WO2009121223A2 - 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 - Google Patents

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 Download PDF

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WO2009121223A2
WO2009121223A2 PCT/CN2008/070653 CN2008070653W WO2009121223A2 WO 2009121223 A2 WO2009121223 A2 WO 2009121223A2 CN 2008070653 W CN2008070653 W CN 2008070653W WO 2009121223 A2 WO2009121223 A2 WO 2009121223A2
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prourokinase
variants
use according
minutes
units
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PCT/CN2008/070653
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French (fr)
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刘建宁
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Liu Jianning
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Priority to EP08715385.4A priority Critical patent/EP2281569B1/en
Priority to US12/935,161 priority patent/US9211317B2/en
Publication of WO2009121223A2 publication Critical patent/WO2009121223A2/zh

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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
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)

Definitions

  • prourokinase and prourokinase variant in facilitating percutaneous coronary intervention in acute myocardial infarction
  • the invention belongs to the field of biomedicine and relates to a plasminogen activator prourokinase (proUK) and prourokinase variant in the application of acute myocardial infarction for facilitating percutaneous coronary intervention.
  • proUK plasminogen activator prourokinase
  • Infarction-related coronary artery is initiated as soon as possible, and effective forward flow is reestablished, and reperfusion of ischemic myocardium is the most important strategy for early treatment of acute myocardial infarction.
  • PCI percutaneous coronary intervention
  • intravenous thrombolysis are the two main methods for opening IRA and reperfusion, thereby reducing mortality.
  • Facilitated PCI refers to the treatment of patients with acute myocardial infarction with thrombolytic drugs before PCI, and then the implementation of PCI for acute myocardial infarction.
  • AHA American Heart Association
  • ACC American Heart Association
  • the American Heart Association (AHA)/American Heart Association (ACC) guidelines for the diagnosis and treatment of acute myocardial infarction suggest that if the first hospital cannot perform direct PCI within 90 minutes, patients with contraindications to thrombolytic therapy should receive immediate thrombolytic therapy. For patients within 3 hours of onset, if unconditional direct PCI or direct PCI is more than 1 hour longer than thrombolysis, thrombolytic therapy should also be preferred [1] .
  • Prourokinase (the urokinase originally contains 411 amino acids with a molecular weight of 46393.65 Daltons and its amino acid sequence is shown in SEQ ID NO. 1) is a serine proteolytic enzyme with dual properties of enzyme and zymogen. After entering the blood circulation intravenously, it acts as a zymogen and does not cause systemic fibrinolysis. As an enzyme, it can dissolve embolic thrombus with high selectivity, and hardly acts on hemostatic thrombus at the wounds of tissues and organs. Hemorrhagic complications during thrombolytic therapy are avoided. Since it also has the effect of inhibiting platelet aggregation, the incidence of reocclusion after successful thrombolysis can be reduced.
  • prourokinase and prourokinase variants in facilitating PCI in acute myocardial infarction.
  • the method is as follows: when the patient has acute myocardial infarction within 6 hours, the first use of prourokinase or prourokinase variant thrombolytic therapy, and then using percutaneous coronary intervention (PCI) surgery, as soon as possible Open the infarct-related coronary artery (IRA) and re-establish an effective forward flow to reperfuse the ischemic myocardium.
  • PCI percutaneous coronary intervention
  • prourokinase is native prourokinase or recombinant human prourokinase, the amino acid sequence of which is set forth in SEQ ID N0.1.
  • prourokinase variant is a protein or polypeptide having a pro-urokinase original amino acid sequence substituted, deleted or added with one or several amino acids and having prourokinase activity; or More than 40% homologous protein; or a protein or polypeptide having more than 90% homology to the native prourokinase protein B chain sequence.
  • prourokinase variant is a lysine (Lys) point mutation at position 300 of the prourokinase amino acid sequence SEQ ID NO. 1 to histidine (His).
  • the dose is 500,000 units to 3 million units per patient; or 30 to 120 minutes intravenously 50,000 units ⁇ 200,000 units / min / patient; or intravenous bolus combined with intravenous drip (per patient)
  • 20% of the total dose of prourokinase is injected intravenously, and the remaining part is intravenously administered 50,000 units to 200,000 units/min for 90 minutes.
  • the total dose of prourokinase or prourokinase variant used does not exceed 6 million units.
  • thrombolysis is carried out using prourokinase or prourokinase variants for a period of from 5 minutes to 120 minutes.
  • the prourokinase or prourokinase variant of the present invention is effective in combination with PCI.
  • the prourokinase or prourokinase variant is a serine proteolytic enzyme having both the enzyme and the zymogen. After entering the blood circulation intravenously, it acts as a zymogen and does not cause systemic fibrinolysis.
  • As an enzyme it can dissolve embolic thrombus with high selectivity, and hardly acts on hemostatic thrombus at the wounds of tissues and organs. Hemorrhagic complications during thrombolytic therapy are avoided. Since it also has the effect of inhibiting platelet aggregation, the incidence of reocclusion after successful thrombolysis can be reduced.
  • Figure 1 is a test result of a dog hemostasis in Example 2. Dogs (number of dogs in each group are shown in brackets in the figures) Initial bleeding time (mean SD), respectively, infusion of saline, tPA, prourokinase and M5 (60 g/kg/min) o
  • the invention is further illustrated by the following examples.
  • Prourokinase may be extracted from natural prourokinase or recombinant human prourokinase, and its amino acid sequence is SEQ ID NO. D o
  • a prourokinase variant is a protein or polypeptide having a pro-urokinase amino acid sequence substituted, deleted or added with one or several amino acids and having prourokinase activity; or more than 40% homology with the entire prourokinase protein sequence Protein; or a protein or polypeptide having more than 90% homology to the natural prourokinase protein B chain sequence.
  • a specific example of a prourokinase variant is a lysine (Lys) point mutation at position 300 of the prourokinase amino acid sequence (SEQ ID N0.1) to histidine (His) 0
  • Methods for the preparation of prourokinase and prourokinase variants can be carried out by genetic engineering methods well known in the art.
  • Example 2 A special case of prourokinase variants by in vitro and in vivo experiments (primary amino acid sequence of urokinase)
  • the lysine point mutation at position 300 of SEQ ID NO. 1 is histidine, Lys300 -> ffls, M5), has a thrombolytic activity at least comparable to or superior to prourokinase, and can be used to facilitate PCI Treatment.
  • Fibrinogen, fibrin monomer, D-dimer and other fibrin analogs are also being investigated for plasminogen activation because they do not promote urokinase-mediated plasminogen activation.
  • fibrin fragment E selectively promotes plasminogen activation of prourokinase, which has a similar effect on M5, whereas D-dimer and soluble fibrin or fibrinogen are almost No promotion.
  • Double-stranded tc-M5 can be inhibited by PAI-1 with a K value of about 1.3 ⁇ 0.3 X 10 7 mol/Ls, which is equivalent to urokinase (1.7+0.4 X 10 7 mol/L -s) o
  • 0-20 g/mL of M5 or 0-8 g/mL of prourokinase was incubated in 1 mL of citrate anticoagulated mixed plasma for 6 hours at 37 ° C, and 0.2 mL of aprotinin was added (10,000 kinins per mL) Release enzyme inhibition unit).
  • the amount of fibrinogen remaining in the blood paddle was measured by the thrombin clotting method and compared with the baseline level.
  • I 125 labeled fibrinogen clots were prepared from 0.2 mL plasma and incubated in 4 mL plasma.
  • a range of fibrin-specific concentrations of prourokinase (0.5-1.5 g/mL) or M5 (0.5-5.0 g/mL) were used to test, blood clot lysis was expressed as a percentage of complete dissolution value over time, fiber The proprotein is measured after the complete blood clot is dissolved; if it is still not completely dissolved at 6 hours, it is measured at 6 hours.
  • M5 maintains fibrin specificity (25% fibrinogen degradation) at concentrations up to 5 g/mL, while the upper limit of prourokinase is 1.5 g/mL.
  • the maximum rate of clotting of prourokinase in blood clots is 41% per hour, while M5 is 64% per hour.
  • the blood clot was prepared by adding 1 mL of fibrinogen labeled with iodine 125 (1.9 Ci, 0.75 mCi/mg protein) and thrombin (10 units) of dog whole blood. After 20 minutes, the blood clot was washed three times with physiological saline, and then cut into small pieces of 1 mm and injected into the femoral vein with a 16-gauge needle. After 15 minutes, blood samples were taken via cannula outside the other femoral vein and baseline radioactivity was determined.
  • tPA intravenous saline or prourokinase or tPA were administered intravenously, and the input rates of prourokinase and tPA were 20 g/kg/min and 10 g/kg/min, respectively. These rates were based on thrombolysis in dogs before the literature. Effective and fibrin specific. tPA input lasts for 60 minutes. The infusion of prourokinase and M5 lasted for 90 minutes. The input rates of M5 were 20, 40, 60 g/kg/min, respectively, and blood samples were taken to determine the radioactivity and fibrinogen.
  • the experimental results show that the blood clot lysis caused by M5 is dose dependent. Since M5 is 4 times more stable in plasma than urokinase, M5 causes rapid clot lysis at 60 g/kg/min, reaching 100% in 45 minutes. M5 causes blood clot lysis to be more effective, as it requires only 600 g/kg of M5 to achieve 50% dissolution, and this dose is 1200 g/kg for prourokinase. At lower doses (40 or 20 g/kg/min), the blood clots caused by M5 dissolve quite or less. This relatively low dose at low doses reflects the longer hysteresis phase of M5. The plasma fibrinogen concentrations of the experimental dogs in the M5 maximal infusion dose group were 72%, 65%, and 52% of baseline levels at 30, 45, and 60 min, respectively.
  • Rhesus monkeys are quite sensitive to human prourokinase/urokinase.
  • Six rhesus monkeys (three females and three males) from 5.8 kg to 8.6 kg were anesthetized with sodium pentobarbital (30 mg/kg), and a polyethylene catheter was inserted into each upper limb vein for blood and infusion.
  • a 2 mL whole blood sample was mixed with radioactive iodine-labeled fibrinogen and thrombin and incubated at 37 degrees for 20 minutes.
  • the whole blood clot was cut into small pieces of 1 mm and washed six times with saline.
  • Blood clots (3.3-10 cpm) were suspended in 5 mL of saline and injected intravenously through the right upper extremity. After 30 minutes, blood samples were taken from the contralateral upper extremity vein to determine baseline radioactivity. At the same time, infusion of normal saline (2 monkeys) or M5 (4 monkeys) was started. M5 was infused at 60 g/kg/min for 60 minutes, and blood samples were taken at a certain time interval for infusion for fibrinogen and fibrinogen determination. The initial bleeding time was measured in the lower abdomen at 0, 30, 45, and 60 minutes with a 5 mm long, 1 mm deep incision; the bleeding time was measured using a standard method of tapping the filter paper every 30 seconds.
  • ECG at least 2 or more limb leads ST segment elevation 0.1mV, or 2 or more adjacent chest lead ST segment elevation 0.2mV;
  • Age 85 years old, gender is not limited.
  • Hemorrhagic stroke occurred at any time in the past, and an ischemic stroke or cerebrovascular event occurred within 1 year.
  • a serious progressive disease such as a malignant tumor
  • a disease with a poor prognosis that causes extreme exhaustion in the patient a serious progressive disease (such as a malignant tumor) or a disease with a poor prognosis that causes extreme exhaustion in the patient;
  • Protokines of prourokinase First, thrombolytic therapy with recombinant human prourokinase, which can be an intravenous bolus, the dose is 500,000 units to 3 million units per patient; it can be 30 to 120 minutes intravenously 50,000 units ⁇ 200,000 units / min / patient; can be intravenous bolus combined with intravenous drip (each patient first intravenous infusion of 20% of the total dose of prourokinase, the rest of the 90 minutes intravenous drip 50,000 units ⁇ 200,000 units / Min), the total dose of prourokinase used does not exceed 6 million units.
  • the prourokinase is treated with thrombolytic therapy for a period of 5 minutes to 90 minutes.
  • PCI Percutaneous coronary intervention
  • the first angiography should use a large field of view (image) to observe all blood vessels, and the exposure time should continue until the contrast agent is emptied.
  • the left coronary artery should be administered at least 3 individuals, and the right coronary artery should be at least 2 individuals, and a large image should be applied. Do not give nitroglycerin through the coronary artery before infarct-related angiography.
  • the angiographic results were determined according to the TIMI blood perfusion grading standard, and those who reached level 2 and 3 were revascularized.
  • TIMI level 0 occlusion of the coronary artery without anterior blood perfusion
  • TIMI Level 1 The distal vessels of the coronary stenosis have a forward blood flow, but cannot fill the distal vascular bed;
  • TIMI Level 2 The distal vessels of the coronary stenosis have a forward perfusion, but more than 3 distal vascular beds are required.
  • the cardiac cycle can be slowly filled with basic filling, and the contrast agent is cleared slowly;
  • TIMI Level 3 The distal vessels of the coronary stenosis have a forward blood perfusion, and the distal vascular bed contrast agent fills completely and rapidly (less than 3 cardiac cycles), and the contrast agent is cleared rapidly.
  • the arterial sheath can be removed at APTT ⁇ 80 seconds, and local compression can be stopped for at least half an hour. Heparin instillation was stopped 48 hours after the end of thrombolysis, and low molecular weight heparin was injected subcutaneously every 12 hours for 5 days.
  • Mild bleeding does not require blood transfusion and does not cause hemodynamic changes. Including subcutaneous hemorrhage, small hematoma, and oozing at the site of acupuncture.
  • Moderate bleeding requires blood transfusion, but does not cause abnormal hemodynamic changes requiring treatment. Including large hematoma, operational bleeding (such as cardiac catheterization), or retroperitoneal hemorrhage confirmed by clinical examination or ultrasonography, did not cause abnormal changes in hemodynamics, but blood transfusion is required.
  • Severe bleeding leads to hemodynamic changes and requires treatment and blood transfusion. Includes acute gastrointestinal bleeding or retroperitoneal bleeding. Intracranial hemorrhage is a severe bleeding.
  • ECG recording 18 lead electrocardiogram should be recorded before thrombolysis, and 12-lead ECG should be reviewed every half hour within 2 hours after thrombolysis (positive posterior wall and right ventricular infarction still record 18-lead ECG). Review the full set of standard ECGs at 3 hours. The ECG was reviewed once a day for the next 7 days, and the ECG was reviewed 1 time before discharge. The lead electrode position should be strictly fixed.
  • CK and CK-MB were checked before and 8, 10, 12, 14, 16, 18, and after the onset. In the future, it will be checked as necessary according to the situation.
  • the opening rate of the infarct-related coronary artery was used as the evaluation criterion for effectiveness.
  • the diagnostic criteria for infarct-related arterial access are:
  • Coronary angiography 90 minutes after the start of thrombolysis showed that the blood flow of the infarct-related blood vessels reached TIMI level 2 or level 3, which was judged to have been reopened.
  • Chest pain is significantly reduced within 2 hours after the start of the input of thrombolytic agent, the degree of reduction is 75%;
  • the CK-MB enzyme peak is advanced to within 14 hours of onset or the CK enzyme peak is within 16 hours.
  • the clinical diagnosis is acute reocclusion of the infarct-related artery.

Description

说明书 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 技术领域
本发明属于生物医药领域， 涉及一种纤溶蛋白酶原激活剂 （plasminogen activator) 尿 激酶原 （proUK) 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。 背景技术
尽快开通梗死相关的冠状动脉 （IRA)， 重新建立起有效的前向血流， 使缺血心肌得 到再灌注是急性心肌梗塞早期治疗的最重要策略。
直接经皮冠状动脉介入 （PCI) 和经静脉给药溶栓是开通 IRA、 进行再灌注， 从而降 低死亡率的两种主要方法。
再灌注治疗急性心肌梗死的最佳时间是胸痛症状发作开始后 1小时之内，缩短从胸痛 发作到缺血心肌得到有效再灌注的时间是降低死亡率与致残率的关键。 若能在患者就诊 90分钟内进行直接 PCI， 其疗效优于溶栓疗法 [1A3]。 但是， 由于患者当地医疗机构的 PCI 设备、是否有及时到位的技术熟练的操作人员等条件及转运患者难免时间延迟的限制， 即 使在医疗服务非常发达的美国， 只有 4%被转诊的患者能达到在就诊后 90分钟内接受直 接 PCI再灌注的治疗目标， 约有 60— 70%患者不能得到及时的直接 PCI治疗 [4]。
研究发现， 在胸痛发作开始后 3小时内到达医院就诊的患者， 接受溶栓疗法组的 30 天死亡率近似甚至低于直接 1^1组[5'6]，直接 PCI延迟的时间达到 62分钟后， 与溶栓疗法 比较， 两者的 4一 6周死亡率无显著差别 [7]。
易化 PCI (facilitated PCI)是指在 PCI之前先用溶栓药对急性心肌梗塞的病人进行治 疗，然后再实施 PCI的急性心肌梗塞治疗方案。美国心脏协会 (AHA)/美国心脏学会 (ACC) 关于急性心肌梗塞的诊治指南建议， 若首诊医院不能在 90分钟内实施直接 PCI， 对于无 溶栓治疗禁忌证的患者应立即进行溶栓治疗；对于发病 3小时内患者，若无条件接受直接 PCI或直接 PCI较溶栓延迟超过 1小时以上， 也应首选溶栓疗法 [1]。 鉴于溶栓疗法的易行 性和有效性， 其仍然是大多数急性心肌梗塞患者接受的再灌注治疗。 这意味着易化 PCI 在急性心肌梗塞的临床治疗中有着重要的应用意义。然而，利用目前批准使用的链激酶和 TPA类等溶栓药 （包括替奈普酶 /TNK、 瑞替普酶 /reteplase、 阿替普酶 /alteplase等） 进行 易化 PCI的有效性和安全性却显著差于直接 PCI[8'9]。 这可能与 TPA类等溶栓药在使用是 会激活凝血系统而具有较高的再闭塞发生率有关。 因此我们急需发现一种可在易化 PCI 使用的新溶栓药。
尿激酶原 （尿激酶原含 411个氨基酸， 分子量为 46393.65道尔顿， 其氨基酸序列见 SEQ ID NO.1 )是具有酶和酶原双重特性的丝氨酸蛋白水解酶原。经静脉进入血液循环后， 作为酶原， 不引起系统性纤溶激活； 作为酶， 它能够高度选择性地溶解栓塞血栓， 而几乎 不作用于组织、器官伤口处的止血性血栓， 因而最大限度地避免了溶栓治疗时的出血性并 发症。 由于其还具有抑制血小板聚集的作用， 可减少溶栓成功后再闭塞的发生率。研究结 果显示， 用 rh-PROUK溶栓治疗急性心肌梗死， 给药开始后 90分钟的梗死相关动脉的开 通率与 TPA相当， 24小时再闭塞率低于 TPA和链激酶， 30天的患者死亡率低于 TPA、 链激酶和尿激酶[1()'11]。 目前没有文献将尿激酶原或尿激酶原变体用于急性心肌梗塞易化 PCI的报导。 发明内容
本发明的目的是提供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。
本发明的目的是通过下列技术方案实现的：
尿激酶原及尿激酶原变体在急性心肌梗塞易化 PCI中的应用。
所述的应用，其方法是当病人急性心肌梗塞发生后的 6小时以内，首先采用尿激酶原 或尿激酶原变体进行溶栓治疗， 然后采用经皮冠状动脉介入 （PCI ) 手术， 以尽快开通梗 死相关的冠状动脉 （IRA)， 重新建立起有效的前向血流， 使缺血心肌得到再灌注。
所述的应用，其中尿激酶原是天然尿激酶原或重组人尿激酶原，其氨基酸序列如 SEQ ID N0.1所示。 所述的应用，其中尿激酶原变体是尿激酶原氨基酸序列经过取代、缺失或添加一个或 几个氨基酸且具有尿激酶原活性的蛋白质或多肽；或者是与天然尿激酶原蛋白质全序列有 40%以上同源性的蛋白质； 或者是与天然尿激酶原蛋白质 B链序列有 90%以上的同源性的 蛋白质或多肽。
所述的应用， 其中尿激酶原变体是尿激酶原氨基酸序列 SEQ ID NO.l的 300位的赖 氨酸 （Lys ) 点突变为组氨酸 （His)。
所述的应用， 其中采用尿激酶原或尿激酶原变体进行溶栓治疗的方式及用量是静脉 推注，剂量是每位病人 50万单位〜 300万单位；或者是 30〜120分钟静脉点滴 5万单位〜 20万单位 /min/病人； 或者是静脉推注与静脉点滴联合使用（每位病人首先静脉推注尿激 酶原总剂量的 20%， 其余部分 90分钟静脉点滴 5万单位〜 20万单位 /min)， 使用的尿激 酶原或尿激酶原变体的总剂量不超过 600万单位。
所述的应用，其中采用尿激酶原或尿激酶原变体进行溶栓治疗，给药时间为 5分钟〜 120分钟。
所述的应用， 其中经皮冠状动脉介入手术的时间是使用尿激酶原或尿激酶原变体进 行溶栓治疗后的半小时至 24小时。
所述的应用，其中经皮冠状动脉介入手术的时间是使用尿激酶原或尿激酶原变体进行 溶栓治疗后的 1天至 7天。 本发明的有益效果：
本发明在重组人尿激酶原 Π期临床试验（国家食品药品监督管理局药物临床研究批件 的批件号： 2003L03626 )研究中， 发现造影组 38例患者中， 28例在采用重组人尿激酶原 溶栓结束后立即施行了 PCI， 其中 1例接受了 PTCA， 其余 27例在 PTCA后置入了支架 (即进行了 PCI)， 30天与半年的死亡率均为零， 再闭塞、 脑卒中、 中度出血与重度出血 的发生率也均为零， 亦未见 "无再流"发生。
临床观察结果证明， 用重组人尿激酶原溶栓后立即进行 PCI治疗急性心肌梗塞的易 化 PCI， 具有优于直接 PCI临床疗效。
本发明尿激酶原或尿激酶原变体与 PCI联合使用效果较好的原因是： 尿激酶原或尿 激酶原变体是具有酶和酶原双重特性的丝氨酸蛋白水解酶原。经静脉进入血液循环后，作 为酶原， 不引起系统性纤溶激活； 作为酶， 它能够高度选择性地溶解栓塞血栓， 而几乎不 作用于组织、器官伤口处的止血性血栓， 因而最大限度地避免了溶栓治疗时的出血性并发 症。 由于其还具有抑制血小板聚集的作用， 可减少溶栓成功后再闭塞的发生率。尿激酶原 或尿激酶原变体治疗急性心肌梗塞的再栓塞率远远低于 tPA，而使得 PCI手术时支架植入 后出现再栓塞的几率下降， 从而导致死亡率显著下降。 附图说明
图 1为实施例 2中狗止血测验结果。 狗 （每组狗的数量见图中括号中数字） 初始出 血时间 （平均值士 SD)， 分别了输注生理盐水、 tPA、 尿激酶原和 M5 ( 60 g/kg/min) o 以下通过实施例对本发明作进一步的阐述。
实施例 1: 尿激酶原及其变体
尿激酶原可以采用提取的天然尿激酶原或重组人尿激酶原， 其氨基酸序列为 SEQ ID NO.D o
尿激酶原变体是尿激酶原氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有 尿激酶原活性的蛋白质或多肽；或者是与天然尿激酶原蛋白质全序列有 40%以上同源性的 蛋白质； 或者是与天然尿激酶原蛋白质 B链序列有 90%以上的同源性的蛋白质或多肽。
尿激酶原变体的一个特例是尿激酶原氨基酸序列 （SEQ ID N0.1 ) 的 300位的赖氨酸 (Lys) 点突变为组氨酸 (His)0
尿激酶原及尿激酶原变体的制备方法可采用本领域公知的基因工程方法。
实施例 2: 通过体内体外实验证明尿激酶原变体的一个特例 （尿激酶原氨基酸序列
SEQ ID NO.1的 300位的赖氨酸点突变为组氨酸， Lys300 ->ffls， M5), 具有至少与尿 激酶原相当或优于尿激酶原的溶栓活性， 可用于易化 PCI的治疗。
(1) M5的体外试验结果
1.1 内在催化活性测验
对于生色底物 S2444 (L-焦谷氨酸 -L-甘氨酸-精氨酸对硝基苯胺盐酸盐） 的水解， 1.0 mol/L的尿激酶原或 10.0 mol/L的 M5与一系列浓度 （0到 2.4 mmol/L) 的 S2444在室温 下于测试缓冲液 (0.05 mol/L Tris-HCl, 0.10mol/L NaCl, and 0.01% Tween 80, pH 7.4) 中共 孵育， 反应速率由 410 nm处的光吸收增加值测定， 并以 Lineweaver-Burk图算得反应常 数。 结果显示， M5的迟滞相是尿激酶原的两倍， 但随后速率的增加更快， 这分别反映了 M5相对较低的内在活性与较高的双链催化活性。
1.2 辅因子对于尿激酶原或 M5导致的纤溶酶原激活作用的促进
我们通过纤溶酶耐受的尿激酶原及 M5变体（Lysl58)来研究了纤维蛋白片段 E对于 尿激酶原或 M5导致的纤溶酶原激活的作用。 室温下测定反应混合物中 （含 1.5 mmol/L S225KD-缬氨酸 -L-亮氨酸 -L-赖氨酸对硝基苯胺二盐酸盐）， 2.0 mol/L Glu-纤维蛋白原， 1.0 nmol/LAlal58-proUK 或 Alal58-M5， 并且含或不含 5.0mol/L的片段 E2) 410nm的光吸 收随时间的增加值。 纤维蛋白原、 可溶性的纤维蛋白单体、 D-二聚体等纤维蛋白类似物 对纤溶酶原激活作用也在被研究之列， 因为它们不能促进尿激酶介导的纤溶酶原激活。实 验结果显示， 纤维蛋白片段 E能选择性地促进尿激酶原的纤溶酶原激活作用， 它对于 M5 也有着相似的作用， 而 D-二聚体和可溶性纤维蛋白或纤维蛋白原对之几乎没有促进。
1.3 PAI-1的抑制作用
双链 tc-M5可以被 PAI- 1抑制， K值约为 1.3±0.3 X 107 mol/L-s,与尿激酶的相当（ 1.7+0.4 X 107 mol/L -s) o
1.4 M5与尿激酶原在人血浆中的稳定性 （惰性）
0-20 g/mL的 M5或 0-8 g/mL的尿激酶原在 lmL枸櫞酸抗凝的混合血浆中 37°C孵 育 6小时后， 加入 0.2 mL抑肽酶 （每 mL 10000激肽释放酶抑制单位）。 以凝血酶可凝蛋 白法测得血桨中剩余的纤维蛋白原量， 并与基线水平相较。
在混合血浆中， M5保持惰性， 在 8 g/mL以下的剂量并不导致纤维蛋白原的降解。 而在 10 g/mL的剂量下，仍有 30.6 %的纤维蛋白原未降解。与之相反，尿激酶原在 2 g/mL 的剂量下就可导致纤维蛋白原的降解， 而在 4 g/mL时， 只有 32.7%的纤维蛋白原得以保 持。
1.5 人血浆尿激酶原与 M5对于血凝块的体外溶解作用
I125标记的纤维蛋白原凝块由 0.2 mL血浆制得， 并在 4 mL血浆中孵育。 一系列的纤 维蛋白特异性浓度的尿激酶原 (0.5-1.5 g/mL) 或 M5 (0.5 -5.0 g/mL) 被用来测试， 血 凝块溶解以完全溶解值对时间的百分比表示， 纤维蛋白原在完全血凝块溶解后测定； 若 6 小时时依然尚未完全溶解， 则在 6小时时测定.
在血浆环境中， M5在高达 5 g/mL的浓度下依然保持着纤维蛋白特异性 （25%纤维 蛋白原降解），而尿激酶原对之的上限是 1.5 g/mL。尿激酶原的血凝块溶解最大速率是每 小时 41%， 而 M5为每小时 64%。 当 M5 (2 g/mL) 与少量 tPA(30 ng/mL)联用时， 迟滞 相缩短了一半， 这与之前的 tPA和尿激酶原联用的结果类似。
(2) M5的体内研究
2.1 狗的血凝块溶解
10-15 kg的雄性杂种狗以戊巴比妥钠麻醉。 血凝块由 lmL加入以碘 125标记的纤维 蛋白原 ( 1.9 Ci, 0.75 mCi/mg protein) 与凝血酶 ( 10单位) 的狗全血制得。 20分钟后， 将 血凝块以生理盐水冲洗三次后切成 lmm小块以 16号针头注入股静脉。 15分钟后， 经由 另一侧股静脉外的插管取得血样，测定基线放射性。然后分别静脉给予生理盐水或尿激酶 原或 tPA， 尿激酶原和 tPA的输入速率分别为 20 g/kg/min和 10 g/kg/min, 这些速度根 据之前的文献报道在狗体内的溶栓有效且具有纤维蛋白特异性。 tPA输入持续 60分钟， 尿激酶原和 M5的输注持续 90分种。 M5的输入速率分别为 20、 40、 60 g/kg/min, 间隔 一定时间， 取血样测定放射度与纤维蛋白原。
实验结果显示， M5导致的血凝块溶解是剂量依赖的。 由于 M5在血浆中的稳定性比 尿激酶原高 4倍， 在以 60 g/kg/min的速度输注时， M5引起了快速的血凝块溶解， 45分 钟内就达到了 100%。M5引起血凝块溶解也更为有效，因为达到 50%溶解只需要 600 g/kg 的 M5， 而此剂量对尿激酶原来说是 1200 g/kg。 在更低的剂量下 （40或 20 g/kg/min)， M5引起的血凝块溶解相当或更少。这种低剂量下的相对低效反映了 M5的更长的迟滞相。 M5的最大输注剂量组的实验狗的血浆纤维蛋白原浓度在 30、 45、 60min分别是基线水平 的 72%， 65%和 52%。
2.2 狗止血测验
在剃去毛的腹部作一 lcm标准化切口， 剥去表皮。 将一暴露的浅表血管以手术刀割 开， 出血点每三十秒以滤纸轻拍， 直到血流停止。 此即初始出血时间 （BT)， 在相邻的 lcm切口处 0、 20、 60分种时各测定一次。全出血也以吸收伤口渗血所需要的标准纱布片 (5 X 5cm)的数量记录， 每张纱布垫吸足血后即行更换。这代表了二次出血， 因为它主要来 自两个之前测定初始出血时间后已止血的出血点。 此实验于每次输注前 60分钟进行。
实验结果 （如图 1 ) 显示， 在 tPA输注组， 初始出血时间从 1.2分钟增加到输注 20 分钟时的 2.4分钟， 并在 60分钟后进一步达到 5分钟； 尿激酶原组也增加到 4分钟。 对 于 M5剂量组， 在 20分钟时未见 BT显著延长， 在 60分钟时 BT增加为 1.5分钟。 反应 了二次出血的、 以吸血所需纱布片数表示的总出血量， 在 tPA组增加了 8倍， 尿激酶原组 增加了 5倍， 而 M5最大剂量组未见增加。
2.3 恒河猴血凝块溶解与止血实验
恒河猴对于人尿激酶原 /尿激酶有着与人相当的敏感度。 5.8公斤至 8.6公斤的六只恒 河猴（三雌三雄）静脉给戊巴比妥钠 （30 mg/kg)麻醉， 每侧上肢静脉中插入一聚乙烯导 管分别用于取血与输注。 2 mL全血样与放射性碘标记的纤维蛋白原和凝血酶混合后在 37 度孵育 20分钟。全血凝块切成 1 mm的小块， 以生理盐水冲洗六次。血凝块（3.3-10 cpm) 悬于 5 mL生理盐水中， 经由右上肢静脉注入。 30分钟后， 经对侧上肢静脉采得血样测定 基线放射度。 与此同时， 开始输注生理盐水 （2猴） 或 M5 (4猴）。 M5以 60 g/kg/min 输注 60分钟，在输注的一定时间间隔取血样进行放射度与纤维蛋白原的测定。 0、 30、 45、 60分钟时在下腹部以 5 mm长， 1 mm深的切口测定初始出血时间； 出血时间的测定采用 以滤纸每隔三十秒轻拍的标准方法进行。 初始出血点止血后的再出血也予以评价。 实验结果显示， 以 60 g/kg/min输注 60 min的 M5在全部的四只实验猴中都引起了 100%的血凝块溶解（对照组仅为 8%)。 输注 30、 45、 60分钟时的纤维蛋白原浓度分别为 基线水平的 78%、 66%和 57%。 初始出血时间 （以基线水平百分比表示） 在 30分钟时减 少为 85%， 45分钟时回到基线水平， 而 60分钟时显著增加到 108%。 BT在生理盐水对照 组也显示了类似的结果。 另外， 与狗中的结果一致， 给予 M5的实验猴中再出血并没有发 生。
实施例 3、 尿激酶原在易化 PCI中的应用：
(1) 入选标准
1.1 缺血性胸痛持续 30分钟， 含服硝酸甘油无效；
1.2 持续性缺血性胸痛发作 12小时；
1.3 心电图至少 2个或 2个以上肢体导联 ST段抬高 0.1mV， 或在相邻 2个或 2个 以上胸导联 ST段抬高 0.2mV;
1.4年龄 85岁， 性别不限。
(2) 排除标准
2.1 非 ST段抬高性急性心肌梗死或不稳定型心绞痛；
2.2妊娠、 哺乳期、 月经期妇女；
2.3既往任何时间发生过出血性脑卒中， 1年内发生过缺血性脑卒中或脑血管事件。 2.4严重进展性疾病 （如恶性肿瘤） 或预后不良使患者极度衰竭的疾病；
2.5 四周内有活动性的内脏出血 （如胃肠道、 泌尿生殖系统出血， 但月经除外）， 或 有未治愈的消化道溃疡；
2.6 颅内肿瘤、 可疑主动脉夹层、 动静脉畸形、 动脉瘤；
2.7 高血压患者经积极降压治疗后， 血压仍 170/110mmHg， （收缩压、 舒张压其中 一项达到此血压标准）；
2.8目前正在使用治疗剂量的抗凝药， 如华法林等；
2.9已知的出血倾向 （止血或凝血功能障碍）；
2.10 新近 （6个月内） 颅脑或脊柱手术史；
2.11 二个月内创伤史， 包括创伤性心肺复苏或较长时间 （〉10min) 的心肺复苏， 或 接受过外科大手术； 6个月内头部外伤史；
2.12 二周以内接受过在不能压迫部位的大血管穿刺；
2.13 高度怀疑左心腔内血栓 （如二尖瓣狭窄并房颤）；
- 1 - 2.14糖尿病或其它疾病引起的眼底出血病史；
2.15 严重肝、 肾功能障碍史 （ALT ^常上限 3倍； 肌酐 >225 mol/L);
2.16 休克；
2.17 感染性心内膜炎、 急性心肌炎、 急性心包炎、 脓毒性血栓性静脉炎、 严重感染 部位存在的动-静脉痿；
2.18 与以前部位相同的再梗死；
2.19 正在参加其它同类药物临床试验。
2.20 医师认为有不适合静脉溶栓的其它疾病、 情况。
( 3 ) 临床应用方法（用法、 用量、 给药时间）：
尿激酶原的易化 PCI: 首先采用重组人尿激酶原进行溶栓治疗， 可以是静脉推注， 剂 量是每位病人 50万单位〜 300万单位； 可以是 30〜120分钟静脉点滴 5万单位〜 20万单 位 /min/病人； 可以是静脉推注与静脉点滴联合使用 （每位病人首先静脉推注尿激酶原总 剂量的 20%， 其余部分 90分钟静脉点滴 5万单位〜 20万单位 /min)， 使用的尿激酶原的 总剂量不超过 600万单位。 尿激酶原进行溶栓治疗， 给药时间为 5分钟〜 90分钟。 接着 进行经皮冠状动脉介入 （PCI) 手术治疗的时间是使用尿激酶原进行溶栓治疗后的半小时 至 24 小时。 在特殊情况下， 经皮冠状动脉介入 （PCI) 手术治疗的时间可以是使用尿激 酶原进行溶栓治疗后的 1天至 7天。
( 4 ) 心血管造影：
部分病人接受心血管造影检查，观察在静脉注射溶栓药物开始后 90分钟时和实施 PCI 手术后冠状动脉造影所显示的梗死相关动脉的再通状况。
4.1 冠状动脉造影：
先行梗死相关冠状动脉造影（根据心电图梗死部位推测），首次造影应采用大视野（小 像）， 以便观察全部血管， 曝光时间应一直持续到造影剂排空为止。 左冠状动脉应至少投 照 3个体位， 右冠状动脉应至少投照 2个体位， 且应用大像。 在作梗死相关动脉造影前， 不要经冠状动脉给硝酸甘油。造影结果依据 TIMI血流灌注分级标准进行判定， 达到 2、 3 级者为血管再通。
TIMI血流灌注判定标准：
TIMI 0级： 闭塞冠状动脉无前向血流灌注；
TIMI 1级： 冠脉狭窄病变远端血管有前向血流灌注， 但不能充盈远端血管床；
TIMI 2级： 冠脉狭窄病变远端血管有前向血流灌注， 但远端血管床需经过 3个以上 心动周期才能缓慢基本充盈， 且造影剂清除缓慢；
TIMI 3级： 冠脉狭窄病变远端血管有前向血流灌注， 远端血管床造影剂充盈完全、 迅速 （小于 3个心动周期）， 同时造影剂清除迅速。
4.2 血管造影术后处理
冠状动脉造影完成后， 若不需要 PTCA和支架置入术等进一步治疗者， 在 APTT < 80秒时方可拔除动脉鞘管， 局部压迫止血至少半小时以上。 按溶栓结束后 48小时停止肝 素静滴， 以后低分子肝素皮下注射每 12小时一次， 共 5天。
(5 ) 临床和实验室观察指标：
5.1 临床症状和体征：
5.1.1 胸痛： 有无减轻及减轻的程度；
按四级评定： 无改变、 轻度缓解 （50%)、 明显缓解 （75% )、 完全缓解；
5.1.2 出血表现： 皮肤、 粘膜、 咳痰、 呕吐物及大小便中有无出血征象；
按以下标准分为轻、 中、 重度三级
轻度： 轻度出血无需输血， 不导致血流动力学改变。 包括皮下出血、 小血肿、 针刺 部位渗血等。
中度：中度出血需要输血，但不会引起需要治疗的血流动力学异常改变。包括大血肿、 操作引起的出血 （如心导管检查）、 或通过临床检查或超声检查证实的腹膜后出血， 未引 起血液动力学的异常改变， 但需要输血。
重度：重度出血导致血流动力学改变并需要治疗及输血。包括急性胃肠道出血或腹膜 后出血。 颅内出血均属重度出血。
5.1.3 血压、 心率、 心律、 心音及心脏杂音的变化；
5.1.4其它并发症
5.2 心电图记录： 溶栓前应记录 18导联心电图， 溶栓开始后 2小时内每半小时复查 一次 12导联心电图 （正后壁、 右室梗死仍记录 18导联心电图）。 3小时时复查全套标准 心电图。此后 7天内每天复查 1次心电图， 出院前复查 1次心电图。导联电极位置应严格 固定。
5.3 血常规、 尿常规、 便潜血： 入选前、 溶栓开始后 2、 6、 12、 24、 48 (是否可以减 少检查次数， 在 2期试验中都达不到此要求） 小时、 7天（住院少于 7天者出院前测定）， 可视大小便情况调整便潜血检查时间点。
5.4 心肌酶： 入选前和发病后 8、 10、 12、 14、 16、 18、 小时各查 CK和 CK-MB— 次， 以后根据情况而在必要时进行检查。
5.5 肝功能、 肾功能、 血糖、 电解质： 在入选前、 溶栓开始后第 7天测定。
(6)疗效评定标准
以梗死相关冠状动脉的开通率作为有效性的评定标准。梗死相关动脉开通的诊断标准 为：
6.1.冠状动脉造影：
溶栓开始后 90分钟冠状动脉造影显示梗死相关血管的血流达到 TIMI 2级或者 3级， 即判断为已再开通。
6.2.临床再通指标：
6.2.1抬高的 ST段在输注溶栓剂开始后的 2小时内迅速回降 50%；
6.2.2胸痛自输入溶栓剂开始后的 2小时内明显减轻， 减轻程度 75%；
6.2.3输入溶栓剂后 2小时内， 出现加速的室性自主心律、 房室或束支阻滞突然改善 或消失、 或者下壁梗死患者出现一过性窦性心动过慢、 窦房阻滞伴有或不伴有低血压。
6.2.4血清 CK-MB酶峰提前到发病 14小时以内或 CK酶峰在 16小时以内。
具备以上 4项指标中 2项或以上者考虑再通，但第 2项与第 3项组合不能判定为再通。
6.3.梗死相关动脉急性再闭塞的诊断标准：
6.3.1 溶栓成功后 48小时内再度发生胸痛， 原梗死部位对应的心电图导联 ST段再次 抬高， 胸痛持续时间 30分钟且含服硝酸甘油不能缓解。
6.3.2血清 CK-MB水平再度升高。
具备以上 2项时， 可临床诊断为梗死相关动脉急性再闭塞。
6.3.3急症冠状动脉造影证实为梗死相关动脉急性再闭塞。
(7) 随访：
对每例受试病人随访 30天内病死率和不良事件发生率。
(8) 观察数据结果：
在重组人尿激酶原 II期临床试验（国家食品药品监督管理局药物临床研究批件的批件 号： 2003L03626 )研究中， 发现造影组 38例患者中， 28例在溶栓结束后立即施行了 PCI， 其中 1例接受了 PTCA， 其余 27例在 PTCA后置入了支架（即进行了 PCI)。 30天与半年 的死亡率均为零， 再闭塞、 脑卒中、 中度出血与重度出血的发生率也均为零， 亦未见 "无 再流"发生。 参考文献
1、 Antman EM, Anbe DT, Amstrong TW, et al. ACC/AHA guidelines for the management of patients with ST-elevation myocardial infarction-executive summary: a report of the American College of Cardiology/ American Heart Association Task Force on Practice Guidelines (Writing Committee to Rivise the 1999 Guidelines for the Management of Patients With Acute Myocardial Infarction). Circulation 2004;110:588-636.
2、 Global Use of Strategies to Open Occluded Coronary Arteries in Acute Coronary Syndromes ( GUSTO lib ) Agioplasty Sub study Investigators. A clinical trial comparing primary coronary angioplasty with tissue plasminogen activator for acute myocardial infarction. N Engl J Med 1997;336:1621-8
3、 Keeley EC, Boura J A, Grines CL. Primary angioplasty versus intravenous thrombolytic therapy for acute myocardial infarction: a quantitative review of 23 randomised trials. Lancet 2003; 361:13-20
4、 Nallamothu BK，Bates ER, Herrin J, et al. Times to treatment in transfer patients undergoing primary percutaneous coronary inervention in the United States: National Registry of Myocardial Infarction (NRMI)-3/4 analysis. Circulation 2005; 111:761-7
5、 Steg PG, Bonnefoy E, Chabaud S, et al. Impact of time to treatment on mortality after prehospital fibrinolysis or primary angioplasty: data from the CAPTIM randomized clinical trial. Circulation 2003;108:2851-6
6、 Widimsky P,Budesinsky T,Vorac D, et al. Long distance transport for primary angioplasty vs immediate thrombolysis in acute myocardial infarction. Final results of the randomezed national multicentre trial-PRAGUE-2. Eur Heart J 2003;24:94-104
7、 Nallamothu BK, Bates ER. Percutaneous coronary intervention versus fibrinolytic therapy in acute myocardiial infarction: is timing (almost) everything? Am J Cardiol 2003;92:824-6
8、 Primary versus tenecteplase-facilitated percutaneous coronary intervention in patients with st- segment elevation acute myocardial infarction ( ASSENT-4 PCI )： randomised trial. Lancet 2006; 367: 569-78
9、 Keeley EC, Boura J A, Grines CL. Comparison of primary and faciliated percutaneous coronary interventions for S-T elevation myocardial infarction: quantitative review of randomised trials. Lancet 2006; 367： 579-88
10、 Weaver WD, Hartmann JR, Anderson JL, Reddy PS, Sobolski JC, Sasahara AA. New recombinant glycosylated prourokinase for treatment of patients with acute myocardial infarction. J Am Coll Cardiol. 1994; 24:1242-8.
11、 Zarich SW, Kowalchuk GJ, Weaver WD, Loscalzo J, Sassower M, Manzo K, Byrnes C, Muller JE, Gurewich V. Sequential combination thrombolytic therapy for acute myocardial infarction: results of the Pro-Urokinase and t-PA Enhancement of Thrombolysis (PATENT) Trial. J Am Coll Cardiol. 1995; 26(2):374-9.

Claims

权利要求书
1、 尿激酶原及尿激酶原变体在急性心肌梗塞易化 PCI中的应用。
2、 根据权利要求 1所述的应用， 其特征在于应用方法是当病人急性心肌梗塞发生后 的 6小时以内， 首先采用尿激酶原或尿激酶原变体进行溶栓治疗， 然后采用经皮冠状动脉 介入手术， 以尽快开通梗死相关的冠状动脉， 重新建立起有效的前向血流， 使缺血心肌得 到再灌注。
3、 根据权利要求 1或 2所述的应用， 其特征在于尿激酶原是天然尿激酶原或重组人 尿激酶原， 其氨基酸序列如 SEQ ID N0.1所示。
4、 根据权利要求 1或 2所述的应用， 其特征在于尿激酶原变体是尿激酶原氨基酸序 列经过取代、缺失或添加一个或几个氨基酸且具有尿激酶原活性的蛋白质或多肽； 或者是 与天然尿激酶原蛋白质全序列有 40%以上同源性的蛋白质； 或者是与天然尿激酶原蛋白质 B链序列有 90%以上的同源性的蛋白质或多肽。
5、根据权利要求 4所述的应用，其特征在于尿激酶原变体是尿激酶原氨基酸序列 SEQ ID N0.1的 300位的赖氨酸点突变为组氨酸。
6、 根据权利要求 2所述的应用， 其特征在于采用尿激酶原或尿激酶原变体进行溶栓 治疗的方式及用量是静脉推注，剂量是每位病人 50万单位〜 300万单位； 或者是 30〜120 分钟静脉点滴 5万单位〜 20万单位 /min/病人； 或者是静脉推注与静脉点滴联合使用， 每 位病人首先静脉推注尿激酶原总剂量的 20%，其余部分 90分钟静脉点滴 5万单位〜 20万 单位 /min， 使用的尿激酶原或尿激酶原变体的总剂量不超过 600万单位。
7、 根据权利要求 2所述的应用， 其特征在于采用尿激酶原或尿激酶原变体进行溶栓 治疗， 给药时间为 5分钟〜 120分钟。
8、 根据权利要求 2所述的应用， 其特征在于经皮冠状动脉介入手术的时间是使用尿 激酶原或尿激酶原变体进行溶栓治疗后的半小时至 24小时。
9、根据权利要求 2所述的应用， 其特征在于经皮冠状动脉介入手术的时间是使用尿激 酶原或尿激酶原变体进行溶栓治疗后的 1天至 7天。
- 1 -
PCT/CN2008/070653 2008-03-31 2008-04-01 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 WO2009121223A2 (zh)

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