CN102131844B - 肽-聚合物缀合物 - Google Patents
肽-聚合物缀合物 Download PDFInfo
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- CN102131844B CN102131844B CN200980130470.7A CN200980130470A CN102131844B CN 102131844 B CN102131844 B CN 102131844B CN 200980130470 A CN200980130470 A CN 200980130470A CN 102131844 B CN102131844 B CN 102131844B
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Abstract
本发明涉及一种聚合物部分与干扰素-β部分、红血球生成素部分或生长激素部分的缀合物。
Description
技术领域
相关申请段落的交叉引用
背景技术
本申请主张2008年7月31日递交的美国临时申请No.61/085,072的权益,其内容在此通过整体引用引入本文。
本发明涉及一种肽-聚合物缀合物。
在细胞生物学及重组蛋白质技术上的发展已导致蛋白质治疗的发展。
发明内容
然而,主要困难仍然存在。大部分蛋白质易受蛋白代谢性降解影响,因此具有短的循环时间。其它缺点包括低水溶解度及引发中和性抗体。
聚合物(例如,聚乙二醇(PEG))接附至蛋白质会阻碍蛋白代谢酶接近蛋白质骨架,此导致蛋白质稳定性提高。此外,也可改良水溶解度及减少免疫抗原性。已需要有效将聚合物接附至蛋白质的方法。
本发明的观点涉及式I的聚合物-多肽缀合物:
式I
其中R1、R2、R3、R4及R5各自独立地为H、C1-10烷基、C2-10烯基、C2-10炔基、芳基、杂芳基、C3-8环烷基或C3-8杂环烷基;A1及A2各自独立地为聚合物部分(例如,聚环氧化烷部分);G1、G2及G3各自独立地为一键结或一连结官能基;P为干扰素-β(INF-β)部分、红血球生成素(EPO)部分或生长激素(GH)部分;m为0或整数1-10;及n为整数1-10。在这些缀合物中,该INF-β部分、EPO部分或GH部分的N-终端键结至G3。
参照上式,该聚合物-多肽缀合物具有一或多个下列特征:A1及A2为具有分子量2-100kD(10-30kD较佳,例如,20kD)的聚环氧化烷部分;G1及G2各为(其中O原子键结至A1或A2及N原子键结至碳原子,如显示在式I中);G1及G2各为尿素、磺酰胺或酰胺(其中N原子键结至碳原子,如显示在式I中);m为4;n为2;及R1、R2、R3、R4及R5各为H。在这些缀合物的某些中,P为rINF-βSer17或在INF-β的N-终端处包含1-4个其它氨基酸残基的经改质的INF-β部分。
本发明的另一个观点涉及式II的聚合物-肽缀合物:
A-G1-L-G2-P
式II
其中A为聚合物部分(例如,聚环氧化烷部分);G1及G2各自独立地为一键结或一连结官能基;L为C2-10伸烯基或C2-10伸炔基;及P为INF-β部分、EPO部分或GH部分。在这些缀合物中,INF-β部分、EPO部分或GH部分的N-终端接附至G2。
参照式II,该聚合物-肽缀合物具有一或多个下列特征:A1及A2为具有分子量2-100kD(10-30kD较佳,例如,20kD)的聚环氧化烷部分,G1及G2各者为一键结,C6为伸烯基,及R1、R2、R3、R4与R5各为H。
本发明的另一个观点涉及式III的聚合物-肽缀合物:
其中R1、R2、R3及R4各者各自独立地为H、C1-10烷基、C2-10烯基、C2-10炔基、芳基、杂芳基、C3-8环烷基或C3-8杂环烷基;n为整数2-10;A为聚合物部分;G为连结官能基;及P为肽部分,该肽部分的N-终端的氮原子键结至在上述式中所显示出的部分的碳原子。
参照式III,该聚合物-肽缀合物具有一或多个下列特征:n为1;A为具有分子量10-40kD或20-30kD的聚环氧化烷部分;G为:
其中O原子键结至A及N原子键结至碳原子;及P为INF部分、EPO部分、GH部分。
于本文中所使用的名称“C1-10烷基”指为包含1至10个碳原子的直链或分支的烃单价基团。该烷基的实施例包括甲基、乙基、正丙基、异丙基、三级丁基及正戊基。类似地,名称“C2-10烯基”指为包含2至10个碳原子及一或多个双键的直链或分支的烃单价基团。名称“C2-10炔基”指为包含2至10个碳原子及一或多个三键的直链或分支的烃单价基团。名称“C2-10伸烯基”指为包含2至10个碳原子及一或多个双键的直链或分支的烃二价基团。名称“C2-10伸炔基”指为包含2至10个碳原子及一或多个三键的直链或分支的烃二价基团。
于本文中所使用的名称“芳基”指为具有至少一个芳香环的烃环系统(单环或双环)。该芳基部分的实施例包括但不限于苯基、萘基及芘基。
于本文中所使用的名称“杂芳基”指为具有至少一个芳香环(其包含至少一个杂原子(例如O、N或S)作为该环系统的部分及剩余为碳)的烃环系统(单环或双环)。该杂芳基部分的实施例包括但不限于呋喃基、吡咯基、噻吩基、唑基、咪唑基、噻唑基、吡啶基、嘧啶基、喹唑啉基及吲哚基。
于本文中所使用的名称“环烷基”指为仅具有碳环原子的部分或完全饱和的单环或双环环系统。实施例包括但不限于环丙烷、环戊烷及环己烷。
于本文中所使用的名称“杂环烷基”指为除了碳外,具有一或多个杂原子(例如,O、N或S)作为环原子的部分或完全饱和的单环或双环环系统。实施例包括但不限于哌啶、哌、吗福啉、硫吗福啉及1,4-氧氮。
于本文所提到的烷基、烯基、炔基、伸烯基、伸炔基、芳基、杂芳基、环烷基及杂环烷基包括经取代及未经取代的部分二者。该取代基的实施例包括C1-C10烷基、C2-C10烯基、C2-C10炔基、C3-C8环烷基、C5-C8环烯基、C1-C10烷氧基、芳基、芳氧基、杂芳基、杂芳氧基、胺基、C1-C10烷基胺基、C1-C20二烷基胺基、芳基胺基、二芳基胺基、羟基胺基、烷氧基胺基、C1-C10烷基磺酰胺、芳基磺酰胺、羟基、卤素、硫、C1-C10烷硫基、芳硫基、氰基、硝基、酰基、酰氧基、羧基及羧酸酯。
名称“聚合物部分”指为衍生自线性、分支或星形聚合物的单价基团。该聚合物部分的分子量可为2-100kD。该聚合物部分的实施例包括但不限于聚环氧乙烷、聚乙二醇、聚环氧异丙烷、聚环氧丁烯、聚乙二醇及其共聚物。也可使用其它聚合物,例如葡萄聚糖、聚乙烯醇类、聚丙烯酰胺类或以碳水化合物为基础的聚合物,只要它们不具抗原性、毒性或引起免疫反应。
名称“多肽部分”指为来自天然发生的多肽或经改质的多肽的单价基团。该天然发生的肽可为INF-α2b、INF-β、GH、EPO及粒性细胞结肠刺激因子或抗体。该经改质的肽可例如为在INF-α2b的N-终端处包含INF及1-4个其它氨基酸残基的肽。此经改质的INF的实施例有IFN代表INF-α2b部分,该胺基在其N-终端处键结至羰基。
名称“干扰素-β”指为高度同源可抑制病毒复制及细胞增生且可调节免疫反应的蛋白质家族。参见Derynck等人,(1980),Nature285(5766):542-7;及Taniguchi等人,(1980),Gene10(1):11-5。其包含天然发生的INF-β及其官能性同等物(即,具有至少80%(例如,85%、90%、95%或99%)与其野生型对应体相同的多肽)二者。INF-β的实施例包括在可商业购得的药物中的活性成分,例如Avonex、Betaseron及Rebif。参见例如(EtemadifarM.等人,ActaNeurol.Scand.,2006,113(5):283-7。
下列列出的为典型人类INF-β蛋白质的氨基酸序列(呈前驱物形式或成熟形式):
mtnkcllqialllcfsttalsmsynllgflqrssnfqcqkllwqlngrle
yclkdrmnfdipeeikqlqqfqkedaaltiyemlqnifaifrqdssstgw
netivenllanvyhqinhlktvleeklekedftrgklmsslhlkryygri
lhylkakeyshcawtivrveilrnfyfinrltgylrn
(参见Genbank登录编号:M28622,1993年4月23日版本;斜体部分表示信号肽)
mnsfstsafgpvafslglllvlpaafpapvppgedskdvaaphrqpltss
eridkqiryildgisalrketcnksnmcesskealaennlnlpkmaekdg
cfqsgfneetclvkiitgllefevyleylqnrfesseeqaravqmstkvl
iqflqkkaknldaittpdpttnaslltklqaqnqwlqdmtthlilrsfke
flqsslralrqm
(参见Genbank登录编号:CAA00839,1993年12月3日版本)
在一个实施例中,该INF-β为突变型rINF-βSer17(重组的INF-β,其中丝氨酸为在天然成熟的INF-β序列中的位置17处的半胱氨酸的取代)。此突变型的氨基酸显示在下列:
synllgflqrssnfqsqkllwqlngrleyclkdrmnfdipeeikqlqqfq
kedaaltiyemlqnifaifrqdssstgwnetivenllanvyhqinhlktv
leeklekedftrgklmsslhlkryygrilhylkakeyshcawtivrveil
rnfyfinrltgylm
在另一个实施例中,该INF-β为经改质的天然INF-β,其中1-4个其它氨基酸残基接附至天然的INF-β的N-终端。
EPO(由肝或肾脏制造)为一种控制红血球生成或红血球制造的糖蛋白激素。其包括天然发生的EPO及其官能性同等物二者。参见美国专利5,621,080及美国专利申请公开20050176627。人类EPO的氨基酸序列(呈前驱物及成熟形式)显示在下列:
mgvhecpawlwlllsllslplglpvlgapprlicdsrvlerylleakeae
nittgcaehcslnenitvpdtkvnfyawkrmevgqqavevwqglallsea
vlrgqallvnssqpweplqlhvdkavsglrslttllralgaqkeaisppd
aasaaplrtitadtfrklfrvysnflrgklklytgeacrtgdr(前驱物)
apprlicdsrvlerylleakeaenittgcaehcslnenitvpdtkvnfya
wkrmevgqqavevwqglallseavlrgqallvnssqpweplqlhvdkavs
glrslttllralgaqkeaisppdaasaaplrtitadtfrklfrvysnflr
gklklytgeacrtgdr(成熟形式)
用来制得本发明的缀合物的EPO蛋白质可为通过合适的物种(例如,人类、鼠、猪或牛)所制造的EPO蛋白质(呈前驱物或成熟形式)。在一个实施例中,该EPO蛋白质具有与上述显示出的氨基酸序列之一有至少80%(例如,85%、90%、95%或99%)相同的氨基酸序列。在另一个实施例中,该EPO为经改质的天然EPO,其中14个其它氨基酸残基接附至天然EPO的N-终端。
名称“生长激素”指为天然发生的人类生长激素(呈前驱物或成熟形式)及其官能性变异体,即,具有与天然发生的人类生长激素至少80%(例如,85%、90%、95%或99%)相同且拥有与人类生长激素相同的生理学活性的氨基酸序列。在一个实施例中,该生长激素为经改质的天然生长激素,其中14个其它氨基酸残基接附至天然的生长激素的N-终端。天然发生的人类生长激素的氨基酸序列(呈前驱物及成熟形式)显示在下列:
matgsrtslllafgllclpwlqegsafptiplsrlfdnamlrahrlhqla
fdtyqefeeayipkeqkysflqnpqtslcfsesiptpsnreetqqksnle
llrisllliqswlepvqflrsvfanslvygasdsnvydllkdleegiqtl
mgrledgsprtgqifkqtyskfdtnshnddallknygllycfrkdmdkve
tflrivqcrsvegscgf(前驱物)
fptiplsrlfdnamlrahrlhqlafdtyqefeeayipkeqkysflqnpqt
slcfsesiptpsnreetqqksnlellrisllliqswlepvqflrsvfans
lvygasdsnvydllkdleegiqtlmgrledgsprtgqifkqtyskfdtns
hnddallknygllycfrkdmdkvetflrivqcrsvegscgf(成熟形式)
mfptiplsrlfdnamlrahrlhqlafdtyqefeeayipkeqkysflqnpq
tslcfsesiptpsnreetqqksnlellrisllliqswlepvqflrsvfan
slvygasdsnvydllkdleegiqtlmgrledgsprtgqifkqtyskfdtn
shnddallknygllycfrkdmdkvetflrivqcrsvegscgf(改质形式)
使用Karlin及Altschul的算法来测量二种氨基酸序列的“同一性百分比”,Proc.Natl.Acad.Sci.USA87:2264-68,1990;如在Karlin及Altschul的Proc.Natl.Acad.Sci.USA90:5873-77,1993中修改。此算法并入Altschul等人的NBLAST及XBLAST程序(2.0版)中,J.Mol.Biol.215:403-10,1990。可使用XBLAST程序,得分=50,字长=3来进行BLAST蛋白质研究,以获得与使用在本发明中的蛋白质分子同源的氨基酸序列。若于二个序列间存在有缺口时,可使用GappedBLAST,如描述在Altschul等人,NucleicAcidsRes.25(17):3389-3402,1997中。当使用BLAST及GappedBLAST程序时,可使用各个程序(例如,XBLAST及NBLAST)的预设参数。
名称“连结官能基”指为二价官能基,其一端连接至聚合物部分及其它端连接至肽部分。实施例包括但不限于-O-、-S-、羧酸盐、羰基、碳酸盐、酰胺、胺基甲酸盐、尿素、砜基、亚砜基、胺基、亚胺基、羟基胺基、膦酸盐或磷酸盐基团。
上述描述的肽-聚合物缀合物可呈自由态形式或呈盐形式(若合适的话)。例如,可在阴离子与于本发明的肽-聚合物缀合物上的正电荷基团(例如,氨基)间形成盐。合适的阴离子包括氯化物、溴化物、碘化物、硫酸盐、硝酸盐、磷酸盐、柠檬酸盐、甲磺酸盐、三氟醋酸盐及醋酸盐。同样地,也可在阳离子与于本发明的多肽-聚合物缀合物上的负电荷基团(例如,羧酸盐)间形成盐。合适的阳离子包括钠离子、钾离子、镁离子、钙离子及铵阳离子(例如四甲基铵离子)。
此外,该肽-聚合物缀合物可具有一或多个双键或一或多个不对称中心。此缀合物可发生如为外消旋盐、外消旋混合物、单一镜像物、各别的非镜像异构物、非镜像异构混合物及顺-或反-或E-或Z-双键异构形式。
本发明的聚合物-肽缀合物的实施例显示在下列:
其中mPEG代表具有分子量20kD的经甲氧基封端的聚乙二醇,及rINF-βSer17、EPO及GH的N-终端接附至显示在上述结构中的最右边的碳。
某些蛋白质具有治疗用途。因此,本发明的缀合物(包括肽部分)可使用来治疗疾病。例如,INF-β为一种用来治疗HCV或HBV感染的免疫调节用药法。参见例如,(JournalofVascularandInterventionalRadiology13(2002):191-196。因此,在本发明的范围内包括使用上述描述的INF-β-聚合物缀合物来治疗肝炎C病毒(HCV)感染或肝炎B病毒(HBV)感染的方法。至于另一个实施例,EPO为一种由肾脏制造以促进在骨髓中形成红血球的激素。其已经使用作为免疫调节用药法,以治疗产生自慢性肾脏病的贫血、AIDS的齐多夫定(zidovudine)治疗的二级贫血症及与癌相关的贫血症。近来的研究已也发现EPO提高神经生成且在中风后恢复上扮演关键性角色。参见例如,P.T.Tsai,JournalofNeuroscience,2006,26:1269。因此,本发明的另一个观点涉及一种通过上述描述的EPO-聚合物缀合物来治疗贫血或提高神经生成的方法。
包含上述描述的INF-β-聚合物缀合物以使用来治疗HCV感染或HBV感染的组合物;及包含上述描述的EPO-聚合物缀合物以使用来治疗贫血或提高神经生成的组合物;和该缀合物的治疗用途及使用来制造用来治疗HCV感染、HBV感染或贫血、或用来提高神经生成的药剂也在本发明的范围内。
在下列描述中提出本发明的一或多个具体实例的细节。将从该描述及从申请专利范围中明了本发明的其它特征、目标及优点。
实施方式
可通过在化学领域熟知的合成方法来制备本发明的肽-聚合物缀合物。例如,可结合具有一或多个活性官能基的连结子分子与二个对在该连结子分子上的官能基具有官能基反应性的聚合物分子。随后,让包含官能基的肽分子与该连结子分子的官能基反应,以形成本发明的肽-聚合物缀合物。于本文中提供二个阐明的合成方法。
下列方法1显示出制备式I的肽-聚合物缀合物的实施例。二胺化合物1(其包含乙缩醛基团)与碳酸N-羟基琥珀酰亚胺酯mPEG(即,化合物2)反应,以形成二-聚乙烯二醇化的(pegylated)化合物3,其随后转换成醛4。此醛化合物与具有自由态胺基的肽H-P经由还原性烷基化反应,以获得本发明的肽-聚合物缀合物。
方法1
下列方法2显示出制备式II的肽-聚合物缀合物的实施例。化合物6具有一聚合物部分及一醛官能基。其可与具有自由态胺基官能基的肽7反应。随后还原(例如,通过氢化或通过NaBH3CN)所产生的产物8,以获得肽-聚合物缀合物9。
A为聚合物部分。
G1为一键结或一连结官能基。
L为伸烯基或伸炔基。
H2N-P’为INF-β、EPO或GH。
方法2
下列方法3为制备式III的肽-聚合物缀合物的实施例。具有乙缩醛基团的化合物10(其可从β-氨基酸制备)与碳酸N-羟基琥珀酰亚胺酯mPEG2反应,以形成聚乙烯二醇化的化合物11,其随后转换成醛12。此醛化合物与具有自由态胺基的肽H-P经由还原性烷基化反应,以获得想要的化合物13。
方法3
上述描述的化学反应包括使用溶剂、试剂、催化剂、保护基团与去保护基团试剂及某些反应条件。它们可额外地包括加入或移除合适的保护基团的步骤(在特别描述于本文的步骤前或后),以最后允许用于肽-聚合物缀合物的合成。此外,可使用另一种程序或顺序来进行多个合成步骤,以获得想要的多肽-聚合物缀合物。在合成合适的肽-聚合物缀合物中有用的合成化学转换及保护基团方法(保护及去保护)在本领域中已知,及包括例如描述在下列中的那些:R.Larock,ComprehensiveOrganicTransformations,VCH出版社(1989);T.W.Greene及P.GM.Wuts,ProtectiveGroupsinOrganicSynthesis,第2版,JohnWileyandSons(1991);L.Fieser及M.Fieser,FieserandFieser’sReagentsforOrganicSynthesis,JohnWileyandSons(1994);及L.Paquette编辑,EncyclopediaofReagentsforOrganicSynthesis,JohnWileyandSons(1995)及其后续版本。
从而合成的肽-聚合物缀合物可通过诸如离子交换色层分析法、凝胶层析色层分析法、电泳、透析、超微滤法或超速离心法的方法进一步纯化。
本发明的肽-聚合物缀合物可呈具医药活性的缀合物形式。再者,其可通过酶促裂解(例如,透过水解)在肽部分与聚合物部分间的连结来活体内释放医药活性肽。牵涉活体内裂解连结的酶的实施例包括氧化酶(例如,过氧化酶类、胺氧化酶类或脱氢酶类)、还原酶(例如,酮基还原酶类)及水解酶(例如蛋白酶、酯酶、硫酸酯酶或磷酸酯酶)。
因此,本发明的一个观点涉及一种给药有效量的一或多种上述描述的肽-聚合物缀合物来治疗病症(例如,HCV或HBV感染或贫血)的方法。特别是,可通过将有效量的一或多种肽-聚合物缀合物给药至患者来治疗的疾病。此患者可由保健专家根据任何合适的诊断方法的结果鉴别。
如于本文中所使用,名称“治疗”定义为将一包含肽-聚合物缀合物的组合物施加或给药至患有病症、该病症的症状、该病症的二级疾病或病症,或关于该病症的体质的患者(人类或动物),其目的为治愈、缓和、解除、补救或改善该病症、该病症的症状、该病症的二级疾病或病症、或关于该病症的体质。“有效量”指为在受治疗的患者上授予治疗效应的肽-聚合物缀合物的量。治疗效应可为目标(即,可由某些测试或标志测量)或主观(即,患者提供迹象或感觉效应)。
为了实施本发明的方法,具有一或多种上述提及的缀合物的组合物可非经肠道、口服、鼻、直肠、局部或颊给药。如于本文中所使用的名称“非经肠式”指为皮下、皮内、静脉内、肌肉内、关节内、动脉内、滑膜内、胸骨内、鞘内、病灶内、腹膜内、气管内或颅内注射,和任何合适的输液技术。
无菌可注射组合物可为在无毒非经肠道的可接受的稀释剂或溶剂中的溶液或悬浮液,例如在1,3-丁二醇中的溶液。在可使用的可接受的媒剂及溶剂当中,其可为甘露醇、水、Ringer溶液及等渗压的氯化钠溶液。此外,常规使用非挥发性油作为溶剂或悬浮媒质(例如,合成的单或双甘油酯)。脂肪酸(如,油酸及其甘油酯衍生物)在注射物质的制备上有用,如天然医药可接受的油,例如橄榄油或蓖麻油,特别是其聚氧乙基化的形式。这些油溶液或悬浮液也可包含一长链醇稀释剂或分散剂、或羧甲基纤维素或类似的分散剂。为了调配的目的,也可使用其它通常使用的界面活性剂,如Tween类或Span类或在医药可接受的固体、液体或其它剂型的制造中通常使用的其它类似的乳化剂或生物效性促进剂。
用于口服给药的组合物可为任何口服可接受的剂形,包括胶囊、锭剂、乳液及水性悬浮液、分散液与溶液。在锭剂的实例中,通常使用的载剂包括乳糖及玉黍蜀粉。典型上也加入润滑剂,例如硬脂酸镁。对以胶囊形式口服给药来说,有用的稀释剂包括乳糖及干玉黍蜀粉。当口服给药水性悬浮液或乳液时,该活性成分可悬浮或溶解在油相中与乳化或悬浮剂结合。若必要时,可加入某些变甜、风味或着色剂。
可根据在医药调配物的领域中的常规技术来制备鼻用气雾剂或吸入组合物。例如,此组合物可使用苄醇或其它合适的防腐剂、提高生物效性的吸收促进剂、碳氟化合物及/或其它在技艺中已知的溶解或分散剂来制备,如为在盐液中的溶液。也可以用于直肠给药的栓剂形式来给药具有一或多种上述描述的化合物的组合物。
例行地使用一医药可接受的载剂与一或多种上述提及的活性缀合物。在该医药组合物中的载剂必需在观念上“可接受”,其与该组合物的活性成分(及能稳定该活性成分较佳)兼容且对欲治疗的患者无毒。可使用一或多种增溶剂作为用来传递上述提及的化合物的医药赋形剂。其它载剂的实施例包括胶态氧化硅、硬脂酸镁、纤维素、硫酸月桂酯钠及D&C黄色#10。
下列实施例仅解释为阐明用且无论如何不以任何方式限制本发明内容的剩余部分。没有进一步详细地阐述,相信本领域技术人员可根据于本文中的描述将本发明使用至其最大程度。于本文中所引用的全部公告其全文藉此以参考方式并入本文。
实施例1:IFN-β-二-PEG聚合物缀合物
二-PEG醛的制备
根据描述在BioconjugateChem.1993,4,568-569中的方法,从购买自美国加利福尼亚州的SunBioInc.的20kDmPEGOH来制备20kDPEGO(C=O)OSu。
将6-(1,3-二氧戊环-2-基)己烷-1,5-二胺在二氯甲烷中的溶液(11.97克包含9.03毫克二胺(47.8微摩尔的溶液)加入至包含20kDPEGO(C=O)OSu(1.72克,86.0微摩尔)的烧瓶。在PEGO(C=O)OSu完全溶解后,加入N,N-二异丙基乙基胺(79微升,478微摩尔)。在室温下搅拌该反应混合物24小时,然后,随着搅拌逐滴加入甲基三级丁基醚(200毫升)。收集所产生的沉淀及在真空中干燥,以获得二-PEG乙缩醛(1.69克,98%),如为白色固体。
1HNMR(400MHz,d6-DMSO)δ7.16(t,J=5.2Hz,1H),7.06(d,J=8.8Hz,1H),4.76(t,J=4.8Hz,1H),4.10-3.95(m,4H),1.80-1.65(m,1H),1.65-1.50(m,1H),1.48-1.10(m,6H)。
将二-PEG乙缩醛(4.0克,0.2毫摩尔)悬浮在pH2.0缓冲液(柠檬酸,40毫升)中。在35℃下搅拌该反应混合物24小时,然后以二氯甲烷(3×50毫升)萃取。在硫酸镁上干燥结合的有机层,浓缩,然后再溶解于二氯甲烷(20毫升)中。随着搅拌将该溶液逐滴加入至甲基三级丁基醚(400毫升)。收集所产生的沉淀及在减压下干燥,以获得二-PEG醛(3.8克,95%),如为白色固体。
1HNMR(400MHz,d6-DMSO)δ9.60(s,1H),7.24(d,J=8.4Hz,1H),7.16(t,J=5.2Hz,1H),4.10-3.95(m,4H),3.95-3.80(m,1H),3.00-2.85(m,2H),2.58-2.36(m,2H),1.46-1.15(m,6H)。
再者,以下列方式制备二-PEG醛:
通过苄氧基羰基保护商业可获得的同源离氨酸(homo-lysine)(AstatechPharmaceuticalCo.,Ltd.)的二个胺基。酯化及还原该N经保护的同源离氨酸以形成一醛化合物。随后,以乙二醇保护该醛基团。然后,通过于钯催化剂存在下氢化来移除该苄氧基羰基保护基团。在温和的碱性条件下,让该N经去保护的化合物与活化的mPEGOH(韩国SunbioChemicalCo.,Ltd.)反应。在pH2.0柠檬酸缓冲液(Sigma-Aldrich)中,于25℃下搅拌所产生的产物72小时,以移除醛保护基团。获得109克的二-PEG聚合物醛(产率:95%)。纯度大于97.7%(通过HLC测量)及大于95%(通过1HNMR分析测量)。
人类rhIFN-βSer17的制备
将编码出人类INF-βSer17的DNA片段克隆进入表达载体pET24a中,以产生一表达质粒rhIFN-βSer17-pET24a。将此表达质粒转化至大肠杆菌中,且选择、培养正转化株(即,携带表现质体的选殖物),并将所产生的大肠杆菌培养物贮存在-80℃下。
在37℃及200rpm下,将10微升上述提及的经贮存的大肠杆菌培养物灌输进入200毫升由Terrific肉汤及甘油所组成的播种培养基中约15小时。将150毫升如此获得的大肠杆菌培养物转移至2.5升包含葡萄糖(10克/升)、MgSO4·7H2O(0.7克/升)、(NH4)2HPO4(4克/升)、KH2PO4(3克/升)、K2HPO4(6克/升)、柠檬酸盐(1.7克/升)、酵母菌萃取物(10克/升)、卡那霉素(50毫克/毫升)、氯霉素(50毫克/毫升)、抗发泡剂及微量元素(包括FeSO4·7H2O(10毫克/升)、ZnSO4·7H2O(2.25毫克/升)、CuSO4·5H2O(1毫克/升)、MnSO4·H2O(0.5毫克/升)、H3BO3(0.3毫克/升)、CaCl2·2H2O(2毫克/升)、(NH4)6Mo7O24(0.1毫克/升)、EDTA(0.84毫克/升)及Cl(50毫克/升))的培养基,并在37℃下培养。当该大肠杆菌培养物的OD600到达120至140时,将IPTG(1M)加入至该培养物以引发rhIFN-βSer17的表达性。在37℃及300rpm下培养所引发的培养物3小时。当需要时,在培养期间,将包含800克/升葡萄糖及20克/升MgSO4的饲育培养基加入至该大肠杆菌培养物。
让如上所述所获得的大肠杆菌培养物接受离心以采集大肠杆菌细胞。将该细胞再悬浮于PBS缓冲液(0.1MNa2HPO4,0.15MNaCl)中及在APV均质化机中破裂。在10,000rpm,4℃下离心如此获得的均质化溶液15分钟。收集沉淀物(包括包涵体),将其再悬浮于PBS中及在室温下搅拌20-30分钟,以形成一悬浮液。将NaOH(6N)加入至该悬浮液以将其pH调整至12,以允许包含在该包涵体中的蛋白质溶解。约2分钟后,以6NHCl将该悬浮液的pH值调整至7.5。然后,让该悬浮液接受离心及收集从而形成的上层液,使用分光光度计来测量其蛋白质浓度。让该上层液与再折叠缓冲液(TEA,pH8.3)混合及没有搅拌在室温下培养24-48小时。然后,使用由MilliporeInc.所提供的TFF系统及PLCCC匣(PLCCCcassette)浓缩及透析其。让所产生的溶液接受超微滤、透析及以SPFF琼脂糖凝胶管柱分馏。使用另一种SPFF琼脂糖凝胶管柱进一步分馏如此获得的馏分A9及A10(包括重组蛋白质rhIFN-βSer17),以丰富化该重组蛋白质(在馏分A8-A10中)。通过凝胶层析进一步纯化(Superdex75HR10/300)这些含rhIFN-βSer17的馏分,以获得具有纯度大于90%的rhIFN-βSer17蛋白质(1毫克/毫升)。该重组蛋白质的生物活性为>2×107IU/毫克蛋白质。
IFN-β-二-PEG聚合物缀合物的制备
将18.9毫克的rhINF-βSer17及1.51克的二PEG醛悬浮在26毫升0.1M磷酸钠缓冲液(pH5.0)中。在此溶液中加入400当量的NaCNBH3(比利时的AcrosOrganics)。在室温下搅拌该反应混合物16小时,然后接受以25mMtris-HCl(pH7.8)透析。通过离子交换管柱纯化粗产物,以获得2毫克的IFN-β-二-PEG聚合物。
人类IFN-β的制备
在250毫升补充以50微升/毫升卡那霉素及50微升/毫升氯霉素的SYN培养基(10克/升的选择的大豆蛋白胨(soytone)、5克/升酵母菌萃取物及10克/升NaCl)中,灌输经转化的大肠杆菌BLR(DE3)-RIL细胞(其携带已操作连结至大肠杆菌启动子的IFN-β的编码序列)。然后,在37℃下,于摇动器培养器中,以220rpm培养细胞过夜(即,16小时)。
将250毫升上述提及的过夜的培养物灌输进入3.0升碱性培养基(10克/升葡萄糖、0.7克/升MgSO4·7H2O、4克/升(NH4)2HPO4、3克/升KH2PO4、6克/升K2HPO4、2克/升柠檬酸盐、10克/升酵母菌萃取物及2克/升异白氨酸)中,并补充以10克/升的基本葡萄糖、0.7克/升的饲育MgSO4、30毫升的饲育微量元素(10克/升FeSO4·7H2O、2.25克/升ZnSO4·7H2O、1克/升CuSO4·5H2O、0.5克/升MnSO4·H2O、0.3克/升H3BO3、2克/升CaCl2·H2O、0.1克/升(NH4)6Mo7O24、0.84克/升EDTA、50毫升/升HCl)、25微升/毫升的卡那霉素及25微升/毫升的氯霉素,并在五升发酵槽(Bioflo3000;NJEdison的BrunswickScientationCo.)中培养。在发酵期间,通过自动化加入37%NH4OH溶液将培养基的pH控制在pH7.1。将溶氧(DO)程度维持在30%。使用程控泵加入饲育溶液(800克/升葡萄糖、20克/升MgSO4、50微升/毫升卡那霉素及50微升/毫升氯霉素),其经设定以便当程度超过40-60时进料。当在发酵培养物中的细胞密度(OD600)到达180至200时,将4毫升1M的异丙基-β-D-1-硫代半乳吡喃糖苷(IPTG)与30毫升的饲育微量元素及25克的酵母菌萃取物一起加入至该发酵培养物以引发IFNβ表达性。在IPTG诱发后5小时,通过离心采集细胞。
以比率大约1∶3(湿重克/毫升)将细胞丸粒悬浮在PBS缓冲液(0.1M磷酸钠,0.15M氯化钠,pH7.4)中,通过微射流均质机破裂,然后在4℃下以10,000rpm离心20分钟。以PBS缓冲液清洗该含包涵体(IB)的丸粒两次,如上所述般离心,及悬浮在1升PBS溶液(0.1M磷酸钠,0.15M氯化钠,pH7.4,3%两性洗涤剂3-14,5mMDTT)中。在搅拌30分钟后,让该悬浮液接受pH调整,使用6.0MNaOH调整至12,同时搅拌以溶解丸粒。然后,以6NHCl将悬浮液的pH调整至pH7.5。在10,000rpm下离心20分钟后,收集包含可溶的IFNβ的上层液。
然后,让该可溶的INF-β接受如下的再折叠。在10升新鲜制备的再折叠缓冲液(100mMTris-HCl(pH7.6),0.5ML-精氨酸,2mMEDTA)中稀释上述提及的上层液,用来形成一再折叠混合物。没有搅拌,培养该混合物48小时。在培养后,对着20mMTris(含有100mMNaCl,0.05%两性洗涤剂3-14,pH7.0)缓冲液来透析该包含再折叠的重组IFN-β的混合物。
将经透析的混合物负载到SP-琼脂糖凝胶管柱(GEAmershamPharmacia)上,其已预先平衡及以20mMTris-HCl,100mMNaCl缓冲液(pH7.0)清洗。以包含20mMTris-HCl缓冲液(pH7.0)及200mMNaCl的溶液冲提IFNβ。根据其在280纳米处的吸收来收集包含IFNβ的馏分。通过疏水性交互作用管柱(GEhealthcare),丁基琼脂糖凝胶快速流(ButylSepharoseFastFlow))(其已预先平衡及以包含1.0M硫酸铵、20mM醋酸钠及0.05%zwittergent(pH4.5)的溶液清洗),来进一步纯化包含在其中的IFNβ。使用包含0.5M硫酸铵及20mM醋酸钠的溶液冲提IFNβ。根据其在280纳米处的吸收收集包含蛋白质的馏分。储备这些馏分及通过BCA蛋白质分析(BCATM蛋白质分析,Pierce)来测量IFNβ浓度。
PEG-IFN-β缀合物的制备
在水(1.46毫升)中的二-PEG醛(296毫克,7.4微摩尔)溶液中,加入2M磷酸钠缓冲液(pH4.0,0.37毫升),两性先涤剂3-14(1.48毫升,10%在水中)及INF-β(14.8毫克在3.7毫升pH4.5包含20mM醋酸钠、0.7%硫酸铵及0.05%清洁剂的缓冲液中)。在室温下搅拌该反应混合物10分钟,接着加入氰基硼氢化物水溶液(400mM,92.5微升,37微摩尔)。在黑暗中搅拌该反应混合物40小时及通过SPHP琼脂糖凝胶色层分析法纯化。根据其滞留时间及在280纳米处的吸收来收集包含想要的PEG-IFNβ缀合物的馏分。通过BCA蛋白质分析(BCATM蛋白质分析,Pierce)来测量缀合物浓度。
在大白鼠中的药物动力学研究
在大白鼠模型中进行药物动力学研究,以比较IFN-β及PEG-IFN-β的血清半衰期。对公大白鼠(250-350克)静脉内给药剂量600微克/千克的IFNβ(n=3)及PEG-IFNβ(n=3)。在给药前及在给药后的0.1、1、2、4、6、10、24、48、72及96小时处,从每只大白鼠收集血液(250微升)。从该血液制备血清样品,及通过酶连接的免疫检定法(ELISA)分析包含在该样品中的IFN-β量。从最后三个时间点的血清浓度计算,IFN-β及PEG-IFN-β的血清半衰期各别为2小时及20小时。
实施例2:EPO-PEG聚合物缀合物
PEG-EPO的制备
在水(2.67毫升)中的二-PEG醛(267毫克,6.1微摩尔)溶液中,加入2M磷酸钠缓冲液(pH4.0,1毫升)及EPO(10毫克在3.03毫升pH7.3包含20mM磷酸钠及150mMNaCl的缓冲液中)。在室温下搅拌该反应混合物10分钟,接着加入氰硼氢化钠水溶液(400mM,100微升,40微摩尔)。在黑暗下搅拌该反应混合物17小时及通过SPToyopearl管柱(Tosoh)纯化。该管柱与20mM醋酸钠缓冲液,pH4.5平衡。将该反应混合物稀释至浓度0.3-0.4毫克/毫升并将其负载到SPToyopearl管柱上。根据其滞留时间及在280纳米处的吸收来收集包含想要的PEG-EPO缀合物的馏分。通过280纳米UV吸收来测量缀合物浓度。
在大白鼠中的药物动力学研究
在大白鼠模型中进行药物动力学研究,以比较EPO及PEG-EPO的血清半衰期。对公大白鼠(250-350克)静脉内给药剂量25微克/千克的EPO(n=5)及PEG-EPO(n=5)。在给药前及给药后0.088、0.75、1.5、3、6、10、24及48小时,从每只大白鼠抽出血液(250微升)。对经PEG-EPO治疗的大白鼠来说,在给药后72及96小时处进一步收集血液样品。从该血液制备血清样品及以酶连接的免疫检定法(ELISA)分析以测量包含在其中的EPO量。结果显示出EPO的血清半衰期为9小时,同时PEG-EPO明显增加(即,38小时)。
EPO-PEG聚合物缀合物的制备
将0.2毫克的EPO(台湾CashmereScientificCompany)及4毫克的二PEG醛(20当量)悬浮在0.1M磷酸盐缓冲液(pH5.0)中。在此溶液中,加入400当量的NaCNBH3。在室温下搅拌该反应混合物16小时。HPLC确认EPO-二-PEG聚合物形成。
实施例3:GH-PEG聚合物缀合物
Met-hGH的制备
在上述描述的发酵程序后,培养经转化能表现出Met-hGH的大肠杆菌BLR(DE3)-RIL细胞,用以表现出Met-hGH。
经由离心采集细胞,且以比率大约1∶3(湿重克/毫升)将细胞丸粒悬浮在TE缓冲液(50mMTris-HCl,1mMEDTA,pH8.0)中。然后,通过微射流均质机破裂该等细胞,然后在10,000rpm下离心20分钟。以TED缓冲液(50mMTris-HCl,1mMEDTA,2%脱氧胆酸盐,pH8.0)清洗含包涵体(IB)的丸粒两次,如上所述般离心,及悬浮在米里Q(MilliQ)水中及在20,000rpm下离心15分钟。将IB悬浮在400毫升50mMTUD溶液(50mMTris-HCl,4M尿素,2.5mMDTT,pH10.0)中,及在20,000rpm下离心该悬浮液20分钟;收集上层液。
在2.0升新鲜制备的再折叠缓冲液(50mMTris-HCl,0.5mMEDTA,5%甘油,10mMGSH/1mMGSSG,pH8.0)中稀释上层液。没有搅拌,培养从而形成的混合物36小时,然后对着20mMTris缓冲液(pH7.0)透析。
将经透析包含Met-hGH的混合物负载到Q-琼脂糖凝胶管柱(GEAmershamPharmacia,Pittsburgh,PA)上,其已预先平衡及以20mMTris-HCl缓冲液,pH7.0清洗。以包含20mMTris-HCl缓冲液,pH7.0及100mMNaCl的溶液冲提Met-hGH。收集、储备包含Met-hGH的馏分(通过其在280纳米处的吸收测量),且将其负载到疏水性交互作用管柱(GEAmershamPharmacia,Pittsburgh,PA)上,其已预先平衡及以20mM醋酸钠缓冲液(pH7.0),在流速5毫升/分钟下清洗。以包含20mM醋酸钠缓冲液及150mM硫酸铵的溶液冲提Met-hGH。收集包含Met-hGH的馏分及接受BCA蛋白质分析(BCATM蛋白质分析,Pierce),以测量Met-hGH浓度。
PEG-Met-hGH缀合物的制备
在水(387微升)中的二-PEG醛(74毫克,1.7微摩尔)溶液中,加入2M磷酸钠缓冲液(pH4.0,374微升)及人类GH(22.4毫克在6.5毫升pH4.5包含20mM醋酸钠及150mMNaCl的缓冲液中)。在室温下搅拌该反应混合物10分钟,接着加入氰硼氢化钠水溶液(400mM,140微升,56微摩尔)。在黑暗下搅拌该反应混合物17小时及通过SPXL琼脂糖凝胶色层分析法纯化。根据其滞留时间及在280纳米处的吸收来收集包含想要的聚合物-蛋白质缀合物的馏分。使用Bradford方法(Pierce,Rockford,IL),通过蛋白质分析工具组来测量缀合物浓度。
在大白鼠中的药物动力学研究
在大白鼠模型中进行药物动力学研究,以比较Met-hGH及PEG-Met-hGH的血清半衰期。对公大白鼠(250-350克)静脉内给药剂量100微克/千克的Met-hGH(n=5)或PEG-Met-hGH(n=5)。在给药前及在给药后0.083、1、2、4、8、12及24小时,从经Met-hGH-治疗的大白鼠收集血液样品;及在给药前及在给药后0.33、1、4、8、12、24、48、72及96小时,从经PEG-Met-hGH-治疗的大白鼠收集。从该血液制备血清样品及以酶连接的免疫检定法(ELISA)分析,以测量hGH浓度。Met-hGH及PEG-Met-hGH的血清半衰期分别为3小时及35小时。
其它具体实例
在本专利说明书中所公开的全部特征可以任何组合结合。在本专利说明书中所揭示的每种特征可由另一种提供相同、同等或类似目的的特征置换。因此,除非其它方面有明确描述,否则所公开的每种特征仅为同等或类似特征的一般系列的实施例。
本领域技术人员可从上述描述中容易地查明本发明的基本特征而不背离其精神及范围,可对本发明制得多种改变及改质,使其适合于不同用途及条件。因此,其它具体实例也在所附权利要求的范围内。
Claims (18)
1.一种下式的肽-聚合物缀合物:
其中
R1、R2、R3、R4及R5各自独立地为H、C1-10烷基、C2-10烯基、C2-10炔基、芳基、杂芳基、C3-8环烷基或C3-8杂环烷基;
A1及A2各自独立地为聚合物部分;
G1、G2及G3各自独立地为一键结或一连结官能基;
P选自于由下列所组成的群:干扰素-β部分、红血球生成素部分及生长激素部分,该P的N-终端的氮原子键结至G3;
m为0或整数1-10;及
n为整数1-10。
2.权利要求1的缀合物,其中P为干扰素-β部分。
3.权利要求2的缀合物,其中P为rINF-βSer17。
4.权利要求2的缀合物,其中P为在N-终端处包含1-4个其它氨基酸残基的经改质的干扰素-β部分。
5.权利要求1的缀合物,其中P为红血球生成素部分。
6.权利要求1的缀合物,其中A1及A2各为具有分子量2-100kD的聚乙二醇部分。
7.权利要求6的缀合物,其中A1及A2各为具有分子量10-30kD的聚乙二醇部分。
8.权利要求7的缀合物,其中G1及G2各为:
其中O键结至A1或A2且N原子键结至碳原子;及G3为一键结。
9.权利要求8的缀合物,其中m为4,n为2及R1、R2、R3、R4及R5各为H。
10.权利要求9的缀合物,其中P为干扰素-β部分。
11.权利要求10的缀合物,其中P为rINF-βSer17。
12.权利要求10的缀合物,其中P为在N-终端处包含1-4个其它氨基酸残基的经改质的干扰素-β部分。
13.权利要求9的缀合物,其中P为红血球生成素部分。
14.权利要求9的缀合物,其中P为生长激素部分。
15.权利要求1的缀合物,其中P为生长激素部分。
16.权利要求1的缀合物,其中该缀合物为:
其中mPEG为具有分子量20kD的经甲氧基封端的聚乙二醇部分。
17.权利要求1的缀合物,其中该缀合物为;
其中mPEG为具有分子量20kD的经甲氧基封端的聚乙二醇部分。
18.权利要求1的缀合物,其中该缀合物为:
其中mPEG为具有分子量20kD的经甲氧基封端的聚乙二醇部分。
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