StreamlineSP扩张柱层析在重组人尿型纤溶酶原激活剂中试生产中的应用

尿型纤溶酶原激活剂 (ｕｒｉｎａｒｙｔｙｐｅｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒ,ｕ ＰＡ)包括单链型和双链型两种形态 ,以单链形态为主的ｕ ＰＡ具有很好的选择性溶血栓作用。1985年 ,Ｈｏｍｅｓ等在大肠杆菌中获得了ｕ ＰＡ的表达并纯化[1] 。近几年 ,用哺乳动物细胞表达糖基化ｕ ＰＡ取得了很大进展[2 ,3] 。本实验室成功地建立了工艺简便、效率高的重组人ｕ ＰＡ的中试生产纯化工艺。工程细胞表达的ｕ ＰＡ ,经阳离子交换层析、亲和层析和凝胶过滤层析 ,纯度大于 95% ,年生产量大于 50ｇ ,收率大于 6 0 %。目前 ,还未见到优于此…     

重组人尿型纤溶酶原激活剂规模生产中纯化过程的质量控制

尿型纤溶酶原激活剂(u-PA)是一种高效、出血副作用和再阻率低的溶栓制剂,由于在溶液中不稳定,在纤溶酶、激肽释放酶、胰酶及理化因素影响下,容易从158Lye-159Ile处断裂成双链,故u-PA有包括单链和双链两种形态.u-PA的国外临床用量最大剂量可达80 mg,因此对产品的质量提出更高的要求.为了保证产品的最终质量,必须在生产过程中加以严格控制,以确保产品的安全有效.我们对u-PA中试生产中纯化阶段的质量控制进行了研究,包括:蛋白含量、活性测定、活性回收率及比活计算、单双链比例测定、纯度分析几项指标的控制,以保证产品的质量.     

重组人尿型纤溶酶原激活剂规模生产中纯化过程研究简报的质量控制

尿型纤溶酶原激活剂 (ｕ＿ＰＡ)是一种高效、出血副作用和再阻率低的溶栓制剂 ,由于在溶液中不稳定 ,在纤溶酶、激肽释放酶、胰酶及理化因素影响下 ,容易从 15 8Ｌｙｅ＿15 9Ｉｌｅ处断裂成双链 ,故ｕ＿ＰＡ有包括单链和双链两种形态。ｕ＿ＰＡ的国外临床用量最大剂量可达 80ｍｇ ,因此对产品的质量提出更高的要求。为了保证产品的最终质量 ,必须在生产过程中加以严格控制 ,以确保产品的安全有效。我们对ｕ＿ＰＡ中试生产中纯化阶段的质量控制进行了研究 ,包括 :蛋白含量、活性测定、活性回收率及比活计算、单双链比例测定、纯度分析几项指…     

重组人尿型纤溶酶原激活剂的冻干及其稳定性观察

由血栓引起的心血管系统疾病严重威胁着人类健康，临床上急需一种溶栓效果好、副作用小的药物。尿型纤溶酶原激活剂（ｕ－ＰＡ）作为一种有前景的溶栓药物已受到国内外普遍重视，我所构建的表达重组人ｕ－ＰＡ的ＣＨＯ工程细胞（ＣＬ－１１Ｇ株）培养上清经纯化可获得含约...     

尿激酶型纤溶酶原激活剂诱发的纤溶反应及其调节因子

目的 研究一些凝血和纤溶因子对尿激酶型纤溶酶原激活剂（uPA)诱发纤溶反应的调节作用及机制。方法 采用凝块溶解测定和纤维蛋白检测板测定观察了一些凝血和纤溶因子对uPA诱导的纤溶反应的影响及相互作用。结果 凝血酶激活的纤溶抑制因子（TAFI）对uPA或单链uPA（scuPA）诱发的纤溶反应具有明显的拮抗作用，凝血酶调节因子（TM）可明显增强其抑制作用，凝血酶对uPA诱导的纤溶反应没有明显的影响，但可抑制scuPA诱导的纤溶反应，该作用亦可被TM所增强或被水蛭素所消除。结论 凝血酶／TM可通过激活TAFI对uPA或scuPA诱导的纤溶反应产生抑制作用，凝血酶对scuPA诱发的纤溶反应的抑制作用亦可通过其对scuPA的裂解失活而实现。     

重组型纤溶酶原激活剂与尿激酶在急性心肌梗死溶栓治疗中的比较

目的比较小剂量重组型纤溶酶原激活剂（rt-PA）与尿激酶（UK）溶栓治疗急性心肌梗死（AMI）的疗效和安全性。方法回顾42例接受rt-PA和49例接受UK溶栓治疗的AMI患者病例资料,对结果进行比较分析。结果冠脉总再通率rt-PA组和UK组分别为85．71％和61．22％（P〈0．05）;患者在发病3h和3—12h内溶栓,冠脉再通率在rt-PA组为94．11％和80．00％（P〉0．05）,在UK组为70．00％和58．97％（P〉0．05）;出血并发症rt．PA组和UK组分别为9．52％和10．20％（P〉0．05）。结论rt-PA的溶栓冠脉再通率显著高于UK,并且两组在发病后3h内较3h后溶栓均能取得更高的冠脉再通率,但无统计学差异。     

纤溶酶原激活剂抑制物-1的研究进展

纤溶酶原转变成有纤溶活性的纤溶酶是纤溶过程决定性的一步?t-PA是体内主要的纤溶酶原激活物之一,而纤溶酶原激活剂抑制物(Plasminogen Activator Inhibitor PAI)是组织型纤溶酶原激活剂(t-PA)和尿激酶纤溶酶原激活剂(u-PA)的快速抑制剂,在纤溶系统中起着关键性调节作用?自1983年Chmielewska J等[1]发现纤溶酶原激活剂抑制物(PAI-1)以来,越来越多的证据表明PAI活性或含量的改变直接影响纤溶系统的活性,是许多疾病特别是血栓形成性疾病发病的危险因素之一[2～4]?1 生物学特性纤溶酶原激活剂抑制物(PAI)包括PAI-1,PAI-2,PAI-3,C…     

小剂量重组组织型纤溶酶原激活物静脉溶栓治疗急性心肌梗死

急性心肌梗死溶栓治疗已成为早期心肌血流再灌注的重要措施。重组组织型纤溶酶原活物（ｒｔ－ＰＡ）是纤维蛋白特异性溶栓剂，每次１００ｍｇ静脉给药的疗效国外已大量报道〔１〕。为探讨小剂量ｒｔ－ＰＡ的治疗效果。本研究采用５０ｍｇｒｔ－ＰＡ于３０ｍｉｎ内静脉输注，观察对急性心肌梗死（ＡＭＩ）的溶栓效果。１　材料与方法１．１　病例选择　ＡＭＩ３６例，其中男３０例，女６例，平均年龄（５４．９±２．０）岁（３６～７０岁）。梗死部位：前间壁、前壁和广泛前壁２４例，下壁、下后壁及下壁与右室１０例，高侧壁和正后壁各１…     

纤溶酶原激活剂及其活性测定

纤溶酶原激活剂 (plasminogen activator,PA)是一种特异性的蛋白水解酶 ,它能将无活性的酶前体转变为纤溶酶 (plasmin)。纤溶酶是一种有效的非特异性的蛋白水解酶 ,能够裂解血液内的纤维蛋白凝块使其成为可溶性的多肽。本文描述了纤港酶原激活系统的一些生化特征 ,及对纤溶酶原激活剂活性的测定方法。纤溶酶原激活剂在血栓性疾病和出血性疾病的诊断具有潜在应用价值。     

纤溶酶原激活剂及其活性测定

纤溶酶原激活剂（plasminogen activator,PA）是一种特异性的蛋白水解酶，它能将无活性的酶前体转变为纤溶酶（plasmin）。纤溶酶是一种有效的非特异性的蛋白水解酶，能够裂解血液内的纤维蛋白凝块使其成为可溶性的多肽。本文描述了纤港酶原激活系统的一些生化特征，及对纤溶酶原激活剂活性的测定方法。纤溶酶原激活剂在血栓性疾病和出血性疾病的诊断具有潜在应用价值。     

Localization of human malignant tumors with radioiodinated recombinant tissue plasminogen activator

Human recombinant tissue plasminogen activator (tPA), labeled with 131I(1.1 to 6.2 mCi) by the iodogen method, was administered intravenously to 15 patients with various soft-tissue malignant tumors after blocking of thyroidal radioiodine uptake. Gamma camera imaging was performed 4 and 24 hr after injection; three patients were also imaged 5 days following injection. We observed accumulation of radioactivity in primary and secondary lesions in 11 patients. In this preliminary study we did not detect any definite association between the magnitude of uptake and type of tumor. Tumors were usually visualized already after 4 hr but the uptake was more intense at 24 hr. The target-to-nontarget ratios at 24 hr, determined by computer analysis of stored images, varied from less than 1.2 to 2.1. This is the first demonstration of accumulation of radiolabeled tPA in malignant tissue. We do not know the mechanism of the uptake but because tPA is known to be avidly bound to fibrin, a component of the stroma of many malignant tumors, it is possible that [131I]tPA is bound to fibrin rather than taken up by the malignant cell; various possible cell uptake mechanisms are discussed. Due to the relatively early maximal uptake of this radiopharmaceutical it will be possible to substitute 123I for 131I, a possibility suggesting a potential clinical use of radioiodinated tPA for the detection of malignant tumors of various origin.     

Thrombolytic therapy with recombinant human tissue-type plasminogen activator: a comparison of two doses

The efficacy and safety of two doses of recombinant human tissue-type plasminogen activator (rt-PA) were compared. Forty patients with peripheral arterial occlusions were treated with intraarterial rt-PA. Group A (n = 21) received 0.1 mg/kg/h, and group B (n = 19) received 0.05 mg/kg/h. Infusion durations varied from 4 to 8 hours. Complete thrombolysis occurred in 20 of 21 patients (95%) in group A and in all 19 patients (100%) in group B. In group A, fibrinogen levels were greater than 75% of baseline in ten of 21 patients (48%) at infusion termination. In group B, fibrinogen levels were greater than 75% of baseline in 12 of 19 patients (63%) at infusion termination. Three of 40 patients (7%) had significant complications resulting from rt-PA infusion. The results demonstrate that over similar infusion times, a dose of 0.05 mg/kg/h is as efficacious and results in less systemic fibrinogenolysis than a dose of 0.1 mg/kg/h.     

In vivo kinetics of99mTc labeled recombinant tissue plasminogen activator in rabbits

Our previous studies demonstrated that^99mTc labeled recombinant tissue plasminogen activator (rt-PA) retained high affinity with fibrinin vitro but showed unexpectedly low uptake in fresh thrombiin vivo. The present study was performed to determine thein vivo kinetics of radiolabeled t-PA in the rabbit. Sequential images and blood samples after the intravenous administration of^99mTc labeled rt-PA in thrombus-bearing rabbits were taken. The radioactivity and immunological level of t-PA and PAI-1 in the solution eluted to each fraction by gel permeation chromatography were measured by means of a well scintillation counter and enzyme-linked immunosorbent assay (ELISA). Most of the radioactivity was eluted in the fraction (Fr. 7) of larger molecular weight than that (Fr. 9) of intact t-PA. The level of intact rt-PA was increased with a regimen involving the preadministration of cold rt-PA which was followed by the administration of hot rt-PA. The level of PAI-1 in plasma showed an increased rebound 15 minutes after the intravenous injection. These results suggest two possible reasons why rt-PA retains high affinity with fibrinin vitro, once radiolabeled, but was ineffective in delineating fresh thrombi with a gamma camera: 1) some plasma components such as PAI-1 combine with circulating radiolabeled rt-PA and form a larger molecule immediately and/or 2) radiolabeled rt-PA is modulated as a consequence of the radiolabeling and forms a larger molecule than intact rt-PA.     

Current indications and results of thrombolysis by intravenous recombinant tissue plasminogen activator

A number of landmark trials have proven the efficacy of thrombolysis by intravenous recombinant tissue plasminogen activator in the acute phase of the ischemic stroke. Despite the recently extended time window of 4.5 hours, the number of people who are being treated in most centers is low. Several reasons seem to account for this, including poor recognition of symptoms, delays in emergency transport, low levels of public awareness, or age limits originally imposed by drug regulatory rules. Trials are ongoing to possibly extend the indications to the treatment. A major effort is to extend the time window by bridging the treatment with neuroprotective approaches, or by identifying subgroups that may particularly benefit from recanalization and reperfusion. Procedures using ultrasounds or alternative intravenous compounds are also being investigated with promising results.     

静注125I-标记人重组单链尿型纤溶酶原激活剂后酸可沉淀放射性在兔组织的分布

用12 5I 标记单链尿激酶型纤溶酶原激活剂 ( 12 5I labelledsingle chainhumanrecombi nanturokinase typeplasminogenactivator ,( 12 5I rh sc uPA)研究单次静脉推注后三氯醋酸 (TCA)可沉淀放射性在兔体内各组织分布的时间过程。方法 :Iodogen法制备12 5I rh sc uPA ,SephacrylS 2 0 0HR分离纯化 ,反相高效液相鉴定放射化学纯度 ,纤维蛋白平板法测定标记物的生物活性。结果 :制备了符合药代动力学研究要求、有体外溶纤维蛋白活性的12 5I rh sc uPA ,放射化学纯度为94 .4 %。检测灵敏度为 5Bq/g新鲜组织 ,各组织标准曲线的相关系数r >0 .999,回收率 >80 %。分布实验表明 ,酸可沉淀放射性按高到低排列依次为肾 >尿 >血清 >肝 >脾 >肾上腺 >骨髓 >心脏 >膀胱 >肺 >生殖腺 >小肠壁 >脂肪 >淋巴结 >胆囊 >胸腺 >肠内容 >肌肉 >肠内粪 >脑。结论 :静脉推注 12 5　I rh sc uPA后溶栓靶器官血液中的酸可沉淀放射性浓度较高 ,药物主要通过泌尿系统排泄。     

Sequential combination of intravenous recombinant tissue plasminogen activator and intra-arterial urokinase in acute ischemic stroke

BACKGROUND AND PURPOSE: Combined intravenous (IV) and intra-arterial (IA) thrombolytic therapy may be faster and easier to initiate than monotherapy, and its recanalization rate may be better as well. The sequential combination of recombinant tissue plasminogen activator (rTPA) and urokinase (UK) has synergistic and complementary effects on clot lysis. We prospectively evaluated the effectiveness and safety of sequential combination of IV rTPA and IA UK in acute ischemic stroke. METHODS: IV rTPA was administered to patients with acute stroke within 3 hours of onset. Those whose condition had not improved at the end of rTPA infusion were further treated with selective IA UK. We evaluated baseline and 30-day National Institutes of Health Stroke Scale (NIHSS) scores and 90-day modified Rankin Scale scores. RESULTS: Thirty patients were initially treated with IV rTPA; 24 were further treated with IA UK. Four patients who had rapid reocclusion following initial successful IA therapy received IV abciximab. Fourteen of 24 patients who underwent angiography had an effective perfusion state of Thrombolysis in Myocardial Infarction grade 3 flow. Median baseline and 30-day NIHSS scores were 18 and 2, respectively. Eighteen patients improved to a modified Rankin scale score of 0 or 1 after 90 days. Symptomatic hemorrhage developed in two patients. CONCLUSION: The strategy of using conventional-dose IV rTPA and the sequential combination of IA UK in patients without an early clinical response to IV treatment was safe and feasible. This strategy achieved high complete arterial recanalization rates and good functional outcomes.     

Characterization of indium-111 labeled recombinant tissue plasminogen activator for the imaging of thrombi

The in vitro functional properties of recombinant tissue plasminogen activator (rt-PA), its biodistribution in mice, and its pharmacokinetics and clot localization properties in dogs have been investigated after labeling rt-PA with ¹¹¹In. The rt-PA was coupled with the bicyclic anhydride of DTPA using standard methodology. Amidolytic and fibrinolytic assays showed retention of protein activity when rt-PA was conjugated with an average of one DTPA group or less per molecule. Size exclusion HPLC showed each preparation to be radiochemically pure with ¹¹¹In bound exclusively to the attached DTPA groups. Biodistribution in mice showed major accumulation of activity in the liver and kidneys. After administration of 0.5–1.0 mg of the labeled protein to dogs, blood activity decreased with a half time of approximately 5 min in agreement with previous reports of rapid blood clearance. Largely because of decreased blood levels, clot: blood ratios of labeled protein increased rapidly, in one study reaching 6.3 after 31 min, and satisfactory images of fibrin thrombi were obtained. The rt-PA may be labeled with ¹¹¹In without destroying the ability of the protein to localize in clot and images of forming clot can be obtained with this agent within 1 h after administration.