CN106102764A - 在出血性综合症情况下能够控制出血和/或取代血小板的基于胶原蛋白的注射制剂 - Google Patents
在出血性综合症情况下能够控制出血和/或取代血小板的基于胶原蛋白的注射制剂 Download PDFInfo
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Abstract
本发明涉及一种注射制剂,其包括具有小于10μm长度的颗粒或纤丝以及至少一种药学上可接受的载剂或赋形剂。所述颗粒或纤丝包括诱导血小板黏附和激活,或者甚至其聚集的蛋白质或肽。所述制剂用于治疗出血。
Description
技术领域
本发明涉及基于胶原蛋白的注射制剂,其用于治疗出血。所述注射制剂由源自胶原蛋白的蛋白质和肽组成,它们本身能够诱导血小板的黏附和激活,以及甚至它们的聚集。本发明也涉及在紧急情况(出血、外科手术)下通过全身/一般途径将源自胶原蛋白的蛋白质和肽紧急注射到用抗血小板药物治疗的患者中。
背景技术
出血被定义为在正常血液之外的血流。其能够由简单出血组成,或者在某些情况下特别是创伤时演变为大量出血,并且导致与血压下降相联系的失血性休克状态。
各种临床情境会导致出血风险,诸如外科手术、创伤、治疗、血小板减少症或者体质或功能缺陷(诸如血友病)(Spahn D等,Critical Care.2013,17:R76)。在严重出血情况下,观察到凝血功能障碍。凝血功能障碍对应于血小板和凝血因子消耗。治疗是必不可少的,而且出血的后果决定于治疗。目前遵循各种治疗策略。
一种方法包括基于新鲜冷冻血浆(FFP)、浓缩红细胞或血小板使用输血支持疗法。但是,基于血小板的方法成功受限于许多因素,包括血小板的可得性、它们保存期短(平均5天)、与存储条件相关的问题、某些类型的样品的污染风险、它们的成本和无效风险,甚或由于异源免疫导致的不耐受。
另一个方法基于使用凝血因子浓缩物,诸如纤维蛋白原浓缩物和凝血素复合物浓缩物(因子II、VII、IX和X)。研究也建议使用因子IIa然而,不推荐该最后的用途,因为其诱导高比例血栓形成。
最后,在紧急情况下的另一种方法由使用凝血酸组成,所述凝血酸是一种强大的纤溶酶原激活拮抗剂,并因而作为抗纤维蛋白溶解药起作用(Fries D.Transfusion.2013;53:91S–95S)。
目前提出的方法中没有任何一种能够在出血发作期间诱导血小板激活和聚集,尽管这些步骤是止血的关键。当血管损伤导致出血时,被称为初期止血的一系列步骤到位。其涉及血小板,即无细胞核、直径约2–5μm的双凸圆盘状细胞。各种角色参与这一过程,其导致形成止血凝块。第一阶段称为黏附,使得循环的血小板募集在损伤位点。这一步骤涉及血管性血友病因子、胶原蛋白和称为胶原蛋白受体(GpVI和α2β1)的血小板受体。第二阶段称为血小板激活,包括致聚因子的释放和诸如P-选择素的蛋白质在血小板表面的表达。这一步骤之后是称为聚集的阶段,其特别涉及纤维蛋白原和血小板受体GpIIb/IIIa以及促凝血磷脂曝露于膜上,使得凝血因子固定到损伤位点并最终形成纤维蛋白/血小板凝块并阻止出血。
目前已经确认,在止血过程中血小板的激活导致出现两种血小板群体,称为致聚和促凝血群体。后者的特征在于在凝血酶生成起点处表面表达磷脂酰丝氨酸。这些被称为超激活的血小板。因此,我们谈及血小板超激活潜力用以描述每个个体在应答诸如凝血酶和胶原蛋白的激动剂中产生这一血小板群体的能力。最近的研究表明,旨在诱导这一激活血小板群体出现的任何治疗策略对于出血性病况的治疗显示巨大潜力(Mazepa M etal.ATVB 2013,33(8):1747–52)。此外,他们的重要性通过确认输注血小板的止血效果依赖于这一群体的数据得到强化。但是,目前提出的方法并不是基于血液注射生理的或天然的血小板激活剂诸如胶原蛋白或者本发明所请求的源自胶原蛋白的蛋白质和肽。
血小板在止血中的主要作用是旨在开发源自血小板的产品或者模拟血小板的产品(合成血小板)的最近的工作的出发点。在所提出的方法中,基于源自细胞(诸如血栓红细胞和血栓细胞)的产品的那些方法与使用下述微米尺寸的颗粒的那些方法之间有明显区别:覆盖源自血小板激活级联反应涉及的蛋白质的肽的颗粒,诸如基于源自纤维蛋白原的肽(RGD肽或者十肽H12)的合成细胞(synthocytes)或FibrocapsTM,或者覆盖与血管性血友病因子结合、与胶原蛋白结合及与GpIIb/IIIa结合的肽的那些颗粒(Lashof–Sullivan Met al.Nanoscale 2013,5,10719–10728)。已经描述了覆盖与血管性血友病因子结合、与胶原蛋白结合及与GpIIb/IIIa结合的肽的那些颗粒具有止血效果,其使得可以设想在出血情境中应用。但是,这一方法具有的主要问题在于这种类型的产品的工业化,所述产品在单一颗粒上组合了许多肽,有必要熟练一些参数并使其达标,诸如结合率和各个肽的比例,以及产品一旦注射后的稳定性,不考虑当前必须注射非常高的剂量(每公斤几十毫克的等级)(Modery–Pawlowski C et al.Biomaterials 2013:516–541)。
因此,需要获得可注入血液的治疗出血的新颖技术手段。优选地,它们能够容易地工业制备,并且能够在低浓度下使用。
随着人口年龄的增长,医生越来越经常面对经抗凝血剂和抗血小板药物治疗的患者。尽管程序化干预已经明确了暂时撤销这些治疗的策略(对于新颖抗血小板药物撤销5-7天,和对于新颖抗促凝血剂撤销5天),但它们使得出血性病况(外科手术或者创伤,特别是颅脑创伤)下患者的急救治疗严重复杂化(Bonhomme F et al.Eur J InternMed.2014Mar;25(3):213–20)。已经提出了急救复原的许多策略,但是,对于新颖抗血小板药物而言,它们主要限于血小板输血(Beynon C et al.Crit Care.2012Jul 26;16(4):228)。需要新颖的注射止血产品,其在出血性发作期间能够不依赖于这些新颖抗血小板药物的作用模式而使得血小板功能恢复。胶原蛋白依赖性血小板激活途径是最具生理性的,并且到目前为止未被市面上的任何抗血小板治疗所修改,这一事实强化了在这些情况下通过全身途径投予源自胶原蛋白的蛋白质或肽的重要性。已经明确,GPVI与胶原蛋白或与本发明的源自胶原蛋白的蛋白质或肽的结合导致血小板内的剧烈信号传导。
在限制为后方勤务的情境下,诸如海外军事战区,出血治疗受控于“损伤控制性复苏”的概念,特别涉及输血(浓缩红细胞、血浆和血小板)。然而,这一情况的特征在于获得这些血液产品,特别是血小板困难,需要涵盖于“远程损伤控制复苏”术语下的改进治疗,最主要的区别在于使用全血(Jenkins DH et al.Shock.2014May;41Suppl 1:3–12)。虽然这一策略具有污染的风险,但它是有前景的,即便目前仍限于军事领域(Murdock AD etal.Shock.2014May;41Suppl 1:62–9)。
本发明因此涉及在以下三种涉及出血的情况下,将源自胶原蛋白的血小板激活蛋白质或肽作为新颖注射止血剂静脉投药的利益:
-在大出血发作期间,特别是非压缩大出血发作期间,需要激活血液中的血小板的能力;
-在远离医疗中心的输血复苏期间作为佐剂,作为基于特别是全血或血小板输血的方法的补充;
-作为目前在市面上没有解毒剂的新颖抗血小板药物(例如普拉格雷、替格瑞洛)的恢复剂,用于特别是颅脑的外科手术或出血。
发明内容
本发明涉及一种注射制剂,其用于治疗出血的用途,其包括:
-具有小于10μm长度的颗粒或纤丝,这样的颗粒或纤丝蛋白或肽诱导血小板的黏附和激活,甚或它们的聚集;和
-至少一种药学上可接受的载剂或赋形剂。
附图说明
图1显示由DLS测量序列ID No.1多肽的蛋白提取物经浓缩50倍后的粒径。
图2显示由ELISA获得的以2IU/dL终浓度使用的纯化血管性血友病因子(Willfactin,LFB)与以不同粒径的颗粒形式存在的、以2μg/mL终浓度使用的序列ID No.1多肽的结合结果,以及与以10μg/mL使用的I型胶原蛋白(SIRCOL胶原蛋白,阳性对照)的结合结果。
图3A表示由流式细胞术获得的、在针对序列ID No.1多肽和I型胶原蛋白(Horm)的血小板表面上的典型P-选择素表达谱。
图3B表示针对各种激动剂获得的平均荧光强度。
图4显示用颗粒形式的序列ID No.1多肽获得的聚集结果。
图5显示由各种显微技术观察到的颗粒形式的序列ID No.1多肽相比纤丝形式的I型胶原蛋白(Horm)的大分子结构。
图6表示由流式细胞术获得的、在小鼠中注射序列ID No.1多肽和I型胶原蛋白(Horm)之后采样的血小板表面上的P-选择素表达谱。
图7显示在小鼠中诱导肺栓塞的模型中,在肾上腺素存在下,将纤丝形式的I型胶原蛋白(Horm)和颗粒形式的序列ID No.1多肽静脉注射到小鼠中的结果。图7A显示注射之后获得的关于小鼠死亡率的结果,并且图7B显示在投药各种测试的胶原蛋白之后至少5分钟注射的锝放射性标记的白蛋白大颗粒的生物分布。
图8显示在小鼠中诱导从尾巴出血模型中将序列ID No.1多肽注射到小鼠中的结果。
序列说明
序列ID No.1:编码能够诱导血小板黏附和激活的重组蛋白的多肽。
具体实施方式
本发明涉及一种注射制剂,其用于治疗出血的用途,其包括:
-具有小于10μm长度的颗粒或纤丝,所述颗粒或纤丝包括诱导血小板的黏附和激活的蛋白或肽;和
-至少一种药学上可接受的载剂或赋形剂。
根据本发明的注射制剂能够刺激血小板活性。在某些实施方案中,所述颗粒或纤丝包括一旦注射入血液中即诱导血小板黏附和激活,以及血小板聚集的蛋白质或肽。后者也能用作佐剂以在血小板浓缩物或全血输血过程中预激活血小板。
诱导血小板黏附、激活和甚至聚集的蛋白质或肽
构成本发明的制剂配方的蛋白质或肽具有诱导血小板黏附和激活,或者还诱导血小板聚集的能力。应当理解这两种或三种活性是一种蛋白质或肽及相同蛋白质或肽的特性。这些蛋白质或肽以具有小于10μm的长度的颗粒或纤丝形式存在。
优选地,所述纤丝具有小于9μm、8μm、7μm、6μm、5μm、4μm、3μm、2μm、1μm或0.5μm的长度。更优选地,所述纤丝具有介于0.5μm和9μm、介于1μm和5μm,或介于2μm和4μm之间的长度。
这些蛋白质或肽能够结合或激活负责血小板黏附的血管性血友病因子,并且能够诱导血小板激活标志物P选择素或磷脂酰丝氨酸的表面表达。在某些实施方案中,所述蛋白质或肽能够诱导富含血小板的血浆或者全血的体外聚集。
在某些实施方案中,所述蛋白质或肽能够刺激血小板并且诱导止血,而不必能够诱导凝血激活,凝血激活依赖于磷脂酰丝氨酸在血小板表面的表达、形成据说是促凝血的表面、并且其结果导致产生凝血酶。这使得全身注射制剂之后诱导的血栓形成风险被限制。不过,会发生这一被称为促凝血血小板的活化血小板亚群体的激活,但其水平低于可通过经典测试检测的产生凝血酶所必须的那种水平。
一个被称为胶原蛋白的蛋白质家族由于在某些胶原蛋白(主要是I型和III型胶原蛋白)的序列中存在能够与循环的血管性血友病因子结合并能够与两种血小板受体GpVI和α2β1结合的肽序列而具有诱导前述三种活性的能力。这导致血小板黏附,导致它们的激活,且然后导致它们的聚集。这样,构成本发明的制剂配方的蛋白质可以从胶原蛋白,并且特别从I型和III型胶原蛋白中选择。
到目前为止,尚未提出使用胶原蛋白治疗出血。这能够通过天然胶原蛋白中仅描述了以纤丝形式存在的那些能够诱导血小板的黏附、激活和聚集这一事实来解释(Clementson K.Thrombosis Research 2012 129 220–224)。但是,含有能够达到若干微米长度的纤维的这种纤丝形式不适合于注射到血液中。本发明的特定形式适合于注射到血液中。
特定形式的胶原蛋白,诸如微球体、微绢丝(microflorettes)和树状大分子能够通过诸如由Pires M和Chmielewski J.J.Am.Chem.Soc.2009131,2706–2712;Przybyla D等,J.Am.Chem.Soc.2013,135,3418–3422;Kojima C等,J.Am.Chem.Soc.2009 131,30652–6053;Slatter D等,Peptides 2012 36,86–93;Kar,Journal of Biological Chemistry2006vol 281,no.44 33283–33290描述的那些方法获得。为了获得这些特定形式,已经使用各种方法,特别基于使用金属或盐,基于在肽序列中增加半胱氨酸(二硫键)或芳香族氨基酸,或者基于不同性质的氨基酸(极性、非极性、酸性、碱性)之间的比例。
除胶原蛋白以外,构成本发明的制剂配方的蛋白质可为重组蛋白质,诸如国际专利申请WO 2010/034718中描述的那些。这些源自胶原蛋白的重组蛋白质能够以与天然胶原蛋白等价的方式诱导血小板聚集。可用于本发明且源自胶原蛋白的其它蛋白质是描述于欧洲专利申请EP2013/071816中的那些。这些蛋白质能够与血管性血友病因子结合,这种结合与血小板在受损害的血管位点起始黏附相关。
这样,构成本发明的制剂配方的蛋白质可以从天然胶原蛋白诸如I型和III型胶原蛋白,或者其序列包括至少一种选自如下的多肽的重组蛋白质中选择:
-序列ID No.1多肽;
-具有由GXY三联体重复形成的序列的多肽,其中G指代甘氨酸,并且X和Y指代任何氨基酸,重复n次,n大于或等于2;
-具有序列ID No.1从25位至184位的序列的多肽;
或者
-其全长与序列ID No.1多肽,或者与具有序列ID No.1从25位至184位的序列的多肽具有至少70%同一性的多肽。
此外,具有每3个氨基酸含一个甘氨酸的序列的天然或合成肽或多肽可以被认为是胶原蛋白衍生物(An B等,Frontiers in chemistry.2014June;2文章40)。这一链的一级结构在氨基酸(例如半胱氨酸)或者使得形成多聚体结构的肽序列(例如MBL(甘露糖-结合血凝素)蛋白质的三聚体化结构域)存在下诱导与胶原蛋白构象类似的肽链构象。
从序列ID No.1的1位至24位的多肽对应于使得宿主细胞分泌重组蛋白的信号肽。这一信号肽可能不存在或者根据本领域技术人员熟知的技术由另一个信号肽代替。本领域技术人员将能够选择适于在各种原核或真核表达系统中表达和分泌多肽的同源信号肽或异源信号肽。优选地,所述多肽在原核细胞或原核有机体中产生,并且特别地在哺乳动物细胞中产生。在本发明的一个特定实施方案中,所述多肽包括信号肽,其使得它们分泌到细胞外介质中。在另一个实施方案中,在切割所述信号肽之后获得成熟多肽。
在某些实施方案中,重组蛋白质的序列包括含有下述肽序列的至少一种多肽:
-GX1X2GER,其中X1和X2独立地表示选自A、R、N、D、Q、E、G、H、I、K、M、F、P、S、T、W、Y、V和O的氨基酸;
-(GPX3)n,其中n介于4和10之间,并且X3表示P或O;和
-GPRGQX4GVMGFX5,其中X4和X5独立地表示P或O。
P表示脯氨酸并且O表示羟脯氨酸。
在某些实施方案中,重组蛋白质的序列包括含有下述肽序列的至少一种多肽:
-GAPGER,
-KPGEPGPK,
-(GPP)n,其中n介于4和10之间,
-RGD。
这样,所述重组蛋白质包括:
(a)具有4个GPO三联体的至少一次重复的肽序列;
(b)对于各种血小板受体具有结合活性并且存在于胶原蛋白天然序列中的肽序列;和
(c)在序列(a)和序列(b)之间由GXY三联体重复形成的结合序列,其中G指代甘氨酸,并且X和Y指代任何氨基酸。
构成本发明的制剂配方的重组蛋白质可以呈现包括至少一种多肽的序列,所述多肽全长与序列ID No.1多肽,或者与具有序列ID No.1从25位至184位的序列的多肽具有至少70%同一性。在某些实施方案中,所述多肽呈现与序列ID No.1多肽至少70%、80%、90%、95%、98%和优选至少99%的同一性。该百分比同一性指代相同氨基酸(两个序列之间非变体或未改变的氨基酸)的百分比。这些多肽可以呈现对于序列ID No.1多肽的至少一个氨基酸的缺失、添加或取代。
测量多肽之间同一性百分比的方法是本领域技术人员已知的。可以使用VectorNTi 9.1.0、比对程序AlignX(Clustal W算法)(Invitrogen INFORMAX,http://www.invitrogen.com)。优选使用系统设定的参数。
重组蛋白质可以通过本领域技术人员熟知的方法,特别是WO 2010/034718和EP2013/071816中描述的那些方法,由细菌和哺乳动物细胞产生。
在某些实施方案中,所述蛋白质也能够特别地通过序列中存在的重复n次(n=3–10)的GPO序列而与受损害血管位点上的胶原蛋白结合。
在某些实施方案中,所述蛋白质可以是聚乙二醇化或者聚硫酸铝化的(添加脯氨酸、丙氨酸和丝氨酸n次)。
构成本发明制剂配方的肽可为上述的那些。这样,所述肽可以选自:
-序列ID No.1多肽;
-具有由GXY三联体重复形成的序列的多肽,其中G指代甘氨酸,并且X和Y指代任何氨基酸,重复n次,n大于或等于2;
-具有序列ID No.1从25位至184位的序列的多肽;
或者
-其全长与序列ID No.1多肽,或者与具有序列ID No.1从25位至184位的序列的多肽具有至少70%同一性的多肽。
所述多肽从天然环境中分离或纯化。所述多肽可以通过各种方法制备。这些方法特别是由合适的宿主细胞生产重组多肽以及它们的后续纯化、由化学合成法生产、或者最终将这些不同的方法组合。这些不同的生产方法是本领域技术人员熟知的。优选地,所述多肽由原核重组细胞或者真核重组细胞产生。这样,所述多肽可以在细菌中或者在哺乳动物细胞中生产。
在本发明的某些实施方案中,所述多肽是糖基化的。所述序列ID No.1多肽特别在存在于102位和141位上(在一个GXY三联体的3位上)的赖氨酸处具有O-糖基化位点。在一个优选的实施方案中,在所述序列ID No.1多肽的93位上的天门氨酸残基是糖基化的。
纤丝形式的蛋白质
构成根据本发明的注射制剂配方的蛋白质可以形成具有小于10μm长度的纤丝形式的结构。包括具有大于10μm长度的纤维的纤丝形式,以及可以达到若干微米长度的纤丝形式不适合于注射到血液中。
可以通过本领域技术人员熟知的方法,特别通过显微镜诸如原子力显微镜(AFM)测量纤丝的长度。
颗粒
构成本发明的制剂配方的颗粒可以包括诱导血小板黏附和激活或者它们的聚集的蛋白质或肽,或者由诱导血小板黏附和激活或者它们的聚集的蛋白质或肽组成。所述蛋白质和肽如上所描述。
在某些实施方案中,可以通过将蛋白质或肽黏附到载体上而获得所述颗粒。这样,构成本发明的制剂配方的颗粒包括载体,在其上黏附诱导血小板黏附、激活或者聚集的蛋白质或肽。
所述载体可以是有机微粒或无机微粒。微粒的实例包括白蛋白,脂类,金属诸如金、钛、铁、银、或其氧化物,石墨烯、二氧化硅、或高分子材料。
可以通过存在于颗粒表面的官能团,或者通过双功能间隔基团或者双功能聚合物,以共价键合实现蛋白质或肽黏附到载体上。所述双功能间隔基团或所述双功能聚合物使得微粒功能化。其在一端呈现使其本身共价地结合至微粒表面的官能团,且其在另一端呈现使其本身共价地结合至蛋白质或肽的官能团。
优选地,所述双功能聚合物为异基双功能聚合物。两种不同官能团的存在使得不希望的偶合(诸如聚合物的两端都连接到微粒上)得到限制。优选地,聚合物末端官能团之间的亲和性也很弱,以便限制聚合物通过这两端之间的相互作用而自凝结。
可以使用各种类型的双功能聚合物以功能化所述微粒。这样,所述双功能聚合物可以选自聚乙二醇(PEG)、聚(乳酸-乙醇酸)共聚物(PLGA)、聚己内酯(PLCL)、聚(乳酸)(PLA)、乙交酯聚合物(PGA)、壳聚糖、葡聚糖等。优选地,所述聚合物是双功能的聚乙二醇,优选为异基双功能的。所述聚合物的分子质量一般从500Da至10,000Da,优选2000–8000Da变化,更优选大约5000Da。所述聚合物的分子质量通常是经选择的,以便在蛋白质或肽通过所述双功能聚合物连接到所述微粒上时,所述聚合物链具有足够的长度使得所述蛋白质或肽的性质不会由于微粒的存在而受到影响。
在某些实施方案中,可以通过蛋白或肽的自组装而获得所述颗粒。在这样的实施方案中,所述颗粒不包括载体而仅由蛋白质或肽组成。可以通过不同方法实现自组装,诸如通过凝聚或者通过聚集,例如通过沉淀、浓缩、添加金属或盐,或者温度变化。
根据动态光散射的测量,本发明的所述颗粒可以具有从0.05μm至6μm,或从1μm至5μm、或从2μm至4μm范围内的平均直径。有利地,本发明的颗粒的平均直径可以介于0.02μm和0.05μm之间,例如介于0.025μm和0.045μm之间,或者例如等于0.035μm。
当所述颗粒包括重组蛋白质或由重组蛋白质组成,所述重组蛋白质的序列包括来自下述多肽的至少一种多肽:
-序列ID No.1多肽;
-具有序列ID No.1从25位至184位的序列的多肽;
或者
-其全长与序列ID No.1多肽,或者与具有序列ID No.1从25位至184位的序列的多肽具有至少70%同一性的多肽;
并且当它们根据DLS测量呈现大于2μm的平均直径时,它们能够诱导血小板的粘附、激活和聚集。对于平均直径小于2μm,例如介于0.02μm和1μm之间、介于0.05μm和0.5μm之间、或者介于0.1μm和0.3μm之间的分子也发现这一效果。
药学上可接受的载剂或赋性剂
根据本发明的药学上可接受的载剂或赋性剂,即将其对于个体投药不会伴随显著副作用的载剂或赋性剂,是本领域技术人员熟知的。
药学上可接受的赋性剂或载剂的实例包括但不限于溶剂、助流剂、悬浮剂、增溶剂、稳定剂、防腐剂、缓冲液、抗氧化剂和螯合剂。
根据本发明的注射制剂可以根据本领域技术人员熟知的方法制备。
根据本发明的注射制剂在出血性综合症中能够通过血小板激活和/或通过代替血小板而控制出血。
本发明因此涉及一种用于治疗出血的方法,其包括通过将有效量的如上文所定义的制剂注射到个体血液中而投药。
因此,在本发明的上下文语境中,很清楚“注射”与将本发明的产品全身投药(有时称为全身性给药)给患者是同义的。
最后,本发明涉及如上所定义的制剂的用途,其用于制备用以治疗出血的药物产品。
实施例
胶原蛋白的产生和自组装为颗粒(由DLS确定粒径的方法)
在转染之前对CHO-S细胞(Invitrogen)进行3周的预培养。37℃下,在含有5%CO2的培养箱中,将细胞在震荡(80rpm)条件下培养在摇动的125mL长颈瓶中补充了4mM L-谷氨酰胺(Lonza)和1×proHT(Lonza)的对CHO细胞特异的培养基中(Power–CHO、EXCEL 302、proCHO4、proCHO5等)。转染前两天,通过完全更换培养基以5×105活细胞/mL接种细胞,并将细胞培养在摇动的125mL长颈瓶中的12.5mL对CHO细胞特异的补充培养基中。
转染当天,通过离心分离5×106活细胞(1000g 5分钟),然后重悬在补充了4mM L-谷氨酰胺(Lonza)和1×proHT(Lonza)的5mL RPMI培养基(Lonza)中。将4mL悬浮液分配在含有9mL补充RPMI培养基的四个25mL摇动长颈瓶中(每个长颈瓶1mL)(每个摇动长颈瓶1×106活细胞)。然后如先前所述用含有编码序列ID No.1多肽的序列的载体转染所述CHO-S细胞。通过用pMAX–GFP载体转染细胞而实施阳性转染对照,并且通过用未加载抗性基因的载体转染细胞和通过不进行任何处理而实施两个阴性转染对照。使用转染剂Fecturine(PolyPlusTransfection)或任何其它的合适转染剂,并根据所述产品的最优商业化流程实施转染。对于Fecturine而言,对106活细胞所选择的转染条件为12μL Fecturine 6μg DNA(DNA/转染剂比例=1/2)。本领域技术人员会知道对于瞬时转染或对于稳定转染如何确定最合适的转染剂。无论转染模式是瞬时转染或稳定转染,于静态条件下在37℃和5%CO2下在转染复合物存在下孵育细胞。转染后四小时,将细胞重悬在对CHO细胞特异的补充培养基中并恢复震荡条件。转染后24小时,通过流式细胞术对于阳性和阴性转染对照样品测量转染效力。
为了在瞬时转染模式中制备序列ID No.1多肽,在D+3天采集初始上清样品(D0对应于转染当天),然后在D+5天停止培养。通过在3000g离心10分钟回收含有由细胞分泌的序列ID No.1多肽的培养上清液,以去除细胞和细胞残渣,然后冻存在–20℃。
为了在稳定转染模式中制备序列ID No.1多肽,在转染之后48小时进行细胞计数。然后将所有细胞在1000g离心5分钟,接着以3×105活细胞/mL接种在对CHO细胞特异的补充培养基+700μg/mL遗传霉素(G418Merck)(选择压力)中。然后通过将所有细胞接种在125mL摇动长颈瓶中的12.5mL终体积完全培养基+700μg/mL G418中而每周传代细胞三次。在选择压力下的第一周的培养物中观察到细胞浓度和/或细胞活力降低。在选择压力下2-3周之后,细胞活力又增加,但仅针对用含有编码序列ID No.1多肽的序列的载体转染的细胞或者用空载体转染的细胞。非转染细胞的活力持续下降直至变为零。一旦培养物达到95%以上的活力,以5×106活细胞/安瓿(10安瓿)进行冷冻保藏。将细胞冷冻于对CHO细胞特异的补充培养基+10%DMSO中。
为了产生序列ID No.1多肽,将经遗传改造用于制备序列ID No.1多肽的细胞解冻并培养在合适的培养基中。将细胞接种在12.5mL终培养基中并接种在125mL摇动长颈瓶中,在Kühner振荡器中以80rpm震荡,调整5%CO2以及湿度介于40%和80%之间。以3×105活细胞/mL培养细胞一周或两周。然后将细胞扩大以获得进行生产必需的量。扩大由每次更换培养基时将细胞以较大体积培养构成,以便保证所有细胞均处于活力浓度。
一旦获得生产必需量的细胞,就能够开展生产。以3×105活细胞/mL接种细胞。能够在各种设备中进行生产:放置于Kühner振荡器中的摇动长颈瓶、CultibagRM(Sartorius)、(Merck Millipore)、(Applikon)和具有相同或较大规格的其它等同设备。根据培养条件,继代培养进行5天或更多天,最多10天。每天监测生产参数、细胞浓度和细胞活力。在制备期间,可以添加组分诸如氨基酸、维他命、葡萄糖、或者对于生产或对于细胞有利的其它任何元素。在这种情况下,所述培养基是“分批补料培养基”或“半连续培养基”,其使得细胞能够最多培养21天。如果在培养期间不添加化合物,则其为“分批培养基”或“不连续”培养基。
可以例如使用存在于其序列中的纯化标签诸如6(his)-标签进行序列ID No.1多肽的纯化。这样,通过离心或深度过滤(Merck Millipore POD或任何其它形式的设备载体),或在具有0.2μm截留量的膜上的切向过滤将存在于含有序列ID No.1多肽的培养基中的细胞和细胞残渣除去。由此获得的上清液在整合了金属诸如镍、钴、锌或铜的柱子上通过亲和层析纯化。为了促进序列ID No.1多肽的连接,向上清液中添加含有0-50mM咪唑、0-500mM NaCl、5-20mM(Na2HPO4·2H2O)和5–20mM(NaH2PO4·H2O)的缓冲溶液,并且将pH调整为介于7和8之间。根据本发明的多肽的洗脱通过使用两种缓冲溶液的混合物(缓冲液1:500mM咪唑、500mM NaCl、10mM(Na2HPO4·2H2O)和10mM(NaH2PO4·H2O);缓冲液2:20mM咪唑、500mM NaCl、10mM(Na2HPO4·2H2O)和10mM(NaH2PO4·H2O))梯度进行,或者通过使用含有50–500mM咪唑、0–500mM NaCl、5–10mM(Na2HPO4·2H2O)和5–10mM(NaH2PO4·H2O)的缓冲溶液等强度进行。可以增加其它纯化步骤,以改善序列ID No.1多肽的纯度。这些步骤可以是过滤、离子交换色谱、亲和色谱、疏水性相互作用色谱、分子体积排阻色谱,或任何其它形式的色谱。
将感兴趣的洗脱组分于天然条件下置于电泳凝胶中迁移,并且用考马斯亮蓝染色,然后根据它们的图谱进行收集并在水中或磷酸缓冲液中或任何其它适于存储本发明的多肽的缓冲溶液中透析。使用kit(TebuBio)根据制造商的说明,或者使用Bradford测试或者任何其它适于定量序列ID No.1多肽的测试确定多肽的浓度。
通过超滤作用,将序列ID No.1多肽的相对于其初始体积浓缩若干倍(50倍),使得蛋白质浓度达到足以诱导序列ID No.1多肽自组装和颗粒形成。以这种方式,在具有10,000Da截留量的超滤柱(Vivaspin 4,Sartorius)上通过在4℃和4000rpm反复离心将含有序列ID No.1多肽的蛋白质提取物浓缩。一旦溶液被浓缩,使用马尔文Zetasizer纳米设备通过动态光散射(DLS)技术测量由此形成的颗粒的粒径。该DLS测量是一种非破坏性的光谱分析技术,其使得能够测量液体中悬浮的颗粒粒径;其特别适于测量诸如序列ID No.1多肽的蛋白质自组装或蛋白质聚集。
图1显示将序列ID No.1多肽蛋白质提取物浓缩50倍之后,由DLS测量颗粒粒径的特征性结果。观察到一系列峰,对应于溶液中存在的不同颗粒粒径。三个峰是显著的:他们对应于1.25μm、3μm和5.5μm的粒径。为了将序列ID No.1多肽的生物活性与由此生成的颗粒粒径联系起来,向溶液中添加盐酸至20mM终浓度。然后之前测量的三个峰消失了,而在大约500nm和1.8μm出现两个峰。直观上看,观察到该组分是“干净的”,外观从粒状变为透亮。
测量血管性血友病因子结合活性随粒径的变化
基于以颗粒形式存在的序列ID No.1多肽的制备物具有与血管性血友病因子结合的能力。在96孔板(Nunc Maxisorp)中通过ELISA技术测量血管性血友病因子对于序列IDNo.1多肽的结合活性。为了这一目的,将含有以2μg/mL溶于磷酸缓冲液或任何其它合适的缓冲溶液中的多肽的100μL体积的溶液添加到每个孔中并在22℃孵育18-20小时。用200μLPBS–0.05%吐温清洗三次之后,将200μL溶于磷酸缓冲液中的1%BSA溶液(Euromedex,经0.45μm过滤)添加到每个孔中并在室温下(22℃)孵育两小时。用200μL PBS–0.05%吐温清洗三次之后,将100μL各种浓度(以IU/dL表示)的纯化血管性血友病因子(Wilfactin100IU/mL,LFB)和/或稀释于磷酸缓冲液或任何其它合适的缓冲溶液中的患者血浆在室温下(22℃)孵育一小时30分钟。清洗三次之后,将100μL含有于磷酸缓冲液或任何其它合适的缓冲溶液中稀释至1:8000的偶联至辣根过氧化物酶的第一抗-vWF抗体(兔抗-人VWF/HRP,DAKO)的溶液在22℃孵育一小时;然后进行如上所述的四次清洗。然后添加125μL四甲基联苯胺溶液(TMB,Sigma)作为过氧化物酶的底物;将平板在黑暗中孵育最多10-45分钟。通过添加125μL 2N HCL(Sigma)而终止反应。即刻在分光光度计(Wallacvictor 3)中测量450nm的吸光度。通过将纯化vWF或血浆用磷酸缓冲液或任何其它合适的缓冲溶液替换而设置空白。
图2显示了由ELISA获得的、2IU/dL终浓度使用的纯化血管性血友病因子(Willfactin,LFB)与以不同粒径的颗粒形式存在、以2μg/mL终浓度使用的序列ID No.1多肽,以及与以10μg/mL使用的I型胶原蛋白(SIRCOL胶原蛋白,阳性对照)的结合结果。
该结果表明,序列ID No.1多肽构成大部分大于2μm的颗粒,能够以与I型胶原蛋白等价的方式与血管性血友病因子结合。当序列ID No.1多肽以小于2μm的形式存在时,血管性血友病因子结合活性极大地增强。这后一结果表明,序列ID No.1多肽自组装为较大颗粒减少了血管性血友病因子的识别位点数量,其可能“隐藏”在较大颗粒内部。这一结果显示,实际上形成粒径介于1μm和5μm之间的颗粒结构的序列ID No.1多肽能够结合血管性血友病因子。
测量由胶原蛋白诱导的P-选择素的表面表达随颗粒的变化
基于以颗粒形式存在的序列ID No.1多肽的制剂具有诱导血小板激活的能力。在全血中能够通过流式细胞术测量P-选择素在血小板表面的表达而确定这一活性。在静息的血小板中,P-选择素储藏在被称为α粒的小颗粒中。当血小板由不同的激动剂激活时,P-选择素从这些小颗粒中释放并能在血小板表面被检测到。
将血样采集于柠檬酸管(BD Vacutainer,BD Biosciences)中然后在使用之前稳定一小时。在玻璃试管中,将50μL血液添加至含有50μg/mL浓度的序列ID No.1多肽(终浓度25μg/mL)的50μL磷酸缓冲液中;在室温下将所述混合物孵育10分钟。
然后通过向每个试管中添加500μL磷酸缓冲液而稳定所述反应。在细胞计数管中,接着将之前描述的20μL反应混合物添加到含有偶联至FITC的抗-CD62P第一抗体(FITC鼠抗人CD62P,BD Biosciences)或偶联至FITC的同型对照抗体(FITC小鼠IgG1κ同型对照,BDBiosciences)的20μL溶液中,并且在黑暗中室温孵育10分钟。然后向每个试管中添加2mL磷酸缓冲液。即刻使用流式细胞仪(LSRII,BD Biosciences)测量520nm的平均荧光强度(MFI)。通过将序列ID No.1多肽由磷酸缓冲液代替而实施阴性对照。通过将序列ID No.1多肽由源自TRAP凝血酶的血小板激活剂(人临床诊断试剂盒:PLT Gp/受体试剂盒,Biocytex)或离子载体激活剂A23187(Calbiochem,Merck;Streptomyces chartreusensis)取代而实施两个阳性对照。通过将所述多肽由以50μg/mL使用的I型胶原蛋白(Horm)取代而设置参照。
图3A对应于针对序列ID No.1多肽和针对I型胶原蛋白(Horm),由流式细胞术获得的P-选择素在血小板表面上的典型表达图谱的代表。图3B中的表格显示针对各种激动剂获得的平均荧光强度。TRAP和钙离子载体用作阳性激活对照。
具有大于2μm的粒径的序列ID No.1多肽能够诱导血小板激活,血小板激活的特征在于表面表达P-选择素。这一激活等价于以相同浓度使用的纤丝状I型胶原蛋白(比1μm长的纤丝)诱导的激活。具有小于2μm的粒径的序列ID No.1多肽(序列ID No.1+20mM HCl)也能够诱导P-选择素的表达并因而激活血小板,但是程度较低(47.3对比70.4MFI)。
测量由胶原蛋白诱导的血小板聚集随着粒径的变化
基于以颗粒形式存在的序列ID No.1多肽的制剂对于人血液血小板具有致聚活性,其可根据参照技术使用血栓-聚集仪(Soderel,Florange,法国)检测(Born,1962)。将富血小板血浆(PRP)与激动剂(290μL PRP中的10μL)接触,并且实时监测(聚集曲线)培养基的清晰度(与聚集体形成相关)。在不存在血小板聚集时,信号保持平坦(培养基保持浑浊)。通过在测量终了时检测试管可能核实聚集存在或不存在。37℃下在持续搅拌(1000rpm)下评估血小板聚集测试反应。将仪器如下校准:富血小板血浆0%聚集(通过低速离心和浓缩获得的制剂,调整至300×109/L)和贫血小板血浆100%聚集(由快速离心获得的制剂)。通过核实对于用作研发抗血小板治疗的参照激动剂(5μM ADP和1μg/mL胶原蛋白)的反应而证实血小板制剂的质量。当蛋白质能够诱导大于40%的不可逆聚集时其被认为是致聚激动剂。
图4显示由以颗粒形式存在的序列ID No.1多肽获得的聚集结果。路径1显示具有大于2μm粒径的序列ID No.1多肽制剂能够在低于1分钟内并且以与纤丝形式的参照胶原蛋白Horm I型胶原蛋白等价的方式(路径4)诱导血小板聚集。与此相反,具有小于2μm粒径的序列ID No.1多肽甚至在10分钟之后都不能诱导血小板聚集(路径2)。这一活性缺失并不是与溶液中存在20mM HCl相关的,因为路径3显示,在存在20mM HCl情况下,由六分钟之后加入的I型胶原蛋白刺激引起正常的血小板聚集。
这一结果显示,序列ID No.1多肽必须形成大于2μm的颗粒形式的结构才能获得致聚活性。
综上所述,这些不同的实例已经显示,序列ID No.1多肽能够使自己形成具有介于1μm和6μm之间的粒径的颗粒形式;这些颗粒具有与血管性血友病因子结合以诱导血小板激活(表面表达P-选择素)的能力,并且仅当粒径大于2μm时导致血小板聚集。
通过各种显微技术与纤丝状I型胶原蛋白(Horm)相比观察以颗粒形式存在的序列ID No.1多肽的结构
存在各种能够以不同的分辨率和不同的比例将分子的大分子结构可视化的显微技术。图5显示了用三种显微技术(透射显微镜、扫描电子显微镜和原子力显微镜)获得的图片,其使得能够观察胶原蛋白类型的蛋白质的结构。
由透射显微镜观察时,将I型胶原蛋白或序列ID No.1多肽在37℃下放置于载玻片上超过2小时,然后使用蔡司反向相衬显微镜用60×物镜观察。使用蔡司AxioCam ICc 1摄像机获得图片。
对于由扫描电子显微镜的观察,选择用于放置的表面是单晶硅(100)。其既是半导体而且还原子级平整,是通过这一方法观察胶原蛋白的显而易见的选择。通过将浓缩至200μg.mL–1的纤丝状I型胶原蛋白或序列ID No.1多肽的10μL样滴放置在硅表面六小时而实施样品制备。然后用蒸馏水漂洗表面并在氮存在下干燥。在Edwards S150离子溅射镀膜仪中通过喷金处理将干燥的表面金属化(在0.3mPa氩氛中25mA电流处理2分钟)。用JEOL 6500F扫描电子显微镜观察样品表面,设定发射电势20keV,以及发射电流60μA。
对于由原子力显微镜(AFM)的观察,在其上观察I型胶原蛋白和序列ID No.1多肽的表面是多晶金表面(通过在云母上外延获得的Au(111)台阶结构,PHASIS,编号:20020022)。以25μg.mL–1的浓度,在超过30分钟内以300s–1的剪切速率下溢进行非原位沉积。为了使得这一流体功能化,将PDMS微流体细胞挤压到金表面并且通过ISMATEC IPC–4蠕动泵将蛋白质溶液加到表面上。使用装配了FESPA探针(Bruker)的Nanoscope IVMultimode原子力显微镜(Digital Instrument,Veeco Inc.,Santa Barbara,CA)观察样品,设定刚性常数k为N.m–1,共振频率f=50–100kHz,以及操作杆长度L=200–250μm。使用Bruker PeakForce QNM模式采集信息,动态轻敲模式(dynamic tapping mode)使得能够在这些生物样品上实现低噪音信息收集。扫描频率固定为1Hz。所获得的图片经WSxM程序处理,以便通过应用压平和补偿功能获得高分辨率。
图5中的观察结果确认了由DLS获得的结果,指示与I型胶原蛋白的纤丝状结构相比序列ID No.1多肽以颗粒形式存在的结构。在用于观察它们的实验条件下,观察到的颗粒的粒径为几十纳米的等级。
将颗粒形式的序列ID No.1多肽和纤丝状形式的I型胶原蛋白(Horm)注射到小鼠中之后测量血小板表面的P选择素表达。
如由流式细胞术测量的血小板表面的P选择素表达所示,基于以颗粒形式存在的序列ID No.1多肽和纤丝状的I型胶原蛋白(Horm)的制剂具有在全血中体外诱导血小板激活的能力。为了证实这些制剂确实能够诱导这一同样的激活,一旦将制剂注射入血液后,就在投药400μg/kg I型胶原蛋白或2mg/kg序列ID No.1多肽之后15分钟,或者在出现第一次栓塞(窒息)迹象的时刻,从小鼠收集血样到柠檬酸管(BD Vacutainer,BD Biosciences)中。将50μL血放置在玻璃试管中然后用250μL磷酸缓冲液稳定。在细胞计数管中,接着将之前描述的20μL反应混合物添加到含有偶联至FITC的抗-CD62P第一抗体(FITC鼠抗人CD62P,BD Biosciences)或偶联至FITC的同型对照抗体(FITC小鼠IgG1κ同型对照,BDBiosciences)的20μL溶液中,并且在黑暗中室温孵育10分钟。然后向每个试管中添加2mL磷酸缓冲液。即刻使用流式细胞仪(LSRII,BD Biosciences)测量520nm的平均荧光强度(MFI)。
图6显示针对两种粒径,在存在肾上腺素或不存在肾上腺素情况下,从注射纤丝状I型胶原蛋白和序列ID No.1多肽之后的小鼠采集的血液中测量的血小板P选择素表达。可以注意到,序列ID No.1多肽在其投药给小鼠之后15分钟或者在出现第一次栓塞(窒息)迹象的时刻确实诱导血小板激活,无论粒径如何。与由纤丝状I型胶原蛋白的激活被肾上腺素急剧增强不同,这一激活不由同时投药的肾上腺素所调控。这一结果证实,如体外实验所示,序列ID No.1多肽一旦投药至血液中确实能够诱导血小板激活。
在小鼠中诱导的肺栓塞模型中测量与序列ID No.1多肽注射到血液中相关的血栓形成风险
本发明描述了源自胶原蛋白的蛋白质或肽通过依赖于血小板激活的机制用于治疗出血的用途。为了在不依赖于出血情境下确定由注射以颗粒形式存在的这些源自胶原蛋白的蛋白质或肽诱导的体内血小板激活是否与血栓形成风险相关,使用通过投药胶原蛋白和肾上腺素在小鼠中诱导的肺栓塞模型。目的是显示由本发明的源自胶原蛋白的蛋白质或肽所诱导的血小板激活本身不会由于一经注射即诱导血栓形成而有害。为了表征这一投药方式的潜在血栓形成效果,除了用于这一模型中的经典标准以外,使用能够通过同位素成像可视化的放射性追踪剂作为观察标准,其由测量动物从肺栓塞到死亡所花费的时间组成。在静脉注射之后,由锝-99m标记的人白蛋白大颗粒在血液中循环并使得闪烁扫描器可以检测到肺或者某些静脉。如果存在血栓形成中心,则这一追踪剂的正常肺分布就会改变并且同位素成像将显示该追踪剂在肺中减少或不存在。
将25–30g体重的雄性SWISS小鼠用于这一肺栓塞模型。在整个实验期间,将小鼠保持在异氟醚麻醉状态。首先,暴露两根颈静脉,然后以150μL的终体积将序列ID No.1多肽(1mg/kg)/肾上腺素(60μg/kg)混合物或纤丝状I型胶原蛋白(400μg/kg)/肾上腺素(60μg/kg)混合物注射到一根静脉中。如果发生了肺栓塞,则小鼠开始窒息并且在注射10分钟内死亡。认为能够存活15分钟以上的就是没有发生肺栓塞。为了实施SPECT数据采集,正好在肺栓塞小鼠心脏停博之后,或者在小鼠存活15分钟之后,将标记了锝-99m的人白蛋白大颗粒(每只小鼠30μCi,终体积100μL)注射到另外一根颈静脉中。实施扫描然后是15分钟的同位素成像数据采集。
图7A中的表格显示在肾上腺素存在下给小鼠投药纤丝状I型胶原蛋白和颗粒形式的序列ID No.1多肽之后获得的关于其中观察到肺栓塞的动物百分比和这一栓塞出现的平均时间的结果。结果显示,100%的动物在注射纤丝状I型胶原蛋之后平均5分钟50秒之后死亡。与此相反,在注射序列ID No.1多肽之后,没有动物发生肺栓塞,即便以高2.5倍的浓度使用(1mg/kg对比400μg/kg)。图7B显示注射放射性标记的白蛋白大颗粒之后获得的图片,其确认在注射纤丝状I型胶原蛋白的小鼠中存在大量肺栓塞且在注射序列ID No.1多肽的小鼠中追踪剂肺分布正常,显示不存在诱导的血栓形成。
这结果确认,对于具有相同血小板激活活性的两种胶原蛋白型蛋白质,一旦其注射到血液中,只有以颗粒形式存在的那些可以使用而不存在诱导血栓形成的风险。
在小鼠中诱导尾巴出血模型中测量注射序列ID No.1多肽的效果
上述呈现的结果确认,与纤丝状I型胶原蛋白不同,以颗粒形式存在的序列IDNo.1多肽一旦注射到小鼠血液中确实能够诱导血小板激活,而且这种激活不具有形成血栓的效果。为了验证将本发明的源自胶原蛋白的蛋白质或肽注射到血液中确实能够通过依赖于血小板激活的机制诱导止血,在小鼠中诱导从尾巴出血模型中评估了投药序列ID No.1多肽对于血液损失的效果。
将25–30g体重的雄性SWISS小鼠用于这一尾巴出血模型。在整个实验期间,将小鼠保持在异氟醚麻醉状态。首先,暴露两根颈静脉之一,然后注射稀释于磷酸缓冲液中的不同分子。将尾巴从末端切成1.5cm小段以将3根动脉和静脉切段。在注射磷酸缓冲液(媒介对照)或序列ID No.1多肽(1.6mg/kg)之后10分钟和在注射肝素(抗凝血剂,阳性出血对照,60IU/kg)之后20分钟进行切段。切段以后,在37℃下将尾巴放置在50mL生理盐水中,以便收集血液超过16分钟而不损伤红细胞。在室温下将试管固定超过一小时,然后在250g离心15分钟。弃去上清并将红细胞沉淀悬浮在3mL特异的裂解缓冲液中(8.3g/L NH4Cl,1g/L KHCO3和37mg/L EDTA)。在孔板检测仪上读取200μL裂解液在550nm的光密度。
构建标准曲线以确定损失血液体积。为了这一目的,使25–30g体重的雄性SWISS小鼠自腹主动脉出血。将血液收集在柠檬酸管中以避免凝结。在含有50mL生理盐水的试管中构建一系列血液体积。然后如上所述实施血液处理(参见图8A)。
图8B中的结果确认,以颗粒形式存在的序列ID No.1多肽无论以天然形式还是以浓缩形式确实能够在这一模型中诱导止血。在序列ID No.1多肽存在下,止血前的平均损失体积是11.6±2.4μL(非浓缩的)和12.6±6.1μL(浓缩10倍)相对于未处理小鼠为42±18.4μL。因此,与对照小鼠相比,血液损失减少大约70%。与此相反,在肝素存在下,血液损失急剧增加,达到平均血液损失257.9±87.5μL。因此,与对照小鼠相比血液损失增加514%,从而验证了该模型。
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Claims (11)
1.一种用于治疗出血的注射制剂,其包括:
-具有小于10μm长度的颗粒或纤丝,所述颗粒或纤丝包括诱导血小板黏附和激活的蛋白质或肽;和
-至少一种药学上可接受的载剂或赋形剂。
2.根据权利要求1所述用途的注射制剂,其中所述蛋白质选自胶原蛋白或者重组蛋白,其序列包括至少一种选自下述的多肽:
-序列ID No.1多肽;
-具有序列ID No.1从25位至184位的序列的多肽;
-其全长与序列ID No.1多肽,或者与具有序列ID No.1从25位至184位的序列的多肽具有至少70%同一性的多肽;
-具有由GXY三联体重复形成的序列的多肽,其中G指代甘氨酸,并且X和Y指代任何氨基酸,重复n次,n大于或等于2。
3.根据权利要求1所述用途的注射制剂,其中所述多肽选自:
-序列ID No.1多肽;
-具有序列ID No.1从25位至184位的序列的多肽;
-其全长与序列ID No.1多肽,或者与具有序列ID No.1从25位至184位的序列的多肽具有至少70%同一性的多肽;或者
-具有由GXY三联体重复形成的序列的多肽,其中G指代甘氨酸,并且X和Y指代任何氨基酸,重复n次,n大于或等于2。
4.根据前述权利要求中的一项所述用途的注射制剂,其中所述颗粒由选自胶原蛋白或重组蛋白的多肽或蛋白质组成。
5.根据权利要求4所述用途的注射制剂,其中所述颗粒由凝聚、聚集或自组装获得。
6.根据权利要求1至3中的任一项所述用途的注射制剂,其中所述颗粒包括所述多肽或蛋白质黏附到其上的载体。
7.根据权利要求6所述用途的注射制剂,其中所述载体是有机微粒或无机微粒。
8.根据权利要求7所述用途的注射制剂,其中所述蛋白质通过共价键合被连接到微粒表面。
9.根据权利要求7或8所述用途的注射制剂,其中所述微粒选择包括白蛋白、脂类、金属和其氧化物、石墨烯、二氧化硅和高分子材料的群组。
10.根据前述权利要求中的任一项所述用途的注射制剂,其中所述颗粒具有从1μm至6μm范围内的平均直径。
11.根据前述权利要求中任一项所述用途的注射制剂,其中所述胶原蛋白或蛋白质是聚乙二醇化或者聚硫酸铝化的。
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FR1362418A FR3014319B1 (fr) | 2013-12-11 | 2013-12-11 | Preparations injectables a base de collagenes capables de controler les saignements et/ou de se substituer a des plaquettes dans le cas de syndromes hemorragiques |
FR1362418 | 2013-12-11 | ||
PCT/EP2014/077385 WO2015086748A1 (fr) | 2013-12-11 | 2014-12-11 | Preparations injectables a base de collagenes capables de controler les saignements et/ou de se substituer a des plaquettes dans le cas de syndromes hemorragiques |
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CN115737814B (zh) * | 2022-10-26 | 2024-06-21 | 中国科学院大学 | 抗血小板药物及制备方法、抗血小板药物捕获剂 |
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WO1998057678A2 (en) * | 1997-06-18 | 1998-12-23 | Cohesion Technologies, Inc. | Compositions containing thrombin and microfibrillar collagen |
CN102224167A (zh) * | 2008-09-24 | 2011-10-19 | 第戎大学医疗中心 | 具有止血活性并能够诱导血小板聚集的重组蛋白 |
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US20060100138A1 (en) * | 2004-11-10 | 2006-05-11 | Olsen David R | Implantable collagen compositions |
US20090299034A1 (en) * | 2007-08-01 | 2009-12-03 | Mabel Alamino Cejas | Collagen-related peptides |
JP5566885B2 (ja) * | 2007-08-01 | 2014-08-06 | エシコン・インコーポレイテッド | コラーゲン関連ペプチド及びその使用 |
NL2009102C2 (en) | 2012-07-02 | 2014-01-06 | Dcprime B V | Method for dc-loading. |
FR2997083B1 (fr) * | 2012-10-19 | 2014-12-12 | Nvh Medicinal | Proteines recombinantes derivees du collagene a activite de liaison au facteur willebrand |
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WO1998057678A2 (en) * | 1997-06-18 | 1998-12-23 | Cohesion Technologies, Inc. | Compositions containing thrombin and microfibrillar collagen |
CN102224167A (zh) * | 2008-09-24 | 2011-10-19 | 第戎大学医疗中心 | 具有止血活性并能够诱导血小板聚集的重组蛋白 |
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KR102329184B1 (ko) | 2021-11-19 |
JP6636443B2 (ja) | 2020-01-29 |
CN106102764B (zh) | 2020-03-06 |
ES2829503T3 (es) | 2021-06-01 |
RU2709738C1 (ru) | 2019-12-19 |
IL246154B (en) | 2019-03-31 |
US20160310577A1 (en) | 2016-10-27 |
WO2015086748A1 (fr) | 2015-06-18 |
CA2933120A1 (fr) | 2015-06-18 |
AU2014363519B2 (en) | 2020-06-25 |
BR112016013386B1 (pt) | 2023-12-26 |
JP2017501217A (ja) | 2017-01-12 |
FR3014319B1 (fr) | 2016-12-09 |
KR20160107172A (ko) | 2016-09-13 |
EP3079713A1 (fr) | 2016-10-19 |
AU2014363519A1 (en) | 2016-07-07 |
BR112016013386A2 (pt) | 2017-09-26 |
EP3079713B1 (fr) | 2020-08-19 |
US9878018B2 (en) | 2018-01-30 |
IL246154A0 (en) | 2016-07-31 |
FR3014319A1 (fr) | 2015-06-12 |
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