CN101172161B - 水溶性聚合物修饰的g-csf偶联物 - Google Patents
水溶性聚合物修饰的g-csf偶联物 Download PDFInfo
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Abstract
本发明涉及一类全新结构、通式为(I)的经水溶性聚合物修饰的G-CSF偶联物或其药学上可接受的盐，和其制备方法，及含通式(I)化合物或其药学上可接受的盐的药物组合物。水溶性聚合物-连接基团-N端-G’ (I)。
Description
技术领域
本发明涉及一类全新结构、通式为(I)的经水溶性聚合物修饰的G-CSF偶联物或其药学上可接受的盐，和其制备方法，和含通式(I)化合物或其药学上可接受的盐的药物组合物。
水溶性聚合物-连接基团-N端-G’
(I)
背景技术
粒细胞集落刺激因子(G-CSF)是由单核细胞及成纤维细胞产生，可刺激粒细胞集落形成，对中性粒细胞有刺激作用。G-CSF与靶细胞膜受体结合，主要刺激粒细胞系造血，亦可使多功能造血干细胞进入细胞周期，促进髓系造血组细胞的增殖、分化和成熟，并驱使中性粒细胞释放至血流，使外周中性粒细胞数量增加，并提高其功能，如吞噬功能，针对肿瘤细胞的抗体依赖细胞细胞毒活性等[Metcalf，Blood 67：257(1986)；Yan et.al，Blood 84(3)：795-799(1994)；Bensinger，et.al，Blood 81(11)：3158-3163(1993)；Neben，et.al，Blood 81(7)：1960-1967(1993)]，因此重组的粒细胞集落刺激因子常用于接受放射治疗或化疗的癌症病人以及骨髓移植后白血病患者的辅助治疗。
市场常使用的人源G-CSF为Neupogen和Neutrogin，以及人源G-CSF的衍生物Neu-wp，G-CSF的衍生物或变体蛋白也有大量文献报道[如US5581476、US5214132、US5362853、US4904584]，这些变体蛋白含多个氨基酸的取代，以求寻找更稳定、活性更高、更适用于临床的G-CSF。
市售的重组人源G-CSF由于其在人体内生物利用度差、半衰期短以及容易被体内蛋白酶破坏，因此需要频繁注射给药，难取得良好的临床治疗效果。研究表明，具有治疗应用作用的蛋白质经聚乙二醇修饰形成聚乙二醇-蛋白质偶联物后，其成为药物的可能性大大提高。这一类聚乙二醇修饰的蛋白质已经在临床上得到了充分的应用[如Katre，Advanced Drug Delivery Systems，10：91(1993)；Inada，et.al，J.Bioact and Compatible Polymers，5：343(1990)]。经过聚乙二醇修饰得到的蛋白偶联物具有更好的物理、化学稳定性，同时还具有更好的蛋白酶介稳定性；另外由于偶联物分子量的增加，其在体内的半衰期得到了延长，在体内形成抗体的可能性亦有所下降，再加上其分布体积减少，这样可使毒性减少。由聚乙二醇修饰的G-CSF或G-CSF变体蛋白已公开在许多文献中，如EP0335423，EP0401384，US5824778，US5985265，WO0044785，WO2001051510，US5824784等。具体而言，在专利US5985265公开的聚乙二醇修饰的G-CSF偶联物中，以修饰在G-CSF的N末端所得偶联物的体外、体内生物活性最好，但用酰化反应进行聚乙二醇修饰氨基选择性差，故通常得到的是一组聚乙二醇修饰在各个位置的混合物，需要进行分离提纯才能得到各个单体，收率低，难于工业化生产；在专利US5824784中用大分子的聚乙二醇醛直接来修饰G-CSF，通过严格控制反应的PH，有选择性的修饰G-CSF的N末端，可以得到相对专一的N末端聚乙二醇修饰的偶联物，但是不可否认，这种反应的选择性是通过边链氨基和N末端氨基的Pka不同而实现的，这在制备生产和质量控制上是有相当难度的；另外，由于大分子醛各批之间醛的含量不一，难以控制大分子醛与蛋白的投料比例，必然影响反应收率和生产成本，同时由于大分子醛与不同氨基偶联产生的偶联物生物活性有差别也会影响到最终产品质量的均一性和活性。
发明内容
针对现有技术的不足，本发明的目的在于提供一类全新结构、通式为(I)的、经水溶性聚合物修饰的G-CSF偶联物或其药学上可接受的盐；本发明的另一目的在于提供制备通式为(I)的偶联物或其药学上可接受的盐的方法；本发明的又一目的在于提供含该类偶联物或其药学上可接受的盐的药物组合物及其用途。
本发明涉及通式为(I)的偶联物或其药学上可接受的盐。
水溶性聚合物-连接基团-N端-G’
(I)
其中，水溶性聚合物包括聚乙二醇、聚丙二醇、聚乳酸、聚合氨基酸等，优选聚乙二醇，聚乙二醇的分子量选自2KD-100KD，优选5KD-40KD；聚乙二醇可以是直线型或分支型的；
连接基团由水溶性聚合物末端的功能团与引入蛋白质G′的N末端的功能团发生专一反应而形成；
G′选自天然G-CSF、基因重组G-CSF或者具有G-CSF功能的基因突变产物，优选编号为SEQ ID NO：1-10的G-CSF衍生物，更优选为天然存在的人源G-CSF的序列或SEQ ID NO：1表示的Met-G-CSF。
具体地，上述通式为(I)的偶联物或其药学上可接受的盐的化学结构式由通式(II)表示：
其中，
R选自C1-4的直链或支链烷基，优选甲基或乙基，更优选甲基；
R1、R2彼此独立地选自氢或C1-4直链或支链烷基，优选氢、甲基或乙基；
X选自O、S、NH、
l选自1-20的整数，优选1-10的整数，更优选1-5的整数；
m选自50-2500的整数，优选100-1000的整数；
n选自1-20的整数，优选1-10的整数，更优选1-5的整数。
G’选自天然G-CSF、基因重组G-CSF或者具有G-CSF功能的基因突变产物。
进一步，本发明涉及上述通式为(I)、(II)的偶联物或其药学上可接受的盐，其特点是G’具有天然存在的人源G-CSF的序列。
进一步，本发明涉及上述通式为(I)、(II)的偶联物或其药学上可接受的盐，其特点是G’为Met-G-CSF(SEQ ID NO：1)。
上述通式为(I)、(II)的偶联物包括：
m为选自400-500的整数。
进一步，本发明通式为(I)、(II)的偶联物可以和酸成盐，所用的酸选自有机酸或无机酸，有机酸包括乙酸、三氟乙酸、丙酸、丁酸、马来酸或者对甲苯磺酸或其混合物，优选为乙酸、三氟乙酸；无机酸包括盐酸、硫酸、磷酸或者甲磺酸或其混合物，优选为盐酸。
另一方面，本发明提供一种制备通式为(I)、(II)的偶联物的方法，包括以下步骤：
1)将通式化合物(III)与G’的N末端氨基还原胺化反应得通式化合物(IV)：
2)将通式化合物(IV)脱去巯基保护基得通式化合物(V)：
3)将通式化合物(V)与mPEG-MAL发生迈克尔加成得偶联物(II)：
其中，
R、R1、R2、G’、X、l、m、n如上所定义；
Z为巯基保护基团，选自：甲酰基、乙酰基、丙酰基、三苯甲基或叔丁基，优选乙酰基。
进一步，本发明还涉及本发明的偶联物或其药学上可接受的盐在制备治疗由于放射性治疗或化疗所致白血球减少症、爱滋病及其他免疫缺陷病、细菌感染疾病的药物中的应用。
另一方面，本发明提供一种药物组合物，其含有药物有效剂量的本发明偶联物或其药学上可接受的盐，和药学上可接受的载体。
进一步，本发明还涉及所述药物组合物在制备治疗由于放射性治疗和化疗所致白血球减少症、爱滋病及其他免疫缺陷病、细菌感染等疾病的药物中的应用。
本发明公开了一类全新的聚乙二醇修饰的G-CSF偶联物及其全新的制备方法，同传统的偶联物和方法相比，存在以下差别：
首先，利用在聚乙二醇和G-CSF之间引入连接基团的新概念，可以
保证在蛋白的N末端反应的专一性，因为用小分子修饰N末端所得的蛋白容易分离、提纯，保证了修饰点的专一性；
其次，由于连接基团中巯基的存在，通过控制反应体系的PH，确保了具有高专一性的迈克尔加成反应的进行；
另外，由于连接基团的存在，其他功能基团亦有可能加入，如亚酰胺基、酯基的引入，这为在体内由偶联物释放G-CSF提供了前提条件。
本发明的偶联物或其药学上可接受的盐具有天然人源G-CSF生理活性，而且具有比G-CSF更长的体内循环半衰期和更好的粒细胞刺激因子活性。
具体而言，本发明所公开的偶联物结构式由(II)表示：
其中，
R选自C1-4的直链或支链烷基，优选甲基或乙基，更优选甲基；
R1、R2彼此独立地选自氢或C1-4直链或支链烷基，优选氢、甲基或乙基；
X选自O、S、NH、
l选自1-20的整数，优选1-10的整数，更优选1-5的整数；
m选自50-2500的整数，优选100-1000的整数；
n选自1-20的整数，优选1-10的整数，更优选1-5的整数。
G’选自天然G-CSF、基因重组G-CSF或者具有G-CSF功能的基因突变产物。
当X为O、S时，聚乙二醇与G-CSF之间的连接基团中存在一个在体内易被酯酶水解的酯键，此时，G-CSF可以以其自由形式与受体作用而体现生物活性；
当X为NH、
时，其生物活性主要由偶联物本身实现，当然，由于亚酰胺键在体内亦有被水解而释放G-CSF的能力，因此也不排除自由G-CSF起作用的可能。
在本发明公开的制备方法中，首先通过还原胺化反应将含有巯基的连接基团与G-CSF的N末端氨基反应得到中间体(IV)。其实可以通过多种反应方式将连接基团与G-CSF中氨基反应而连接(如各种活化酯与氨基的反应)，但考虑到醛基与N末端氨基反应的高选择性，因此在本发明中由醛基与G-CSF中的N末端氨基发生还原胺化反应引入连接基团。所用的还原剂选自本领域技术人员所熟知的各种还原剂，优选氰基硼氢化钠、三乙酰氧基硼氢化钠。
在中间体(V)的制备中，根据巯基保护基团的不同，选择不同的反应条件。当保护基团为三苯甲基或叔丁基时，使用酸性条件(如三氟乙酸、盐酸、甲磺酸等)脱保护基团；当保护基团为乙酰基、丙酰基等时，使用本领域技术人员熟知的各种方法、试剂脱保护基团，优选在PH＝5-7时用盐酸羟胺脱保护基团。
在通式化合物(II)的制备中，反应通过控制反应PH值，利用经典的迈克尔加成来实现，该反应具有很好的选择性，可达到99％以上。反应结束后可用反相高效液相制备柱、离子交换柱或凝胶柱分离纯化。
在本发明中，“C1-4烷基”指直链或支链饱和脂肪族碳氢基，如甲基、乙基、丙基、异丙基、丁基等。
在本发明中，优选人源G-CSF，由于没有其内部序列的改变，这样会减少蛋白的体内抗原性，最低限度的降低体内中和抗体的形成，提高药效。G-CSF以及G-CSF的变体蛋白可由文献报道的方法用传统的基因表达的方法获得，包括但不限于Met-G-CSF、Trp-G-CSF、Asp-G-CSF、Glu-G-CSF，优选以大肠杆菌表达来源的Met-G-CSF(序列见SEQ ID NO：1)。
本发明所得化合物通常以剂量单元给药。所说的剂量单元可以更好的表述为将活性化合物与制药赋形剂混合所得的药物组合物。
在本发明所提供的药物组合物中，通式(I)、(II)偶联物或其药学上可接受的盐为活性化合物。本发明所提供的药物组合物可以作为癌症病人正在进行化疗或放射性治疗以及骨髓移植病人的辅助治疗，以防止由于体内免疫能力下降而造成的感染；也可用于慢性或相对的白细胞减少患者的治疗；亦可用于急性髓细胞性白血病患者、爱滋病或其他缺陷性疾病的治疗；同时可用于抗真菌，尤其是对全身性或侵袭性念珠菌感染的治疗。
通式(I)、(II)偶联物或其药学上可接受的盐给药的剂量为5-500ug/kg，其中“ug”指通式(I)、(II)偶联物或其药学上可接受盐的计量单位，“kg”指哺乳动物体重的计量单位。当然，如我们所知道的，给药的剂量及给药的频率依赖于许多因素，如病人的性别、年龄，病人所需预防或治疗疾病的种类等。
将本发明所得偶合物或其药学上可接受盐与传统的制药载体混合可制备成各种单元形式，选用不同的方式给药，如皮下、肌内、静脉。
附图的详细说明
图1：本发明化合物1的MALDI-TOF-TOF图；
图2：本发明化合物2的MALDI-TOF-TOF图；
图3：PEG-G-CSF，G-CSF对环磷酰胺处理的小鼠外周血白细胞计数的影响图；
图4：PEG-G-CSF，G-CSF对环磷酰胺处理的小鼠外周血红细胞计数的影响图；
图5：PEG-G-CSF，G-CSF对环磷酰胺处理的小鼠外周血血小板计数的影响图
具体实施方式
为了更详细地说明本发明，给出下述例子。但本发明的范围并非限定于此。
实施例一
Met-G-CSF的还原胺化
Met-G-CSF(PH＝5.0)：40mg/140ml
7.0mg
氰基硼氢化钠： 176mg
取Met-G-CSF原液透析转换为0.1M的乙酸/乙酸钠溶液，取140毫升，约含蛋白40毫克。取小分子醛(7.0mg)，溶解于乙腈(300ml)中，加入到蛋白液中，再加入氰基硼氢化钠(176mg)，室温搅拌反应3小时。
将反应液用含2mM EDTA的0.1M的PBS溶液(PH＝6.2)于4℃透析。
实施例二
脱巯基保护基-乙酰基
将实施例一中所制备的蛋白液中加入10ml配置好的0.1M的盐酸羟胺(PH＝6.3)，使其浓度为50mM，室温搅拌反应30分钟，以脱乙酰基使巯基游离。
实施例三
mPEG-Met-G-CSF(39KD)的制备
将实施例二中所制备的蛋白溶液中加入mPEG-MAL(400mg，20KD)，室温下搅拌反应60分钟，HPLC中控反应结束后，直接进行制备纯化，所得化合物为本发明化合物1，经MALDI-TOF-TOF确证结构正确，见图谱1。
中控条件：
分析柱：Jupiter C4，5u，150*4.6
流动相：A：0.05％TFA/H2O B：0.05％TFA/CH3CN
梯度条件：
0’ 30’ 35’ 36’
A 60％ 20％ 20％ 60％
B 40％ 80％ 80％ 40％
分离纯化条件：
使用SP Sepharose H.P填装柱子，1.6cm×12cm，体积约24ml，流速：4ml/min，
柱子的前处理：先用0.5M NaOH溶液洗5倍体积；
再用纯化水洗至中性；
再用20mM HAc/NaAc，pH4.0洗5倍体积至平衡。
上样：用泵直接将脱盐后样品吸入柱中；
洗脱：先用20mM HAc/NaAc，pH4.0洗脱10倍柱体积，以除掉过量的PEG；
再设置盐梯度：0～50％，50min，4ml/min，将产杂质、产物以及未反应的蛋白依次洗脱收集。
实施例四
mPEG-Met-G-CSF(59KD)的制备
制备和分离、纯化方法同实施例三，不同之处在于将mPEG-MAL(400mg，20KD)替换为mPEG-MAL(800mg，40KD)，所得化合物为本发明化合物2，MALDI-TOF-TOF见图谱2。
实施例五
mPEG-Met-G-CSF(39KD)即化合物1注射剂的制备
化合物1：10克
在无菌配料间里，称取醋酸钠(0.12克)、聚山梨酯20(35毫克)、山梨醇(50克)，加注射用水(1000毫升)，搅拌，使之溶解，加入化合物1(10克)搅拌均匀，加注射用水至3000毫升，在无菌条件下，用0.22μm微孔滤膜过滤后分装，加灭菌塞并轧外盖，即得。
试验例一
PEG-G-CSF和G-CSF升高小鼠外周血白细胞计数的作用比较
注明：PEG-G-CSF指本发明化合物1。
1试验目的
评价并比较PEG-G-CSF和G-CSF升高环磷酰胺处理小鼠外周血白细胞计数的疗效。
2材料及方法：
PEG-G-CSF，G-CSF，环磷酰胺(CTX)，由江苏豪森药业股份有限公司提供；使用前以生理盐水稀释。
昆明种小鼠，购自中科院上海实验动物中心，体重18-22g，♀，各组动物数：10只。
动物经适应后，腹腔注射环磷酰胺，第2天皮下注射PEG-G-CSF，G-CSF。PEG-G-CSF 0.5、1.0mg/kg皮下注射1次；G-CSF每天皮下注射1次，连续4次，剂量分别为0.1、0.2mg/kg。给药结束后断颈处死小鼠，取血。血细胞计数用ABC全自动血球计数仪计数。
3结果
腹腔注射CTX明显降低小鼠外周血白细胞、红细胞、血小板计数(均P＜0.01与对照比，图3-5)，说明CTX是一个较强的骨髓抑制剂。
PEG-G-CSF单次皮下注射使环磷酰胺处理后的小鼠外周血白细胞计数升高，0.5mg/kg给药时，使白细胞计数回复到正常水平；1.0mg/kg给药时，外周血白细胞计数甚至高于正常水平(P＜0.01与对照比，图3)。但PEG-G-CSF对外周血红细胞和血小板计数没有明显的升高作用(图4，3)，说明PEG-G-CSF作用的特异性。
G-CSF连续皮下注射同样升高环磷酰胺处理小鼠的外周血白细胞计数。0.1mg/kg给药时，白细胞计数升到正常水平；0.2mg/kg给药时，白细胞计数高于正常水平(P＜0.01与对照相比，图3)，有明显的剂量依赖性。但G-CSF对外周血红细胞、血小板计数没有明显影响(图4，5)。
综合以上结果，在给药总剂量相当的情况下比较，PEG-G-CSF单次皮下注射与G-CSF多次皮下注射对小鼠外周血白细胞计数的疗效相当。
4结论
PEG-G-CSF和G-CSF均明显升高环磷酰胺处理小鼠的外周血白细胞计数；在给药总剂量相当的情况下，本发明化合物1单次皮下注射与G-CSF多次皮下注射对小鼠外周血白细胞计数的疗效相当。
天然G-CSF或基因重组GCSF以及具有G-CSF生理功能的基因突变产物氨基酸序列表
<110>江苏恒瑞医药股份有限公司
<120>水溶性聚合物修饰的G-CSF偶联物
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Claims (12)
1.式(II)所示的偶联物或其药学上可接受的盐：
其中，
R选自甲基或乙基；
R1、R2彼此独立地选自氢、甲基或乙基；
X为
l选自1-20的整数；
m选自50-2500的整数；
n选自1-20的整数；
G’选自天然G-CSF、基因重组G-CSF或者具有G-CSF功能的基因突变产物。
2.根据权利要求1所述的偶联物或其药学上可接受的盐，其特征在于G’选自SEQ ID NO：1-10所示的G-CSF衍生物。
3.根据权利要求1所述的偶联物或其药学上可接受的盐，其特征在于所述的G’为天然存在的人源G-CSF的序列。
4.根据权利要求1所述的偶联物或其药学上可接受的盐，其特征在于所述的G’为序列编号SEQ ID NO：1表示的Met-G-CSF。
5.根据权利要求1所述的偶联物或其药学上可接受的盐，其特征在于所述的偶联物包括：
m为选自400-500的整数；和
m为选自400-500的整数。
6.根据权利要求1所述的偶联物或其药学上可接受的盐，其特征在于所述的偶联物和酸成盐，所用的酸选自有机酸或无机酸。
7.根据权利要求6所述的偶联物或其药学上可接受的盐，其特征在于所述的有机酸选自乙酸、三氟乙酸、丙酸、丁酸、马来酸或者对甲苯磺酸或其混合物。
8.根据权利要求6所述的所述的偶联物或其药学上可接受的盐，其特征在于所述无机酸选自盐酸、硫酸、磷酸或者甲磺酸或其混合物。
9.一种制备权利要求1中所述偶联物的方法，包括：
1)将通式化合物(III)与G’的N端氨基还原胺化反应得通式化合物(IV)：
2)将通式化合物(IV)脱去巯基保护基得通式化合物(V)：
3)将通式化合物(V)与mPEG-MAL发生迈克尔加成得化合物(II)：
其中，
R选自甲基或乙基；
R1、R2彼此独立地选自氢、甲基或乙基；
X为
l选自1-20的整数；
m选自50-2500的整数；
n选自1-20的整数；
G’选自天然G-CSF、基因重组G-CSF或者具有G-CSF功能的基因突变产物；
Z为巯基保护基团，选自：甲酰基、乙酰基、丙酰基、三苯甲基或叔丁基。
10.根据权利要求1中所述的偶联物或其药学上可接受的盐在制备治疗由于放射性治疗或化疗所致白血球减少症、爱滋病及其他免疫缺陷病、细菌感染疾病的药物中的应用。
11.一种药物组合物，其含有药物有效剂量的如权利要求1中所述的偶联物或其药学上可接受的盐，和药学上可接受的载体。
12.根据权利要求11所述的药物组合物在制备治疗由于放射性治疗或化疗所致白血球减少症、爱滋病及其他免疫缺陷病、细菌感染疾病的药物中的应用。
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| TW097113755A TWI491410B (zh) | 2006-10-30 | 2008-04-16 | 水溶性聚合物修飾的ｇ－ｃｓｆ偶聯物 |
| HK08107818.7A HK1112673A1 (en) | 2006-10-30 | 2008-07-15 | G-csf conjugates modified by water-soluble polymers |
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| CN102485742A (zh) * | 2010-12-02 | 2012-06-06 | 山东新时代药业有限公司 | 一种聚乙二醇单修饰的重组人粒细胞集落刺激因子的制备及分离纯化方法 |
| CN106986928B (zh) * | 2016-04-15 | 2020-04-24 | 江苏恒瑞医药股份有限公司 | 一种聚乙二醇化重组人粒细胞刺激因子的纯化方法 |
| CN107129531B (zh) * | 2016-04-15 | 2020-09-11 | 江苏恒瑞医药股份有限公司 | 一种聚乙二醇化重组人粒细胞刺激因子的纯化方法 |
| US11732016B2 (en) | 2017-12-27 | 2023-08-22 | Council Of Scientific & Industrial Research | Polypeptide exhibiting granulocyte-colony stimulating factor activity |
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