CN1134265C - 用作为免疫佐剂的磷腈聚电解质 - Google Patents
用作为免疫佐剂的磷腈聚电解质 Download PDFInfo
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- CN1134265C CN1134265C CNB94193067XA CN94193067A CN1134265C CN 1134265 C CN1134265 C CN 1134265C CN B94193067X A CNB94193067X A CN B94193067XA CN 94193067 A CN94193067 A CN 94193067A CN 1134265 C CN1134265 C CN 1134265C
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- phosphonitrile
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- oxygen base
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Classifications
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- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G79/00—Macromolecular compounds obtained by reactions forming a linkage containing atoms other than silicon, sulfur, nitrogen, oxygen, and carbon with or without the latter elements in the main chain of the macromolecule
- C08G79/02—Macromolecular compounds obtained by reactions forming a linkage containing atoms other than silicon, sulfur, nitrogen, oxygen, and carbon with or without the latter elements in the main chain of the macromolecule a linkage containing phosphorus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
本发明公开了一种免疫佐剂,即可溶性聚磷腈聚电解质。在一种实施方案中,所述聚合物佐剂是下述聚(有机磷腈),它具有(i)电离的或可电离的侧基,该侧基含有例如羧酸、磺酸或羟基部分;和(ii)在使用条件下易于水解的侧基以赋予聚合物以生物降解性。
Description
本申请是1993年7月12日提交的美国专利申请序列号08/090,841的部分继续申请。
发明背景
本申请涉及用于生物医学的聚合物领域,具体地说,描述了可用作免疫佐剂的聚合物。
疫苗的开发
各种抗原刺激动物中抗体的产生,从而对随后的感染起到保护作用。然而,某些抗原不能刺激有效的免疫应答。
相对较弱的抗原的致免疫性常常是通过同时施用所述抗原与一种佐剂来增强的,所述佐剂是这样一种物质,它在单独施用时不产生致免疫性,而当与抗原结合时会诱发粘膜和/或系统免疫。人们习惯性地认为佐剂例如矿物油乳剂或氢氧化铝在注射部位形成抗原储存,并缓慢释放抗原。Allison和Byars在“Vaccines:New Approachesto Immunological Problems:,R.W.Ellis,ed.,P.431,Butterworth-Heinemann,Oxford(1992)中的最近的研究表明,佐剂通过释放细胞因子从而刺激特异性的、有时是非常窄范围的免疫应答来增强免疫应答。不幸的是,许多免疫佐剂例如弗氏完全佐剂是有毒的,因而仅可用于动物研究目的,而不能用于人的接种。弗氏完全佐剂含有经加热灭活的结核分支杆菌在含有表面活性剂的矿物油中的悬浮液,并且在动物的免疫接种部位引起肉芽肿损伤。弗氏佐剂也可以使接种疫苗者对结核病的试验呈阳性。
已发现某些合成的聚电解质当与抗原结合时会提供免疫刺激性。例如,Petrov等人在Jhurnal Vses.Khim.Ob-va im.D.I.Mendeleeva,33:22-42(1988)中研究了聚丙烯酸(PAA)、丙烯酸与N-乙烯基吡咯烷酮的共聚物(CP-AAVPD)、聚-2-甲基-5-乙烯基吡啶(PMVP)、聚-4-乙烯基-N-乙基溴化吡啶鎓(PVP-R2)及类似化合物与抗原轭合时的佐剂活性。含有许多上述同样的聚电解质的聚电解质复合物的免疫调节作用最近还由Petrov等人在Sov.Med.Rev.D.Immunol.,4:1-113(1992)中进行了综述。然而,这些聚合物的毒性和生物降解性还无人研究,而且可能阻碍这些聚合物作为人用佐剂的应用。
具有刺激对非抗原性或弱抗原性分子的免疫应答能力的无毒佐剂或载体可用于开发和施用疫苗。
因此,本发明的一个目的是提供一种可以以最小的毒性安全地施用于人和动物的佐剂。
本发明的另一目的是提供一种可溶的并且可生物降解的佐剂。
本发明的另一目的是提供一种具有抗有机体例如流感病毒或破伤风梭状芽胞杆菌的保护作用的疫苗。
本发明的再一目的是提供一种快速而有效的合成用作佐剂的聚合物例如聚磷腈(polyphosphazene)的方法。
本发明的概要
本发明公开了用作佐剂的一种合成的水溶性聚磷腈(polyphosphazene)。在一个优选的实施方案中,所述磷腈是一种可生物降解的、并且当施用于动物例如人时具有最低毒性的聚电解质。
在一种实施方案中,所述聚合物佐剂是一种聚(有机磷腈),它具有(i)电离的或可电离的侧基,该侧基含有例如羧酸、磺酸或羟基部分;和(ii)在液用条件下易于水解的测基以赋予聚合物以生物降解性。适宜的可水解基团包括例如氯、氨基酸、氨基酸酯、咪唑、甘油和葡糖基。
可用作免疫佐剂的聚磷腈的两个例子有:聚[二(羧基合苯氧基)(carboxylatophenoxy)磷腈-共-二(羧甲氨基合)(glycinato)磷腈-共(羧基合苯氧基)(羧甲氨基合)磷腈]和聚[二(羧基合苯氧基)磷腈-共-二(氯)磷腈-共-(羧基合苯氧基)-(氯)磷腈]。
疫苗组合物是通过在施用前使聚合物佐剂与抗原混合成轭合来制备的。另一种可选择的方式是,聚合物和抗原可以分别施用到同一部位。
当用多价离子交联时,聚合物的溶解性变小,导致聚合物从施用部位的较慢的释放。
本发明的详细描述
本文所用术语氨基酸是指天然的及合成的氨基酸,并且包括但不限于丙氨酰基、缬氨酰基、亮氨酰基、异亮氨酰基、脯氨酰基、苯丙氨酰基、色氨酰基、甲硫氨酰基、甘氨酰基、丝氨酰基、苏氨酰基、半胱氨酰基、酪氨酰基、天冬酰胺酰基、谷氨酰胺酰基、天冬氨酰基、谷氨酰基、赖氨酰基、精氨酰基和组氨酰基。术语氨基酸酯是指天然或合成的氨基酸的脂族、芳基或杂芳族羧酸酯。
本文所用术语烷基是指饱和的直链、支链或环状烃基或其混合物,典型的是C1-C20的饱和的直链、支链或环状烃基,具体地讲,包括甲基、乙基、丙基、异丙基、丁基、异丁基、叔丁基、戊基、环戊基、异戊基、新戊基、己基、异己基、环己基、3-甲基戊基、2,2-二甲基丁基、2,3-二甲基丁基、庚基、辛基、壬基和癸基。
术语(烷基或二烷基)氨基分别指具有一个或两个烷基取代基的氨基。
本文所用术语链烯基和炔基分别指具有至少一个双键或三键的C2-C20直链或支链烃基。
本文所用术语芳基是指苯基或取代的苯基,其中的取代基为卤素、烷基、烷氧基、烷硫基、卤代烷基、羟基烷基、烷氧基烷基、亚甲二氧基、氰基、C(O)(低级烷基)、-CO2H、-SO3H、-PO3H、-CO2烷基、酰氨基、氨基、烷氨基和二烷基氨基,而且其中的芳基可以最多具有3个取代基。
术语脂族基是指烃基,典型的是C1-C20烃基,所述烃基可以含有可以为直链、支链或环状或这三种形式的结合的一种或混合的烷基、链烯基或炔基。
本文所用术语卤素包括氟、氯、溴和碘。
术语芳烷基是指具有烷基取代基的芳基。
术语烷芳基是指具有芳基取代基的烷基,包括苄基、取代的苄基、苯乙基或取代的苯乙基,其中所述取代基为如上对芳基所定义者。
本文所用术语杂芳基或杂芳族基是指在芳环上包括至少一个硫、氧或氮的、并且任选被如上对于芳基所定义的取代基取代的芳族基。上述芳族基的非限制性实例为呋喃基、吡啶基、嘧啶基、噻吩基、异噻唑基、咪唑基、四唑基、吡嗪基、苯并呋喃基、苯并噻吩基、喹啉基、异喹啉基、苯并噻吩基、异苯并呋喃基、吡唑基、吲哚基、异吲哚基、苯并咪唑基、嘌呤基、carbozolyl、噁唑基、噻唑基、异噻唑基、1,2,4-噻二唑基、异噁唑基、吡咯基、吡唑基、喹唑啉基、哒嗪基、吡嗪基、噌啉基、2,3-二氮杂萘基、喹喔啉基、黄嘌呤基、次黄嘌呤基、喋啶基、5-氮杂胞嘧啶核苷基、5-氮杂尿嘧啶基、三唑并吡啶基、咪唑并吡啶基、吡咯并嘧啶基和吡唑并嘧啶基。
术语“可药用阳离子”是指带有正电荷并且可以作为抗衡离子施用到磷腈聚电解质中的有机或无机部分。
本文所用术语杂烷基是指在碳链中或在碳链末端包含一个杂原子例如氧、硫或氮(其价键由氢或氧来满足)的烷基。
提供了用作免疫佐剂的合成聚合物。所述聚合物佐剂是一种至少部分可溶于水(典型的溶解度至少为0.001%(重量))、含水缓冲盐溶液或含水醇溶液的聚磷腈。所述聚磷腈优选合有带电荷的侧基,该带电荷的侧基或者以与其抗衡离子处于平衡状态的酸或碱的形式存在、或以其离子型盐的形式存在。
所述聚合物优选为生物降解性的,并且当给动物(包括人)施用时具有最低毒性。
聚磷腈聚电解质的选择
聚磷腈类化合物是这样的聚合物,其骨架由交替的磷和氮组成,所述的磷和氮由交替的单键和双键分开。每个磷原子与两个侧基(“R”)共价键合。聚磷腈类化合物中的重复单元具有下列通式:其中n为整数。
取代基(“R”)可以是在聚合物内可以变化的各种部分的任何一种,包括但不限于脂族基、芳基、芳烷基、烷芳基、羧酸基、杂芳族基、碳水化合物(包括葡萄糖)基、杂烷基、卤素、(脂族基)氨基(包括烷基氨基)、杂芳烷基、二(脂族基)氨基(包括二烷基氨基)、芳基氨基、二芳基氨基、烷基芳基氨基,-氧基芳基,包括但不限于-氧基苯基CO2H、-氧基苯基SO3H、-氧基苯基羟基和-氧基苯基PO3H;-氧基脂族基,包括-氧基烷基、-氧基(脂族基)CO2H、-氧基(脂族基)SO3H、-氧基(脂族基)PO3H和-氧基(脂族基)羟基(包括-氧基(烷基)羟基):-氧基烷芳基、-氧基芳烷基、-硫基芳基、-硫基脂族基(包括-硫基烷基)、-硫基烷芳基、-硫基芳烷基、-NHC(O)O-(芳基或脂族基)、-O-[(CH2)xO]y-CH2)-O-[(CH2)xO]y(CH2)xNH(CH2)xSO3H和-O-[(CH2)xO]y-(芳基或脂族基),其中x为1-8,并且y为1-20的整数。所述基团可以通过例如氧、硫、氮或碳原子键合到磷原子上。
一般而言,当聚磷腈具有一种以上类型的侧基时,所述基团会在整个聚合物中无规则地变化,因而该聚磷腈是无规共聚物。磷原子可以与两个类似的基团或两个不同基团键合。带有两种或更多种类型的侧基的聚磷腈可以通过使聚(二氯磷腈)与所需的一种亲核试剂或多种亲核试剂以所需比例反应来制备。在聚磷腈中所得侧基的比率可由许多因素来决定,所述因素包括用于制备聚合物的起始原料的比率、所述亲核取代反应进行的温度以及所用的溶剂体系。尽管确定所得聚合物中各基团的准确取代方式是困难的,但是本领域技术人员可以容易地确定聚合物中各基团的比率。
其中A和B可以独立地在聚合物中变化,并且可以为:
(i)在使用条件下易于水解的基团,包括但不限于氯、氨基酸、氨基酸酯(通过氨基键合)、咪唑、甘油或葡糖基;或
(ii)在使用条件下不易水解的基团,包括但不限于脂族基、芳基、芳烷基、烷芳基、羧酸、杂芳族基、杂烷基、(脂族基)氨基-(包括烷基氨基-)、杂芳烷基、二(脂族基)氨基(包括二烷基氨基-)、芳基氨基-、二芳基氨基-、烷基芳基氨基-,-氧基芳基,包括但不限于-氧基苯基CO2H、-氧基苯基SO3H、-氧基苯基羟基和-氧基苯基PO3H;-氧基脂族基,包括-氧基烷基、-氧基(脂族基)CO2H、-氧基(脂族基)SO3H、-氧基(脂族基)PO3H和-氧基(脂族基)羟基(包括-氧基(烷基)羟基);-氧基烷芳基、-氧基芳烷基、硫基芳基、-硫基脂族基(包括-硫基烷基)、-硫基烷芳基、或硫基芳烷基;
其中聚合物含有至少1%或1%以上、优选10%或10%以上、更优选80-90%或80-90%以上、但低于100%的在使用条件下不易水解的重复单元,并且
其中n为整数4或4以上的整数,并且优选在10与20,000至300,000之间。
不言而喻,某些基团,例如非咪唑杂芳族基在中性含水条件下(例如血液中)以极缓慢的速率水解,因此,就本发明的目的而言,通常认为它们是不可水解的基团。然而,在某些条件下(例如正如所发现的那样,在低pH条件下,例如在胃中),正常的不可水解的基团(例如非咪唑杂芳族化合物)的水解速率可以增加到可以影响聚合物的生物降解性能的程度。本领域普通技术人员采用已知技术可以容易地确定在使用条件下测基是否以相当大的速率水解。本领域普通技术人员还可以确定具有本发明所述各种结构的聚磷腈类化合物的水解速率,并且可以选择可提供适用于目的用途的所需生物降解特性的聚磷腈。
聚合物的水解降解程度将随着易于水解的侧基的百分率和可水解基团的水解速率而变化。所述可水解基团在含水环境下被羟基取代,所提供的P-OH键赋予聚合物以水解不稳定性。
在其它实施方案中,所述免疫佐剂为:(i)一种非生物降解性聚磷腈,其中聚合物中的侧基没有一个(或基本上没有)在使用条件下易于水解,或(ii)一种完全可生物降解的聚磷腈,其中所有的基团均在使用条件下易于水解(例如聚[二(羧甲氨基)磷腈])。
磷腈聚电解质在本发明中被定义为含有可提供阴离子型、阳离子型或双亲型聚磷腈的电离的或可电离的侧基的聚磷腈类化合物。离子型基团可以为盐、或已经或可以至少部分解离的酸或碱的形式。任何可药用的单价阳离子均可以用作该盐的抗衡离子,包括但不限于钠、钾和铵离子。磷腈聚电解质也可以含有非离子型侧基。磷腈聚电解质在使用条件下可以是生物降解性或非生物降解性的。电离的或可电离的侧基优选在使用条件下不易水解。
优选的磷腈聚电解质免疫佐剂含有包括羧酸、磺酸或羟基部分在内的侧基。尽管酸性基团通常位于不可水解的侧基上,但是它们也可以、或以结合方式位于可水解基团上。具有作为侧链的羧酸基团的磷腈聚电解质的实例如下式所示:
其中n为整数,优选在10与10,000至300,000之间的整数。该聚合物的化学名称为聚[二(羧基合苯氧基)磷腈]或聚[双(羧基合苯氧基)磷腈](PCPP)。
磷腈聚电解质优选是可生物降解的,以防止在身体的较远部位(例如脾脏)处聚合物分子的最终沉积和累积。本文中所用的术语可生物降解的是指这样的聚合物,一旦将其置于大约25-37℃的pH6-8的生理溶液中,在所需应用可接受的期间内、一般少于约五年并且最优选少于约一年的期间内降解。
最优选的是所述聚合物为包括在使用条件下不水解的、包括羧酸部分在内的侧基以及在使用条件下易于水解的侧基的聚(有机磷腈)。优选的带有水解敏感性基团的磷腈聚电解质的实例为聚[二(羧基合苯氧基)磷腈-共-二(氨基酸)磷腈-共-(羧基合苯氧基)(氨基酸)磷腈],具体包括聚[二(羧基合苯氧基)磷腈-共-二(羧甲氨基)磷腈-共-(羧基合苯氧基)(羧甲氨基合)磷腈]和聚[二(羧基合苯氧基)磷腈-共-二(氯)磷腈-共-(羧基合苯氧基)(氯)磷腈]。
聚磷腈的毒性是用本领域技术人员公知的细胞培养实验测定的。例如,聚[二(羧基合苯氧基)磷腈]的毒性是在细胞培养物中按下述方法测定:通过用聚[二(羧基合苯氧基)磷腈]涂覆细胞培养皿,然后将小鸡胚胎成纤维细胞接种到涂覆后的佩特里细胞菌养皿上。在小鸡胚胎成纤维细胞接种后三天,细胞变平,并形成纺锤状细胞。在相衬显微镜下,观察到核分裂图象。这些观察提供了聚[二(羧基合苯氧基)磷腈]对于复制细胞无毒性的证据。
用作免疫佐剂的交联的聚磷腈类化合物可以通过使磷腈聚电解质与金属多价阳离子例如锌、钙、铋、钡、镁、铝、铜、钴、镍或镉结合来制备。
磷腈聚电解质的合成
聚磷腈类化合物(包括磷腈聚电解质)可以通过聚(二氯磷腈)与各种化学试剂或试剂混合物按照本领域技术人员已知的方法进行大分子亲核取代反应来制备。优选地,磷腈聚电解质通过使聚(二氯磷腈)与适合的一种或多种取代氯的亲核试剂反应来制备。聚合物中可水解侧链与不可水解的侧链的所需比例可以根据需要通过调节与聚(二氯磷腈)反应的相应的亲核试剂的用量及反应条件来获得。优选的具有免疫活性的聚磷腈类化合物的分子量大于1,000。
例如,聚[(羧基合苯氧基)(羧甲氨基合)磷腈](PC-GlPP)的制备方法如下:使聚(二氯磷腈)的氯原子与对羟基苯甲酸丙酯和甘氨酸乙酯盐酸盐进行亲核取代反应(PC-GlPP合成法)。然后,将所得的聚[(芳氧基)(羧甲氨基合)磷腈]酯水解成相应的聚(羧酸)。其他的聚磷腈类化合物可以按下述文献所述方法制备:Allcock,H.R.等人,Inorg.Chem.11,2584(1972);Allcock,H.R.等人,Macromolecules 16,715(1983);Allcock,H.R.等人,Macromolecules 19,1508(1986);Allcock,H.R.等人,Biomaterials 19,500(1988);Allcock,H.R.等人,Macromolecules 21,1980(1988);Allcock,H.R.等人,Inorg.Chem.21(2),515521(1982);Allcock,H.R.等人,Macromolecules 22:7579(1989);U.S.P.Nos.4,440,921,4,495,174,4,880,622,授予Allcock,H.R.等人;U.S.P.No.4,946,938,授予Magill,等人,U.S.P.No.5,149,543,授予Cohen等人,和出版物Grolleman等人,J.Controlled Release 3,143(1986),上述文献的方法及其中所公开的聚合物并入本文作为参考。
抗原的选择
抗原可衍生自细胞、细菌或病毒粒子、或其部分。正如本文所定义的那样,抗原可以是蛋白质、肽、多糖、糖蛋白、糖脂、核酸或其混合物,抗原在动物例如哺乳动物、鸟或鱼中引起致免疫应答。正如本文所定义的那样,致免疫应答可以是体液或细胞介导的。如果欲进行致免疫应答的物质的抗原性差,则可将其通过标准共价结合技术例如采用几种市售试剂盒中的一种而轭合到载体例如白蛋白或半抗原上。
在一种实施方案中,用聚合物传递核酸,该核酸将抗原编码到粘膜表面,在此表面上核酸得以表达。
优选的抗原的实例包括病毒蛋白例如流感蛋白、人免疫缺陷病毒(HIV)蛋白和B型肝炎蛋白,以及细菌蛋白和脂多糖例如革兰氏阴性细菌细胞壁和淋病奈瑟菌(Neisseria gonorrhea)蛋白。
致免疫组合物的制备
将抗原与聚合物结合以同时施用
致免疫组合物或疫苗是通过使聚合物佐剂与抗原结合来制备的。将大约0.5~0.0001份抗原加到1份聚合物中,优选通过搅拌聚合物和抗原的溶液直至得到溶液或悬浮液,优选在25℃搅拌10分钟或10分钟以上。聚合物优选通过一种将抗原均匀地分散到佐剂中的方法与抗原结合。液化聚合物的方法包括将聚合物溶于水基溶剂中,该溶剂的pH范围优选为7.1至7.7,以及熔化该聚合物。后者只有当抗原在聚合物熔化温度下是稳定的时候才是有用的。然后,将抗原与聚合物混合。固体形式(例如当抗原被冷冻干燥时)的聚合物和抗原也可例如通过挤压成型用物理方法混合在一起。聚合物也可以用于包封抗原,例如采用美国专利5,149,543(授予Cohen等人)的方法(该文献内容并入本文)或通过喷雾干燥聚合物和抗原的溶液来进行。另外,含有抗原和佐剂的微球体可以通过简单地将各组分在水溶液中混合然后通过机械力使聚合物与该物质一起凝结从而形成微粒来制备。如果必要,微粒可以利用电解质、pH变化、有机溶剂、加热或冷冻方法进行稳定化,从而形成聚合物基质包封的生物材料。
在一种优选的实施方案中,在搅拌下,将大约1份聚合物溶于10份3%Na2CO3水溶液中,然后缓慢加入10-90份磷酸盐缓冲剂(pH7.4)。
聚合物-抗原轭合物
聚合物也可以按照本领域技术人员公知的方法、通常通过抗原上氨基或羧基与聚合物上的可电离测基之一的共价键连而与抗原共价轭合,从而形成水溶性轭合物。
交联的聚合物佐剂
在另一优选的实施方案中,将聚合物用多价离子交联,优选采用含有与聚磷腈的带电荷的侧基电性相反的多价离子(例如若聚合物具有酸性侧基,则为多价阳离子,或若聚合物具有碱性侧基,则为多价阴离子)的水溶液。
优选地,聚合物被二价和三价金属离子(例如钙、铜、铝、镁、锶、钡、锡、锌和铁)、有机阳离子(例如聚(氨基酸))或其他聚合物(例如聚(乙烯亚胺)、聚(乙烯胺)和多糖类)交联。
聚合物-佐剂混合物的添加剂
本领域技术人员将会理解,致免疫疫苗组合物可以含有其他生理学上可接受的成分,例如水、盐水或矿物油,例如DrakeolTM、MarkolTM和角鲨烯,从而形成乳剂。
聚合物-抗原疫苗的施用
致免疫组合物可以作为疫苗按照本领域技术人员已知的引起免疫应答的任何方法来施用,包括胃肠外、口服或通过经膜或经粘膜途径施用。优选地,将疫苗经胃肠外(静脉内、肌内、皮下、腹膜内等)施用,优选皮下施用。粘膜表面施用途径的非限制性实施有鼻内(或一般而言,与鼻有关的淋巴(样)组织)、呼吸道、阴道和直肠途径。
正如下列实施例所证明的那样,剂量是由抗原负载量、测定剂量的标准技术、对每一抗原的施用方法(基于施用聚合物-抗原所引起的抗体效价)决定的。
尽管在优选实施方案中,聚合物抗原混合物是同时施用的,而在另一可供选择的实施方案中,聚合物和抗原分别施用到同一部位或邻近部位。聚合物用于吸引免疫系统的细胞至该部位,在此它们处理抗原。
参考下列非限制性实施例将进一步理解聚磷腈佐剂和合成方法。实施例1:聚[(羧基合苯氧基)(羧甲氨基合)磷腈]的合成
聚[(羧基合苯氧基)(羧甲氨基合)磷腈]制备如下。将聚(二氯磷腈)(5.0,0.0425mol)溶于300ml四氢呋喃(THF)中。向溶解的聚合物中滴加对羟基苯甲酸丙酯的钠盐(通过使羟基苯甲酸丙酯(30.6g,0.17mol)与60%的氢化钠(6.12,0.15mol)的THF溶液反应来制备)。加入钠盐后,将反应混合物在回流条件下搅拌2天,并由31P NMR监测。
将甘氨酸乙酯盐酸盐(23.63g,0.17mol)悬浮于50ml含有三乙胺(23.69,0.17mol)的甲苯中,并回流3.5小时。将反应混合物在冰浴中冷却,三乙胺盐酸盐从溶液中沉淀出来。过滤溶液,将其在0℃下加入到聚合物混合物中。使反应混合物温热至室温,并搅拌2天。聚合物经反复沉淀入100%乙醇中来纯化。
将所得聚合物(0.5g,1.33mmol)溶于无水THF(20ml)中。将该溶液缓慢加入到叔丁醇钾和水在无水THF中的混合物中。在最初5分钟内,将该混合物冷却至0℃;然后将其于室温下搅拌40小时。加入大过量的冰水(300ml),溶液经蒸发浓缩。聚合物经用盐酸酸化溶液至pH5.5来分离。反应条件和在水中用凝胶渗透色谱法测定的所得聚合物的重均分子量列于下面表1中。表1.聚[(羧基合苯氧基)(羧甲氨基合)磷腈]的合成编号 聚合物浓度 叔丁醇钾 水的浓度 反应时间 MW
%w/v mol/l 的浓度 mol/l 小时 kDa1 0.42 0.30 0.1 42 802 0.42 0.15 0.05 18 1303 0.42 0.04 0.05 5 170
聚合物结构是用1H和31P NMR(JEOL FX90Q NMR分光计)
和元素微量分析来证实的。实施例2:聚[(二(羧基合苯氧基)磷腈]的合成
聚[二(羧基合苯氧基)磷腈]是通过用对羟基苯甲酸丙酯的钠盐对聚(二氯磷腈)进行化学修饰、接着按Allcock,H.R.& Kwon,S.(1989)Macromolecules 22,75-79所述方法将酯基水解为羧酸来制备的,上述文献的内容并入本文。实施例3:聚[(羧基合苯氧基)(氯)磷腈]的合成
聚[(羧基合苯氧基)(氯)磷腈]的制备如下。将聚[二(氯)磷腈](5.0g,0.0425mol)溶于300ml四氢呋喃(THF)中。向该溶解的聚合物中滴加对羟基苯甲酸丙酯的钠盐(通过使羟基苯甲酸丙酯(15.52g,0.0864mol)与60%氢化钠(3.06g,0.0765mol)的THF溶液反应来制备)。加入钠盐后,将反应混合物在回流条件下搅拌2天,并用31P NMR监测。聚合物通过反复沉淀入水、乙醇和己烷中来纯化。
将聚[(羟基苯甲酸丙酯)(氯)磷腈](2.0g)溶于200ml无水THF中。将20g叔丁醇钾溶于200ml THF中。将该碱性溶液冷却至0℃。向丁醇盐/THF溶液中加入水(1ml),并搅拌5分钟。然后将聚合物溶液滴加到碱的水溶液中。将反应混合物温热至室温,并搅拌40小时。40小时后,将反应混合物倾入到冰水混合物中,并蒸发THF。然后将水溶液对水渗析2天。渗析完成后,渗析液用HCl酸化,得到的白色沉淀聚[(羧基苯氧基)(氯)磷腈](含有钾作为抗衡离子)用水洗涤,并从溶液中滤出。元素分析数据:“P”-10.39;“N”-4.52;“C”-47.26;“Cl”-1.44,“K”-2.61。实施例4:磷腈聚电解质的降解
在37℃下、在空气重力保温箱(Imperial II Incubator,Lab-Line Instruments.Inc.)中、在旋转摇动器(ORBIT Shaker,LabLine Instruments,Inc.,Melrose Park,Ill.)的温和搅拌下,于含有50mg聚合物在5ml 13mM HEPES缓冲盐水溶液(pH7.4)中的悬浮液的小瓶中体外研究聚[(羧基苯氧基)(羧甲氨基)磷腈]的降解。聚磷腈的分子量是由Perkin-Elmer Series 10液相色谱仪(利用紫外光)和折光指数检测器采用Ultragel 2000柱(WatersChromatography Division,Millipore Corporation,Milford,MA)来测定。用13mM Hepes缓冲盐水溶液(pH7.4)作洗脱剂。色谱图用GPC 5和CHROM 2软件(Perkin-Elmer)进行处理以计算重均分子量和数均分子量(采用聚丙烯酸作标准物)。聚合物分子量随时间的下降列于表2中。表2.聚[(羧基苯氧基)(羧甲氨基)磷腈]的降解
时间 重均分子量 数均分子量
天 kDa kDa
0 132.0 70.0
15 40.6 13.8
60 6.3 1.5
180 6.0 0.9
240 0.9 0.5
聚[(羧基苯氧基)(氯)磷腈]的降解是在37℃下、在保温箱-摇动器(New Brunswick Scientific,G24)中、在含有0.2%聚合物在磷酸盐缓冲盐水溶液(pH7.4)中的溶液的小瓶中进行体外研究。聚磷腈的分子量是用利用紫外光的Waters色谱仪(Water 486,Millipore Corporation,Milford,MA)和折光指数检测器(Waters410,Millipore Corporation,Milford,MA)采用Ultragel线性柱(Waters Chromatography Division,Millipore Corporation,Milford,MA)进行测定的。用磷酸盐缓冲盐水溶液(pH7.4)作为洗脱剂。色谱图用Millenium 2.0软件进行处理以计算重均分子量和数均分子量(采用聚丙烯酸和聚甲基丙烯酸作标准物(1,250Da-1,100,000Da))。聚合物分子量随时间的降低示于表3。基苯氧基表3.聚[(羧基合苯氧基)(氯)磷腈]的降解
时间 重均分子量 数均分子量
天 kDa kDa
0 120.0 42.0
1 113.0 42.0
6 105.0 41.0
8 100.0 39.0
10 96.0 39.0
14 95.0 39.0
18 86.0 35.0
28 73.0 31.0
35 67.0 30.0
59 59.0 28.0
91 51.0 23.0
实施例5:用混有聚磷腈佐剂的破伤风类毒素免疫后的抗体效价
抗体效价在用混有各种浓度的聚磷腈佐剂的破伤风类毒素接种后
的、7-8周龄的雌性BALB/c小鼠中测定。
含有在聚磷腈中的破伤风类毒素的致免疫组合物的制备如下。将
100mg聚[二(羧基合苯氧基)磷腈]溶于1ml Na2 CO3中,并加入
1ml磷酸盐缓冲盐水(PBS)(pH7.2)。将1.4ml破伤风类毒素
(2.2mg/ml或1000LF/ml,Connaught Laboratories,Inc.,
Swiftwater,PA)与0.6ml含有0.025%Brij的溶液(10μl 10%
Brij 58,Sigma Chemical Co.,St.Louis,MO)一起加到聚合物中。
用掺有含有0.5%聚磷腈、0.05%聚磷腈或0.005%聚磷腈的dH2O稀释液
的单次剂量的25μg破伤风类毒素对5只一组的小鼠进行皮下免疫。
另一组小鼠用单次皮下给药剂量的在弗氏完全佐剂(SIGMA,St.Louis,
MO)中的25μg破伤风类毒素进行免疫。从用CO2麻醉后的小鼠的眶后窦取血样,用ELISA免疫测定法分析抗破伤风类毒素IgG。
正如表4所示,抗原聚合物溶液以剂量依赖性方式产生抗破伤风类毒素ELISA抗体。0.5%PCPP使对破伤风类毒素的免疫应答比在水中对破伤风类毒素的免疫应答提高了100多倍。浓度为0.05%及0.005%的PCPP也比在水中的破伤风类毒素产生较高的抗体效价,尽管没有用0.5%PCPP所得到的高。此外,0.5%PCPP浓度作为佐剂与弗氏完全佐剂一样浓。
另一种含有各种剂量的在聚磷腈中的破伤风类毒素的致免疫组合物的制备如下。将100mg聚[二(羧基合苯氧基)磷腈]溶于1mlNa2CO3中,并加入1ml磷酸盐缓冲盐水(PBS)(pH7.6)。然后,将破伤风类毒素(2.2mg/ml)(Connaught Laboratories,Swiftwater,PA)以1∶10的比例在水中稀释,并将适当的体积与0.1%聚磷腈混合。
将3只一组的小鼠用单次剂量的每一致免疫组合物进行皮下免疫。接种21天后,从用CO2麻醉后的小鼠的眶后窦取出血样,并用ELISA免疫测定法分析抗破伤风类毒素IgG(表5)。正如所期望的那样,用可溶性PCPP在所有时间点均有明确的抗原剂量依赖性应答。配制到0.1%PCPP中的25μg破伤风类毒素剂量所引起的ELISA效价大大高于相同量的抗厚在水中所引起的ELISA效价,并且高于25μg破伤风类毒素在弗氏完全佐剂中所引起的ELISA效价。应该注意的是,在PCPP中的5、1及0.2μg抗原剂量水平在第25周时ELISA效价仍在升高,而用弗氏完全佐剂的制剂的ELISA效价却早就达到峰值。实施例6:用混有聚磷腈佐剂的流感病毒免疫后的抗体效价
将含有流感病毒(流感)(Influenza Branch,Center for DiseaseControl,Atlanta,GA)和0.1%PCPP的致免疫组合物配制成制剂。流感是在蛋中按照标准方法生长的,并用蛋白质、血细胞凝集作用和蚀斑测定法定量。流感用加入最终稀释度为1∶4000的38%甲醛溶液进行福尔马林灭活。
采用下列流感免疫测定法测定流感效价:将一个96孔ELISA微滴度板用10μg/ml在碳酸盐缓冲液(pH9.6)中的流感细胞溶解产物涂敷,每孔100ml,并于37℃下保温2小时。该板用0.05%吐温20/PBS(Sigma,St.Louis,MO)洗涤,并向每孔中加入100μl2.5%牛血清白蛋白/磷酸盐缓冲盐水(BSA/PBS)作为阻断手段。然后将板于37℃保温1小时,用0.05%吐温20/PBS洗涤。向所有孔中加入50μl 1%BSA/PBS。通过将5μl血清加入到635μl 1%BSA/PBS中而将血清样品稀释成1∶128。将50μl待测的稀释后的血清样品加入一排孔的第一个孔中(1∶256稀释度)。试验阳性和阴性对照组。两倍连续稀释的血清样品按下述方法制备:从一排孔的第一个孔中取出50μl、并在搅拌下将该50μl加到第二孔中;然后从第二个孔中取出50μl并将其在搅拌下加入到第三个孔中,并对该排其余孔类似操作,从最后的孔或第12个孔中弃去50μl。然后,将该板在37℃保温1小时,并用0.05%吐温20/PBS洗涤。向每孔中加入100μl OPD溶液(邻苯二胺二盐酸盐(Sigma,St.Lonis,MO)在含有0.05%过氧化氢(20.8μl 30%H2O2/12.5ml柠檬酸盐缓冲液)的0.05M磷酸盐-柠檬酸盐缓冲液(pH5.0)中的0.4mg/ml溶液(1片OPD/12.5ml柠檬酸盐缓冲液)。显色30分钟,然后加入50μl 2M H2SO4/孔来停止。在OD490处读出吸光度,终点效价通过查出每一血清样品的稀释度来测定,所述每一血清样品的OD490大于或等于相同稀释度的阴性对照组的OD490数值的两倍。
流感血细胞凝集作用抑制抗体测定是用加热失活的小鼠血清进行的,该小鼠血清已与10%小鸡红血细胞(Spafas)保温30分钟以除去非特异性抑制剂。向一96孔微滴度板中加入2倍稀释的血清,向每孔中加入相同体积的8HA单位的病毒悬浮液,并在室温下保温30分钟。向每一孔中加入0.5%小鸡红血细胞悬浮液,并在室温下保温45-60分钟。HAI效价表示为完全抑制红细胞的血细胞凝集作用的最高稀释度的倒数。
流感抗原特异性中和抗体的诱导是通过蚀斑减少测定的。该测定法测量导致细胞培养物中流感感染性降低50%的抗体的量。将血清样品在56℃加热灭活30分钟。血清的两倍稀释是在DMEM(JRHBiochemicals)中从1∶50开始进行的,并将0.5ml每个稀释液加入到0.5ml效价为400pfu/ml的流感病毒中。在37℃保温1小时后,使250μl各样品吸收MDCK细胞的融合单分子层1小时。抽吸掉吸附混合物,并将细胞涂上含有10μg/ml胰蛋白酶的MEM/0.6%琼脂糖混合物。3天后观察到蚀斑。去除琼脂糖管塞,单分子层用结晶紫(J.J.Baker)染色。计数蚀斑,50%效价降低通过与不含血清的对照感染中蚀斑数目的50%相比较来测定。
将小鼠用在0.1%PCPP中配制的各种流感剂量或5μg流感在水或弗氏完全佐剂中进行皮下接种。在如所期望的那样,在接种用0.1%PCPP配制的流感抗原后,在所有时间点均存在剂量依赖性ELISA免疫应答(表6)。在该试验中,在0.1%聚磷腈中的5μg流感诱发的抗流感应答远高于在弗氏完全佐剂中的5μg流感诱发的抗流感应答。此外,在PCPP制剂中的所有抗原剂量引起的免疫应答在第25周时仍然在上升,而弗氏完全佐剂制剂在较早的时间点时就诱导峰值效价。这与在破伤风类毒素试验中观察到的结果相似。特别值得注意的是,在PCPP中的0.04μg剂量直至第25周才诱发可检测的抗体水平。这可以认为是抗原缓释的证据。
0.1%PCPP溶液诱导官能性抗体的能力是在血细胞凝集抑制(表7)和中和(表8)测定法中测定的。同样,PCPP制剂在血细胞凝集测定法和中和测定法中诱导非常高的抗体活性,而在这些测定法中,弗氏完全佐剂制剂几乎没有或没有可检测的活性。
流感疫苗施用于人体时不加明矾,因为该佐剂对免疫应答具有非常小的正面作用。在小鼠潜能试验中,诱发大于40个单位的HAI抗体效价的抗原剂量是对人体有保护作用的预示。因此,在0.1%PCPP中的0.04μg全部流感抗原能够诱发保护水平的抗体,这是用5μg不加佐剂的抗原无法达到的。
还测定了在此应答中产生的抗体同型(表9)。尽管用PCPP配制的流感抗原主要诱发IgG1应答,但是也检测到了显著的IgG2a和IgG2b应答。该应答的水平大于对用弗氏完全佐剂配制的流感抗原所观察到的应答的水平。在该试验中没有检测到IgG3抗体。实施例7:用混有聚磷腈佐剂的H.流感b型多糖抗原免疫后的抗体效价
前面所讨论的所有抗原均为蛋白质抗原。下面我们将研究衍生自流感嗜血杆菌B型(Hib)的PRP多糖的致免疫性。多糖抗原一般不给出记忆性IgG应答,除非它们与蛋白质抗原轭合。轭合到破伤风类毒素上的Hib用明矾或0.1%PCPP进行配制(表10)。将小鼠皮下注射在明矾或PCPP制剂中的2μg PRP。抗体应答是通过测定在每一时间点的特异性抗PRP IgG水平来追踪的。加入明矾佐剂的免疫原在第4周时间点处引发可检测的抗体水平,在第8周时引发峰值效价。随后在到20周的时间内,抗体效价迅速降低。用PCPP佐剂的Hib抗原在第4周时间点比用明矾佐剂时所观察到的抗体水平高6倍。用PCPP佐剂的抗原也在第8周引发峰值效价,但是这些效价比用明矾佐剂时引发的效价大约高10倍。尽管用PCPP佐剂的抗体效价随周数增加而降低,但是它们在第20周时的数值仍比用明矾佐剂在峰值第8周时间点时所达到的数值高2倍。实施例8:用混合有聚电解质的破伤风类毒素抗原免疫后的抗体效价
我们比较了PCPP的效果与用两种其他聚电解质即聚甲基丙烯酸(PMA)和聚丙烯酸(PAA)所达到的效果。将小鼠注射1μg配制于0.1%聚合物溶液中的TT。在第3、6和9周测定抗TT ELISA效价。从表11中所汇编的数据可清楚地看出,0.1%PCPP所刺激的抗体应答比用任何分子量的PMA和PAA所观察到的抗体应答都高。应该注意到的是,抗体应答有某种随着PMA和PAA聚合物的分子量增加而上升的趋势。然而,分子量为1.3×105道尔顿的0.1%PMA不能刺激等同于0.1%PCPP所刺激的免疫应答。实施例9:用混有各种分子量的聚磷腈的流感抗原免疫后的抗体效价
在上面实施例中所描述的试验中所用的PCPP溶液多分散成含有2,000至大约10,000,000道尔顿的分子。因此,检验PCPP分子量对佐剂性质的影响是有意义的。将PCPP通过顺序酸沉淀法和FPLC柱色谱法进行分级,以便获得具有较窄多分散性(1.37-2.01)和峰值平均分子量为3,000-1.8×106的PCPP级分。对这些级分的HPLC分析示于表12中。将0.1%浓度的每一上述级分与5μg福尔马林灭活的流感病毒混合并皮下注射到小鼠中。还制备了相同体积的每一上述制剂的掺合物,使得最终PCPP浓度为0.1%,并且灭活的流感病毒的用量为5μg。对小鼠注射该PCPP重组制剂。作为对照,对另一组小鼠注射配制于0.1%未分级的PCPP中的流感。表13中所示数据证明,在增加的PCPP分子量与增加的对流感抗原的抗体应答之间存在正性相互关系。由分子量为1.8×106的0.1%PCPP所引发的应答与重组级分的PCPP制剂和未分级的PCPP所引发的应答基本上是不可区分的。实施例10:用混有各种浓度的三种不同的磷腈聚合物佐剂的破伤风类毒素或流感免疫后的抗体效价
将100mg聚[二(羧基苯氧基)磷腈](聚合物1)、聚[(羧基苯氧基)(羧甲氨基)磷腈](聚合物2)或聚[(羧基苯氧基)(氯)磷腈](聚合物3)溶于1ml Na2CO3中并将3ml PBS加入到聚合物溶液中。
抗体效价是在几组雌性BALB/c小鼠中测定的,每组3只小鼠,7-8周龄,皮下注射过混有聚合物1或聚合物2的5μg流感。如表14所示,在聚合物1或聚合物2中的流感与在弗氏完全佐剂中的相同剂量的抗原所引发的血清IgG效价一样高或更高。这些血清IgG效价在免疫后21周得到保持。
抗体效价是在几组雌性BALB/c小鼠中测定的,每组3只小鼠,7-8周龄,皮下注射过混有聚合物1或聚合物3的1μg破伤风类毒素。如表15所示,在聚合物1或聚合物3中的破伤风类毒素诱发的血清IgG效价在免疫后25周得到保持。表4施用混有各种浓度的聚磷腈佐剂或弗氏佐剂的破伤风类毒素后的ELISA抗体效价
在以下各周的抗 TT ELISA 效价
3 5 7 9TT/水 1024 2048 2048 4096TT/0.5%PCPP 65536 262144 524288 524288TT/0.05%PCPP 16384 32768 32768 65536TT/0.005%PCPP 4096 8192 32768 32768TT/CFA 16384 131072 262144 262144小鼠用25μg破伤风类毒素(TT)免疫。表5施用混有聚磷腈佐剂或弗氏佐剂的各种剂量的破伤风类毒素后的ELISA抗体效价
在下列各周的抗 TT ELISA 效价
3 6 9 17 2525μgTT/0.1%PCPP 16384 65536 131072 >524288 2621445μgTT/0.1%PCPP 4096 16384 32768 65536 1310721μgTT/0.1%PCPP 2048 16384 16384 32768 655360.2μgTT/0.1%PCPP 512 1024 1024 2048 409625μgTT/水 2048 2048 8192 8192 1638425μgTT/cFA 16384 131072 262144 131072 131072表6施用混有聚磷腈佐剂或弗氏佐剂的流感后的ELISA抗体效价
在下列各周的抗-TT ELISA效价
3 6 9 17 25 375μg flu/0.1%PCPP 2048 16384 16384 32768 65536 163841μg flu/0.1%PcPP 4096 16384 16384 21768 131072 163840.2μg flu/0.1%PCPP <256 4096 4096 16384 65536 81920.05μg flu/0.1%PCPP <256 <256 <256 <256 4096 10245μg flu/水 256 256 256 <256 <256 <2565μg flu/CFA 512 4096 4096 2048 1024 2048表7施用混有聚磷腈佐剂或弗氏佐剂的流感后的血细胞凝集作用抑制抗体效价
在下列各周的HAI效价
3 6 9 17 25 375μg flu/0.1%PCPP 160 1280 1280 2560 640 12801μg flu/0.1%PCPP 320 1280 1280 2560 2560 12800.2μg flu/0.1%PCPP 40 640 640 2560 1280 12800.05μg flu/0.1%PCPP 阴性 40 80 160 160 1605μg flu/水 阴性 阴性 20 阴性 阴性 阴性5μg flu/CFA 阴性 80 40 40 40 40表8施用混有聚磷腈佐剂或弗氏佐剂的流感后的蚀斑中和抗体效价
在下列各周的HAI效价
3 6 9 17 25 375μg flu/0.1%PPP 200 400 400 200 400 16001μg flu/0.1%PPP 400 - 200 400 400 4000.2μg flu/0.1%PPP <100 - 100 100 400 4000.05μg flu/0.1%PPP <100 - <100 <100 - <1005μg flu/水 <100 <100 <100 <100 - <1005μg flu/CFA <100 <100 <100 <100 - <100表9施用混有聚磷腈佐剂或弗氏佐剂的流感后的流感特异性抗体同型
在下列各周的抗体同型
3 6 9
IgG1 IgG2A IgG2B IgG1 IgG2A IgG2B IgG1 IgG2A IgG2A5μg flu/0.1%PCPP 8192 <256 <256 131072 <256 256 131072 256 10241μg flu/0.1%PCPP 8192 <256 <256 65536 256 1024 65536 1024 40960.2μg flu/0.1%PCPP 256 <256 <256 16384 1024 2048 16384 1024 10245μg flu/水 <256 1024 <256 256 1024 <256 256 512 <2565μg flu/CFA 2048 <256 <256 16384 1024 512 16384 1024 256所有样品的IgG3抗体效价均小于256。表10施用混有聚磷腈佐剂或明矾佐剂的H.流感b型多糖后的ELISA抗体效价
抗PRP IgG(μg/ml)
明矾 聚磷腈wk4 wk8 wk12 wk16 wk20 wk4 wk8 wk12 wk16wk205.48 10.00 5.97 5.45 3.84 32.16 109.31 50.80 35.5118.60
*10只小鼠的几何平均效价
免疫剂量=2μg PRP/小鼠
wk=周表11施用混有聚电解质的破伤风类毒素后的ELISA抗体效价
在下列各周的抗flu ELISA效价
3 6 9TT/PBS 512 512 256TT/CFA 4096 4096 4096TT/0.1%PCPP 16384 16384 65536TT/0.1%PMA MW 7,000 1024 1024 1024TT/0.1%PMA MW 25,000 512 512 512TT/0.1%PMA MW 70,000 512 1024 1024TT/0.1%PMA MW 110,000 512 1024 1024TT/0.1%PMA MW 350,000 1024 1024 512TT/0.1%PAA MW 2,000 1024 512 512TT/0.1%PAA MW 35,000 512 512 1024TT/0.1%PAA MW 500,000 4096 4096 4096TT/0.1%PMA MW 1,300,000 4096 4096 4096表12在HPLC上测定的分级的聚磷腈的分子量和多分散性
聚磷腈分级-HPLC
峰值平均分子量 多分散性
3,000 1.62
25,500 1.37
72,000 1.54
331,000 1.64
464,000 1.57
634,000 1.56
1,846,000 2.01表13施用混有各种分子量的聚磷腈的流感后的ELISA抗体效价PBS <64 PCPP MW464 <64
128 512
<64 256
<64 256
<64 512PCPP MW 3,000 <64 PCPP MW 634,6000 512
<64 512
<64 1024
<64 1024
<64 1024PCPP MW 25,500 256 PCPP MW 1024
1,846,000
<64 1024
<64 2048
128 8192
<64 4096PCPP MW 72,000<64 重组的PCPP级分 51
<64 1024
<64 2048
<64 2048
256 128PCPP MW 331,000256 未分级的PCPP 8192
<64 2048
128 8182
2048 2048
256 2048小鼠用每剂量5μg福尔马林灭活的流感病毒免疫。所有PCPP溶液均以0.1%浓度施用。表14施用混有聚[二(羧基苯氧基)磷腈](聚合物1)、聚[(羧基苯氧基)(羧甲氨基)磷腈](聚合物2)或弗氏佐剂的流感后的ELISA抗体效价
在下列各周的抗flu ELISA效价
3 5 7 9 13 17 21Flu/0.1%聚合物 14096 8192 65535 32768 8192 32768 32768Flu/0.1%聚合物 21024 1024 4096 2048 1024 4096 4096Flu/弗氏完全佐剂 256 1024 16384 1024 512 4096 4096小鼠用5μg完全福尔马林灭活的流感病毒粒子皮下免疫。表15施用混有聚[二(羧基苯氧基)磷腈](聚合物1)或聚[(羧基苯氧基)(氯)磷腈](聚合物3)的破伤风类毒素后的ELISA抗体效价
在下列各周的抗flu ELISA效价
3 6 9 13 17 21 25TT/0.1%聚合物 14096 2048 4096 4096 8192 16384 16384TT/0.1%聚合物 32048 512 2048 1024 1024 2048 2048小鼠皮下免疫1μgTT。
从上述详细描述可见,本发明的改良和变体、聚合物佐剂和合成方法及在疫苗组合物中的应用对本领域技术人员来说是显而易见的。这些改良和变体应在所附权利要求书的范围内。
Claims (24)
1.一种组合物,该组合物包含一种合成的水溶性磷腈聚电解质,该电解质由一种聚[二(羧基合苯氧基)磷腈-共-二(氨基酸)磷腈-共-(羧基合苯氧基)(氨基酸)磷腈]组成。
2.权利要求1所述的组合物,其中磷腈聚电解质用多价阳离子交联。
3.权利要求2所述的组合物,其中多价阳离子选自钙、铜、铝、镁、锶、钡、锡、锌、铁、聚(氨基酸)、聚(乙烯亚胺)、聚(乙烯胺)、和多糖类。
4.权利要求1所述的组合物,其中磷腈聚电解质是可生物降解的。
5.聚[二(羧基合苯氧基)磷腈-共-二(羧甲氨基合)磷腈-共-(羧基合苯氧基)(羧甲氨基)磷腈]。
6.聚[二(羧基合苯氧基)磷腈-共-二(氯)磷腈-共-(羧基合苯氧基)(氯)磷腈]。
7.一种权利要求1-4之任一的组合物或权利要求5-6之任一的水溶性聚磷腈聚电解质在制备一种用于与一种抗原结合而在动物体内引起免疫应答佐剂中的应用。
8.权利要求7所述的应用,其中抗原与聚磷腈聚电解质轭合。
9.权利要求7所述的应用,其中抗原和聚磷腈分别施用到邻近的部位。
10.权利要求7所述的应用,其中首先使抗原与聚磷腈结合,并将结合物施用于动物。
11.权利要求7所述的应用,其中施用方法是胃肠外法。
12.权利要求7所述的应用,其中施用方法是口服法。
13.权利要求7的应用,其中聚磷腈电解质具有下式结构:
其中A和B可以在聚合物中独立地变化,并且可以为:
(i)在使用条件下易于水解的基团;或
(ii)在使用条件下不易水解的基团,选自下列基团:脂族基、芳基、芳烷基、烷芳基、羧酸、杂芳族基、杂烷基、(脂族基)氨基-、杂芳烷基、二(脂族基)氨基芳基氨基-、二芳基氨基-、烷基芳基氨基-、-氧基芳基、-氧基苯基CO2H、-氧基苯基SO3H、-氧基苯基羟基、-氧基苯基PO3H、-氧基脂族基、-氧基烷基、-氧基(脂族基)CO2H、-氧基(脂族基)SO3H、-氧基(脂族基)PO3H、-氧基(脂族基)羟基、-氧基烷芳基、-氧基芳烷基、-硫基芳基、-硫基脂族基、-硫基烷芳基、-硫基芳烷基、-NHC(O)O-(芳基或脂族基)、-O[(CH2)xO]y-CH2)-O-[(CH2)xO]y(CH2)xNH(CH2)xSO3H或-O-[(CH2)xO]y-(芳基或脂族基),其中x为1-8,y为1-20的整数;并且其中n为4或4以上的整数。
14.权利要求13所述的应用,其中聚合物含有至少10%或10%以上在使用条件下不易水解的重复单元。
15.权利要求13所述的应用,其中聚合物含有至少90%或90%以上在使用条件下不易水解的重复单元。
16.权利要求7所述的应用,其中聚磷腈聚电解质是可生物降解的。
17.权利要求16所述的应用,其中聚磷腈聚电解质含有选自氨基酸、氨基酸酯、氯、咪唑、甘油和葡糖基的可水解侧链。
18.权利要求7所述的应用,其中聚磷腈聚电解质被多价阳离子交联。
19.权利要求7所述的应用,其中聚磷腈聚电解质与抗原物理混合。
20.权利要求7所述的应用,其中抗原选自衍生自细胞、细菌或病毒粒子或其部分的化合物,其中所述化合物选自蛋白质、肽、多糖、糖蛋白、糖脂、核酸或其混合物。
21.权利要求20所述的应用,其中抗原选自流感蛋白、人免疫缺陷病毒(HIV)蛋白、B型肝炎蛋白、细菌蛋白和细菌脂多糖。
22.权利要求7所述的应用,其中施用方法是与鼻有关的淋巴(样)组织给药。
23.权利要求7所述的应用,其中施用方法是呼吸道给药。
24.权利要求7所述的应用,其中施用方法是直肠给药。
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US08/090,841 US5562909A (en) | 1993-07-12 | 1993-07-12 | Phosphazene polyelectrolytes as immunoadjuvants |
US08/090,841 | 1993-07-12 |
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CN1128954A CN1128954A (zh) | 1996-08-14 |
CN1134265C true CN1134265C (zh) | 2004-01-14 |
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US (3) | US5562909A (zh) |
EP (1) | EP0710117B1 (zh) |
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CN (1) | CN1134265C (zh) |
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WO2020145489A1 (ko) * | 2019-01-10 | 2020-07-16 | 서울대학교산학협력단 | 하이드로겔에 의해 캡슐화된 불활성화 균체를 포함하는 경구 투여용 백신 조성물 |
IT201900007060A1 (it) | 2019-05-21 | 2020-11-21 | St Superiore Di Sanita | Cellule tumorali ingegnerizzate e loro usi |
US20200383932A1 (en) * | 2019-06-05 | 2020-12-10 | The Florida International University Board Of Trustees | Biotherapy for viral infections using biopolymer based micro/nanogels |
IT201900012540A1 (it) | 2019-07-22 | 2021-01-22 | Humanitas Mirasole Spa | Inibitori di CHI3L1 e loro usi |
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US3893980A (en) * | 1974-03-29 | 1975-07-08 | Pennsylvania Res Corp | Poly(amino acid alkyl ester phosphazenes) |
FR2321896A1 (fr) * | 1975-08-29 | 1977-03-25 | Anvar | Agents adjuvants immunologiques actifs en solution aqueuse |
US4055520A (en) * | 1976-07-14 | 1977-10-25 | Armstrong Cork Company | Polyphosphazene blends |
US4026839A (en) * | 1976-08-30 | 1977-05-31 | Armstrong Cork Company | Polyphosphazene polymer/silicone rubber blends and foams therefrom |
US4209014A (en) * | 1977-12-12 | 1980-06-24 | Canadian Patents And Development Limited | Dispensing device for medicaments |
US4353888A (en) * | 1980-12-23 | 1982-10-12 | Sefton Michael V | Encapsulation of live animal cells |
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US4440921A (en) * | 1982-06-21 | 1984-04-03 | Research Corporation | Coupling of polyorganophosphazenes to carboxylic acid |
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GB8826116D0 (en) * | 1988-11-08 | 1988-12-14 | Danbiosyst Ltd | Adhesive drug delivery composition |
US4975280A (en) * | 1989-01-23 | 1990-12-04 | Ethyl Corporation | Bioerodable sustained release implants |
US4946938A (en) * | 1989-08-01 | 1990-08-07 | The University Of Pittsburgh | A process for the catalytic synthesis of polyphosphazenes |
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US5053451A (en) * | 1990-01-19 | 1991-10-01 | The Pennsylvania Research Corporation | Ionically cross-linkable polyphosphazene: poly(bis(carboxylatophenoxy) phosphazene) and its hydrogels and membranes |
US5126147A (en) * | 1990-02-08 | 1992-06-30 | Biosearch, Inc. | Sustained release dosage form |
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-
1993
- 1993-07-12 US US08/090,841 patent/US5562909A/en not_active Expired - Fee Related
- 1993-11-04 US US08/147,781 patent/US5529777A/en not_active Expired - Lifetime
-
1994
- 1994-07-08 WO PCT/US1994/007665 patent/WO1995002415A1/en active IP Right Grant
- 1994-07-08 DK DK94923378T patent/DK0710117T3/da active
- 1994-07-08 BR BR9407051A patent/BR9407051A/pt not_active Application Discontinuation
- 1994-07-08 ES ES94923378T patent/ES2166376T3/es not_active Expired - Lifetime
- 1994-07-08 CN CNB94193067XA patent/CN1134265C/zh not_active Expired - Fee Related
- 1994-07-08 AT AT94923378T patent/ATE207505T1/de active
- 1994-07-08 SG SG1996007424A patent/SG46659A1/en unknown
- 1994-07-08 DE DE69428795T patent/DE69428795T2/de not_active Expired - Lifetime
- 1994-07-08 PT PT94923378T patent/PT710117E/pt unknown
- 1994-07-08 AU AU73261/94A patent/AU691824B2/en not_active Ceased
- 1994-07-08 JP JP7504624A patent/JPH09500629A/ja active Pending
- 1994-07-08 KR KR1019960700227A patent/KR100352336B1/ko not_active IP Right Cessation
- 1994-07-08 EP EP94923378A patent/EP0710117B1/en not_active Expired - Lifetime
- 1994-07-08 NZ NZ269396A patent/NZ269396A/en not_active IP Right Cessation
- 1994-07-08 CA CA2166208A patent/CA2166208C/en not_active Expired - Fee Related
- 1994-07-11 US US08/273,285 patent/US5494673A/en not_active Expired - Lifetime
- 1994-07-11 KR KR1019960700228A patent/KR100353496B1/ko not_active IP Right Cessation
- 1994-07-12 WO PCT/US1994/007710 patent/WO1995002628A1/en active Application Filing
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Publication number | Publication date |
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NZ269396A (en) | 1999-09-29 |
EP0710117B1 (en) | 2001-10-24 |
KR960703620A (ko) | 1996-08-31 |
SG46659A1 (en) | 1998-02-20 |
DE69428795D1 (de) | 2001-11-29 |
KR100353496B1 (ko) | 2003-03-03 |
US5562909A (en) | 1996-10-08 |
WO1995002415A1 (en) | 1995-01-26 |
ATE207505T1 (de) | 2001-11-15 |
AU7326194A (en) | 1995-02-13 |
CN1128954A (zh) | 1996-08-14 |
CA2166208C (en) | 2010-04-20 |
AU691824B2 (en) | 1998-05-28 |
ES2166376T3 (es) | 2002-04-16 |
WO1995002628A1 (en) | 1995-01-26 |
EP0710117A1 (en) | 1996-05-08 |
DK0710117T3 (da) | 2002-02-18 |
US5529777A (en) | 1996-06-25 |
KR960703617A (ko) | 1996-08-31 |
PT710117E (pt) | 2002-04-29 |
US5494673A (en) | 1996-02-27 |
EP0710117A4 (en) | 1996-08-07 |
JPH09500629A (ja) | 1997-01-21 |
CA2166208A1 (en) | 1995-01-26 |
KR100352336B1 (ko) | 2003-04-16 |
BR9407051A (pt) | 1996-08-13 |
DE69428795T2 (de) | 2002-06-13 |
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