CN1211578A - 聚乙二醇－干扰素结合物 - Google Patents

聚乙二醇－干扰素结合物 Download PDF

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CN1211578A
CN1211578A CN97105433A CN97105433A CN1211578A CN 1211578 A CN1211578 A CN 1211578A CN 97105433 A CN97105433 A CN 97105433A CN 97105433 A CN97105433 A CN 97105433A CN 1211578 A CN1211578 A CN 1211578A
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R·卡拉西韦茨
C·纳林
P·罗森
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Abstract

本发明涉及具有生理活性的水溶性聚乙二醇同干扰素的结合物,以及可用来制备这些结合物的新型聚乙二醇化合物。

Description

聚乙二醇-干扰素结合物
本申请是申请号为93116479.6、申请日为1993年8月25日、发明名称为“聚乙二醇-干扰素结合物”的发明专利申请的分案申请。
许多天然的和重组的蛋白质都具有医疗和医药效用。一旦被纯化并制成制剂，它们能够对各种临床适应征进行非肠道使用。然而，非肠道使用的蛋白质可能具有免疫原性，可能相对来说不易溶于水，也可能药理半寿期短。结果，病人体内的蛋白质很难达到有效治疗的血液水平。
这些问题可通过将蛋白质同聚合物(如聚乙二醇)结合来克服。Davis等人在美国专利4,179,337中公开了将聚乙二醇(PEG)结合到蛋白质(如酶和胰岛素)上，从而得到其中蛋白质免疫原性较低，但能保持大部分生理活性的结合物。Nakagawa等人公开了将PEG结合到小岛活化蛋白质上以减少其副作用和免疫原性。Veronese等人在Applied Biochem.and Biotech,11:141-152(1985)上公开了用氯甲酸苯酯类来活化聚乙二醇以修饰核糖核酸酶和超氧化物歧化酶。Katre等人在美国专利4,766,106和4,917,888上也公开了通过聚合物结合使蛋白质增溶。将PEG和其它聚合物同重组蛋白质结合以减弱蛋白质的免疫原性和提高它们的半寿期。参见Nitecki等人的美国专利4,902,502；Enzon公司的国际申请PCT/US90/03133；Nishimura等人的欧洲专利申请154,316；Tomasi的国际申请PCT/US85/02572。
以往形成PEG/蛋白质结合物的方法以及由所述方法得到的结合物存在几个问题。其一是形成这些蛋白质-PEG结合物的某些方法可能使蛋白质失活，从而使得到的结合物的生物活性可能较差。另外，在形成这些PEG-蛋白质结合物时所用的某些连接剂可能易在体内水解断开。当给药以后发生这样的断裂时，这些结合物便失去了由PEG带来的有利性能。
本发明的一个实施方案是由独特的连接剂将干扰素(IFN)的氨基连接到PEG上的新型干扰素-PEG结合物。本发明特别涉及通式Ⅰ的具有生物活性的干扰素结合物：
Figure A9710543300041
其中R为低级烷基；R1、R2、R3和R4为H或低级烷基；m选自≥1的整数到干扰素中可及的氨基的数目；W为O或NH；x为1-1000之间的整数，y和z为0-1000之间的整数，x、y和z三者之和为3-1000；
附带条件，R1、R2、R3和R4中至少有一个是低级烷基。
式Ⅰ中的-NH-基团由干扰素分子中可及的氨基衍生而来的，这一点是不言而喻的。
更具体地说，两种不同的干扰素结合物具有下列化学式：其中R为低级烷基；R1、R2、R3、R4为H或低级烷基；m为最大可到干扰素中可及氨基数的数字；x、y和z选自使结合物具有形成结合物的蛋白质的至少一部分生物活性的任意数字组合；附带条件是，R1、R2、R3和R4中至少有一个是低级烷基。
图1：用实施例7的化合物进行PEG修饰的时间进程。干扰素(5mg/ml)与相对于蛋白质过量10倍、20倍或40倍的试剂一起在25mMTricine(pH 10.0)、0.5M KSCN、100mM NaCl中保温所示时间。在不同时刻取等分试样，用甘氨酸停止反应，在15％的SDS-PAGE凝胶上分析。标记“Ⅰ”为干扰素。
图2：用实施例5的化合物进行PEG修饰的时间进程。象图1中那样，干扰素与过量3倍或10倍的试剂一起保温所示的时间。在所示时刻取等分试样，用甘氨酸停止反应，在15％SDS-PAGE凝胶上分析。“S”为蛋白质分子量标准物的标记，“Ⅰ”为干扰素的标记。
图3：用实施例1(左侧)和实施例3(右侧)的化合物进行PEG修饰的比较。干扰素与3倍过量的试剂一起保温0.25、1.5或24小时。取出等分试样，用甘氨酸停止反应并在15％SDS-PAGE凝胶上分析。“S”为蛋白质分子量标准物的标记，“Ⅰ”为干扰素的标记。
按照本发明，式ⅠA和式ⅠB的IFN结合物可如下制得：使末端羟基或氨基已被活化的连接基团取代的活化PEG缩合，然后，这些试剂可与IFN中的一个或更多个氨基反应。只同一个氨基缩合形成单PEG化的结合物是本发明的优选实施方案。所以，本发明也涉及那些可用来制备本发明的干扰素结合物的新型活化化合物(试剂)。这些化合物具有下列通式：
Figure A9710543300051
其中R为低级烷基；R1、R2、R3、R4和R5为H或低级烷基；W为NH或O；x为1-1000的整数，y和z为0-1000的整数，x、y和z三者之和为3-1000；附带条件是，假如W为NH或W为O,R5为H，则R1、R2、R3和R4中至少有一个为低级烷基；其中，R为低级烷基；R1、R2、R3和R4为H或甲基；x为1-1000的整数，y和z为0-1000的整数，x、y和z三者之和为3-1000。
更具体地说，式Ⅱ包括下列两种类型的化合物：
Figure A9710543300062
在ⅡA、ⅡB和Ⅲ中，R、R1、R2、R3、R4和R5均如上所述；x、y和z选自能使聚合物在同蛋白质结合后允许蛋白质保持至少一部分结合前生物活性水平的任何数字组合；加上式Ⅱ中所提到的附带条件。
按照本发明，通过使用式ⅡA、ⅡB或Ⅲ的活化的PEG试剂制备结合物，在蛋白质(如干扰素(IFN))的游离氨基和PEG之间形成了一个连键，使所得的结合物保持了蛋白质的至少一部分生物活性，但免疫原性降低。另外，通过使用式ⅡA、ⅡB或Ⅲ中的任何一种活化的聚乙二醇而在本发明结合物中形成的连接基团，使所得的蛋白质结合物不易在体内水解断开，也不易出现先有技术PEG蛋白质结合物中所存在的缺点。
按照本发明，R、R1、R2、R3、R4和R5可以是低级烷基，优选甲基。低级烷基指含1-6个碳原子的低级烷基，如甲基、乙基、正丙基、异丙基等。通常，优选的烷基是含1-4个碳原子的低级烷基，其中甲基是最优选的。R1、R2、R3、R4和R5也可以是氢，但R1、R2、R3和R4不能同时为氢。
按照本发明，x、y和z可以选自能使所得结合物保持形成结合物的IFN的至少一部分生物活性的任何数字组合。显然，x、y和z的总和以及m与结合物所保持的IFN生物活性量成反比。x、y和z的数值代表形成结合物的聚乙二醇中的二醇单元数。m代表IFN中包含的能与活化的PEG混合物反应的游离氨基或可及氨基数。m以及x、y和z的值越大，结合物的分子量越大。按照本发明，x、y和z是使除了蛋白质部分以外的结合物分子量在约300-30,000道尔顿的任何数字。对于IFN，m优选1-3。一个特别优选的实施方案是m为1的单PEG化的结合物，它的制备条件能够以高收率得到IFN中仅有一个游离氨基同式ⅡA或ⅡB或Ⅲ的PEG试剂反应的IFN结合物。按照m为1的优选实施方案，x、y和z是使形成结合物的二醇的平均分子量为大约300-30,000道尔顿，优选大约1,000-10,000道尔顿，尤其是大约1,000-5,000道尔顿的任何数字。特别优选的实施方案是分子量大约为2,000道尔顿。
至于单元数x、y和z,x为1-1000的整数，y和z为0-1000的整数，x、y和z三者之和为3-1000。
在式ⅠA和ⅠB结合物的一个优选实施方案中，x和y为5-500,z为0-4。在一个特别优选的实施方案中，所用的二醇是一个二醇混合物，其中x为10-100,y为1-10,z为0。最优选的是式ⅠA的干扰素结合物，其中m为1,R、R2和R4为CH3；R1为H；x大约为19，y大约为2，z为0。这对应于PEG单元的平均分子量约为1000道尔顿。
为了避免有关PEG分子中单元数的任何疑问，聚乙二醇聚合物用分子量表征优于指出PEG聚合物中用x、y和z来表示的自重复单元(SRU)数。这些数值由于起始PEG化合物的潜在不均一性而可能难于估计，因为这些起始PEG化合物通常由平均分子量定义而不是由它们所含的自重复单元数定义。不同分子量的起始PEG化合物可由本领域已知的方法制得或由供应商处购得。
假如由分子量测定得到的或由供应商标示的x、y和z值不是整数(一般是这种情况)，它们的值则必须按常用方法取舍而化为整数，从而使可能构成聚合物混合物的主要部分的聚合物分子中的上述数值为整数。
式ⅡA、ⅡB或Ⅲ中的任何一个试剂同含一个以上游离氨基的IFN反应时，可能得到IFN同PEG试剂混合物的不同反应产物的混合物。这些反应产物是由于PEG试剂同一个或更多个游离氨基反应而形成的。这可由式IA和IB中的m表示。例如，当IFN含有三个游离氨基时，活化的PEG试剂可同其中的一个、二个或所有三个游离氨基反应。这种情形下，混合物包含所有三种情况下所形成的结合反应产物。由于这个混合物中的不同结合反应产物依m值的不同(即1、2或3)而具有大不相同的分子量，这些反应产物可用传统方法(如色谱法)分离。为确定m以及x、y和z是否选取适当，分离的结合反应产物可用与筛选IFN母体同样的方法筛选有生物活性的结合反应产物，从而确定结合反应产物是否仍保持了用未形成结合物的IFN的一部分生物活性。
按照优选的实施方案，m为1。即使有两个或更多个游离氨基也能得到m为1的结合物。活化的PEG试剂首先同IFN中包含的游离氨基之一发生反应。通过控制试剂(如IFN)的浓度和反应条件，按照胺缩合的标准方法，可控制蛋白质中包含的游离氨基的聚乙二醇化程度。在包含一个或更多个游离氨基的蛋白质中，若有一个游离氨基较其它氨基活泼，可选择反应条件使蛋白质同活化的PEG化合物反应以形成m为1的式ⅠA或ⅠB化合物。形成蛋白质的氨基酸中所含的其它游离氨基可通过延长缩合反应时间或利用其它更剧烈的反应条件进一步同PEG反应。
本说明书和权利要求书中所用的术语干扰素和IFN包括所有类型的干扰素(即：具有干扰素活性的分子)，例如，α、β、γ和ω干扰素以及这些类型的所有亚类，如α1、α2、α2A、α2B或α2C以及不同类型和/或亚类干扰素的杂种分子或嵌合体。干扰素可以是任何来源的，可以从天然来源、组织培养或由重组DNA技术得到。制备或分离天然或重组干扰素的方法在本领域中是公知的，并在如下公开号的专利申请中有所描述：EP43980、EP211148、EP140127、DE3028919、USP4503035和USP4414150。
使用R1、R2、R3和R4中至少有一个为低级烷基尤其是甲基的式ⅡA、ⅡB和Ⅲ的试剂(烷基取代试剂)，其优点在于当用烷基取代试剂时，同相应的非取代试剂相比可以出人意料地提高结合物即PEG化蛋白质的产率。在相同反应时间内，烷基取代试剂同相应的非取代试剂相比，制备结合物时，前者可得到至少两倍量的结合物。
当给予病人由上述的取代试剂制备的式ⅠA和ⅠB结合物用于治疗时，这些结合物同由相应的非取代试剂制备的结合物相比，在病人血流中的体内半寿期意外地提高。虽然体内半寿期同结合物的分子量成正比，由取人试剂制得的结合物出人意料地同由非取代试剂制得的较高分子量的结合物具有同样长的半寿期。治疗剂在病人血流中的半寿期较长能提高对病人给药的效率。例如，由烷基取代试剂制得的结合物可以比由相应的非取代试剂制得的结合物给药次数少，或者用量较少。为了提高同聚乙二醇结合的生物性蛋白质的给药效率，形成这些蛋白质结合物时使用了较高分子量的聚乙二醇。然而，结合的活性IFN的生物效能随分子量增大而减小。但是，通过使用本发明的由取代试剂制得的结合物，相对于使用相应的非取代结合物提高了给药效率，但分子量增加较少。
在另外一个实施方案中，本发明也涉及制备新型结合物的方法。
式ⅠA的结合物可如下制备：
Figure A9710543300101
其中，R、R1、R2、R3、R4和R5，m和x、y和z同上；附带条件是R1、R2、R3、R4中任何一个或更多个都可以是低级烷基。
在这一反应中，PEG-胺在烃或氯代烃熔剂中同式Ⅳ化合物混合以制备式ⅡA化合物。式ⅡA化合物可在水性介质中同蛋白质的一个或更多个游离氨基缩合以制备式ⅠA的结合物。这个反应可在水性介质中胺缩合的常规条件下进行。该反应通常在pH7-10的标准缓冲水溶液中进行，以制备式ⅠA的结合物。依蛋白质中的游离氨基数和反应时间而定，该反应可制备不同分子量的PEG蛋白质结合物的混合物。PEG蛋白质结合物接下来可用常规方法如高效液相色谱(HPLC)或凝胶电泳法分出各个组分。用HPLC或凝胶电泳法依分子量来分离化合物的任何常规方法均可使用。这个混合物的分离可按照本文所述的所得产物的分子量来进行。
式ⅠB的IFN结合物可按下列反应路线来制备：
Figure A9710543300111
Figure A9710543300121
其中，R、R1、R2、R3、R4、R5、m、x、y、z和附带条件如上所述。
式Ⅳ化合物由光气和2-羟基吡啶(当R5为低级烷基时为取代的2-羟基吡啶)用酰卤和醇缩合的任何常规方法制备。
PEG醇同式Ⅳ化合物的缩合利用醇和碳酸酯缩合的常规条件进行以制备式ⅡB化合物。式ⅡB化合物通过蛋白质上的一个或更多个游离氨基同蛋白质缩合，以制备式ⅠB化合物。该反应可按照上述的式ⅡA化合物缩合制备式ⅠA结合物的方法进行。依蛋白质中与式ⅡB化合物发生反应的游离氨基数的不同，式ⅠB的结合物可由不同分子量结合物的混合物组成。该结合物混合物可用前面描述过的方法来分离。
式ⅠB化合物也可用如下反应路线来制备：
Figure A9710543300131
其中，R、R1、R2、R3、R4、R5、m、x和y均如上所述。
在该反应路线中，PEG-醇同式Ⅳ化合物缩合以制备式Ⅲ化合物。在该反应中，式ⅡB化合物作为中间产物形成之后与另一摩尔的PEG-醇反应生成式Ⅲ化合物。在该反应进行时，PEG-醇的用量至少为每摩尔式Ⅳ化合物用2摩尔PEG-醇。在这一步骤中，醇同碳酸酯缩合的任何常规方法均可使用。式Ⅲ化合物同干扰素反应形成式ⅠB结合物是按照上述的式ⅡA化合物转化为式ⅠA化合物的方法进行的。依蛋白质所含的游离氨基数，式Ⅲ化合物同蛋白质缩合得到各种结合物的混合物，该混合物可用前面描述的分离ⅠA结合物的方法分离成各个组分。
按照本发明，已发现本发明的干扰素结合物同用于形成结合物的蛋白质具有同样的效用。这样，这些结合物同形成它们的蛋白质具有相同形式的治疗活性，可以按与蛋白质本身相同的方式使用，而不会产生在给予病人蛋白质本身时可能引起的不良免疫反应。因此，本发明也包括以式Ⅰ化合物或其盐为主要成分的药物组合物以及制备这些组合物的方法。
本发明用来控制或预防疾病的药物组合物包括通式Ⅰ的干扰素结合物和治疗惰性的、治疗上可接受的无毒的载体物质。欲使用的药物组合物可制成制剂，并按与合适的医疗惯例一致的方式给药，并应考虑所要治疗的疾病、病人的个体状况、蛋白质结合物的释放部位、给药方法以及操作者已知的其它因素。
下述实施例代表本发明的说明性实施方案，但本发明不受这些实施例的限制。
在这些实施例中所用的Jeffamine M-2070是由环氧丙烷和环氧乙烷衍生而来的、平均分子量为2070的单甲氧基聚氧化烯丙胺聚合物，其骨架由聚乙二醇组成，并平均包含30％无规结合的环氧丙烷基团。
Jeffamine M-1000是平均分子量为1000的单甲氧基聚氧化烯丙胺聚合物，它由环氧丙烷和环氧乙烷衍生而来，其骨架由聚乙二醇组成，并包含14％专一性结合的环氧丙烷基团，其中x平均为18.6,y平均为1.6,z为0(这里所用的x、y和z的意义如上所述)。
这些实施例中所用的所有试剂均在4℃下于棕色瓶中干燥贮存备用。每次修饰反应都使用新鲜的等分试剂。
                    实施例
                    实施例1
α,α′-氧亚甲基双[ω-甲氧基聚(氧-1,2-亚乙基)]SRU 111的制备
从含1.5克MPEG(甲氧基聚乙二醇，分子量为5000)的80ml干燥甲苯的悬浮液中蒸去50ml溶剂。溶液冷却后加入30.5mg碳酸二-2-吡啶酯。然后将得到的混合物回流24小时。将溶液冷却，得到的沉淀过滤后，先用少量甲苯洗涤，再用乙醚洗涤，得到的固体在高真空下干燥得到0.6克α,α′-氧亚甲基双[ω-甲氧基聚(氧-1,2-亚乙基)]SRU 111白色粉末。经PEG修饰的干扰素由下述的方法1制备。
经PEG修饰的干扰素-α的制备
方法1：浓度为每ml5g蛋白质的重组干扰素-α在含5mM乙酸钠(pH5.0)、120mM NaCl的缓冲液中渗析。加入硫氰酸钾达最后浓度为0.5M，加入1/10体积pH11.9的1M Tricine-氢氧化钠调节pH值，得到最终pH为10.0的溶液。PEG试剂以固体或DMSO溶液(DMSO的体积小于总体积的10％)形式按3∶1摩尔比加入到蛋白质中。在室温下进行修饰反应30分钟到4小时，然后加入1ML-甘氨酸(pH6.3)到最后浓度为20mM以终止进一步的修饰。加入3.5M硫酸铵、50mM磷酸钠，pH7.0，使硫酸铵的最后浓度达到1.1M(对于PEG-1000为1.0M硫酸铵)，使经PEG修饰的蛋白质沉淀，沉淀经离心收集，洗涤后再溶于pH5.0的25mM乙酸铵中。经PEG修饰的蛋白质在疏水交换柱(如75×7.5mm)如BioRad TSK Phenyl-5-PW或ToyopearlPhenyl-650M上用色谱法纯化，使用pH7.0的50mM磷酸钠溶液中硫酸铵浓度递减的梯度。另一种方法是，PEG-IFN在用25mM乙酸钠(pH5.0)、200mM NaCl平衡的Sephacryl S-200柱(如90cm×3.2cm柱)(Pharmacia)上进行凝胶过滤纯化。经PEG修饰的蛋白质用SDS-PAGE鉴定。从柱中洗脱出相应于结合有一个PEG(PEG1-IFN)或两个PEG(PEG2-IFN)的IFN的蛋白质，合并这些蛋白质并浓缩后，通过测定280nm处的光吸收或用比色分析法(Pierce)测定蛋白质浓度。PEG-IFN在4℃下于含50mM磷酸钠(pH7.0)、0.3M硫酸铵的缓冲液中贮存。
方法2：大约6mg/ml蛋白质浓度的干扰素α-2a用5mM乙酸钠(pH5.0)、0.12M氯化钠渗析。以1.0mg-1ml为消光系数测定280nm处的光吸收，以此来测定蛋白质浓度。蛋白质溶液以蛋白质：试剂为1∶3的摩尔比与修饰试剂混合。用1/10体积的pH 10.7的0.1MNa2B4O7-NaOH调节pH到10.0，从而引发修饰反应。在室温下保温1小时后，通过加入1/20体积pH7.5的1M甘氨酸终止反应。3-5分钟后，加入1/20体积的pH4.0的1M乙酸钠使pH降至5.0-6.0。
含PEG-干扰素、已停止反应的试剂和未修饰干扰素的溶液用pH4.5的40mM乙酸铵稀释4倍后，移至一个羧甲基纤维素柱(Whatman CM-52，每mg蛋白质约0.5ml树脂)上，用5倍体积pH4.5的40mM乙酸铵洗柱后，PEG-干扰素和未修饰干扰素用氯化钠在pH4.5的40mM乙酸铵中的线性梯度(0-0.5M)洗脱，含蛋白质的级分用280nm处的光吸收来鉴定，含PEG-干扰素的级分由SDS-PAGE来鉴定。
PEG-干扰素在装有Sephacryl S-200树脂(Pharmacia LKB)的柱上用排阻-凝胶过滤色谱法进一步纯化。从柱中洗脱出的级分用SDS-PAGE来分析，合并含PEG-干扰素的峰物质。
                    实施例2
α,α-氧亚甲基双[ω-甲氧基聚(氧-1,2-亚乙基)]SRU 28.3的制备
按实施例1中描述的方法，将MPEG(分子量1300)转化为α,α-氧亚甲基双[ω-甲氧基聚(氧-1,2-亚乙基)SRU28.3，经PEG修饰的干扰素用这个试剂按实施例1中所述方法1来制备。
                    实施例3
α-甲基-ω-[2-[[2-吡啶基氧)羰基]氧]乙氧基]-聚(氧-1,2-亚乙基)SRU111.7的制备
从含1克分子量为5000的MPEG的30ml干燥CH2Cl2溶液中蒸出10ml溶剂。溶液冷却到室温后加入132mg(0.6mM)碳酸二-2-吡啶酯和4mg DMAP。得到的溶液搅拌14小时后真空下除去溶剂。残余物用乙醚研制并将得到的沉淀过滤。然后将产物溶于7ml干燥的甘醇二甲醚中，加热使其溶解，使得到的溶液冷却，并在室温下放置数小时。然后将得到的沉淀过滤并用2×5ml干燥的甘醇二甲醚洗涤，固体在氮气流下于真空炉中干燥，得到0.7克α-[(2-吡啶基氧)羰基]-ω-甲氧基聚(氧-1,2-亚乙基)SRU111.7。
元素分析C9H11NO4(CH2CH2O)111.7的计算值：C,54.57；H,9.02；N,0.28。实测值：C,54.51；H,9.19；N,0.28。
经PEG修饰的干扰素用这个试剂按实施例1中所述方法1来制备。
                    实施例4
α-[(2-吡啶基氧)羰基]-ω-甲氧基聚(氧-1,2-亚乙基)SRU225的制备
分子量为10,000的MPEG按实施例3中所述方法转化为α-[(2-吡啶基氧)羰基]-ω-甲氧基聚(氧-1,2-亚乙基)SRU225。
元素分析C9H11NO4(CH2CH2O)225的计算值：C,54.54；H,9.08；N,0.14。实测值：C,54.54；H,9.12；N,0.11。
经PEG修饰的干扰素用这个试剂按实施例1中所述方法1来制备。
                    实施例5
α-甲基-ω-[2-[2-[[(2-吡啶基氧)羰基]氧]丙氧基]丙氧基]聚(氧-1,2-亚乙基)SRU64.7的制备
从含0.5克α-2-[2-(羟基丙氧基)丙基]-ω-甲氧基聚(氧-1,2-亚乙基)SRU64.7的40ml干燥CH2Cl2溶液中蒸去15ml溶剂。然后向溶液中加入108mg碳酸二-2-吡啶酯、4mgDMAP和几粒4A分子筛。混合物搅拌过夜，过滤后减压除去溶剂。残余物通过排阻色谱法纯化。
这个试剂对应于式ⅡB-1化合物：其中n约为64，这相当于聚合物的分子量约为3000道尔顿。
经PEG修饰的干扰素用这个试剂按实施例1中所述方法1来制备。
                    实施例6
α-甲基-ω-[2[2-[[(2-吡啶基氧)羰基]氧]丙氧基]丙氧基]聚(氧-1,2-亚乙基)SRU110的制备
α-2-[2-(羟基丙氧基)丙基]-ω-甲氧基聚(氧-1,2-亚乙基)SRU110按实施例5中所述方法转化为α-甲基-ω-[2-[2-[[(2-吡啶基氧)羰基]氧]丙氧基]丙氧基]聚(氧-1,2-亚乙基)SRU110。
这个试剂(ⅡB-2)除了n大约为110，对应于5000道尔顿外，同实施例5所述的化合物(ⅡB-1)相当。
经PEG修饰的干扰素用这个试剂按实施例1中所述方法1来制备。
实施例7
甲基环氧乙烷同环氧乙烷的聚合物2-[[(2-吡啶基氧)羰基]氨基]丙基甲基醚(MO/O=10/32)的制备
从含1克Jeffamine M-2070(Texaco Chemical Co.)的40ml干燥CH2Cl2溶液中蒸出15ml溶剂。溶液冷却到0℃并加入215mg碳酸二-2-吡啶酯。得到的溶液在0℃下再搅拌4小时，之后减压除去溶剂。残余物用串连的两个Phenomenex排阻色谱柱(500和1000)纯化。该产物在紫外光谱上显示232nm和310nm两个吸收带。
这个试剂对应于下式化合物：
Figure A9710543300191
其中，R5为H，R2为H或甲基，并且，作为平均分布，当R2为H时n为32，当R2为甲基时n为10。
经PEG修饰的干扰素用这个试剂按实施例1中所述方法2来制备。
                    实施例8
甲基环氧乙烷同环氧乙烷的聚合物2-[[(3-甲基-2-吡啶基氧)羰基]氨基]丙基甲基醚(MO/O=10/32)的制备
按实施例7中所述方法，1克Jeffamine M-2070同碳酸双(3-甲基-2-吡啶基)酯反应得到甲基环氧乙烷同环氧乙烷的聚合物2-[[(3-甲基-2-吡啶基氧)羰基]氨基]丙基甲基醚(MO/O=10/32)。
除了R5为CH3外，这个试剂(ⅡA-2)同实施例7中所述化合物(ⅡA-1)相当。
经PEG修饰的干扰素用这个试剂按实施例1中所述方法1来制备。
                    实施例9
甲基环氧乙烷同环氧乙烷的聚合物2-[[(2-吡啶基氧)羰基]氨基]丙基甲基醚嵌段(MO/O=1.6/18.6)的制备
按实施例7中所述方法，0.6克Jeffamine M-1000(TexacoChemical Co.)同155.6mg碳酸二-2-吡啶酯反应得到甲基环氧乙烷同环氧乙烷的聚合物-[[(2-吡啶基氧)羰基]氨基]丙基甲基醚嵌段(MO/O=1.6/18.6)。
得到下式化合物：其中聚合物中各单元的平均分布如所示。
经PEG修饰的干扰素用该试剂按实施例1中所述方法1来制备。
干扰素的抗病毒活性：对干扰素及经PEG修饰的干扰素的抗病毒活性进行了测定(Rubenstein等人，(1981)J.Virol.37:755-758；Familletti等人，(1981)Methocls Enzymol.78:387-394)。所有的测定都相对于对照物进行了标准化。测定中所用的干扰素标准物的比活性为每mg蛋白质2×108单位。
修饰干扰素所用条件是以所述的最优化程序为基础的。PEG修饰过程中，由SDS-PAGE分析不同培养时间内干扰素转化为单PEG-干扰素的转化率(化学反应性)以及不同种类PEG-干扰素结合物的分布(位点选择性)。在SDS-PAGE中，观察到经PEG修饰的物质是凝胶上的迁移较慢的条带。单PEG-干扰素和双PEG-干扰素的产率都足以使它们能够用疏水性相互作用色谱法从反应混合物中纯化出来。测定纯化的PEG-干扰素的抗病毒活性并与未修饰的干扰素-α2a标准物比较。所用聚合物的分子量以及一些PEG化的衍生物的抗病毒活性如表1所示。
                 表1
PEG试剂的物理性质及其蛋白质结合物的生物活性
                                    抗病毒活性
实施例化合物                            (％对照物)
             聚合物分子量     单PEG      双PEG
      4         10000          25          2
      3          5000          40          4
      1          5000          40          ND
      6          5000          40          ND
      5          3000          60          ND
      7          2070          45          ND
      2          1300          70          ND
      9          1000          100         40ND未测

Claims (4)

1．一种具有下式的化合物：
Figure A9710543300021
其中R为低级烷基；R1、R2、R3、R4和R5为H或低级烷基；W为NH或O；x为1-1000的整数，y和z为0-1000的整数，x、y和z三者之和为3-1000；附带条件是，假如W为NH或W为O，R5为H，则R1、R2、R3和R4中至少有一个为低级烷基。
2．权利要求1的化合物，其中选择x、y和z，使得所述化合物的分子量在大约300-30000道尔顿的范围内。
3．权利要求1的化合物，其中R为甲基。
4．权利要求3的化合物，其中x为10.0-100.0,y为1.0-10.0,z为0.0-4.0。
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CN100337689C (zh) * 2002-07-24 2007-09-19 弗·哈夫曼-拉罗切有限公司 聚亚烷基二醇酸加合物

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