CN106110311A - 凝血蛋白缀合物 - Google Patents
凝血蛋白缀合物 Download PDFInfo
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- CN106110311A CN106110311A CN201610586352.9A CN201610586352A CN106110311A CN 106110311 A CN106110311 A CN 106110311A CN 201610586352 A CN201610586352 A CN 201610586352A CN 106110311 A CN106110311 A CN 106110311A
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Abstract
本发明涉及使水溶性聚合物缀合于凝血蛋白的氧化的碳水化合物部分的材料和方法,所述方法包括使所述氧化的碳水化合物部分与活化的水溶性聚合物在允许缀合的条件下接触。更具体地,本发明涉及上述材料和方法,其中所述水溶性聚合物含有活性氨氧基,且其中肟键合在所述氧化的碳水化合物部分与所述水溶性聚合物上的活性氨氧基之间形成。在本发明的一个实施方案中,所述缀合在亲核性催化剂苯胺存在下进行。另外,所产生的肟键合可通过用NaCNBH3还原形成烷氧基胺键合来稳定。
Description
本申请是申请号为201080037002.8、申请日为2010年7月26日、名称为“凝血蛋白缀合物”的发明专利申请的分案申请。
本申请要求2010年5月21日提交的美国临时申请第61/347,136号及2009年7月27日提交的美国临时申请第61/228,828号的权益,全部申请以全文引用的方式并入本文中。
技术领域
本发明涉及使水溶性聚合物缀合于凝血蛋白的材料和方法。
背景技术
治疗性多肽例如凝血蛋白(包括因子IX(FIX)、因子VIII(FVIII)、因子VIIa(FVIIa)、冯威利布兰德因子(Von Willebrand Factor,VWF)、因子FV(FV)、因子X(FX)、因子XI(FXI)、因子XII(FXII)、凝血酶(FII)、蛋白质C、蛋白质S、tPA、PAI-1、组织因子(TF)及ADAMTS 13蛋白酶),会被蛋白水解酶快速降解并被抗体中和。这会缩短其半衰期及循环时间,从而限制其治疗效果。必需相对较高剂量及频繁施用来达到并维持这些凝血蛋白的期望治疗或预防效果。因此,难以实现适当的剂量调节,且对频繁静脉内施用的需要给患者的生活方式造成限制。
多肽药物的聚乙二醇化在循环中对所述药物进行保护,且改善其药效学及药物动力学特性(Harris和Chess,Nat Rev Drug Discov.2003:2:214-21)。聚乙二醇化过程使乙二醇重复单元(聚乙二醇(PEG))连接至多肽药物。PEG分子具有大流体动力学体积(球状蛋白大小的5-10倍),具有高度水溶性且为水合的、无毒性、非免疫原性的,并快速自身体内清除。分子的聚乙二醇化可增强药物对酶促降解的抗性,增加体内半衰期、减少给药频率、降低免疫原性、增强物理及热稳定性、增强溶解性、增强液体稳定性及减少聚集。第一种聚乙二醇化药物在20世纪90年代早期获得FDA批准。此后,FDA批准了数种聚乙二醇化药物用于口服、注射及局部施用。
聚唾液酸(PSA)(也称为多聚乙酰神经氨糖酸(colominic acid,CA))为天然存在的多糖。其为含α(2→8)酮苷键合的N-乙酰基神经氨糖酸的同聚物且在其非还原末端含有邻二醇基团。其带负电且为人体的天然组成部分。其可容易地由细菌以大量及预定生理特征制造(美国专利第5,846,951号)。因为细菌产生的PSA在化学及免疫方面与人体所产生的PSA相同,所以细菌PSA即使与蛋白质偶联也是非免疫原性的。不同于某些聚合物,聚唾液酸为生物可降解的。多聚乙酰神经氨糖酸与过氧化氢酶及天门冬酰胺酶的共价偶联已显示增强在蛋白水解酶或血浆存在下的酶稳定性。用聚唾液酸化天门冬酰胺酶与未经修饰的天门冬酰胺酶进行的体内比较研究揭示聚唾液酸化增加酶的半衰期(Femandes和Gregoriadis,Int Biochimica Biophysica Acta 1341:26-34,1997)。
通过在水溶性聚合物与治疗性蛋白质之间形成共价键联来制备缀合物可利用多种化学方法进行。举例而言,Roberts等人(Adv Drug Deliv Rev 2002:54:459-76)回顾了PEG衍生物与肽或蛋白质的偶联。一种使水溶性聚合物与治疗性蛋白质偶联的方法为使聚合物经由蛋白质的碳水化合物部分缀合。可容易地用过碘酸钠(NaIO4)氧化蛋白质中碳水化合物的邻羟基(OH)以形成活性醛基(Rothfus和Smith,J Biol Chem 1963:238:1402-10;van Lenten和Ashweli,J Biol Chem 1971:246:1889-94)。接着,可通过使用含有例如活性酰肼基的试剂使聚合物与碳水化合物的醛基偶联(Wilchek M和Bayer EA,MethodsEnzymol 1987:138:429-42)。新近技术为使用含有与醛反应形成肟键合的氨氧基的试剂(WO 96/40662、WO2008/025856)。
描述使水溶性聚合物缀合于治疗性蛋白质的其它实例描述于以下文献中:WO 06/071801,教导氧化冯威利布兰德因子中的碳水化合物部分,并接着使用酰肼化学与PEG偶联;美国公开第2009/0076237号,教导氧化rFVIII,并接着使用酰肼化学与PEG及其它水溶性聚合物(例如PSA、HES、葡聚糖)偶联;WO 2008/025856,教导氧化不同凝血因子(例如rFIX、FVIII及FVIIa),并接着使用氨氧基化学经由形成肟键合与例如PEG偶联;及美国专利第5,621,039号,教导氧化FIX,并接着使用酰肼化学与PEG偶联。
最近,描述了一种改良方法,所述方法包括以过碘酸盐适度氧化唾液酸以产生醛,接着在催化量的苯胺存在下与含氨氧基的试剂反应(Dirksen A和Dawson PE,Bioconjugate Chem.2008:19,2543-8;及Zeng Y等人,Nature Methods 2009:6:207-9)。苯胺催化作用显著加速肟连接,从而允许使用极低浓度的所述试剂。
纵使存在可用于使水溶性聚合物缀合于治疗性蛋白质的方法,但仍需要开发改良蛋白质的药效学和/或药物动力学性质同时使与各种试剂相关的成本降至最低的使水溶性聚合物缀合于蛋白质的材料和方法。
发明概述
本发明提供使聚合物缀合于蛋白质的材料和方法,其改善蛋白质的药效学和/或药物动力学性质同时使与各种试剂相关的成本降至最低。
在本发明的一个实施方案中,使水溶性聚合物缀合于凝血蛋白的氧化的碳水化合物部分的方法包括使氧化的碳水化合物部分与活化的水溶性聚合物在允许缀合的条件下接触;所述凝血蛋白选自由以下所组成的组:因子IX(FIX)、因子VIII(FVIII)、因子VIIa(FVIIa)、冯威利布兰德因子(VWF)、因子FV(FV)、因子X(FX)、因子XI(FXI)、因子XII(FXII)、凝血酶(FII)、蛋白质C、蛋白质S、tPA、PAI-1、组织因子(TF)及ADAMTS 13蛋白酶或其生物活性片段、衍生物或变异体;所述水溶性聚合物含有活性氨氧基且选自由以下所组成的组:聚乙二醇(PEG)、分支PEG、聚唾液酸(PSA)、碳水化合物、多糖、普鲁兰(pullulane)、壳聚糖、透明质酸、硫酸软骨素、硫酸皮肤素、淀粉、葡聚糖、羧甲基-葡聚糖、聚环氧烷(PAO)、聚亚烷基二醇(PAG)、聚丙二醇(PPG)、聚噁唑啉、聚丙烯酰基吗啉、聚乙烯醇(PVA)、聚羧酸酯、聚乙烯吡咯烷酮、聚磷腈、聚噁唑啉、聚乙烯-共-顺丁烯二酸酐、聚苯乙烯-共-顺丁烯二酸酐、聚(1-羟基甲基亚乙基-羟基甲基缩甲醛)(PHF)、磷酸2-甲基丙烯酰氧基-2’-乙基三甲基铵(MPC);且所述碳水化合物部分通过用包含氧化剂的缓冲液孵育而氧化,所述氧化剂选自由以下所组成的组:过碘酸钠(NaIO4)、四乙酸铅(Pb(OAc)4)及过钌酸钾(KRuO4);其中肟键合在氧化的碳水化合物部分与水溶性聚合物上的活性氨氧基之间形成。
在本发明的另一实施方案中,根据上述方法的水溶性聚合物为PSA。在相关实施方案中,PSA包含约5-500或10-300个唾液酸单元。在又一实施方案中,根据上述方法的凝血蛋白为FIX。在另一实施方案中,根据上述方法的凝血蛋白为FVIIa。在又一实施方案中,根据上述方法的凝血蛋白为FVIII。在又一实施方案中,提供一种上述方法,其中氧化剂为过碘酸钠(NaIO4)。在另一实施方案中,根据上述方法的凝血蛋白的氧化的碳水化合物部分位于凝血蛋白的活化肽中。
在本发明的又一实施方案中,提供一种上述方法,其中所述PSA通过使活化的氨氧基连接基团与氧化的PSA反应来制备;
其中所述氨氧基连接基团选自由以下所组成的组:
下式的3-氧杂-戊烷-1,5-二羟基胺连接基团:
及
下式的3,6,9-三氧杂-十一烷-1,11-二羟基胺连接基团:
其中所述PSA通过用氧化剂孵育来氧化以在PSA的非还原末端形成末端醛基。在又一实施方案中,提供一种上述方法,其中活化的氨氧基连接基团包含1-50个乙二醇单元。
在又一实施方案中,提供一种上述方法,其中氨氧基连接基团为3-氧杂-戊烷-1,5-二羟基胺。在相关实施方案中,氧化剂为NaIO4。
在本发明的另一实施方案中,提供一种上述方法,其中所述氧化的碳水化合物部分与所述活化的水溶性聚合物的接触在包含选自由苯胺和苯胺衍生物所组成的组的亲核性催化剂的缓冲液中进行。
在本发明的又一实施方案中,提供一种上述方法,其进一步包括通过在包含选自由氰基硼氢化钠(NaCNBH3)和抗坏血酸(维生素C)所组成的组的还原化合物的缓冲液中孵育缀合的凝血蛋白来还原缀合的凝血蛋白中的肟键合的步骤。在相关实施方案中,还原化合物为氰基硼氢化钠(NaCNBH3)。
在本发明的另一实施方案中,提供一种由上述方法产生的经修饰凝血蛋白。
在本发明的又一实施方案中,提供一种经修饰FIX,其包含FIX分子或其生物活性片段、衍生物或变异体;及至少一个结合至FIX分子的氨氧基PSA,其中所述氨氧基PSA经由一个或多个碳水化合物部分连接至FIX。
在本发明的另一实施方案中,提供一种经修饰FVIIa,其包含FVIIa分子或其生物活性片段、衍生物或变异体;及至少一个结合至FVIIa分子的氨氧基PSA,其中所述氨氧基PSA经由一个或多个碳水化合物部分连接至FVIIa。
在本发明的又一实施方案中,提供一种经修饰FVIII,其包含FVIII分子或其生物活性片段、衍生物或变异体;及至少一个结合至FVIII分子的氨氧基PSA,其中所述氨氧基PSA经由一个或多个碳水化合物部分连接至FVIII。
在本发明的又一实施方案中,提供一种经修饰FIX,其包含FIX分子或其生物活性片段、衍生物或变异体;及至少一个结合至FIX分子的氨氧基PEG,其中所述氨氧基PEG经由一个或多个碳水化合物部分连接至FIX。
在本发明的另一实施方案中,提供一种经修饰FVIIa,其包含FVIIa分子或其生物活性片段、衍生物或变异体;及至少一个结合至FVIIa分子的氨氧基PEG,其中所述氨氧基PEG经由一个或多个碳水化合物部分连接至FVIIa。
在本发明的又一实施方案中,提供一种经修饰FVIII,其包含FVIII分子或其生物活性片段、衍生物或变异体;及至少一个结合至FVIII分子的氨氧基PEG,其中所述氨氧基PEG经由一个或多个碳水化合物部分连接至FVIII。
在又一实施方案中,提供一种包含活性氨氧基连接基团的水溶性聚合物;所述水溶性聚合物选自由以下所组成的组:聚乙二醇(PEG)、分支PEG、聚唾液酸(PSA)、碳水化合物、多糖、普鲁兰、壳聚糖、透明质酸、硫酸软骨素、硫酸皮肤素、淀粉、葡聚糖、羧甲基-葡聚糖、聚环氧烷(PAO)、聚亚烷基二醇(PAG)、聚丙二醇(PPG)、聚噁唑啉、聚丙烯酰基吗啉、聚乙烯醇(PVA)、聚羧酸酯、聚乙烯吡咯烷酮、聚磷腈、聚噁唑啉、聚乙烯-共-顺丁烯二酸酐、聚苯乙烯-共-顺丁烯二酸酐、聚(1-羟基甲基亚乙基-羟基甲基缩甲醛)(PHF)、磷酸2-甲基丙烯酰氧基-2’-乙基三甲基铵(MPC);所述活性氨氧基连接基团选自由以下所组成的组:下式的3-氧杂-戊烷-1,5-二羟基胺连接基团:
及
下式的3,6,9-三氧杂-十一烷-1,11-二羟基胺连接基团:
在又一实施方案中,提供一种上述方法,其中活化的氨氧基连接基团包含1-50个乙二醇单元。
附图
图1显示凝血因子IX的一级结构。
图2显示氧化的rFIX与氨氧基-PSA的偶联。
图3显示水溶性二氨氧基连接基团3-氧杂-戊烷-1,5-二羟基胺和3,6,9-三氧杂-十一烷-1,11-二羟基胺的合成。
图4显示氨氧基-PSA的制备。
图5显示采用SDS-PAGE和考马斯亮蓝染色对PSA-rFIX缀合物进行的分析性表征。
图6显示采用利用抗FIX和抗PSA抗体进行检测对PSA-rFIX缀合物进行的分析性表征。
图7显示天然rFIX和PSA-rFIX缀合物相对于输注后时间的活性。
图8显示PSA-rFVIII和Advate相对于输注后时间的含量。
发明详述
治疗性蛋白质的药理学和免疫性质可通过化学修饰及与聚合化合物缀合来改善,所述聚合化合物为例如聚乙二醇(PEG)、分支PEG、聚唾液酸(PSA)、碳水化合物、多糖、普鲁兰、壳聚糖、透明质酸、硫酸软骨素、硫酸皮肤素、淀粉、葡聚糖、羧甲基-葡聚糖、聚环氧烷(PAO)、聚亚烷基二醇(PAG)、聚丙二醇(PPG)、聚噁唑啉、聚丙烯酰基吗啉、聚乙烯醇(PVA)、聚羧酸酯、聚乙烯吡咯烷酮、聚磷腈、聚噁唑啉、聚乙烯-共-顺丁烯二酸酐、聚苯乙烯-共-顺丁烯二酸酐、聚(1-羟基甲基亚乙基-羟基甲基缩甲醛)(PHF)、磷酸2-甲基丙烯酰氧基-2’-乙基三甲基铵(MPC)。所得缀合物的性质一般高度取决于聚合物的结构及大小。因此,在本领域中,通常优选具有确定且狭窄大小分布的聚合物。合成聚合物例如PEG可容易地制成具有狭窄大小分布,而PSA可以使得产生具有狭窄大小分布的最终PSA制剂的方式纯化。另外,具有确定聚合物链及狭窄大小分布的聚乙二醇化试剂市场有售且可以合理的价格购得。
例如通过聚唾液酸化来添加可溶性聚合物为一种改善凝血蛋白例如FIX以及其它凝血蛋白(例如VWF、FVIIa(参见例如US 2008/0221032A1,以引用的方式并入本文中)及FVIII)的性质的方法。
凝血蛋白
如本文所述,本发明涵盖以下凝血蛋白,包括(但不限于)因子IX(FIX)、因子VIII(FVIII)、因子VIIa(FVIIa)、冯威利布兰德因子(VWF)、因子FV(FV)、因子X(FX)、因子XI、因子XII(FXII)、凝血酶(FII)、蛋白质C、蛋白质S、tPA、PAI-1、组织因子(TF)及ADAMTS 13蛋白酶。如本文所使用的术语“凝血蛋白(blood coagulation protein)”是指展现与特定天然凝血蛋白相关的生物活性的任何因子IX(FIX)、因子VIII(FVIII)、因子VIIa(FVIIa)、冯威利布兰德因子(VWF)、因子FV(FV)、因子X(FX)、因子XII(FXII)、凝血酶(FII)、蛋白质C、蛋白质S、tPA、PAI-1、组织因子(TF)及ADAMTS 13蛋白酶。
凝血级联分为三个不同的部分:内源途径、外源途径及共同途径(Schenone等人,Curr OpinHematol.2004:11:272-7)。所述级联涉及一系列丝氨酸蛋白酶(酶原)及蛋白质辅因子。需要时,非活性酶原前体转化为活性形式,从而转化级联中的后续酶。
内源途径需要凝血因子VIII、IX、X、XI及XII。当前激肽释放酶、高分子量激肽原、因子XI(FXI)及因子XII(FXII)暴露于带负电的表面时,发生内源途径的引发。还需要自血小板分泌的钙离子及磷脂。
当血管的血管内腔受损时,引发外源途径。膜糖蛋白组织因子暴露,并随后结合于循环的因子VII(FVII)及少量预先存在的其活化形式FVIIa。这种结合有利于FVII完全转化成FVIIa,及接着在钙和磷脂存在下因子IX(FIX)转化成因子IXa(FIXa),及因子X(FX)转化成因子Xa(FXa)。FVIIa与组织因子的缔合通过使FVII的基质(FIX和FX)结合位点更靠近且诱导构型变化(这增强FVIIa的酶促活性)来增强蛋白水解活性。
FX的活化为两个途径的共点。因子Va(FVa)和Xa连同磷脂和钙一起使凝血酶原转化成凝血酶(凝血酶原酶复合物),凝血酶随后裂解纤维蛋白原形成纤维蛋白单体。所述单体聚合形成纤维蛋白链。因子XIIIa(FXIIIa)使这些链相互共价键合以形成刚性网状物。
FVII转化成FVIIa也受若干蛋白酶催化,所述蛋白酶包括凝血酶、FIXa、FXa、因子XIa(FXIa)及因子XIIa(FXIIa)。为对级联的早期进行抑制,组织因子途径抑制剂靶向FVIIa/组织因子/FXa产物复合物。
A.多肽
在一个方面,本发明的起始物质为可来源于人类血浆或由如专利中所述的重组工程改造技术制备的凝血蛋白,所述专利为美国专利第4,757,006号;美国专利第5,733,873号;美国专利第5,198,349号;美国专利第5,250,421号;美国专利第5,919,766号;及EP 306968。如本文所述,术语凝血蛋白是指展现与天然凝血蛋白相关的生物活性的任何凝血蛋白分子。在本发明的一个实施方案中,凝血蛋白分子为全长凝血蛋白。
预期的凝血蛋白分子包括全长蛋白质、全长蛋白质的前体、全长蛋白质的生物活性次单元或片段以及任何这些凝血蛋白形式的生物活性衍生物及变异体。因此,凝血蛋白包括满足以下条件者:(1)具有在至少约25个、约50个、约100个、约200个、约300个、约400个或更多氨基酸的区域内与本文所述的参考核酸或氨基酸序列编码的多肽的氨基酸序列具有大于约60%、约65%、约70%、约75%、约80%、约85%、约90%、约91%、约92%、约93%、约94%、约95%、约96%、约97%、约98%或约99%或更高同一性的氨基酸序列;和/或(2)特异性结合于针对包含如本文所述的参考氨基酸序列的免疫原、其免疫原性片段和/或其保守性修饰变异体产生的抗体,例如多克隆或单克隆抗体。
根据本发明,术语“重组凝血蛋白”包括经由重组DNA技术获得的任何凝血蛋白。在某些实施方案中,所述术语涵盖如本文所述的蛋白质。
如本文所使用的“内源性凝血蛋白”包括起源于意欲接受治疗的哺乳动物的凝血蛋白。所述术语还包括自所述哺乳动物中所存在的转基因或任何其它外来DNA转录的凝血蛋白。如本文所使用的“外源性凝血蛋白”包括并非起源于意欲接受治疗的哺乳动物的凝血蛋白。
如本文所使用的“血浆来源的凝血蛋白”或“血浆性(plasmatic)”包括自哺乳动物获得的血液中可见的具有参与凝血途径的性质的蛋白质的所有形式。
如本文所使用的“生物活性衍生物”或“生物活性变异体”包括分子的与所述分子具有实质上相同的功能和/或生物性质(例如结合性质)和/或相同结构基础(例如肽主链或基础聚合单元)的任何衍生物或变异体。
“类似物”、“变异体”或“衍生物”为与天然存在的分子在结构上实质上类似且具有相同生物活性的化合物,但在某些情况下,程度不同。举例而言,多肽变异体是指与参考多肽共享实质上类似的结构且具有相同的生物活性的多肽。变异体或类似物与衍生出所述类似物的天然存在多肽相比基于一个或多个突变而在氨基酸序列组成方面不同,所述一个或多个突变涉及(i)多肽的一个或多个末端处和/或天然存在多肽序列的一个或多个内部区域(例如片段)中缺失一个或多个氨基酸残基,(ii)在多肽的一个或多个末端处(典型地为“添加”或“融合”)和/或天然存在多肽序列的一个或多个内部区域(典型地为“***”)中***或添加一个或多个氨基酸,或(iii)天然存在多肽序列中的一个或多个氨基酸被取代成其它氨基酸。举例而言,“衍生物”是指已经修饰(例如化学修饰)的与参考多肽共享相同或实质上类似的结构的多肽。
变异体或类似物多肽包括***变异体,其中向本发明的凝血蛋白氨基酸序列添加一个或多个氨基酸残基。***可位于蛋白质的任一个或两个末端,和/或可位于凝血蛋白氨基酸序列的内部区域中。任一个或两个末端具有额外残基的***变异体包括例如融合蛋白及包括氨基酸标签或其它氨基酸标记的蛋白质。在一个方面,凝血蛋白分子视情况含有N末端Met,尤其当分子重组表达于例如大肠杆菌(E.coli)的细菌细胞中时。
在缺失变异体中,在如本文所述的凝血蛋白多肽中一个或多个氨基酸残基经移除。缺失可在凝血蛋白多肽的一个或两个末端实现,和/或移除凝血蛋白氨基酸序列内部的一个或多个残基。因此,缺失变异体包括凝血蛋白多肽序列的片段。
在取代变异体中,凝血蛋白多肽的一个或多个氨基酸残基经移除并用替代残基置换。在一个方面,取代在性质上为保守性的,且所述类型的保守性取代在本领域为熟知的。或者,本发明包涵也是非保守性的取代。示例性保守性取代描述于Lehninger[Biochemistry,第2版;Worth Publishers公司,New York(1975),第71-77页]中,且紧接着陈述如下。
保守性取代
或者,示例性保守性取代紧接着陈述如下。
保守性取代II
B.聚核苷酸
本发明的编码凝血蛋白的核酸包括例如(但不限于)基因、前mRNA、mRNA、cDNA、多态性变异体、等位基因、合成及天然存在的突变体。
本发明的编码凝血蛋白的聚核苷酸还包括(但不限于)满足以下条件者:(1)在严格杂交条件下与编码本文所述的参考氨基酸序列的核酸及其保守性修饰变异体特异性杂交;(2)具有在至少约25个、约50个、约100个、约150个、约200个、约250个、约500个、约1000个或更多核苷酸的区域(至多成熟蛋白质的1218个核苷酸的全长序列)内与本文所述的参考核酸序列具有大于约95%、约96%、约97%、约98%、约99%或更高核苷酸序列同一性的核酸序列。示例性“严格杂交”条件包括在42℃下于50%甲酰胺、5×SSC、20mM Na·PO4,pH6.8中杂交;及在55℃下以1×SSC洗涤30分钟。应了解,此等示例性条件的变化可基于待杂交的序列的长度及GC核苷酸含量进行。本领域的标准式适于确定适当杂交条件。参见Sambrook等人,Molecular Cloning:A Laboratory Manual(第2版,Cold Spring HarborLaboratory Press,1989)§§9.47-9.51。
“天然存在的”聚核苷酸或多肽序列典型地来自哺乳动物,包括(但不限于)灵长类动物,例如人类;啮齿动物,例如大鼠、小鼠、仓鼠;牛、猪、马、绵羊或任何哺乳动物。本发明的核酸和蛋白质可为重组分子(例如为异源的且编码野生型序列或其变异体,或为非天然存在的)。
在本发明的某些实施方案中,上述多肽和聚核苷酸例如为以下凝血蛋白。
因子VIIa
FVII(也称为稳定因子或前转化素)为在止血及凝血中具有关键作用的维生素K依赖性丝氨酸蛋白酶糖蛋白(Eigenbrot,Curr Protein Pept Sci.2002:3:287-99)。
FVII在肝脏中合成且以48kD的单链糖蛋白形式分泌。FVII与所有维生素K依赖性丝氨酸蛋白酶糖蛋白共享类似的由以下组成的蛋白质结构域结构:具有9-12个残基负责蛋白质与脂膜交互作用的氨基端γ-羧基谷氨酸(Gla)结构域、羧基端丝氨酸蛋白酶结构域(催化结构域)、及两个含有介导与组织因子交互作用的钙离子结合位点的表皮生长因子样结构域。γ-谷氨酰羧化酶催化分子氨基端部分中Gla残基的羧化。羧化酶的作用依赖于维生素K的还原形式,所述维生素K被氧化成环氧化物形式。需要维生素K环氧化物还原酶使维生素K的环氧化物形式转化成还原形式。
大比例的FVII以酶原形式在血浆中循环,且此形式的活化导致精氨酸152与异亮氨酸153之间的肽键裂解。所得活化的FVIIa由经由单个二硫键(Cys 135与Cys 262)连接的NH2衍生的轻链(20kD)及COOH端衍生的重链(30kD)组成。轻链含有膜结合Gla结构域,而重链含有催化结构域。
遗传和环境因素决定的FVII血浆浓度为约0.5mg/mL(Pinotti等人,Blood.2000:95:3423-8)。不同FVII基因型的平均FVII含量可相差数倍。健康女性的血浆FVII含量在妊娠期间升高,而且血浆FVII含量会随年龄而增加,且在女性及患高甘油三酯血症者中较高。FVII在所有促凝血因子中半衰期最短(3-6h)。健康个体的平均FVIIa血浆浓度为3.6ng/mL,且FVIIa的循环半衰期与其它凝血因子相比相对较长(2.5h)。
遗传性FVII缺陷为一种罕见常染色体隐性出血病症,据估算总群体的发病率为每500,000个人1例(Acharya等人,J Thromb Haemost.2004:2248-56)。抑制剂所致的后天性FVII缺陷也极罕见。还已报导缺陷的发生与例如头孢菌素、青霉素及口服抗凝血剂的药物相关的病例。此外,已报导后天性FVII缺陷自发发生,或伴随其它病状(例如骨髓瘤、败血症、再生障碍性贫血)、伴随白细胞介素-2及抗胸腺细胞球蛋白疗法而发生。
参考聚核苷酸和多肽序列包括例如GenBank登录号J02933(基因组序列)、M13232(cDNA)(Hagen等人,PNAS 1986:83:2412-6)及P08709(多肽序列)(参考文献全文并入本文中)。已描述FVII的多种多态性现象,例如参见Sabater-Lleal等人(Hum Genet.2006:118:741-51)(参考文献全文并入本文中)。
因子IX
FIX为通过在钙离子、磷脂和FVIIIa存在下将FX转化成其活性形式来参与凝血内源途径的维生素K依赖性血浆蛋白质。FIX的主要催化能力为作为对FX内的特定精氨酸-异亮氨酸键具有特异性的丝氨酸蛋白酶。FIX的活化经由FXIa发生,FXIa引起自FIX切除活化肽,从而产生包含两个经由一个或多个二硫键保持在一起的链的活化FIX分子。FIX的缺陷为隐性X连锁B型血友病的病因。
A型和B型血友病分别为以FVIII和FIX多肽的缺陷为特征的遗传性疾病。缺陷的基础病因经常为FVIII和FIX基因突变的结果,FVIII与FIX基因两者均位于X染色体上。血友病的传统疗法通常涉及静脉内施用来自正常个体的汇集血浆或半纯化凝血蛋白。这些制剂可能受病原体或病毒污染,所述病原体或病毒为例如感染性朊病毒、HIV、细小病毒、A型肝炎及C型肝炎。因此,迫切需要不需要使用人类血清的治疗剂。
FIX活性降低的程度与B型血友病的严重程度成正比。B型血友病的当前治疗由以下组成:用血浆来源的FIX或重组FIX置换缺陷的蛋白质(所谓的FIX替代或置换治疗或疗法)。
FIX的聚核苷酸和多肽序列可见于例如UniProtKB/Swiss-Prot登录号P00740、美国专利第6,531,298号和图1中。
因子VIII
凝血因子VIII(FVIII)以极低浓度在血浆中循环且非共价结合于冯威利布兰德因子(VWF)。止血期间,FVIII与VWF分离且通过在钙和磷脂或细胞膜存在下增强活化速率而充当由活化因子IX(FIXa)介导的FX活化的辅因子。
FVIII可以具有结构域结构A1-A2-B-A3-C1-C2的约270-330kD的单链前体形式合成。当自血浆纯化(例如“血浆来源”或“血浆性”)时,FVIII由重链(A1-A2-B)和轻链(A3-C1-C2)构成。轻链的分子质量为80kD,而归因于B结构域内存在蛋白水解,重链的分子质量在90-220kD范围内。
FVIII还以重组蛋白质形式合成以在治疗上用于止血病症。已设计各种体外测定来测定重组FVIII(rFVIII)作为治疗性药品的潜在功效。这些测定模拟内源性FVIII的体内作用。FVIII的体外凝血酶处理使得其促凝血活性快速增加,接着降低,如体内测定所测量。此活化及失活与重链与轻链中的特异性受限蛋白水解一致,所述蛋白水解改变FVIII中不同结合抗原决定基的可用性,例如允许FVIII自VWF解离并结合于磷脂表面或改变与某些单克隆抗体的结合能力。
FVIII的缺陷或功能异常导致最常见的出血病症,A型血友病。管理A型血友病的所选治疗为血浆来源或rFVIII浓缩物的替代疗法,FVIII含量低于1%的重度A型血友病患者一般采用防治性疗法,目的在于在各剂量之间保持FVIII高于1%。考虑到各种FVIII产物在循环中的平均半衰期,此结果通常可通过每周给予FVIII两至三次来达成。
参考聚核苷酸和多肽序列包括例如UniProtKB/Swiss-Prot P00451(FA8_HUMAN);Gitschier J等人,Characterization of the human Factor VIII gene,Nature,312(5992):326-30(1984);Vehar GH等人,Structure of human Factor VIII,Nature,312(5992):337-42(1984);Thompson AR.Structure and Function of the Factor VIIIgene and protein,Semin Thromb Hemost,2003:29:11-29(2002)。
冯威利布兰德因子
冯威利布兰德因子(VWF)为在血浆中循环的呈一系列大小在约500kD至20,000kD范围内的多聚体的糖蛋白。VWF的多聚形式由经由二硫键键合在一起的250kD多肽次单元构成。VWF介导最初的血小板于受损血管壁内皮下层上的黏附。仅较大多聚体展现止血活性。假定内皮细胞分泌大型多聚形式的VWF,且具有低分子量的那些VWF形式(低分子量VWF)由蛋白水解裂解而产生。具有大分子质量的多聚体储存于内皮细胞的韦伯潘力氏小体(Weibel-Pallade body)内且在受刺激后释放。
VWF由内皮细胞和巨核细胞合成为在很大程度上由重复结构域组成的原VWF前体。信号肽裂解后,原VWF经由其C端区的二硫键合二聚化。所述等二聚体充当多聚化的原聚体,所述多聚化受自由端末端之间的二硫键合控制。组装成多聚体之后,通过蛋白水解移除前肽序列(Leyte等人,Biochem.J.274(1991),257-261)。
自VWF的克隆cDNA预测的初级转译产物为含2813个残基的前体多肽(原VWF前体)。原VWF前体由含22个氨基酸的信号肽和含741个氨基酸的前肽组成,其中成熟VWF包含2050个氨基酸(Ruggeri Z.A.和Ware,J.,FASEB J.,308-316(1993))。
VWF的缺陷为特征在于明显程度不同的出血表型的冯威利布兰德病(VonWillebrand disease,VWD)的病因。第3型VWD为最严重的形式,其中VWF完全缺陷,且第1型VWD与VWF的定量损失相关且其表型可能极轻。第2型VWD与VWF的定性缺陷相关且可能与第3型VWD一样严重。第2型VWD具有许多亚形式,其中一些与高分子量多聚体的损失或减少相关。第2a型冯威利布兰德病(VWD-2A)的特征在于损失中型与大型多聚体。VWD-2B的特征在于损失最高分子量的多聚体。本领域已知其它与VWF相关的疾病和病症。
原VWF前体的聚核苷酸和氨基酸序列分别可以GenBank登录号NM_000552和NP_000543获得。
本领域描述了根据本发明的其它凝血蛋白,例如Mann KG,Thromb Haemost,1999:82:165-74。
C.凝血蛋白的制备
凝血蛋白的制备包括本领域已知的用于以下的任何方法:(i)通过遗传工程改造制备重组DNA,(ii)通过例如(但不限于)转染、电穿孔或显微注射将重组DNA引入原核生物或真核生物细胞中,(iii)孵育所述经转化细胞,(iv)表达凝血蛋白,例如组成性表达或在诱导后表达,及(v)分离所述凝血蛋白,例如自培养基中分离或通过收集经转化细胞以获得经纯化的凝血蛋白。
在其它方面,通过于以产生药理学上可接受的凝血蛋白分子为特征的适合原核生物或真核生物宿主***中表达来制备凝血蛋白。真核生物细胞的实例为哺乳动物细胞,例如CHO、COS、HEK 293、BHK、SK-Hep及HepG2。
多种载体可用于制备凝血蛋白且选自真核生物和原核生物表达载体。用于原核生物表达的载体的实例包括质体,例如(但不限于)pRSET、pET及pBAD,其中用于原核生物表达载体的启动子包括(但不限于)以下中的一个或多个:lac、trc、trp、recA或araBAD。用于真核生物表达的载体的实例包括:(i)用于在酵母中表达时,载体为例如(但不限于)pAO、pPIC、pYES或pMET,使用的启动子为例如(但不限于)AOX1、GAP、GAL1或AUG1;(ii)用于在昆虫细胞中表达,载体为例如(但不限于)pMT、pAc5、pIB、pMIB或pBAC,使用的启动子为例如(但不限于)PH、p10、MX、Ac5、OpIE2、gp64或polh,及(iii)用于在哺乳动物细胞中表达,载体为例如(但不限于)pSVL、pCMV、pRc/RSV、pcDNA3或pBPV,且在一个方面,载体来源于病毒***,所述等病毒***为例如(但不限于)痘苗病毒、腺伴随病毒、疱疹病毒或逆转录病毒,使用的启动子为例如(但不限于)CMV、SV40、EF-1、UbC、RSV、ADV、BPV及β-肌动蛋白。
D.施用
在一个实施方案中,本发明的缀合的凝血蛋白可通过注射(例如静脉内、肌内或腹膜内注射)施用。
给人类或测试动物施用包含本发明的缀合的凝血蛋白的组合物,在一个方面,所述组合物包含一种或多种药学上可接受的载体。术语“药学上”或“药理学上可接受”是指分子实体和组合物当如下文所述使用本领域熟知的途径施用时为稳定的,抑制蛋白质降解(例如聚集和裂解产物),且另外不产生过敏反应或其它不良反应。“药学上可接受的载体”包括任何及所有临床上有用的溶剂、分散介质、包衣剂、抗细菌剂和抗真菌剂、等张剂和吸收延迟剂及其类似物,包括以上所公开的那些药剂。
如本文所使用的“有效量”包括适于治疗患有如本文所述的出血病症的哺乳动物的剂量。
组合物可如下施用:口服、局部、经皮、肠胃外、通过吸入喷雾、经***、经直肠或通过颅内注射。如本文所使用的术语肠胃外包括皮下注射、静脉内、肌内、脑池内注射或输注技术。还涵盖如下施用:静脉内、皮内、肌内、***内、腹膜内、鞘内、眼球后、肺内注射和/或外科手术植入特定部位。一般而言,组合物基本上不含致热原以及可能对接受者有害的其它杂质。
通过治疗医师选择的剂量水平和方式进行组合物的单次或多次施用。预防或治疗疾病时,适当剂量将视以下因素而定:如上所述的待治疗疾病的类型、疾病的严重程度及病程、施用药物是用于预防性目的或治疗性目的、先前疗法、患者的临床病史及对药物的反应及主治医师的判断。
本发明还涉及一种包含有效量的如本文所定义的缀合的凝血蛋白的药物组合物。药物组合物可进一步包含药学上可接受的载体、稀释剂、盐、缓冲剂或赋形剂。药物组合物可用于治疗以上定义的出血病症。本发明的药物组合物可为溶液或冻干产品。可对药物组合物的溶液进行任何适合的冻干过程。
作为另一方面,本发明包括包含以有利于本发明组合物用于施用个体的方式包装的本发明组合物的试剂盒。在一个实施方案中,该试剂盒包括包装于容器(例如密封瓶或容器)中的本文所述的化合物或组合物(例如包含缀合的凝血蛋白的组合物),以及附着于容器上或包括在包装中的描述化合物或组合物在实施所述方法中的用途的标签。在一个实施方案中,该试剂盒含有具有包含缀合的凝血蛋白的组合物的第一容器和具有用于第一容器中的组合物的生理学上可接受的复原溶液的第二容器。在一个方面,化合物或组合物以单位剂型包装。该试剂盒可进一步包括适于根据特定施用途径施用组合物的装置。优选地,该试剂盒含有描述治疗性蛋白质或肽组合物的使用的标签。
水溶性聚合物
在一个方面,所提供的凝血蛋白衍生物(即,缀合的凝血蛋白)分子结合于水溶性聚合物,包括(但不限于)聚乙二醇(PEG)、分支PEG、聚唾液酸(PSA)、碳水化合物、多糖、普鲁兰、壳聚糖、透明质酸、硫酸软骨素、硫酸皮肤素、淀粉、葡聚糖、羧甲基-葡聚糖、聚环氧烷(PAO)、聚亚烷基二醇(PAG)、聚丙二醇(PPG)、聚噁唑啉、聚丙烯酰基吗啉、聚乙烯醇(PVA)、聚羧酸酯、聚乙烯吡咯烷酮、聚磷腈、聚噁唑啉、聚乙烯-共-顺丁烯二酸酐、聚苯乙烯-共-顺丁烯二酸酐、聚(1-羟基甲基亚乙基-羟基甲基缩甲醛)(PHF)、磷酸2-甲基丙烯酰氧基-2’-乙基三甲基铵(MPC)。在本发明的一个实施方案中,水溶性聚合物由分子量范围为以下的唾液酸分子组成:350至120,000Da、500至100,000Da、1000至80,000Da、1500至60,000Da、2,000至45,000Da、3,000至35,000Da及5,000至25,000Da。水溶性聚合物的偶联可通过与蛋白质直接偶联或经由连接分子偶联来进行。化学连接基团的一个实例为含碳水化合物选择性酰肼和硫氢基反应性顺丁烯二酰亚氨基的MBPH(4-[4-N-顺丁烯二酰亚氨基苯基]丁酸酰肼)(Chamow等人,J Biol Chem 1992:267:15916-22)。其它示例性及优选的连接基团描述如下。
在一个实施方案中,衍生物保留天然治疗性凝血蛋白产物的全部功能活性,且与天然治疗性凝血蛋白产物相比,提供延长的体内半衰期。在另一实施方案中,相对于天然凝血蛋白,衍生物保留至少20、21、22、23、24、25、26、27、28、29、30、31、32、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、110、120、130、140或150百分比(%)的生物活性。在相关方面中,以显色活性与凝血因子抗原值的比率(凝血因子Chr:凝血因子Ag)测定衍生物和天然凝血蛋白的生物活性。在本发明的又一实施方案中,相对于天然凝血蛋白的体内半衰期,构建体的半衰期降至或增至0.5倍、0.6倍、0.7倍、0.8倍、0.9倍、1.0倍、1.1倍、1.2倍、1.3倍、1.4倍、1.5倍、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍或10倍。
A.唾液酸和PSA
如本文所使用的“唾液酸部分”包括可溶于水溶液或悬浮液中且在施用药物有效量的PSA-凝血蛋白缀合物之后对哺乳动物几乎不产生负面影响(例如副作用)的唾液酸单体或聚合物(“多糖”)。在一个方面,聚合物的特征在于具有1、2、3、4、5、10、20、30、40、50、60、70、80、90、100、200、300、400或500个唾液酸单元。在某些方面中,不同唾液酸单元组合于一条链中。
在本发明的一个实施方案中,多糖化合物的唾液酸部分具有高亲水性,且在另一实施方案中,整个化合物具有高亲水性。亲水性主要由唾液酸单元的侧接羧基以及羟基赋予。糖单元可含有其它官能基,例如胺、羟基或硫酸酯基或其组合。这些基团可存在于天然存在的糖化合物上或引入衍生的多糖化合物中。
天然存在的聚合物PSA可以显示宽大小分布(例如Sigma C-5762)及高多分散性(PD)的多分散制剂形式获得。因为多糖通常在具有共纯化内毒素的固有风险的细菌中产生,所以长唾液酸聚合物链的纯化可使内毒素含量增加的机率升高。具有1-4个唾液酸单元的短PSA分子还可合成制备(Kang SH等人,Chem Commun.2000:227-8;Ress DK及LinhardtRJ,Current Organic Synthesis.2004:1:31-46),从而将高内毒素水平的风险降至最低。然而,目前可制造大小分布狭窄且多分散性低而且亦无内毒素的PSA制剂。在一个方面,特别可用于本发明的多糖化合物为由细菌产生的多糖化合物。这些天然存在多糖中的一些称为糖脂。在一个实施方案中,多糖化合物实质上不含末端半乳糖单元。
B.聚乙二醇(PEG)和聚乙二醇化
在某些方面中,凝血因子(例如FVIII、FVIIa、FIX或其它凝血因子分子)通过多种化学方法中的任一种缀合于水溶性聚合物(Roberts JM等人,Advan Drug Delivery Rev2002:54:459-76)。举例而言,在一个实施方案中,通过使用N-羟基丁二酰亚胺(NHS)酯使PEG缀合于FVIII、FVIIa或FIX的自由氨基来修饰所述蛋白质。在另一实施方案中,使用顺丁烯二酰亚胺化学或PEG酰肼或PEG胺与事先氧化之后的FVIII、FVIIa或FIX的碳水化合物部分的偶联,来使水溶性聚合物(例如PEG)与自由SH基团偶联。
在一个方面,通过使水溶性聚合物与凝血因子(例如FVIII、FVIIa或FIX)在形成稳定键下直接偶联(或经由连接***偶联)进行缀合。另外,在本发明的某些方面中,使用可降解的、可释放的或可水解的连接***(Tsubery等人,J Biol Chem 2004:279:38118-24/Greenwald等人,J Med Chem 1999:42:3657-67/Zhao等人,Bioconj Chem 2006:17:341-51/WO2006/138572A2/US7259224B2/US7060259B2)。
在本发明的一个实施方案中,通过使用含活性N-羟基丁二酰亚胺酯(NHS)(例如丁二酸丁二酰亚胺酯、戊二酸丁二酰亚胺酯或丙酸丁二酰亚胺酯)的聚乙二醇衍生物经由赖氨酸残基修饰凝血因子(例如FVIII、FVIIa或FIX)。这些衍生物与FVIII、FVIIa或FIX的赖氨酸残基在温和条件下通过形成稳定的酰胺键而反应。在本发明的一个实施方案中,PEG衍生物的链长为5,000Da。各种实施方案中使用链长为500至2,000Da、2,000至5,000Da、5,000以上至10,000Da或10,000以上至20,000Da、或20,000以上至150,000Da的其它PEG衍生物,包括线性和分支结构。
氨基聚乙二醇化的替代方法为(但不限于)通过形成氨基甲酸乙酯键与PEG碳酸酯化学缀合,或通过还原胺化与醛或酮反应从而形成仲酰胺键。
在本发明的一个实施方案中,使用可购得的PEG衍生物对凝血因子(例如FVIII、FVIIa、FIX或其它凝血因子)分子进行化学修饰。在替代方面中,这些PEG衍生物具有线性或分支结构。含NHS基团的PEG衍生物的实例列于下文。
以下PEG衍生物为可自Nektar Therapeutics(Huntsville,Ala.;参见www.nektar.com/PEG试剂目录;Nektar Advanced PEGylation,价格表2005-2006)购得的PEG衍生物的非限制性实例:
mPEG-丙酸丁二酰亚胺酯(mPEG-SPA)
mPEG-α-甲基丁酸丁二酰亚胺酯(mPEG-SMB)
mPEG-CM-HBA-NHS(CM=羧甲基;HBA=羟基丁酸)
分支PEG衍生物(Nektar Therapeutics)的结构:
分支PEGN-羟基丁二酰亚胺(mPEG2-NHS)
Kozlowski等人(BioDrugs 2001:5:419-29)详细描述具有分支结构的这种试剂。
可自NOF Corp.(Tokyo,Japan;参见www.nof.co.jp/english:目录2005)购得PEG衍生物的其它非限制性实例。
线性PEG衍生物(NOF Corp.)的一般结构:
X=羧甲基
X=羧戊基
x=丁二酸酯基
x=戊二酸酯基
分支PEG衍生物(NOF Corp.)的结构:2,3-双(甲基聚氧乙烯-氧基)-1-(1,5-二氧代-5-丁二酰亚氨基氧基戊氧基)丙烷
2,3-双(甲基聚氧乙烯-氧基)-1-(丁二酰亚氨基羧基戊氧基)丙烷
这些丙烷衍生物显示1,2取代型甘油主链。在本发明中,还涵盖基于1,3取代甘油结构或US2003/0143596A1中所述的其它分支结构的分支PEG衍生物。
还涵盖Tsubery等人(J Biol Chem 2004:279:38118-24)和Shechter等人(WO04089280A3)所述的含可降解(例如可水解)连接基团的PEG衍生物。
令人惊讶的是,本发明的聚乙二醇化FVIII、FVIIa、FIX或其它凝血因子展现功能活性以及延长的体内半衰期。另外,聚乙二醇化rFVIII、FVIIa、FIX或其它凝血因子似乎更能抵抗凝血酶失活。
C.连接方法
凝血蛋白可经由本领域熟练技术人员已知的各种技术中的任一种共价连接至多糖化合物。在本发明的各个方面中,唾液酸部分可例如通过美国专利第4,356,170号(以引用的方式并入本文中)中所述的方法结合至凝血蛋白(例如FIX、FVIII、FVIIa或VWF)。
本发明还已知且涵盖用于使PSA与多肽偶联的其它技术。举例而言,美国公开第2007/0282096号描述使例如PSA的胺或酰肼衍生物缀合于蛋白质。另外,美国公开第2007/0191597号描述含有用于与基质(例如蛋白质)的还原末端反应的醛基的PSA衍生物。这些参考文献均以全文引用的方式并入本文中。
美国专利第5,846,951号(其以全文引用的方式并入本文中)第7栏第15列至第8栏第5列公开了各种方法。示例性技术包括经由凝血蛋白或多糖中的任一个上的羧基与凝血蛋白或多糖的胺基之间的肽键,或凝血蛋白或多糖的羧基与凝血蛋白或多糖的羟基之间的酯键的键合。凝血蛋白共价键结至多糖化合物的另一键合是经由凝血蛋白上的自由氨基与通过过碘酸盐氧化在多糖的非还原末端形成的醛基之间反应所形成的希夫碱(JenningsHJ和Lugowski C,J Immunol.1981:127:1011-8;Fernandes AI和Gregoriadis G,BiochimBiophys Acta.1997:1341:26-34)。在一个方面,所产生的希夫碱系经用NaCNBH3特异性还原形成仲胺而稳定。替代方法为在事先氧化之后通过用NH4Cl还原胺化在PSA中产生末端自由氨基。双官能试剂可用于连接两个氨基或两个羟基。举例而言,用试剂如BS3(辛二酸双(磺基丁二酰亚氨基)酯/Pierce,Rockford,IL)使含氨基的PSA与蛋白质的氨基偶联。另外,使用异双官能交联试剂如磺基-EMCS(N-ε-顺丁烯二酰亚氨基己酰氧基)磺基丁二酰亚胺酯/Pierce)来例如连接氨基与硫醇基。
在另一方法中,制备PSA酰肼且使之与事先氧化且产生醛官能团之后的蛋白质的碳水化合物部分偶联。
如上所述,治疗性蛋白质的自由氨基与唾液酸残基的1-羧基反应形成肽键,或在1-羧酸基团与凝血蛋白上的羟基或其它适合活性基团之间形成酯键合。或者,羧基与去乙酰化的5-氨基形成肽键合,或凝血蛋白分子的醛基与唾液酸残基的N-去乙酰化5-氨基形成希夫碱。
或者,多糖化合物以非共价方式与凝血蛋白缔合。举例而言,在一个方面,多糖化合物和药物活性化合物经由疏水性相互作用连接。其它非共价缔合包括带相反电荷的离子相互吸引的静电相互作用。
在各种实施方案中,凝血蛋白与多糖化合物以化学计算量(例如1:1、1:2、1:3、1:4、1:5、1:6、1:7、1:7、1:8、1:9或1:10等)连接或缔合。在各种实施方案中,1-6、7-12或13-20个多糖连接至凝血蛋白。在其它实施方案中,1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20个或更多个多糖连接至凝血蛋白。
在各种实施方案中,对凝血蛋白进行修饰以引入糖基化位点(即除天然糖基化位点以外的位点)。所述修饰可使用本领域已知的标准分子生物技术完成。此外,凝血蛋白可在经由一个或多个碳水化合物部分缀合于水溶性聚合物之前在体内或体外糖基化。这些糖基化位点可充当使蛋白质与水溶性聚合物缀合的靶点(美国专利申请第20090028822号、美国专利申请第2009/0093399号、美国专利申请第2009/0081188号、美国专利申请第2007/0254836号、美国专利申请第2006/0111279号及DeFrees S.等人,Glycobiology,2006,16,9,833-43)。
D.氨氧基键合
在本发明的一个实施方案中,应用羟胺或羟胺衍生物与醛(例如由过碘酸钠氧化后的碳水化合物部分上的醛)形成肟基的反应来制备凝血蛋白缀合物。举例而言,首先用氧化剂(例如过碘酸钠(NaIO4))氧化糖蛋白(例如本发明的凝血蛋白)(Rothfus JA和SmithEL.,J Biol Chem 1963,238,1402-10;及Van Lenten L和Ashwell G.,J Biol Chem 1971,246,1889-94)。糖蛋白的过碘酸盐氧化是基于1928年描述的经典马拉普拉德反应(Malaprade reaction),即用过碘酸盐氧化邻二醇以形成活性醛基(Malaprade L.,Analytical application,Bull Soc Chim France,1928,43,683-96)。这种氧化剂的其它实例为四乙酸铅(Pb(OAc)4)、乙酸锰(MnO(Ac)3)、乙酸钴(Co(OAc)2)、乙酸亚铊(TlOAc)、硫酸铈(Ce(SO4)2)(US 4,367,309)或过钌酸钾(KRuO4)(Marko等人,J Am Chem Soc 1997,119,12661-2)。“氧化剂”是指能够在生理反应条件下氧化碳水化合物中的邻二醇从而产生活性醛基的适度氧化剂。
第二步为使含氨氧基的聚合物与氧化的碳水化合物部分偶联以形成肟键合。在本发明的一个实施方案中,此步骤可在催化量的亲核性催化剂苯胺或苯胺衍生物存在下进行(Dirksen A和Dawson PE,Bioconjugate Chem.2008;Zeng Y等人,Nature Methods 2009:6:207-9)。苯胺催化作用显著加速肟连接,从而允许使用极低浓度的试剂。在本发明的另一实施方案中,所述肟键合可因经NaCNBH3还原形成烷氧基胺键合而稳定(图2)。
在本发明的一个实施方案中,使水溶性聚合物缀合于凝血蛋白的各反应步骤分别且依次进行(即在个别反应步骤之间分离起始物质(例如凝血蛋白、水溶性聚合物等)、试剂(例如氧化剂、苯胺等)及反应产物(例如凝血蛋白上氧化的碳水化合物、活化的氨氧基水溶性聚合物等))。
关于氨氧基技术的其它信息可见于以下参考文献中,各参考文献全文并入本文中:EP1681303A1(HAS化的***);WO 2005/014024(聚合物与蛋白质经由肟连接基团连接的缀合物);WO96/40662(含氨氧基连接化合物及其在缀合物中的应用);WO 2008/025856(经修饰蛋白质);Peri F等人,Tetrahedron 1998,54,12269-78:Kubler-Kielb J和Pozsgay V.,J Org Chem 2005,70,6887-90;Lees A等人,Vaccine 2006,24(6),716-29;及Heredia KL等人,Macromoecules 2007,40(14),4772-9。
在本发明的各种实施方案中,根据本文所述的氨氧基技术连接至凝血蛋白(例如FVIII、FVIIa或FIX)的氧化的碳水化合物部分的水溶性聚合物包括(但不限于)聚乙二醇(PEG)、分支PEG、聚唾液酸(PSA)、碳水化合物、多糖、普鲁兰、壳聚糖、透明质酸、硫酸软骨素、硫酸皮肤素、淀粉、葡聚糖、羧甲基-葡聚糖、聚环氧烷(PAO)、聚亚烷基二醇(PAG)、聚丙二醇(PPG)、聚噁唑啉、聚丙烯酰基吗啉、聚乙烯醇(PVA)、聚羧酸酯、聚乙烯吡咯烷酮、聚磷腈、聚噁唑啉、聚乙烯-共-顺丁烯二酸酐、聚苯乙烯-共-顺丁烯二酸酐、聚(1-羟基甲基亚乙基-羟基甲基缩甲醛)(PHF)、磷酸2-甲基丙烯酰氧基-2’-乙基三甲基铵(MPC)。
以下实施例不旨在限制,而仅示例本发明的特定实施方案。
实施例
实施例1
制备同双官能连接基团NH
2
[OCH
2
CH
2
]
2
ONH
2
根据Boturyn等人(Tetrahedron 1997:53:5485-92)在两步骤有机反应中采用伯胺的经改进的加百利合成(Gabriel-Synthesis)合成含有两个活性氨氧基的同双官能连接基团NH2[OCH2CH2]2ONH2
(3-氧杂-戊烷-1,5-二羟基胺)(图3)。在第一步骤中,使一分子2,2-氯二乙基醚与两分子内式-N-羟基-5-降冰片烯-2,3-二甲酰亚胺在二甲基甲酰胺(DMF)中反应。通过在乙醇中肼解自所得中间物制备所需同双官能产物。
实施例2
制备同双官能连接基团NH
2
[OCH
2
CH
2
]
4
ONH
2
根据Boturyn等人(Tetrahedron 1997:53:5485-92)在两步骤有机反应中采用伯胺的经改进的加百利合成来合成含有两个活性氨氧基的同双官能连接基团NH2[OCH2CH2]4ONH2
(3,6,9-三氧杂-十一烷-1,11-二羟基胺)(图3)。在第一步骤中,使一分子双-(2-(2-氯乙氧基)-乙基)-醚与两分子内式-N-羟基-5-降冰片烯-2,3-二甲酰亚胺在DMF中反应。通过在乙醇中肼解自所得中间物制备所需同双官能产物。
实施例3
制备氨氧基-PSA
将500mg自印度血清研究所(Serum Institute of India)(Pune,India)获得的氧化PSA(MW=18.8kD)溶解于8ml 50mM乙酸钠缓冲液(pH 5.5)中。接着,添加100mg 3-氧杂-戊烷-1,5-二羟基胺。在室温下振荡2小时后,添加44mg氰基硼氢化钠。再在4℃下振荡4小时后,将反应混合物装载于Slide-A-Lyzer(Pierce,Rockford,IL)透析盒(3.5kD膜,再生纤维素)中并用PBS(pH 7.2)透析4天。在-80℃下冷冻产物。图4图解根据此程序制备氨氧基-PSA。
制备氨氧基PSA的替代程序
将1000mg自印度血清研究所(Pune,India)获得的氧化PSA(MW=20kD)溶解于16ml50mM磷酸盐缓冲液(pH 6.0)中。随后,向反应混合物中添加170mg 3-氧杂-戊烷-1,5-二羟基胺。在室温下振荡2小时后,添加78.5mg氰基硼氢化钠且使反应进行18小时过夜。随后使用由再生纤维素制成的截留值为5kD的膜(Millipore)对反应混合物进行超滤/透滤程序(UF/DF)。
实施例4
使氨氧基-PSA与rFIX偶联并纯化缀合物
向溶解于6.3ml 50mM乙酸钠缓冲液(pH 6.0)中的12.6mg rFIX中添加289μl过碘酸钠水溶液(10mM)。在4℃下在黑暗中振荡混合物1小时并在室温下通过添加6.5μl 1 M甘油淬灭15分钟。通过超滤/透滤(UF/DF)采用Vivaspin(Sartorius,Goettingen,Germany)浓缩器(30kD膜,再生纤维素)移除低分子量污染物。接着,向UF/DF渗余物中添加43mg氨氧基-PSA,且在4℃下振荡混合物18小时。利用疏水性相互作用层析(HIC)移除过量PSA试剂。冷却的反应混合物的导电率上升至180mS/cm,且将其装载于经50mM HEPES、3M氯化钠、6.7mM氯化钙、0.01%Tween 80(pH 6.9)预平衡的5ml HiTrap Butyl FF(GE Healthcare,Fairfield,CT)HIC柱(1.6×2.5cm)上。用2.4柱体积(CV)的50mM HEPES、6.7mM氯化钙、0.005%Tween80(pH 7.4)以5ml/min的流动速率洗脱缀合物。通过测量总蛋白质(BCA)和FIX显色活性对所述制剂进行分析性表征。对于PSA-rFIX缀合物,测定出每毫克蛋白质的比活性为80.2IU(与天然rFIX相比为其56.4%)。结果概述于表1中。
表1
图5图解通过SDS-PAGE利用考马斯亮蓝染色对PSA-rFIX缀合物进行的分析性表征。图6显示SDS-PAGE及随后采用抗FIX和抗PSA抗体进行的蛋白质印迹法。
实施例5
氨氧基-PSA与rFIX在苯胺作为亲核性催化剂存在下的偶联
向溶解于1.4ml 50mM乙酸钠缓冲液(pH 6.0)中的3.0mg rFIX中添加14.1μl过碘酸钠水溶液(10mM)。在4℃下在黑暗中振荡混合物1小时并在室温下通过添加1.5μl 1M甘油淬灭15分钟。借助于尺寸排阻层析(SEC)采用PD-10脱盐柱(GE Healthcare,Fairfield,CT)移除低分子量污染物。混合1.2mg溶解于1.33ml 50mM乙酸钠缓冲液(pH 6.0)中的氧化rFIX与70μl苯胺(200mM储备水溶液)并在室温下振荡45分钟。接着,添加4.0mg氨氧基-PSA,且在室温下振荡混合物2小时并再在4℃下振荡16小时。在1小时后、2小时后和18小时后反应结束时抽取样品。接着,借助于HIC移除过量PSA试剂和游离rFIX。冷却的反应混合物的导电率上升至180mS/cm,且将其装载于经50mM HEPES、3M氯化钠、6.7mM氯化钙、0.01%Tween 80(pH 6.9)预平衡的5ml HiTrap Butyl FF(GE Healthcare,Fairfield,CT)HIC柱(1.6×2.5cm)上。用20CV达到50mM HEPES、6.7mM氯化钙、0.005%Tween 80(pH 7.4)的线性梯度以5ml/min的流动速率洗脱缀合物。
实施例6
使氨氧基-PSA与rFIX偶联且用NaCNBH
3
还原
向溶解于5.25ml 50mM乙酸钠缓冲液(pH 6.0)中的10.5mg rFIX中添加53μl过碘酸钠水溶液(10mM)。在4℃下在黑暗中振荡混合物1小时并在室温下通过添加5.3μl 1M甘油淬灭15分钟。借助于UF/DF采用Vivaspin(Sartorius,Goettingen,Germany)浓缩器(30kD膜,再生纤维素)移除低分子量污染物。接着,向UF/DF渗余物中添加35.9mg氨氧基-PSA,且在室温下振荡混合物2小时。随后添加53μl氰基硼氢化钠水溶液(5M),且反应再进行16小时。随后,借助于HIC移除过量PSA试剂。冷却的反应混合物的导电率上升至180mS/cm,且将其装载于经50mM HEPES、3M氯化钠、6.7mM氯化钙、0.01%Tween 80(pH 6.9)预平衡的5mlHiTrap Butyl FF HIC(GE Healthcare,Fairfield,CT)柱(1.6×2.5cm)上。用2.4CV的50mMHEPES、6.7mM氯化钙、0.005%Tween 80(pH 7.4)以5ml/min的流动速率洗脱缀合物。
实施例7
使氨氧基-PSA(连接基团:NH
2
[OCH
2
CH
2
]
4
ONH
2
)与rFIX偶联且纯化缀合物
向溶解于2.8ml 50mM乙酸钠缓冲液(pH 6.0)中的5.6mg rFIX中添加102μl过碘酸钠水溶液(10mM)。在4℃下在黑暗中振荡混合物1小时并在室温下通过添加2.9μl 1M甘油淬灭15分钟。借助于UF/DF采用Vivaspin(Sartorius,Goettingen,Germany)浓缩器(30kD膜,再生纤维素)移除低分子量污染物。随后,向UF/DF渗余物中添加19mg氨氧基-PSA,且在4℃下振荡混合物18小时。借助于HIC移除过量PSA试剂。冷却的反应混合物的导电率上升至180mS/cm,且将其装载于经50mM HEPES、3M氯化钠、6.7mM氯化钙、0.01%Tween 80(pH6.9)预平衡的5ml HiTrap Butyl FF(GE Healthcare,Fairfield,CT)HIC柱(1.6×2.5cm)上。用2.4CV的50mM HEPES、6.7mM氯化钙、0.005%Tween 80(pH 7.4)以5ml/min的流动速率洗脱缀合物。
实施例8
使氨氧基-PSA与rFVIII偶联
向溶解于11ml Hepes缓冲液(pH 6)(50mM Hepes,5mM CaCl2,150mM NaCl,0.01%Tween)中的11mg rFVIII中添加57μl 10mM过碘酸钠。在4℃下在黑暗中振荡混合物30分钟并在4℃下通过添加107μl 1M甘油水溶液淬灭30分钟。随后,添加19.8mg氨氧基-PSA(18.8kD),且在4℃下振荡混合物过夜。通过添加含有8M乙酸铵的缓冲液(8M乙酸铵、50mMHepes、5mM CaCl2、350mM NaCl、0.01%Tween 80,pH 6.9)来增加离子强度以得到2.5M乙酸铵的最终浓度。接着,将反应混合物装载于经平衡缓冲液(2.5M乙酸铵,50mMHepes,5mMCaCl2,350mM NaCl,0.01%Tween 80,pH 6.9)平衡的HiTrap Butyl FF(GE Healthcare,Fairfield,CT)柱上。用洗脱缓冲液(50mM Hepes,5mM CaCl2,0.01%Tween 80,pH 7.4)洗脱产物,且通过离心过滤使用MWCO为30,000的Vivaspin(Sartorius,Goettingen,Germany)装置浓缩洗出液。
实施例9
血友病小鼠中的PK研究
用每公斤体重10ml体积剂量的含rFIX或PSA-rFIX(根据实施例4制备)的调配缓冲液(10mM组氨酸、260mM甘氨酸、29mM蔗糖、0.005%Tween 80,pH 6.8)注射FIX缺陷小鼠。物质注射后5分钟、3小时、9小时、16小时、24小时及48小时处死各组的6只小鼠,且通过心脏穿刺收集血液。制备含柠檬酸盐的血浆,且冷冻储存直至分析FIX活性。
用显色FIX测定(Biophen FIX测定,Hyphen Biomed,Neuville-sur-Oise,France)测定FIX活性,且构筑消除曲线(图7)。PSA-rFIX的实际FIX活性剂量为123IU FIX/kg,且rFIX的实际FIX活性剂量为143IU FIX/kg。用程序R(The R Foundation for StatisticalComputing,2008)计算药物动力学参数。rFIX的体内回收率为13%,且PSA-rFIX的体内回收率为29%。PSA-rFIX的剂量调节的AUC相对于rFIX增至6.4倍,终端半衰期增至1.2倍,且相比于rFIX,PSA-rFIX的MRT为1.7倍长(表2)。
表2
实施例10
使凝血蛋白聚唾液酸化
如本文所述的聚唾液酸化可扩展至其它凝血蛋白。举例而言,在本发明的各个方面中,对凝血蛋白例如FVIII、FVIIa和VWF重复用氨氧基-PSA进行如以上实施例5、6和9中所述的聚唾液酸化。
实施例11
制备同双官能连接基团NH
2
[OCH
2
CH
2
]
6
ONH
2
根据Boturyn等人(Tetrahedron 1997:53:5485-92)在两步骤有机反应中采用伯胺的经改进的加百利合成来合成含有两个活性氨氧基的同双官能连接基团NH2[OCH2CH2]6ONH2
(3,6,9,12,15-五氧杂-十七烷-1,17-二羟基胺)。在第一步骤中,使一分子二氯化六乙二醇(hexaethylene glycol dichloride)与两分子内式-N-羟基-5-降冰片烯-2,3-二甲酰亚胺在DMF中反应。通过在乙醇中肼解自所得中间物制备所需同双官能产物。
实施例12
采用顺丁烯二酰亚氨基/氨氧基连接***使rFIX聚唾液酸化
A.制备修饰试剂
通过使用顺丁烯二酰亚氨基/氨氧基连接***制备氨氧基-PSA试剂(Toyokuni等人,Bioconjugate Chem 2003:14,1253-9)。使用两步骤程序制备含自由末端SH-基团的PSA-SH(20kD):a)根据WO05016973A1通过用NH4Cl对氧化的PSA进行还原胺化制备PSA-NH2,及b)如US7645860所述通过使末端伯氨基与2-亚氨基硫杂环戊烷(尤特奇试剂(Traut'sreagent)/Pierce,Rockford,IL)反应引入硫氢基。使用10倍摩尔过量的连接基团及50mg/ml的PSA-SH浓度使PSA-SH与连接基团的顺丁烯二酰亚氨基在pH 7.5下在PBS缓冲液中偶联。在室温下在平缓振荡下孵育反应混合物2小时。随后,移除过量连接试剂,且通过透滤将氨氧基-PSA缓冲液更换至氧化缓冲液(50mM磷酸钠,pH 6.0)中。采用Pellicon XL5kD再生纤维素膜(Millipore,Billerica,MA)更换缓冲液25次。
B.在用NaIO4事先氧化之后修饰rFIX
在50mM磷酸钠缓冲液(pH 6.0)中采用该缓冲液中的100μM过碘酸钠对rFIX进行氧化。在4℃下在黑暗中振荡混合物1小时并在室温下通过添加甘油至最终浓度为5mM来淬灭15分钟。借助于尺寸排阻层析(SEC)采用PD-10脱盐柱(GE Healthcare,Fairfield,CT)移除低分子量污染物。随后,在氧化的rFIX中掺入苯胺以获得10mM的最终浓度,并与氨氧基-PSA试剂混合以达成5倍摩尔过量的PSA。在室温下在黑暗中在平缓振荡下保温反应混合物2小时。
C.纯化缀合物
借助于HIC移除过量的PSA试剂和游离rFIX。反应混合物的导电率上升至180mS/cm,且将其装载于经50mM HEPES、3M氯化钠、6.7mM氯化钙、0.01%Tween 80(pH 6.9)预平衡的填充有48ml丁基-琼脂糖FF(GE Healthcare,Fairfield,CT)的柱上。接着,用40CV的线性梯度的60%洗脱缓冲液(50mM Hepes,6.7mM氯化钙,pH 7.4)洗脱缀合物。最后,收集含PSA-rFIX的洗脱份,且使用由再生纤维素制成的30kD膜(Millipore)进行UF/DF。通过测量总蛋白质(BCA)和FIX显色活性对所述制剂进行分析性表征。对于由两种变异体制备的PSA-rFIX缀合物,测定到比活性大于天然rFIX的50%。
实施例13
制备氨氧基-PSA试剂
根据实施例3制备氨氧基-PSA试剂。使用5kD膜(再生纤维素,Millipore)用缓冲液(pH7.2)(50mM Hepes)透滤最终产物,在-80℃下冷冻并冻干。冻干后,将所述试剂溶解于适当体积的水中且用于经由碳水化合物修饰来制备PSA-蛋白质缀合物。
实施例14
聚唾液酸化的rFVIII在FVIII缺陷敲除小鼠模型中的药物动力学
根据实施例8制备PSA-FVIII缀合物。缀合物显示比活性为6237IU/mg(FVIII活性由显色测定法测定;总蛋白质由Bradford测定法测定),且聚唾液酸化度为6.7(摩尔PSA/摩尔FVIII),如由间苯二酚测定(Svennerholm L,Biochim Biophys Acta 1957:24:604-11)所测量。
使用Bi等人(Nat Genet 1995:10:119-21)详细描述的FVIII缺陷小鼠作为重度人类A型血友病的模型。各组(每组6只小鼠)经由尾静脉接受剂量为每公斤体重200IU FVIII的根据实施例8制备的PSA-rFVIII或天然rFVIII(ADVATE,Baxter Healthcare公司)的快速注射(200IU FVIII/kg)。注射后5分钟、3小时、6小时、9小时、16小时、24小时、32小时及42小时,通过麻醉后心脏穿刺自各别组制备含柠檬酸盐的血浆。通过使用显色测定测量血浆样品中的FVIII活性程度。此实验的结果概述于表3中且图解于图8中。用2.10.1版本R(一种统计计算用语言和环境。R Foundation for Statistical Computing,Vienna,Austria.http://www.R-project.org.)进行所有计算。结果,平均滞留时间(MRT)自5.4小时(Advate对照组)增至11.1小时(PSA-rFVIII缀合物)。
表3:
实施例15
氨氧基-PSA试剂的详细合成
根据Botyryn等人(Tetrahedron 1997:53:5485-92)在实施例1中概述的两步骤有机合成中合成3-氧杂-戊烷-1,5-二羟基胺。
步骤1:
向内式-N-羟基-5-降冰片烯-2,3-二甲酰亚胺(59.0g;1.00当量)于700ml无水N,N-二甲基甲酰胺中的溶液中添加无水K2CO3(45.51g;1.00当量)及2,2-二氯二乙基醚(15.84ml;0.41当量)。在50℃下搅拌反应混合物22小时。在减压下蒸发混合物至干燥。将残余物悬浮于2L二氯甲烷中,并用饱和NaCl水溶液萃取两次(每次1L)。二氯甲烷层经Na2SO4干燥,并随后在减压下蒸发至干燥并在高真空中干燥,得到64.5g呈黄白色固体状的3-氧杂戊烷-1,5-二氧基-内式-2’,3’-二羧基二酰亚胺降冰片烯(中间物1)。
步骤2:
向中间物1(64.25g;1.00当量)于800ml无水乙醇中的溶液中添加31.0ml水合肼(4.26当量)。随后使反应混合物回流2小时。通过在减压下蒸发溶剂浓缩混合物至起始体积的一半。滤掉出现的沉淀。在减压下蒸发剩余乙醇层至干燥。在真空中干燥含粗产物3-氧杂-戊烷-1,5-二羟基胺的残余物,得到46.3g。通过柱层析(硅胶60;用二氯甲烷/甲醇混合物9+1进行等度洗脱)进一步纯化粗产物,得到11.7g纯最终产物3-氧杂-戊烷-1,5-二羟基胺。
实施例16
使用PSA酰肼使rFIX聚唾液酸化
通过使用使氧化PSA与己二酸二酰肼(ADH)反应制备的PSA酰肼试剂使rFIX聚唾液酸化。
步骤1:制备PSA酰肼
将500mg自印度血清研究所(Pune,India)获得的氧化PSA(MW=20kD)溶解于8ml50mM乙酸钠缓冲液(pH 5.5)中。随后添加100mg己二酸二酰肼(ADH)。平缓振荡溶液2小时。随后添加44mg氰基硼氢化钠。再在4℃下孵育反应物4小时后,将反应混合物装载于Slide-A-Lyzer(Pierce,Rockford,IL)透析盒(3.5kD膜,再生纤维素)中并用PBS(pH 7.2)透析4天。在-80℃下冷冻产物。
步骤2:使PSA酰肼与rFIX反应且纯化缀合物
通过使用如步骤1中所述的PSA酰肼试剂使rFIX聚唾液酸化。在4℃下在黑暗中在平缓振荡下用NaIO4(浓度:80μM)氧化rFIX(浓度:1mg/ml)1小时。通过添加甘油终止反应,且使用由再生纤维素制成的30kD膜(Vivaspin)对氧化的FIX进行UF/DF。随后使用200倍摩尔过量的试剂及1mg/ml的蛋白质浓度在pH 6.5下使氧化的rFIX聚唾液酸化。在室温下在黑暗中在平缓振荡下保温rFIX及聚唾液酸化试剂2小时。最后,利用HIC纯化PSA-rFIX缀合物。通过添加含乙酸铵的缓冲液(50mM Hepes,350mM NaCl,5mM氯化钙,8M乙酸铵,0.01%Tween80,pH 6.9)使反应混合物的导电率上升至130mS/cm,且将其装载于经50mM Hepes、2.5M乙酸铵、350mM氯化钠、5mM氯化钙、0.01%Tween 80(pH 6.9)预平衡的HiTrap Butyl FF柱(5ml,GE Healthcare,Fairfield,CT)上。接着,用50mM Hepes、5mM氯化钙、0.01%Tween 80(pH 7.4)洗脱缀合物。最后,收集含PSA-rFIX的洗脱份,且使用由再生纤维素制成的30kD膜(Vivaspin)进行UF/DF。对于PEG-rFIX缀合物,测定到比活性大于天然rFIX的50%(显色测定)。
实施例17
使用PSA酰肼在苯胺作为亲核性催化剂存在下使rFIX聚唾液酸化
将123mg rFIX溶解于60ml磷酸盐缓冲液(50mM NaPO4,pH 6.5)缓冲液中。随后添加1.2ml过碘酸钠水溶液(10mM),且在4℃下在黑暗中在平缓搅拌下保温混合物1小时。接着,通过添加600μl 1M甘油水溶液在室温下淬灭反应15分钟。接着采用Pellicon XLUltracel 30 kD膜对混合物进行UF/DF。
用59.6ml磷酸盐缓冲液(50mM NaPCU,pH 6.0)进一步稀释含氧化的rFIX的UF/DF渗余物(63.4ml),且与6.5ml苯胺水溶液(200mM)混合并在室温下保温30分钟。随后添加12.3ml PSA-酰肼试剂(根据实施例16制备),得到5倍摩尔试剂过量。在室温下在黑暗中在平缓振荡下孵育此混合物2小时。
借助于HIC移除过量的PSA-酰肼试剂和游离rFIX。反应混合物的导电率上升至180mS/cm,且将其装载于经50mM HEPES、3M氯化钠、6.7mM氯化钙、0.01%Tween 80(pH 6.9)预平衡的填充有48ml丁基-琼脂糖FF(GE Healthcare,Fairfield,CT)的柱上。接着,用50mMHepes、5mM氯化钙、0.01%Tween 80(pH 7.4)洗脱缀合物。最后,收集含PSA-rFIX的洗脱份,且使用由再生纤维素制成的30kD膜(Millipore)进行UF/DF。通过测量总蛋白质(BCA)和FIX显色活性对所述制剂进行分析性表征。对于PSA-rFIX缀合物,测定到比活性大于天然rFIX的50%。
实施例18
使rFIX聚唾液酸化并使用两步骤程序纯化
将140mg rFIX溶解于62ml磷酸盐缓冲液(50mM NaPO4,pH 6.0)缓冲液中。随后添加1.92ml过碘酸钠水溶液(10mM),且在4℃下在黑暗中在平缓搅拌下保温混合物1小时,且在室温下通过添加64μl 1M甘油水溶液淬灭15分钟。接着采用Pellicon XL Ultracel 30kD膜对混合物进行UF/DF。
用73.8ml磷酸盐缓冲液(50mM NaPCU,pH 6.0)进一步稀释含氧化的rFIX的UF/DF渗余物(69.4ml),与8.2ml苯胺水溶液(200mM)混合并在室温下保温30分钟。随后添加12.3ml氨氧基试剂(根据实施例3制备),得到2.5倍摩尔试剂过量。在室温下在黑暗中在平缓振荡下保温此混合物2.5小时。
借助于阴离子交换层析(AIEC)移除游离rFIX。用20ml缓冲液A(50mM Hepes,5mMCaCl2,pH 7.5)稀释反应混合物,且装载于经缓冲液A预平衡的Q-琼脂糖FF 26/10柱(GEHealthcare,Fairfield,CT)上。随后用缓冲液B(50mM Hepes,1M NaCl,5mM CaCl2,pH 7.5)洗脱柱。游离rFIX在导电率介于12-25mS/cm之间时洗脱,且缀合物在导电率介于27-45mS/cm之间时洗脱。接着通过添加缓冲液C(50mM Hepes,5M NaCl,5mM CaCl2,pH 6.9)使含缀合物的洗脱份的导电率上升至190mS/cm,且将其装载于经缓冲液D(50mM Hepes,3M NaCl,5mMCaCl2,pH 6.9)预平衡的丁基琼脂糖FF 26/10柱(GE Healthcare,Fairfield,CT)上。用5CV缓冲液D洗除游离PSA-试剂。接着,用100%缓冲液E(50mM Hepes,5mM CaCl2,pH 7.4)洗脱缀合物。使用由再生纤维素制成的10kD膜(88cm2,截留值为10kD/Millipore)进行UF/DF来浓缩含缀合物的洗脱份。用含有150mM NaCl和5mM CaCl2的组氨酸缓冲液(pH7.2)进行最后的透滤步骤。通过测量总蛋白质(BCA)和FIX显色活性对所述制剂进行分析性表征。对于PSA-rFIX缀合物,测定到比活性大于天然rFIX的50%。
实施例19
使氨氧基-PSA与rFVIIa偶联且纯化缀合物
混合10mg rFVIIa于5ml反应缓冲液(50mM Hepes,150mM氯化钠,5mM氯化钙,pH6.0)中的溶液与NaIO4水溶液(最终浓度:100μM),且在4℃下在黑暗中在平缓搅拌下保温1小时,且通过添加半胱氨酸水溶液(最终浓度:1mM)淬灭15分钟。接着,对反应混合物进行UF/DF。向渗余物(10ml)中添加30倍摩尔过量的氨氧基试剂(根据实施例1制备)。在室温下在黑暗中在平缓振荡下进行偶联反应2小时。通过HIC移除过量氨氧基试剂。通过添加含乙酸铵的缓冲液(50mM Hepes,350mM NaCl,5mM氯化钙,8M乙酸铵,0.01%Tween 80,pH 6.9)使反应混合物的导电率上升至130mS/cm,且将其装载于经50mM Hepes、2.5M乙酸铵、350mM氯化钠、5mM氯化钙、0.01%Tween 80(pH 6.9)预平衡的HiTrap Butyl FF柱(5ml,GEHealthcare,Fairfield,CT)上。接着,用50mM Hepes、5mM氯化钙、0.01%Tween 80(pH7.4)通过20CV的100%洗脱缓冲液的线性梯度洗脱缀合物。最后,收集含PSA-rFVIIa的洗脱份,且使用由再生纤维素制成的30kD膜(Vivaspin)进行UF/DF。通过测量总蛋白质(BCA)和FVIIa显色活性(Staclot测定,Diagnostica Stago,Asnieres,France)对所述制剂进行分析性表征,且显示比活性大于rFVIIa起始物质的20%。
实施例20
氨氧基-PSA与rFVIIa在苯胺作为亲核性催化剂存在下的偶联
向溶解于1.4ml 50mM乙酸钠缓冲液(pH 6.0)中的3.0mg rFVIIa中添加14.1μl过碘酸钠水溶液(10mM)。在4℃下在黑暗中振荡混合物1小时并在室温下通过添加1.5μl 1M甘油淬灭15分钟。借助于尺寸排阻层析(SEC)采用PD-10脱盐柱(GE Healthcare,Fairfield,CT)移除低分子量污染物。混合溶解于3ml 50mM乙酸钠缓冲液(pH 6.0)中的3mg氧化rFVIIa与苯胺(一种亲核性催化剂,最终浓度:10mM)并在室温下振荡30分钟。接着,添加氨氧基-PSA以得到5倍摩尔过量,且在室温下振荡混合物2小时。接着,借助于HIC移除过量PSA试剂和游离rFIX。冷却的反应混合物的导电率上升至180mS/cm,且将其装载于经50mM HEPES、3M氯化钠、6.7mM氯化钙、0.01%Tween 80(pH 6.9)预平衡的5ml HiTrap Butyl FF(GEHealthcare,Fairfield,CT)HIC柱(1.6×2.5cm)上。用20CV的50mM HEPES、6.7mM氯化钙、0.005%Tween 80(pH 7.4)的线性梯度以5ml/min的流动速率洗脱缀合物。
实施例21
制备氨氧基-PEG试剂
使用分支PEG-醛(MW 40kD)与如实施例1所述制备的二氨氧基连接基团偶联。此PEG-醛试剂可获自NOF(NOF Corp.,Tokyo,Japan)。将500mg PEG-醛溶解于8ml 50mM乙酸钠缓冲液(pH 5.5)中。随后,添加100mg 3-氧杂-戊烷-1,5-二羟基胺。在室温下振荡2小时后,添加44mg氰基硼氢化钠。再在4℃下振荡4小时后,将反应混合物装载于Slide-A-Lyzer(Pierce,Rockford,IL)透析盒(3.5kD膜,再生纤维素)中并用PBS(pH 7.2)透析4天。在-80℃下冷冻产物。
实施例22
用氨氧基PEG试剂使rFIX聚乙二醇化
通过使用含氨氧基的线性20kD聚乙二醇化试剂使rFIX聚乙二醇化。此类型试剂的实例为来自NOF的CA系列(NOF Corp.,Tokyo,Japan)。在4℃下在黑暗中在平缓振荡下在反应缓冲液(50mM Hepes,150mM氯化钠,5mM氯化钙,pH 6.0)中在2mg/ml的蛋白质浓度下用NaIO4(最终浓度:100μM)氧化rFIX 1小时且通过添加甘油水溶液(最终浓度:1mM)淬灭15分钟。接着,对反应混合物进行UF/DF。向渗余物中添加3倍摩尔过量的氨氧基试剂和苯胺(亲核性催化剂,最终浓度:10mM)。在室温下在黑暗中在平缓振荡下进行偶联反应2小时。最后,通过在Q-琼脂糖FF上进行的离子交换层析纯化PEG-rFIX缀合物。将每毫升凝胶1.5毫克蛋白质加载于经50mM Tris(pH 8.0)预平衡的柱上。用20CV 50mM Tris和1M氯化钠(pH 8.0)洗脱缀合物,并随后使用30kD膜进行UF/DF。通过测量总蛋白质(BCA)和FIX显色活性对所述制剂进行分析性表征。对于PEG-rFIX缀合物,测定到比活性大于天然rFIX的75%。
实施例23
用氨氧基PEG试剂使rFVIII聚乙二醇化
通过使用含氨氧基的线性20kD聚乙二醇化试剂使rFVIII聚乙二醇化。此类型试剂的实例为来自NOF的CA系列(NOF Corp.,Tokyo,Japan)。在4℃下在黑暗中在平缓振荡下在反应缓冲液(50mM Hepes,150mM氯化钠,5mM氯化钙,pH 6.0)中在1mg/ml的蛋白质浓度下用NaIO4(最终浓度:100μM)氧化rFVIII 1小时且通过添加半胱氨酸水溶液(最终浓度:1mM)淬灭15分钟。接着,对反应混合物进行UF/DF。向渗余物中添加20倍摩尔过量的氨氧基试剂和苯胺(亲核性催化剂,最终浓度:10mM)。在室温下在黑暗中在平缓振荡下进行偶联反应2小时。最后,通过在Q-琼脂糖FF上进行的离子交换层析纯化PEG-rFVIII缀合物。将每毫升凝胶1.5mg蛋白质加载于经含5mM CaCl2的50mM Hepes缓冲液(pH 7.4)预平衡的柱上。用含5mM CaCl2和500mM氯化钠的50mM Hepes缓冲液(pH 7.4)洗脱缀合物,并随后使用30kD膜进行UF/DF。通过FVIII显色测定对缀合物进行的分析性表征和测定总蛋白质(BCA测定)显示比活性大于rFVIII起始物质的60%。
实施例24
用氨氧基PEG试剂使rFVIIa聚乙二醇化
通过使用含氨氧基的线性20kD聚乙二醇化试剂使rFVIIa聚乙二醇化。此类型试剂的实例为来自NOF的CA系列(NOF Corp.,Tokyo,Japan)。在4℃下在黑暗中在平缓振荡下在反应缓冲液(50mM Hepes,150mM氯化钠,5mM氯化钙,pH 6.0)中在2mg/ml的蛋白质浓度下用NaIO4(最终浓度:100μM)氧化rFVIIa 1小时且通过添加甘油水溶液(最终浓度:1mM)淬灭15分钟。接着,对反应混合物进行UF/DF。向渗余物中添加5倍摩尔过量的氨氧基试剂和苯胺(亲核性催化剂,最终浓度:10mM)。在室温下在黑暗中在平缓振荡下进行偶联反应2小时。最后,通过在Q-琼脂糖FF上进行的离子交换层析纯化PEG-rFVIIa缀合物。将每毫升凝胶1.5毫克蛋白质加载于经含1mM CaCl2的20mM Hepes缓冲液(pH 7.4)预平衡的柱上。用含1mM CaCl2和500mM氯化钠的20mM Hepes缓冲液(pH 7.4)洗脱缀合物,并随后使用30kD膜进行UF/DF。通过测量FVIIa活性(Staclot测定,Diagnostica Stago,Asnieres,France)和总蛋白质(BCA测定)对缀合物进行的分析性表征显示比活性大于rFVIIa起始物质的25%。
实施例25
用PEG-酰肼试剂使rFIX聚乙二醇化
通过使用含酰肼基的线性20kD聚乙二醇化试剂使rFIX聚乙二醇化。此类型试剂的实例为来自NOF的HZ系列(NOF Corp.,Tokyo,Japan)。在4℃下在黑暗中在平缓振荡下在反应缓冲液(50mM Hepes,150mM氯化钠,5mM氯化钙,pH 6.0)中在2mg/ml的蛋白质浓度下用NaIO4(最终浓度:100μM)氧化rFIX 1小时且通过添加甘油水溶液(最终浓度:1mM)淬灭15分钟。接着,对反应混合物进行UF/DF。向渗余物中添加50倍摩尔过量的酰肼试剂和苯胺(亲核性催化剂,最终浓度:10mM)。在室温下在黑暗中在平缓振荡下进行偶联反应2小时。最后,通过在Q-琼脂糖FF上进行的离子交换层析纯化PEG-rFIX缀合物。将反应混合物装载于经50mM Tris缓冲液(pH 8.0)预平衡的柱(每毫升凝胶1.5mg蛋白质)上。用20CV Tris缓冲液(pH 8.0)(50mM Tris,1M NaCl)洗脱缀合物,并随后使用30kD膜进行UF/DF。通过测量总蛋白质(BCA)和FIX显色活性对所述制剂进行分析性表征。对于PEG-rFIX缀合物,测定到比活性大于天然rFIX的50%(显色测定)。
实施例26
在2mM苯胺存在下使rFVIII聚唾液酸化
将rFVIII转移至反应缓冲液(50mM Hepes,350mM氯化钠,5mM氯化钙,0.01%Tween80,pH 6)中,稀释至蛋白质浓度为1mg/ml且在4℃下在黑暗中在平缓振荡下在反应缓冲液(50mM Hepes,150mM氯化钠,5mM氯化钙,pH 6.0)中用NaIO4(最终浓度:100μM)氧化1小时,且通过添加半胱氨酸水溶液(最终浓度:1mM)淬灭15分钟。接着,对反应混合物进行UF/DF。向渗余物中添加20倍摩尔过量的氨氧基试剂和苯胺(亲核性催化剂,最终浓度:2mM)。在室温下在黑暗中在平缓振荡下进行偶联反应2小时。借助于HIC移除过量氨氧基试剂。通过添加含乙酸铵的缓冲液(50mM Hepes,350mM氯化钠,5mM氯化钙,8M乙酸铵,0.01%Tween80,pH 6.9)使反应混合物的导电率上升至130mS/cm,且将其装载于经50mMHepes、2.5M乙酸铵、350mM氯化钠、5mM氯化钙、0.01%Tween 80(pH 6.9)预平衡的填充有53ml丁基-琼脂糖FF(GE Healthcare,Fairfield,CT)的柱上。接着,用50mM Hepes、5mM氯化钙、0.01%Tween80(pH 7.4)洗脱缀合物。最后,收集含PSA-rFIX的洗脱份,且使用由再生纤维素制成的30kD膜(Millipore,Billerica,MA)进行UF/DF。通过测量总蛋白质(BCA)和FVIII显色活性对所述制剂进行分析性表征。对于PSA-rFVIII缀合物,测定到比活性为天然rFVIII的80%。
实施例27
在10mM苯胺存在下使rFVIII聚唾液酸化
将rFVIII转移至反应缓冲液(50mM Hepes,350mM氯化钠,5mM氯化钙,0.01%Tween80,pH 6)中,稀释至蛋白质浓度为1mg/ml且在4℃下在黑暗中在平缓振荡下在反应缓冲液(50mM Hepes,150mM氯化钠,5mM氯化钙,pH 6.0)中用NaIO4(最终浓度:100μM)氧化1小时,且通过添加半胱氨酸水溶液(最终浓度:1mM)淬灭15分钟。接着,对反应混合物进行UF/DF。向渗余物中添加20倍摩尔过量的氨氧基试剂和苯胺(亲核性催化剂,最终浓度:10mM)。在室温下在黑暗中在平缓振荡下进行偶联反应2小时。借助于HIC移除过量氨氧基试剂。通过添加含乙酸铵的缓冲液(50mM Hepes,350mM氯化钠,5mM氯化钙,8M乙酸铵,0.01%Tween80,pH 6.9)使反应混合物的导电率上升至130mS/cm,且将其装载于经50mMHepes、2.5M乙酸铵、350mM氯化钠、5mM氯化钙、0.01%Tween 80(pH 6.9)预平衡的填充有53ml丁基-琼脂糖FF(GE Healthcare,Fairfield,CT)的柱上。接着,用50mM Hepes、5mM氯化钙、0.01%Tween80(pH 7.4)洗脱缀合物。最后,收集含PSA-rFIX的洗脱份,且使用由再生纤维素制成的30kD膜(Millipore,Billerica,MA)进行UF/DF。通过测量总蛋白质(BCA)和FVIII显色活性对所述制剂进行分析性表征。对于PSA-rFVIII缀合物,测定到比活性为天然rFVIII的80%。
实施例28
使用分支PEG使凝血蛋白聚乙二醇化
凝血蛋白(例如实施例22-25中所述的FIX、FVIII和FVIIa)的聚乙二醇化可扩展至实施例21中所述的由醛和含活性氨氧基的适合连接基团制成的分支或线性聚乙二醇化试剂。
Claims (22)
1.一种使水溶性聚合物缀合于凝血蛋白的氧化的碳水化合物部分的方法,所述方法包括使所述氧化的碳水化合物部分与活化的水溶性聚合物在允许缀合的条件下接触;
所述凝血蛋白选自由以下所组成的组:因子IX(FIX)、因子VIII(FVIII)、因子VIIa(FVIIa)、冯威利布兰德因子(VWF)、因子FV(FV)、因子X(FX)、因子XI(FXI)、因子XII(FXII)、凝血酶(FII)、蛋白质C、蛋白质S、tPA、PAI-1、组织因子(TF)及ADAMTS 13蛋白酶或其生物活性片段、衍生物或变异体;
所述水溶性聚合物含有活性氨氧基且选自由以下所组成的组:聚乙二醇(PEG)、分支PEG、聚唾液酸(PSA)、碳水化合物、多糖、普鲁兰、壳聚糖、透明质酸、硫酸软骨素、硫酸皮肤素、淀粉、葡聚糖、羧甲基-葡聚糖、聚环氧烷(PAO)、聚亚烷基二醇(PAG)、聚丙二醇(PPG)、聚噁唑啉、聚丙烯酰基吗啉、聚乙烯醇(PVA)、聚羧酸酯、聚乙烯吡咯烷酮、聚磷腈、聚噁唑啉、聚乙烯-共-顺丁烯二酸酐、聚苯乙烯-共-顺丁烯二酸酐、聚(1-羟基甲基亚乙基-羟基甲基缩甲醛)(PHF)、磷酸2-甲基丙烯酰氧基-2’-乙基三甲基铵(MPC);且
所述碳水化合物部分通过用包含氧化剂的缓冲液孵育而氧化,所述氧化剂选自由以下所组成的组:过碘酸钠(NaIO4)、四乙酸铅(Pb(OAc)4)及过钌酸钾(KRuO4);其中肟键合在所述氧化的碳水化合物部分与所述水溶性聚合物上的活性氨氧基之间形成。
2.根据权利要求1所述的方法,其中所述水溶性聚合物为PSA。
3.根据权利要求2所述的方法,其中所述PSA包含约10-300个唾液酸单元。
4.根据权利要求1至3中任一项所述的方法,其中所述凝血蛋白为FIX。
5.根据权利要求1至3中任一项所述的方法,其中所述凝血蛋白为FVIIa。
6.根据权利要求1至3中任一项所述的方法,其中所述凝血蛋白为FVIII。
7.根据权利要求1至6中任一项所述的方法,其中所述氧化剂为过碘酸钠(NaIO4)。
8.根据权利要求4至7中任一项所述的方法,其中所述凝血蛋白的氧化的碳水化合物部分位于凝血蛋白的活化肽中。
9.根据权利要求2所述的方法,其中所述PSA通过使活化的氨氧基连接基团与氧化的PSA反应来制备;
其中所述氨氧基连接基团选自由以下所组成的组:
a)下式的3-氧杂-戊烷-1,5-二羟基胺连接基团:
及
b)下式的3,6,9-三氧杂-十一烷-1,11-二羟基胺连接基团:
其中所述PSA通过用氧化剂孵育来氧化以在所述PSA的非还原末端形成末端醛基。
10.根据权利要求7所述的方法,其中所述氨氧基连接基团为3-氧杂-戊烷-1,5-二羟基胺。
11.根据权利要求7所述的方法,其中所述氧化剂为NaIO4。
12.根据权利要求1至9中任一项所述的方法,其中所述氧化的碳水化合物部分与所述活化的水溶性聚合物的接触在包含选自由苯胺及苯胺衍生物所组成的组的亲核性催化剂的缓冲液中进行。
13.根据权利要求2所述的方法,其进一步包括通过在包含选自由氰基硼氢化钠(NaCNBH3)及抗坏血酸(维生素C)所组成的组的还原化合物的缓冲液中孵育缀合的凝血蛋白来还原所述缀合的凝血蛋白中的肟键合的步骤。
14.根据权利要求11所述的方法,其中所述还原化合物为氰基硼氢化钠(NaCNBH3)。
15.一种经修饰凝血蛋白,其为利用根据权利要求第1至12项中任一项所述的方法产生。
16.一种经修饰FIX,其包含:
(a)FIX分子或其生物活性片段、衍生物或变异体;及
(b)至少一个结合至(a)的FIX分子的氨氧基PSA,其中所述氨氧基PSA经由一个或多个碳水化合物部分连接至所述FIX。
17.一种经修饰FVIIa,其包含:
(a)FVIIa分子或其生物活性片段、衍生物或变异体;及
(b)至少一个结合至(a)的FVIIa分子的氨氧基PSA,其中所述氨氧基PSA经由一个或多个碳水化合物部分连接至所述FVIIa。
18.一种经修饰FVIII,其包含:
(a)FVIII分子或其生物活性片段、衍生物或变异体;及
(b)至少一个结合至(a)的FVIII分子的氨氧基PSA,其中所述氨氧基PSA经由一个或多个碳水化合物部分连接至所述FVIII。
19.一种经修饰FIX,其包含:
(a)FIX分子或其生物活性片段、衍生物或变异体;及
(b)至少一个结合至(a)的FIX分子的氨氧基PEG,其中所述氨氧基PEG经由一个或多个碳水化合物部分连接至所述FIX。
20.一种经修饰FVIIa,其包含:
(a)FVIIa分子或其生物活性片段、衍生物或变异体;及
(b)至少一个结合至(a)的FVIIa分子的氨氧基PEG,其中所述氨氧基PEG经由一个或多个碳水化合物部分连接至所述FVIIa。
21.一种经修饰FVIII,其包含:
(a)FVIII分子或其生物活性片段、衍生物或变异体;及
(b)至少一个结合至(a)的FVIII分子的氨氧基PEG,其中所述氨氧基PEG经由一个或多个碳水化合物部分连接至所述FVIII。
22.一种水溶性聚合物,其包含活性氨氧基连接基团;所述水溶性聚合物选自由以下所组成的组:聚乙二醇(PEG)、分支PEG、聚唾液酸(PSA)、碳水化合物、多糖、普鲁兰、壳聚糖、透明质酸、硫酸软骨素、硫酸皮肤素、淀粉、葡聚糖、羧甲基-葡聚糖、聚环氧烷(PAO)、聚亚烷基二醇(PAG)、聚丙二醇(PPG)、聚噁唑啉、聚丙烯酰基吗啉、聚乙烯醇(PVA)、聚羧酸酯、聚乙烯吡咯烷酮、聚磷腈、聚噁唑啉、聚乙烯-共-顺丁烯二酸酐、聚苯乙烯-共-顺丁烯二酸酐、聚(1-羟基甲基亚乙基-羟基甲基缩甲醛)(PHF)、磷酸2-甲基丙烯酰氧基-2’-乙基三甲基铵(MPC);所述活性氨氧基连接基团选自由以下所组成的组:
a)下式的3-氧杂-戊烷-1,5-二羟基胺连接基团:
及
b)下式的3,6,9-三氧杂-十一烷-1,11-二羟基胺连接基团:
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