CN113646336A - 安全的牛肝素、制备方法和应用 - Google Patents
安全的牛肝素、制备方法和应用 Download PDFInfo
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- CN113646336A CN113646336A CN201980094221.0A CN201980094221A CN113646336A CN 113646336 A CN113646336 A CN 113646336A CN 201980094221 A CN201980094221 A CN 201980094221A CN 113646336 A CN113646336 A CN 113646336A
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Abstract
本发明涉及用于放大规模生产安全的牛肝素的制备方法,及其生产方法和应用,所述安全的牛肝素由不同选择的未分级的牛肝素聚合物组成,其具有低6‑O‑脱硫酸化的葡糖胺含量和猪样抗凝血活性以及鱼精蛋白中和。这种安全的牛肝素(SB肝素)具有与猪肝素(临床使用参考)可比的结构和功能,作为安全的药学产品可预防临床使用障碍,允许其作为可互换的药物使用。
Description
技术领域
本发明涉及新的且安全的牛肝素的制备,其是来自牛肠黏膜的安全的抗凝血药剂(medication),涉及药物发现与制造的生物技术医学领域。
概述
安全的牛肝素,其是来自牛肠黏膜的具有低的6-O-脱硫酸化的葡糖胺(6-O-desulfated glucosamine)含量和猪样的(porcine-like)抗凝血活性以及鱼精蛋白中和的未分级的肝素,以及其制备方法和应用。
背景技术
糖胺聚糖(GAGs)是线性复杂的杂多糖,其被发现作为细胞外基质(EMC)、细胞表面和细胞内空间(intracellular space)的组分。GAGs的历史可以追溯到19世纪,当时硫酸软骨素(CS)被首次鉴定为软骨的组分,并且它的结构被进一步阐明(DAVIDSON和MEYER,1954)。CS是由葡萄糖醛酸和N-乙酰半乳糖胺的二糖重复单元组成的聚合物,其可在N-乙酰半乳糖胺[→4-β-GlcA-(1→3)-β-GalNAc-(4S)(6S)-1→]的4位或6位被硫酸化。随后的研究表明,CS在自然界中广泛分布,与进一步描述的其他GAGs相似。
奇怪的是,在20世纪初,对于不同组织提取物中促凝血的磷脂的研究发现了抗凝血的GAG。狗肝脏酒精提取物产生了具有出乎意料的抗凝血作用的“脂溶性”级分。所述抗凝血作用与名为肝素(heparin)(hepar是希腊词中的肝脏)的碳水化合物的存在有关(Howell和Holt,1918)(Howell,1925)。后来,NMR分析技术的使用表明肝素由艾杜糖醛酸和N-乙酰葡糖胺的重复二糖单元组成,其主要在N-、6-O-、2-O-位硫酸化[→4-β-IdoA-(2S)-1-(1→4)-α-GlcNAc-(NS)(6S)-1→](Cifonelli和Dorfman,1962)。
尽管有关结构和生物学功能的信息有限,但对抗凝血药物的需求推动了晶体形式的肝素在临床试验中进行测试并进入医药市场(Wardrop和Keeling,2008),证明了其作为抗凝血药物的临床相关性和需求。
后来在色谱法和NMR分析技术上的发展可以阐明用于特定GAG-蛋白质结合相互作用所需的基序(motifs)。与生长因子结合的肝素的HPLC和NMR分析表明,2-O硫酸化的艾杜糖醛酸(Habuchi等人,1992)对于特定的生长因子相互作用(bFGF结合而不是用于FGF-2,FGF-2反而需要6-O硫酸化的N-乙酰葡糖胺(Maccarana等人,1993))是必不可少的。另一方面,除了其他结构修饰外,介导肝素-抗凝血酶III结合和抑制的多糖结合位点需要罕见的N-乙酰葡糖胺上的3-O-硫酸化(Lindahl等人,1980)。这些早期的实例显示了肝素结构调节其相互作用并决定其生物学功能的显著意义。
尽管狗肝脏是肝素的最初来源,但从更实际的观点来看,针对生产的分析表明,从不同组织中提取的肝素制备物显示出在牛肠和肺中高浓度的这种GAG,其被选为临床使用的早期肝素来源。牛未分级肝素(UFH)最初于1939年在美国卫生系统市场上上市,并且此后销售了超过50年。
在80年代早期/中期,由于牛海绵状脑病(BSE)污染的风险,来自牛来源的UFH肝素自发地从主要市场撤出,使得猪黏膜肝素几乎成为全球肝素市场的专有的供应。
然而,这种挽救生命的药物在世界范围内的大量使用使肝素生产面临短缺风险,因为其来源仅限于猪源。此外,由于肝素主要由一种动物来源(猪)和地理区域(中国)生产,因此原材料供应变化导致短缺的风险更高。
随着BSE分析和纯化技术知识的进展,BSE污染的风险被更好地理解。最近的研究已经显示,肝素纯化的不同步骤能够从粗肝素中彻底地去除牛海绵状脑病致病因子(agent)(Bett等人,2017),证明目前这是可控的风险。BSE致病因子检测显示了相似的进展,促进了牛源的纯化的肝素的质量控制。
最近的市场需求与该领域的科学进展相结合,导致市场必须重新引入牛肝素这一常识。监管机构公开宣布了市场重新引入牛肝素的需求(Szajek等人,2016)。
然而,尽管降低了BSE的风险,但牛肝素制备物仍然显示较低的质量和活性,这是对于其重新引入的持续问题。从牛黏膜中提取和纯化的肝素与猪肝素(参考药物)相比具有结构差异。与猪肝素相比,牛黏膜肝素显示出较低的6-O-硫酸化率和较高的N-乙酰化率(葡糖胺残基)(Aquino等人,2010)。与这些结构差异相关的是观察到在体外用全血浆或纯化的凝血因子Xa和IIa其抗凝血活性较低,用离体的治疗后的小鼠血浆其抗凝血活性较低,以及在体内在静脉血栓形成的动物模型中其抗凝血活性较低。这些结果已被其他研究证实(Santos等人,2014,Tovar等人,2016)。与用猪肝素治疗的患者相比,在透析患者的血浆中也证明了牛肝素的抗凝血活性较低(Tovar等人,2013)。
尽管具有较低的抗凝血活性,牛肝素样品还具有较高的出血倾向,如动物模型中较高的失血率所显示。此外,据观察,需要更高浓度的鱼精蛋白(肝素解毒剂)去中和牛肝素抗凝血活性(Aquino等人,2010)。结合两种观察表明了在用牛肝素替代猪肝素后在巴西卫生系统中观察到的临床检测到的出血事件增加的机制的决定因素(Melo等人,2008;Junqueira等人,2011)。因此,与猪肝素相比,这种结构和功能的差异给其临床使用带来了关键风险。
综上所述,尽管卫生系统的需求和BSE预防取得发展,但牛肝素在市场上的重新引入受到其较低的抗凝血活性和较高的出血特性的约束。因此,确定未分级牛肝素的多分散异质群体的结构/功能是必不可少的,目标是可规模化的生产具有与市场上的参考产品(猪肝素)相似的临床活性的新一批(poll)的高质量牛肝素,避免临床施用期间的变化(其已显示对患者的生存具有有害影响)。此外,在SPACENET和USPTO上的专利检索确实表明了任何现有专利(具有能够生产具有与猪肝素相似的结构和功能活性的牛肝素的创新)暴露了在该领域知识的缺乏(表1)。
表1.显示在SPACENET和USPTO上检索后,该领域最相关的专利。已经公布了改善肝素纯化的不同途径;然而,没有能够将牛肝素制备物转化为猪样的临床质量的途径。
发明公开内容
本发明提供了从牛肠黏膜中获得具有低6-O-脱硫酸化的葡糖胺含量的未分级肝素制备物的工艺(process),使肝素制备物具有与猪黏膜肝素(市场参考产品)可比的结构和抗凝血活性,这里称为安全的牛肝素(SB肝素)。
肠黏膜牛肝素级分的详细分析表明了高的6-O-脱硫酸化的葡糖胺含量,这与下述有关:较低的抗凝血活性,且需要较高的鱼精蛋白(解毒剂)中和浓度。
本发明工艺包含用精制离子交换步骤分级肠黏膜衍生的牛肝素。离子交换步骤用足够的盐洗脱浓度进行,以使牛肝素具有与猪肝素可比的结构和抗凝血活性。
如下文所述,与传统的肠黏膜牛肝素相比,这些牛肝素制备物含有低的6-O-脱硫酸化的葡糖胺含量。
附图说明
图1显示了与猪肝素相比,未分级的牛肝素的结构。图1A显示了猪和牛的一维NMR图谱;图1B显示了来自猪和牛肝素的不同商业制备物的每个一维峰的百分比计算;图1C显示了猪和牛肝素的预测结构。
图2显示了与猪肝素相比,未分级的牛肝素的功能(活性和鱼精蛋白中和)。图2A显示了猪和牛肝素在体外的抗凝血活性;图2B显示了猪和牛肝素在体内的抗凝血活性;图2C显示了猪和牛肝素的鱼精蛋白中和。
图3显示了猪和牛衍生的肝素之间基于它们的活性和出血倾向的比较。图3A显示了在大鼠腔静脉中使用淤滞(stasis)和高凝性血栓形成模型的静脉抗血栓形成的活性。施用不同剂量的牛或猪肝素并允许其循环5分钟。然后,将促凝血酶原激酶(5mg kg-1体重)缓慢静脉内注射,并结扎0.7cm分离的腔静脉段。淤滞20分钟后,将形成的血栓干燥并称重。结果表示为血栓重量的%,100%代表不存在任何血栓形成抑制(不施用肝素时的血栓重量)。图3B显示了在血管内施用肝素之前和之后5分钟从颈动脉收集的枸橼酸化的血样(citrated blood samples)。然后,测定血浆的离体aPTT。结果表示为施用不同肝素剂量(T1)和施用对照(盐水处理的动物)(T0)后凝血时间的比率。测定中未检测到高于10的值。图3C显示不同剂量的肝素被注入大鼠。5分钟后,将大鼠尾巴从尖端切下3mm,并在室温下浸入40ml蒸馏水中。60分钟后通过测量水中的血红蛋白确定失血。结果以失血的μL表示。面板(Panel)A和C中的插图表示基于抗凝血活性(IU kg-1)的剂量对比反应的曲线。为了清晰,面板中仅显示了一个标准误差(SE)条。对于牛肝素对比猪肝素,所有结果都使用Mann-Whitney秩和检验,表示为平均SE,n=5,*p<0.01和**p<0.05。面板A和C插图中的箭头指示达到完全抑制血栓形成所需的肝素剂量(100IU kg-1体重)。
图4显示了肝素通过鱼精蛋白的中和过程。肝素(0.1IU ml-1)与浓度增加的鱼精蛋白一起孵育,并且然后与10nM抗凝血酶和2nM因子Xa在40μl TS/PEG缓冲液中混合。在37℃下孵育60秒后,用显色底物测定剩余的因子Xa活性(A405nm min-1)。
图5显示了在与HPLC系统偶联的离子交换色谱1mL柱上对SB肝素生产的优化。
图6显示了在与HPLC系统偶联的离子交换色谱10mL柱上对放大规模SB肝素生产的优化。
图7显示了结构(一维氢核磁共振图谱)和功能(全血浆(aPTT)和纯化的凝血因子(凝血酶和Xa)测定。
本发明的实施方式
本发明涉及牛黏膜衍生的未分级的肝素制备物(其呈现与猪黏膜肝素可比的结构和抗凝血活性),其制备方法和用途,由市售可获得的肝素制备。“安全的牛肝素”或“SB肝素”是指具有低6-O-脱硫酸化的葡糖胺含量的制备物,其具有猪黏膜肝素样结构和抗凝血活性。
在本发明的上下文中,肝素的抗凝血活性涉及抗凝血酶III(AT)对Xa和IIa凝血因子的抑制。
一方面,本发明涉及制备具有与猪肝素的临床治疗作用相似的临床治疗作用(包括抗凝血活性和鱼精蛋白中和)的牛肝素的方法。
通过核磁共振(NMR)的1H和13C一维(1D)和二维(2D)图谱分析市售可获得的猪和牛肝素制备物表明,猪肝素主要由三硫酸化的二糖单元组成,牛肝素由高度2-硫酸化的艾杜糖醛酸残基组成,但牛肝素缺乏葡糖胺单元上的6-O-硫酸化。
对具有不同结构组成的肝素的详细分析指示,牛肝素的葡糖胺上缺乏6-O-硫酸化的二糖的存在与较低的抗凝血活性、较高的流血作用和较低的鱼精蛋白中和率有关。
更具体地,经确定当与猪肝素相比时牛肝素在全人血浆实验(aPTT)中具有较低的抗凝血活性(图2A)。aPTT是用人血浆、各种肝素浓度、诱导凝血的aPTT试剂(牛磷脂试剂)和CaCl2进行的,这在凝血计上记录。结果表示为存在(Ti)或不存在(To)肝素时凝血时间的比率。使用基于5th国际肝素标准(229IU mg-1)的平行标准曲线以IU mg-1估计抗凝血活性,该标准获自国家生物标准和控制研究所(波特斯巴,UK)。牛和猪肝素的溶液按重量基础制备,并在通过咔唑反应检查时显示出相似的己糖醛酸含量。
事实上,使用具有纯化的凝血因子的测定证实了与猪肝素相比牛肝素较低的抗凝血活性(图2B和2C)。因子Xa和凝血酶(抗Xa和抗IIa测定)在存在显色底物(S2238用于凝血酶,S-2222用于因子Xa)的情况下使用,并且酶标仪(microplate reader)在405nm处记录吸光度300秒。吸光度的变化率与溶液中剩余的凝血酶或因子Xa的活性成比例。使用基于国际肝素标准(229单位mg-1)的平行标准曲线,抗IIa和抗Xa活性以单位mg-1报告。
在另一方面,使用具有兔脑促凝血酶原激酶作为形成血栓的刺激物的大鼠在体内证实了与猪肝素相比,牛肝素具有较低的抗凝血/抗血栓形成的活性(图3A)。按照动物护理的机构指南,且使实验大鼠(两种性别,~200g体重,每剂量5只动物)麻醉,并将不同剂量的肝素注入右颈动脉并允许其循环5分钟。分离下腔静脉,且缓慢地静脉内注射脑促凝血酶原激酶(5mg kg-1体重);1分钟后,使用远端和近端缝合线夹住0.7cm分离的腔静脉。淤滞20分钟后,小心地取出封闭段内形成的血栓,用磷酸盐缓冲盐水冲洗,在60℃下干燥1h,并且称重。平均血栓重量通过每组的平均重量获得,然后表示为重量的百分比,100%代表不存在任何血栓形成抑制(不施用肝素时的血栓重量)。
与猪肝素相比,注射牛肝素的大鼠血浆显示出较低的抗凝血活性,这些结果通过血管内施用肝素之前和之后5分钟从颈动脉收集的枸橼酸化的血样得到证实(图3B)。测定血浆的离体aPTT。结果表示为施用不同肝素剂量(T1)和对照(盐水处理的动物)(T0)后凝血时间的比率。
在抗凝血疗法期间出血倾向是一个重要的方面,在其临床使用期间呈现为具有死亡风险的重要副作用。对大鼠血管内施用牛肝素后流血倾向的分析显示,与施用牛肝素相比,其失血率更高(图3C)。重要的是要注意,分析显示两种肝素在体重基础上具有相同的剂量依赖性流血诱导。然而,基于抗凝血活性的曲线(插图)清楚地显示,牛肝素在诱导流血方面的效力是猪肝素的两倍。由于在临床上使用肝素是基于其抗凝血活性施用,牛肝素的出血倾向是猪肝素的两倍,使其对健康构成极大风险。
在肝素的临床使用期间,在体外循环结束或检测到药物过量时,肝素的中和需要适当剂量的鱼精蛋白。具有不同化学和生物学特性的肝素,诸如牛肝素和猪肝素,可能表现出不同的鱼精蛋白中和曲线。将鱼精蛋白以相似剂量添加到牛肝素或猪肝素中(以IU基础),并基于抗Xa活性评估肝素中和,证明牛肝素需要比猪肝素显著更高剂量的鱼精蛋白以实现中和(图4)。这种鱼精蛋白中和的变化是市售牛肝素产品的关键方面,因为在临床规程期间难以解释这种变化,从而给患者的健康带来潜在风险。
这些发现在肝素的起源、结构和功能之间建立了新的相关性。更具体地,它确定了含有6-O-脱硫酸化的葡糖胺的肝素片段与较低的抗凝血活性和鱼精蛋白中和以及较高的出血效应有关。
基于这些发现,发展了从肠黏膜中纯化的牛肝素中去除含有6-O-脱硫酸化的葡糖胺的肝素链的创新工艺,使纯化的牛肝素具有与猪肝素可比的结构和功能。令人惊讶的是,对不同纯化策略的检验表明,可以使用具有离子交换色谱的优化的色谱规程进行去除含有6-O-脱硫酸化的葡糖胺的肝素链。更具体地,从牛黏膜中生产这些新的富含三硫酸化的肝素是使用可规模化的纯化规程完成的,该规程使用基于合成的甲基丙烯酸酯(methacrylate)的聚合物基质,该基质具有携带三甲基铵乙基(trimethylammoniumethyl)的功能配体的长线性聚合物链。
本发明显示,使用离子交换色谱精制纯化步骤能够生产安全的牛肝素(SB肝素),其是纯化的牛肝素,具有低的含有6-O-脱硫酸化的葡糖胺的肝素聚合物和高的三硫酸化二糖单元,具有与猪肝素可比的结构和功能。由于其与市场参考(猪肝素)的相似性,这被认为是用于临床的安全的牛肝素,允许在临床医学中互换使用。
为了去除含有6-O-脱硫酸化的葡糖胺的肝素聚合物,使用来自MERCK的离子交换柱TMAE HICAP与HPLC系统偶联(优化显示在图5和6中)。使用市售可获得的肠黏膜牛肝素作为起始材料,其比活性(specific activity)为大约100IU/mL。该肝素粉在运行缓冲液(20mM Tris,具有0.02-0.1M NaCl,pH 7.2)中稀释,并以3.5ml/min的流速应用到TMAE柱上。键合肝素的洗脱通过初始冲洗步骤,然后是盐浓度范围为0.5M至2.0M NaCl的两个逐步条件进行。第一个洗脱步骤产生了含有低抗凝血和高6-O-脱硫酸化的葡糖胺的肝素,然后将其丢弃。收集第二个峰;通过对蒸馏水透析去除盐并冻干。然后将纯化的牛肝素的结构和功能与猪肝素和市售可获得的牛肝素进行比较分析。
对纯化牛肝素的分析表明,这种纯化方法使牛肝素具有与猪肝素相似的结构和抗凝血活性。通过核磁共振进行的结构分析证明,从SB肝素制备方法中去除了含有6-O-脱硫酸化的葡糖胺的肝素聚合物,如缺乏峰C所代表的二硫酸化的6-O-脱硫酸化的葡糖胺二糖残基所证明(图7A)。因此,这种创新的生产工艺对于去除肠牛肝素中的6-O-脱硫酸化的葡糖胺聚合物是有效的,生产出具有与猪肝素结构相似的肝素制备物。
更重要地,纯化的牛肝素显示出与猪肝素可比的抗凝血活性(图7B)。使用全人血浆进行体外抗凝血测定表明,纯化的牛肝素的抗凝血活性略低于猪肝素。然而,用纯化的凝血因子Xa和IIa进行的抗凝血测定显示,纯化的牛肝素的抗凝血活性略高于猪肝素,但在统计学上可以认为是相似的。使用纯化的凝血因子进行的测定是美国药典接受的作为抗凝血活性的官方决定因素的测定。
因此,本发明公开内容是通过使用单个精制纯化预放大步骤,使肠的牛肝素生产出具有与猪肝素相似结构和功能的制备物,一旦它可以在临床医学中互换使用,便可被认为是安全的牛肝素(SB肝素)。
在本发明的方面,使用与季氨基乙基功能配体连接的单珠载体(monobeadssupport)的实验室规模纯化规程也生产了具有猪样结构和功能的富含三硫酸化的牛肝素。这些结果指示,在优化工艺后,其他离子交换树脂可用于生产含低6-O-脱硫酸化的葡糖胺的肝素。此外,在本发明的方面,该纯化步骤可用于纯化工艺的任何步骤。
实施例
以下实施例旨在说明而非限制本发明。
起始材料是来自肠黏膜的牛肝素(Extrasul S.A.),其比活性为大约100IU/mL,且结构富含6-O-脱硫酸化的葡糖胺。将30mg量的牛肝素稀释在3mL运行缓冲液(20mM Tris pH7.2,具有100mM NaCl)中。
TMAE HICAP 1mL柱在具有10倍柱体积运行缓冲液的HPLC系统中平衡,并以3.5mL/min的流速应用3mL牛肝素样品。用5倍柱体积的运行缓冲液冲洗后,先用5倍柱体积的NaCl步骤(0.93M NaCl),然后用5倍柱体积冲洗去除含有6-O-脱硫酸化的葡糖胺的聚合物。用第二个5倍柱体积的2M NaCl步骤洗脱并收集富含三硫酸化的肝素聚合物。收集的样品对蒸馏水透析并冻干。
纯化的样品在蒸馏水中稀释,并且浓度通过咔唑反应定量糖醛酸来确定。然后将样品提交给核磁共振结构分析,其表明了猪肝素样的结构,该结构缺乏先前在市售可获得的牛肝素中观察到的6-O-脱硫酸化的葡糖胺峰。使用纯化的凝血因子测定(Xa和凝血酶)表明活性为大约190mL IU/mL,在统计学上与180IU/mL的猪肝素活性相似。基于活性(IU单位)的回收率为85%,这是肝素制备物生产的决定因素。
这里显示了改进牛肝素生产的工艺,产生了安全的牛肝素(SB肝素),其结构和功能与猪肝素(市场参考)可比,产率为85%。这项创新允许廉价生产高质量的牛肝素,其在临床医学中可以与猪肝素互换使用。这对于整个世界的肝素生产(预防短缺风险)和清真市场具有至关重要的卫生系统价值。
参考文献
Aquino,R.S.,Pereira,M.S.,Vairo,B.C.,Cinelli,L.P.,Santos,G.R.,Fonseca,R.J.andP.A.(2010)'Heparins from porcine and bovine intestinalmucosa:Are they similar drugs?',Thromb Haemost,103(5),pp.1005-15.
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Claims (11)
1.安全的牛肝素(sb肝素)钠,其包含牛肠黏膜肝素的制备物。
2.具有低6-O-脱硫酸化的葡糖胺和猪样的临床疗法特性的未分级肝素,其包含牛肠黏膜肝素的制备物。
3.具有与猪肝素可比的抗凝血活性和鱼精蛋白中和的肝素,其包含在牛肠黏膜肝素的制备物中。
4.如权利要求1-3所述的牛肝素,其中所述结构组成含有高三硫酸化的二糖单元和低6-O-脱硫酸化的葡糖胺聚合物。
5.如权利要求1-3所述的牛肝素,其中大约190UI/mL的活性在统计学上与猪肝素相似。
6.如权利要求1-5所述的牛肝素的制备,其包含步骤:
-牛肠黏膜在运行缓冲液中的增溶;
-通过用不同树脂的离子色谱法纯化;
-过滤后冲洗并干燥以获得所述安全的牛肝素(SB肝素)钠;猪样肝素;以及高抗凝血的牛肝素。
7.如权利要求6所述的牛肝素的制备方法,其包含生产性色谱系统的大规模生产的步骤;使用盐排除方法。
8.根据权利要求1-5所述的牛肝素,其包含抗凝血、抗血栓形成和清真药剂。
9.根据权利要求1-5所述的牛肝素,其包含注射抗凝血、抗血栓形成和清真药剂。
10.根据权利要求1-5所述的牛肝素,其包含抗炎和/或抗转移的药剂,炎症和癌症治疗。
11.根据权利要求1-5所述的牛肝素,其包含用于不同类型的低分子量肝素钠生产的原料来源。
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WO2016137471A1 (en) * | 2015-02-26 | 2016-09-01 | Nantpharma, Llc | Method for enhanced heparin quality |
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Title |
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