iodogen), Sigma公司产品。
Na125I，Amersham International PIC产品， 无还原剂， 放化纯度 99.4%， 比活性 577.2Tbq/yg。
交联葡聚糖凝胶 SephadexG- 150， Pharmacia公司产品。
牛凝血酶， 中国医学科学院血液研究所科技公司生产。 白色冻干粉， 每 瓶 400 IU， 用生理盐水溶解并稀释至所需浓度。
肝素钠，中国惠兴生化试剂有限公司（上海）产品。 lg/瓶，效价 150U/mg。 戊巴比妥钠， SERVA公司产品， 上海行知化工厂分装]， 批号 921019。 其它试剂均为市售。
实验动物
金黄地鼠 140只，体重 126.0±16.6g，雌雄各半，卫生部北京生物制品 研究所提供。
仪器
LKB-1282 Y晶体闪烁计数仪， LKB公司产品。
JC - 1000 PC医用多探头 γ计数器， 西安凯普机电有限责任公司制造。 SJ-8900 Y污染探测仪， 三佳仪器公司产品。
BSJ-160部分收集仪， 上海沪西仪器厂生产。
WZ- 50C2微量输注泵， 浙江大学医学仪器有限公司产品。
实验方法
1251 -标记人纤维蛋白原 （125I- hFG) 的制备
采用 Iodogen法， 即 20 μ g Iodogen用氯仿溶解后， 平铺于 1.5ml锥形 具塞塑料管内， 氮气吹干后， 加入 100 l0.05mol/L磷酸缓冲液（pH7.4)、 5mg人纤维蛋白原和 0.5mCiNa125I， 室温振荡反应 30分钟后， 再加入 50μ1 4%碘化钠（含 5mg人纤维蛋白原）载体溶液终止反应。将反应化合物加至经 预处理后的 SephadexG- 150凝胶柱去除游离放射性碘， 柱体积 1.2X 12cm， 洗脱液为 0.05mol/L磷酸缓冲液（pH7.4), 流速为 0. lml/min。 分别收集洗 脱液， 每 200μ1收集 1管， 以紫外监测仪监测蛋白质洗脱情况， 留取层析 行为与非标记纤维蛋白原相同的放射性峰洗脱液，合并混合后，分装，置- 20 °C保存备用。
对金黄地鼠肺栓塞模型的溶栓实验 6名健康志愿者各取静脉血 5ml， 分别用 3. 8%枸橼酸钠抗凝（全血与抗 凝剂之比为 9 : 1 )， 3000rpm离心 10 min制成贫血小板血浆（血小板计数 <5 X lOVml ), 混合 6例志愿者的贫血小板血浆。 取上述混合血浆 0. 6ml， 冰浴 (4°C) 5min后，加入 20 μ 1 125I-hFG (约 2 μ Ci )，充分混匀，然后加入 0. 5mol/L 氯化钙与凝血酶 （20IU/ml )混合物 50 μ 1， 迅速混匀， 吸入至内径为 5醒 聚乙烯管内， 37°C温育 30min形成血浆凝块，将血栓凝块慢慢推出至平皿中， 均匀剪成 6段， 每段长约 1. 5cm, 用 30ml生理盐水 （Normal Saline, NS) 连续洗涤 30min， 每次 5min， 以除去游离1251-标记物。
模型制备
金黄地鼠经腹腔注射 3%巴比妥钠 （60mg/kg)麻醉后， 暴露右侧颈外静 脉， 近心端插入充满肝素生理盐水的聚乙烯导管， 前端至头臂静脉， 用于注 入1251-纤维蛋白标记的血浆凝块及给药用。将制备的 1251-纤维蛋白标记的血 浆凝块用 1. 5ml生理盐水注入体内， 并用 Y探测仪探测动物胸前壁， 确认已 造成肺动脉 1251-血浆凝块栓塞。 动物预先腹腔注射碘化钾 （5mg) 使甲状腺 摄碘能力饱和。
分组与给药
动物分成 4组， 每组 10只。 于栓子注入体内形成肺栓塞后 lOmin开始 给药， 采用先静脉推注总剂量的 10% (体积 0. lml， 推注时间 lmin)， 剩余 90%静脉恒速输注（体积 0. 9ml， 输注时间 lh)。 对照组给予生理盐水， 受试 药 3个剂量组给予注射用重组人尿激酶原 （pro-uk), 剂量分别为 1、 3、 10 X 104IU/kg。 上述各组每只动物给药总体积均为 lml。 120min处死动物。
血栓溶解测定
实验结束后，取心、肺测量残存血凝块放射性，按下式计算溶栓百分数： 血栓舊％ =注入栓子誦 存检子放射量遍
注入栓子放射总量 并同时取甲状腺、 肝、 肾、 尿液、 血液、 肌肉等组织测定其放射性， 计 算回收率。 以溶解率超过 50%作为溶栓治疗有效至标准来判断各剂量组有效
各实验组的放射性平均回收率为 101. 6 ± 10. 8%,甲状腺放射性为注入血 栓总放射性 0. 22±0. 31%， 表明实验方法是可靠的。
对照组动物残存在肺内的放射性主要分布在肺门处，其血栓溶解百分数 为 15. 4±3. 5%， 表明血栓有一定的自溶。 与对照组相比， pro- uk低、 中、 高 3个剂量组残存在肺内的放射性均明显减少， 并且主要分布在肺叶边缘， 其血栓溶解百分数分别为 19. 7 ±6. 9%(P>0. 05)、 42. 9± 15. 2% (P<0. 001)、 65. 5 ± 11. 5% (P) 0. 001)， 溶栓有效率分别为 0%、 30%、 80%。 实施例 2.重组人尿激酶原对大鼠视网膜中央动脉血栓的溶解作用 实验材料
药品与试剂
受试物： 注射用重组人尿激酶原 (ΡΙΌ-Uk)天津天士力制药股份有限公 司、 军事医学科学院生物工程研究所提供， 白色冻干粉， 20 X 104IU/瓶。 临 用前以注射用水溶解， 并用生理盐水稀释至所需浓度供动物静脉给药用。
四氯四碘突光素二钠 (Tetrachlorotetraiodo- fluorescein sodium salt,虎红），上海试剂三厂产品。以生理盐水配制成 4%溶液并经滤过膜（ Φ 0. 22 μ πι， 法国 Millipore SA. )过滤， 4°C保存备用。 '
其它试剂均为市售。
实验动物
Wistar大鼠， 体重 298. 3± 12. 3 (280〜330) g， 腹腔注射 20%乌拉坦 lg/kg麻醉后， 右侧卧为固定于手术台上， 左眼上下睑缝线， 使眼睑张幵。 于球结膜靠近角膜缘的上、 下、 外三处， 以 6/0无损伤缝合丝线固定牵引， 使眼球微凸出眶外。 从眼球下侧入路、剪开球结膜、 分离、 结扎并切断下直 肌， 暴露出眼球缩肌， 将眼球縮肌沿纤维方向钝性分开， 即可暴露出视神经 腹外侧面， 仔细分离出视网膜中央动脉。 眼底置多普勒流量计探头， 测定视 网膜中央动脉供血区的相对血流量值。
模型制备
动物经左股静脉给予 4%四氯四碘荧光素二钠 (虎红 )40mg/kg，体积 ml/kg (假手术组动物给等体积 NS)， 5min后，采用 SQ- III型血栓形成实验装置（冷 光源， λ 560nm，△ λ 60nm，光强度 lW/cm2)特定波长的光束照射视网膜膜中 央动脉 15min， 血管内的四氯四碘荧光素二钠可大量吸收能量， 发生能级跃 迁， 并将能量传递给氧分子， 使血液中的氧转变成氧自由基 （单线态氧）， 氧自由基再氧化破坏血管内皮细胞， 使内皮细胞损伤和 /或基底膜暴露， 引 起血小板粘附与聚集， 从而激活内、外源性凝血系统， 同时， 氧自由基又能 损伤血小板膜， 由于损伤的血小板可释放一系列促凝血因子并为凝血提供磷 脂表面， 因而可大大加速血栓的形成。 视网膜中央动脉血栓形成后， 其支配 区眼底视网膜的组织血流量出现下降。
分组与给药
动物按体重随机分为 4组， 每组 10只。 血栓形成并稳定 45min后， 经 右股静脉开始给药， 采用先静脉推注总剂量的 10% (体积 0. 2ml， 推注时间 lmin), 剩余 90%静脉恒速输注 （体积 0. 8ml， 输注时间 lh)。 假手术组和模 型对照组给予生理盐水， 受试药 3个剂量组给予 pro-uk， 即量分别为 2. 5、 5. 0、 10. O X 104IU/kg。 上述各组每只动物给药总体积均为 2. 0ml。
测定指标
记录造型前正常视网膜多普勒相对流量值， 及血栓形成（0min)、 给药 前后 5、 10、 15、 20、 30、 45、 60、 90、 120min的相对流量值。 以给药后相 对流量值达到正常相对流量值 30%以上所需时间作为血管再闭塞时间， 并计 算再通率、 再闭塞率。
统计学处理
所有剂量资料均数士标准差 土 s )表示。 剂量资料采用配对 t-检验 比较给药前后均数差异显著性， 非配对 t-检验比较不同组间均数差异显著 性； 计数资料采用精确 Fisher检验进行统计学处理。
实验结果
对大鼠视网膜中央动脉血栓溶解的影响
模型对照组在 120min内， 10只动物栓塞的视网膜中央动脉未出现再通。 静脉给予 pro-uk 2. 5、 5. 0、 10. O X 104IU/kg能明显溶解血栓， 栓塞的血管 分别有 5/10 (50%)、 7/10 (70%)、 9/10 (90%) 出现血管再通， 血管再通时 间分别为 34. 6±33. 4、 38. 8±28. 6、 11. 5±7. 0min。
对大鼠视网膜中央动脉支配区相对血流量的影响
假手术组 120min内视网膜流量无明显变化， 与手术前相比较无明显差 异； 模型对照组造型后流量明显降低， 约占正常值的 50%左右， 与造型前比 较差异有显著意义， 造型后 120min内流量基本保持稳定， 与给药前比较无 明显差异， 各时间点与假手术组比较差异有显著意义； pro- uk 2. 5 X 104IU/kg，静脉给药后可使 90〜120min血流量明显增加，与模型对照组比较 差异有显著意义； pro-uk 5. 0 X 104IU/kg,静脉给药后可使 5、 15、 45、 90min 血流量与模型对照组比较显著增加； pro- uk 10.0 X104IU/kg， 静脉给药后可 使 5〜120min血流量与模型对照组比较显著增加。

Claims

权利要求
1.尿激酶原在制备治疗肺栓塞或视网膜动脉栓塞的药物中的用途。
2.权利要求 1的用途，其中尿激酶原是天然人尿激酶原或重组人尿激酶 原。
3.权利要求 1的用途，其中尿激酶原与可药用载体或者赋形剂配制成可 注射形式。
4.治疗肺栓塞或视网膜动脉栓塞的方法， 包括将尿激酶原给予患者以消 除肺栓塞或视网膜动脉栓塞。
5.权利要求 4的方法，其中尿激酶原是天然人尿激酶原或者重组人尿激 酶原。
6.权利要求 4 的方法， 其中尿激酶原与可药用载体或者赋形剂一起给 药。
7.权利要求 6的方法，其中尿激酶原与可药用载体或者赋形剂配制成可 注射形式。
8.权利要求 7的方法， 其中尿激酶原通过静脉给药。
PCT/CN2005/000141 2004-02-04 2005-02-01 Utilisation de la prourokinase pour le traitement du syndrome pulmonaire thrombo-embolique ou de l'occlusion de l'artere centrale de la retine WO2005074979A1 (fr)

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CN101549150A (zh) * 2008-03-31 2009-10-07 刘建宁 尿激酶原及尿激酶原变体在急性心肌梗塞易化经皮冠状动脉介入中的应用
CN113975381A (zh) * 2020-07-26 2022-01-28 刘建宁 尿激酶原及其变体在病毒引起的凝血疾病中的应用

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US4944943A (en) * 1988-02-13 1990-07-31 Basf Aktiengesellschaft Mixture of a substance having thrombolytic activity and of an antithrombotic substance
CN1062016C (zh) * 1996-09-27 2001-02-14 中国人民解放军军事医学科学院生物工程研究所 重组人糖基化尿激酶原的制备方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4944943A (en) * 1988-02-13 1990-07-31 Basf Aktiengesellschaft Mixture of a substance having thrombolytic activity and of an antithrombotic substance
CN1062016C (zh) * 1996-09-27 2001-02-14 中国人民解放军军事医学科学院生物工程研究所 重组人糖基化尿激酶原的制备方法

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