CN101528197A - 药物应用的干燥重建囊泡制剂 - Google Patents
药物应用的干燥重建囊泡制剂 Download PDFInfo
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- CN101528197A CN101528197A CNA2007800397245A CN200780039724A CN101528197A CN 101528197 A CN101528197 A CN 101528197A CN A2007800397245 A CNA2007800397245 A CN A2007800397245A CN 200780039724 A CN200780039724 A CN 200780039724A CN 101528197 A CN101528197 A CN 101528197A
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- liposome
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- protein
- rehydration
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Abstract
本发明涉及干燥重建囊泡(DRV)组合物及其水基制剂,其包含一种或多种治疗剂(例如亲水性蛋白质)。更具体地,其涉及包括至少一种脂质和融合促进剂的DRVs,其在重建后在水相中形成包封活性剂的大多层脂质体。
Description
技术领域
1本发明涉及干燥重建囊泡(DRV)组合物及其水基制剂,其包含一种或多种治疗剂(例如亲水性蛋白质)。更具体地,其涉及包括至少一种脂质和融合促进剂(fusion promoting agent)的DRVs,其在重建后在水相中形成包封活性剂的大多层脂质体。
背景技术
2已知脂质体用作生物和治疗活性化合物的载体,其促进这些化合物递送至身体。脂质体通常包括封闭的脂滴,所述脂滴具有在水性介质中典型地包含化合物的核。在特定的实施方式中,化合物化学地结合至脂质组分或仅包含在脂质体的水性内室(aqueous insidecompartment)之内。存在不同类型的脂质体:多囊泡脂质体(MVLs),其在各脂质体颗粒内具有多个非同心水性内室(internal aqueouschamber);多层囊泡(MLVs),其具有一系列基本上球形的壳,直径达5μm或以上,所述壳由被水层所间散的脂质双层形成;大多层囊泡(LUVs),直径从600nm至1μm或以上,其具有围绕大的、非结构化的水相的脂质双层;和小单层囊泡(SUVs),其在结构上类似于LUVs,除了其直径小于约0.2μm之外。
3脂质体的各种制备方法在本领域是已知的,其中几种描述于Liposome Technology 2nd Edition in G.Gregoriadis,CRC Press Inc.,Boca Raton(1993)中。脂质体技术的主要挑战为将活性剂高水平的装入脂质体中,以在处理和贮存期间使该装入稳定。另一挑战在于使活性剂的释放速率适于脂质体制剂的具体目标。虽然在脂质体中生物材料的封装对于人类中的药物递送具有显著的潜力,但商业规模上封装材料的生产通常是成问题的。
4大多数肠胃外应用的药物应用集中在小脂质体以避免不期望的副作用,例如如对于大脂质体而言的栓塞。此外,使用小脂质体,看起来更容易制造稳定的产品。
5对于小规模或工业规模上制造MLV包封的材料存在几种已知的方法(Rao,″Preparation of Liposomes on the Industrial Scale.Problems And Perspectives,″in Liposome Technology 2nd Editionin G.Gregoriadis,CRC Press Inc.,Boca Raton,pp 49-65(1993))。在大多数情况下,薄的脂质膜从有机溶剂沉积到容器的壁上,添加要包封材料的水性溶液,并搅拌容器。该方法引起活性剂包封入MLVs。除了生物试剂的降解和脂质体悬浮的贮存不稳定性之外,此类方法的主要缺点是包封的变化,以及通常低的和不可再生的将生物制剂包埋入脂质体。
6如EP0678017中描述的方法生产冷冻和解冻的多层囊泡(FATMLVs)。FATMLV方法需要在要包埋的材料的存在下完成冷冻和解冻。然而,使敏感材料例如蛋白质进行此类苛刻的物理操作导致材料的失活或降解。此外,频繁的冷冻和解冻循环对大规模生产是不可行的,需要高的技术运行费用。
7已知脂质体及其内含物在水性分散体中可能相对不稳定。因此,通过脱水增加脂质体制剂的短的贮存时间的尝试已经成为几种制备方法的焦点。
8改进的被动包埋和包括活性剂的脂质体的贮存已经通过使用脱水-复水方法(EP0485143、WO90/03795、EP0678017及其参考文献)实现,所述方法中将预成型的脂质体加至包含活性剂的水溶液,或与冻干的蛋白质混合,然后将混合物脱水并且随后在水性介质中复水。当将该溶液干燥成高粘度脂质混合物时,单独的脂质体融合形成MLVs,其在壳层(lamellae)之间包封活性剂。复水时,形成其中包封材料的脂质囊泡。由于脂质体的不稳定性、活性剂的泄露或要包封的材料的物理失活或降解,该方法导致相当低的依赖于要包封的药物的包封率。
9已知脱水和形成干燥脂质粉末之前添加糖类防腐剂(例如填充剂)可以保护涉及冷冻干燥的脂质体。填充剂描述于EP0678017、WO90/03795、WO97/42936、WO92/02208、EP190315和Liposomes第二版,A Practical Approach,由Vladimir P.Torchilin和VolkmarWeissig编辑,Oxford University Press(2002)。它们用来防止冷冻干燥期间囊泡的破坏和活性剂的泄露,以及避免小单层囊泡融合成大多层结构。
10WO97/42936描述了冷冻干燥MLVs的制备方法,除了作为膜稳定剂的山梨醇外,所述MLVs包封两性药物组合物。
11WO90/03795描述了在脂质体制备的干燥期间使用防冻剂例如糖类(例如蔗糖、甘露醇、乳糖、海藻糖、麦芽糖)和至少一种蛋白质(例如白蛋白、明胶或酪蛋白)以防止脱水产物在冷冻干燥和随后的重建中受损,以维持脂质体双层的完整性(例如观察到几乎没有或没有融合或聚集)、以及以避免脂质体的泄露。不添加任何防冻剂,干燥和复水后脂质体完全塌陷并形成这样的MLVs,其内含物极大损失,并且其大尺寸防止对于全身应用的合适分配。
12等描述了使用防冻剂例如多元醇和多糖和蛋白质或氨基酸以保护膜双层的结构和完整性,并且以通过脱水和冷冻保护囊泡融合和聚集(Y.等.(1988)Influence of Freezing andFreeze-drying on the Stability of Liposomes Dispersed in AqeousMedia.Acta Pharm.Technol.34:129-139)。
13EP0560138描述了用于包裹亲脂物质例如硝苯地平(Nifedipin)的干燥重建脂质体,以及用于制备包含以下的脂质体的方法:磷脂、抗氧化剂、防冻剂和pH稳定剂。然而,公开的方法对于活性剂例如蛋白质是有害的。防冻剂例如还原糖(例如葡萄糖)通过化学反应改性蛋白质并且导致例如平均直径40至200nm的小囊泡的形成。
14美国5,290,563公开了将外源物质例如蛋白过敏原(protidic allergen)和/或过敏原提取物包封入包括至少一种离子脂质的脂质体而不添加冷冻保护剂的脂质体。此类外源物质的存在稳定脂质体。
15Kim等教导通过从悬浮于水中的氯仿-醚内孢囊(spherules)中蒸发有机溶剂来制备脂质体(Kim,S.等,(1983)Preparation ofmultivesicular liposomes.Biochim.Biophys.Acta 728:339-348)。
Cruz等及其参考文献教导作为用于蛋白质的载体系统的脂质体。该公开的方法与上述例如使用有机溶剂的方法具有类似的缺点,导致低的包封率或不能用于大规模制造(Cruz,M.E.等.(1989)Liposomes as carrier systems for proteins:factors affectingprotein encapsulation.Liposomes in the Therapy of InfectiousDiseases and Cancer 417-426)。
16WO2007/067784通常涉及脂质体药物组合物,其包含一种或多种疏水性治疗剂(例如药物)。然而,WO2007/067784未解决包封蛋白质的脂质体的问题。实际上,其教导使用防冻剂,其稳定脂质膜和/或防止复水后MLVs的形成。
17因此,本发明的目的在于提供干燥形式的包封蛋白质的脂质体的制备方法,所述脂质体能够复水并且用于大规模制造。
18本发明的另一目的在于提供脂质体制剂,其能够复水,脱水时贮存较长时间,并且重建后转变成多层囊泡的分散体,所述多层囊泡具有包封在脂质体的水相中的活性剂。
19本发明的另一目的在于消除用于制造包封活性剂的MLVs的几步冷冻和解冻的需要,以防止活性剂的破坏或失活。
20本发明的另一目的在于提供在活性剂的存在下使脂质体脱水和作为干燥脂质制剂贮存的方法,随后所述干燥脂质制剂通过添加水溶液可以复水以形成具有高的活性剂包封率的多层脂质体,由此干燥产品的方便重建是可行的。
21本发明的另一目的在于提供干燥重建MLVs的大规模生产方法,其简单、可行并且廉价。
22本发明的另一目的在于提供用于治疗例如以下疾病的大小超过1μm的干燥重建囊泡:骨和/或软骨疾病例如骨软骨缺损和骨关节炎。
23本发明的另一目的在于提供脂质体的制造方法,其能够获得亲水性蛋白质的高的包封率,其能够避免有机溶剂或清洁剂,其能够易于大规模的实现,其生产贮存期间稳定的产品而不破坏蛋白质,其允许缓释蛋白质并且其提供足够大以避免从施用位置被快速清除的脂质体。
24一方面,本发明涉及干燥药物组合物,其包括包含冷冻干燥活性剂的囊泡,所述干燥药物组合物包括a)至少一种脂质,b)至少一种活性剂,c)融合促进剂,和d)不含膜稳定剂,其中干燥药物组合物的复水引起平均脂质体直径大于1μm的多层脂质体的形成,所述脂质体包封活性剂。
25根据本发明的干燥药物组合物可以长时间稳定地贮存。优选地,本发明的干燥药物组合物为冷冻干燥组合物。由于以干燥或冷冻干燥形式的组合物稳定,其能够通过添加水溶液容易地重建。水溶液的添加引起平均脂质体直径大于1μm、优选约1.5μm或以上的多层脂质体的形成。在这些多层脂质体中,以优选至少40%,特别地优选至少50%、更优选至少55%、更优选至少60%以及最优选至少80%的高包封率包封活性剂。
26本发明的干燥药物组合物的组分包括至少一种脂质、至少一种活性剂、至少一种融合促进剂并且不包含膜稳定剂。优选地所述至少一种活性剂为蛋白质,特别是亲水性蛋白质或其活性片段。特别优选的是为骨和/或软骨再生剂,优选CD-RAP的蛋白质。优选的融合促进剂为碱性氨基酸,具体地,选自精氨酸、组氨酸、赖氨酸或瓜氨酸。此外,已经发现提供不包含膜稳定剂(即,具体地,不存在保护性糖、糖醇或糖苷)的干燥药物组合物是有利的。此外,在本发明的一些优选实施方式中,组合物还包括无机或有机阴离子,例如琥珀酸根、富马酸根、柠檬酸根、苹果酸根、磷酸根、乙酸根或盐酸根。
27干燥药物组合物优选为无菌组合物。
28不使用脂质体包封,即使大的蛋白质也从施用位置被快速地清除,例如通过滑膜从滑液中清除,并因此将不能充分地获得诱导缺陷组织如软骨或骨的再生。为了延长活性剂例如生长因子在施用位置,例如盘(disc)或其周围和/或关节内的局部停留时间,发明人能够提供蛋白质(例如亲水性蛋白质如CD-RAP或BMP)的脂质体制剂,所述脂质体制剂包括大多层囊泡(MLVs),具有高的包封率和受控的蛋白质的释放。
29发明人提供了包括冻干的蛋白质脂质体组合物的肠胃外用药物组合物,其长期贮存时稳定抗分解,并且其能够重建以产生包括亲水性蛋白质的大多层脂质体。此处所用的干燥重建囊泡为例如干燥颗粒产品,添加水性介质时其分散以形成包括生物活性成分的多层脂质体制剂。有利地是,通过使用根据本发明的干燥脂质体,避免了稳定性问题例如活性剂的聚集或氧化。此外,实现了亲水性成分例如蛋白质的高包封率,所述蛋白质例如包括BMPs和/或CD/RAP的骨和/或软骨再生剂。与现有技术相反,现在发明人能够提供稳定的具有活性剂微小变化的药物组合物,其形成稳定的冻干块(lyo cake),其能够通过水性介质容易快速地重建,具有活性剂的可再生的包裹体包埋(inclusion entrapment)、提高的贮存稳定性和当复水时显著增加的多层脂质体尺寸分布。
30本发明复水的脂质体在注射入例如滑液、关节腔内、盘或盘周围之后,拥有长时期的原位抗性(prolonged resistance in situ),因此克服了本领域现有技术治疗软骨疾病的限制。由于脂质体外的活性剂的存在,蛋白质具有即时作用,并且随着脂质体的降解,具有延迟或缓释效应。长时间活性剂的存在能够对细胞(例如软骨形成细胞和滑膜细胞)、蛋白聚糖的生产有持续的有利效应,从而确保再生效应或降低由活性剂介导的疾病进程。当将本发明所得的制剂给药到受损关节(例如骨关节)时,所得的制剂可以发挥对关节结构的保护性效应和/或由活性剂介导的抗炎和/或再生效应、脂质体的润滑效应、粘度补充(visculosupplementary)效应和/或滑液的替代效应。
31同样在本发明的范围内为干燥脂质体组合物的制备方法,所述脂质体组合物用水溶液复水后形成平均脂质体直径大于1μm的包封活性剂的多层脂质体,该方法包括以下步骤:a)在不存在有机溶剂下将脂质、脂质混合物或脂质膜脱水,b)形成优选具有50至200nm平均直径的小单层囊泡,c)添加活性剂的水溶液,d)在步骤c)之后、之前或一起,添加融合促进剂和任选的无机或有机阴离子,和e)不添加膜稳定剂,将所述脂质分散体脱水。
32在步骤b)中制备的小单层囊泡优选具有至少50、特别至少60和更优选至少70nm的平均直径,以及优选200nm、特别150nm和更优选120nm的最大直径。
33本发明方法的特别优点为可以在步骤b)和c)之后进行无菌过滤。因此,可以提供无菌干燥药物组合物。
34步骤a)和d)的制备之后,制剂可以例如在4℃、优选在室温下的真空中贮存。
35由于MLV脂质体的尺寸和由于许多活性剂例如蛋白质的热敏感性,已知的此类药物制剂的无菌过滤或最终的灭菌是不可行的。使用根据本发明的无菌制造方法克服了这些困难。
36因此本发明进一步提供了可给药的脂质体组合物的制备方法,所述脂质体组合物包括平均脂质体直径大于1μm的包封活性剂的多层脂质体,所述方法包括以下步骤:a)在不存在有机溶剂下将脂质、脂质混合物或脂质膜脱水,b)形成优选具有50至200nm平均直径的小单层囊泡,c)添加活性剂的水溶液,d)在步骤c)之后、之前或与c)一起,添加融合促进剂和任选的无机或有机阴离子,e)不添加膜稳定剂,将所述脂质分散体脱水、f)用水溶液复水并且形成平均脂质体直径大于1μm的包封活性剂的多层囊泡,其中在步骤b)和/或c)之后进行无菌过滤步骤。
37特别的,在步骤f)中使用无菌水溶液,可以获得包括MLV脂质体的无菌组合物。
38还包括提供试剂盒,所述试剂盒包括如本文特别是权利要求1中所述的干燥药物组合物和用于使干燥药物组合物复水的水溶液。
39如果需要冷冻干燥的话现有技术中描述了冻干保护剂(lyoprotectants)。二糖,例如蔗糖、乳糖和海藻糖是最优选的冻干保护剂。应该避免单糖例如葡萄糖或山梨醇或具有低分子量的赋形剂例如氨基酸和无机盐,这是因为它们在冷冻状态的低的转变和坍陷温度(Liposomes第二版,A Practical Approach,edited by VladimirP.Torchilin and Volkmar Weissig,Oxford University Press,第157(2002)页)。然而,发明人发现使用碱性氨基酸,例如精氨酸作为填充剂在冷冻干燥期间不保护囊泡,但是,令人惊奇地是,促进了融合过程,所述融合过程对于获得具有1μm或更大直径的多层囊泡是关键的。除了此效应,这些氨基酸的使用令人惊奇地维持冷冻干燥期间配方蛋白质(formulated protein)的稳定性。这两种效应的联合-一方面使脂质膜去稳定而另一方面使药物稳定迄今为止是全然未知的。
40与现有技术的脂质体例如Kim等描述的MVLs(Kim,S.等(1983)Preparation of multivesicular liposomes.Biochim.Biophys.Acta 728:339-348)相反,该脂质体的优点在于避免了有机溶剂例如氯仿。本发明的脂质体制剂优选不含有机溶剂,并且具体地,包含低于2wt%、优选低于1wt%、更优选低于0.1wt%以及最优选0%的有机溶剂。此外,本发明提供建立大规模制造方法以制造干燥重建囊泡,所述干燥重建囊泡复水时形成用于药物应用的MLVs。
41本发明方法的优点为可以避免额外的脂质体纯化步骤,其描述于现有技术方法中以除去未包埋入水性脂质体核或在脂质体壳之间的材料。未引入的蛋白质的部分,其优选非共价地连接至脂质体的表面,能够在将药物组合物给药至需要其的患者时,起始迅速地释放活性剂例如游离蛋白质,这相当是优点。
42本发明方法的另一优点为,与由现有技术方法制造的MLVs的较低包封率相比,使用本发明方法的MLVs中的蛋白质(例如CD-RAP)的高包封率。此外,使用本发明的方法,形成多层脂质体而不是现有技术方法的大单层脂质体,所述现有技术方法例如用蛋白质溶液重建冷冻干燥脂质粉末。
43术语″多层囊泡(MLVs)″是指包含形成两个或以上壳的多个脂质双层的脂质体,特别是两相多层脂质囊泡。两相脂质囊泡包括复数个间隔分开的包括脂质体形成组分和任选的生物活性剂的脂质双层。脂质囊泡包括形成在脂质双层之间的外围水溶液室和包括任选地包含活性剂的水溶液的中心室。
44此处术语″包封、包裹或包埋″用于在脂质体的水性核心中或在两个相邻的壳之间配置物质、特别是亲水性物质。在脂质体内部包埋的材料的量可以通过本领域已知的方法例如以下方法确定:通过在Liposomes第二版,A Practical Approach,P.Torchilin和VolkmarWeissig编辑,Oxford University Press(2002)中所述的离心来纯化、如在Liposomes,A Practical Approach edited by R.R.C.New,IRL Press(1990)中所述的透析或如本发明实施例中所述的那些方法。
45关于“不添加膜稳定剂”,其是指没有物质以抑制脂质体片段或囊泡的融合(例如抑制多层囊泡的形成)的量添加或存在。膜稳定剂的实例为在内表面和/或外表面的以保护性浓度的保护性糖、糖醇或糖苷。在50mM的糖例如海藻糖浓度下,囊泡通常与脱水前同样大小。在125mM或以上的糖浓度,例如海藻糖浓度下,在脱水前后的囊泡间几乎没有可辨别的结构差异。然而,可以包含少量的此类膜稳定剂,如果在干燥时它们不抑制脂质体的融合例如MLVs的形成的话。在创造性的组合物中膜稳定剂优选以≤5%(w/v),优选≤2.5%(w/v),更优选≤1%(w/v),更优选≤0.1%(w/v)以及最优选0%(w/v)的量存在。
46″活性剂、生物活性剂或生物活性化合物″是指任何具有治疗、生物、药理学、制药学(例如,治疗、控制、改善、预防、延迟一种或多种疾病、紊乱或状况或其症状的发作、降低其发展风险)和/或美容效应的试剂。治疗效应可以是局部或全身的,并且可以是客观或主观的。优选活性剂为蛋白质,特别是亲水性蛋白质,更优选骨和/或软骨再生蛋白质。
47“膜稳定剂”是指当以一定浓度或浓度范围添加时,防止干燥时脂质体破坏或包封的活性剂泄露的试剂。其可以为保护性的糖、糖醇或糖苷,特别是,单糖或二糖或氨基糖苷。保护性的糖和糖苷包括赋形剂例如海藻糖、甘露糖、蔗糖、葡萄糖、乳糖、右旋糖、链霉素和二氢链霉素。
48“融合促进剂”促进或能够实现脂质体组装成MLVs的融合。此外,融合促进剂优选指稳定活性剂例如蛋白质的天然结构的赋形剂或组分。此外,融合促进剂优选在干燥时不保护SUVs或不防止SUV脂质双层的破坏,例如通过形成冰晶。融合促进剂可为无定形或部分结晶的物质或缓冲物质,其在脱水过程例如冷冻干燥期间引起脂质膜的断裂、破裂或打开,以能够实现活性剂的包封,同时通过随后的复水形成MLVs。此类物质包括氨基酸、特别是碱性氨基酸,并且优选精氨酸、组氨酸、瓜氨酸、赖氨酸和其相应的盐例如磷酸盐、硫酸盐或盐酸盐或其混合物。优选地,融合促进剂以干燥脂质体制剂复水(干燥重建囊泡,DRVs)后能够实现等渗条件的量添加。此外,融合促进剂优选对活性剂,例如要包封的蛋白质没有负面影响。
49术语“退变性盘疾病(degenerative disc disease)(DDD)”为慢性过程,其特征部分在于在髓核中的蛋白聚糖和水含量的逐步损失,其可以在例如以下多种疾病中出现:先天性下背痛(idiopathiclow back pain)、盘突出、盘内破裂或盘开裂(fissured discs)、神经根病、椎管狭窄、髓核突出诱导的坐骨神经痛(herniated nucleuspulposus-induced sciatica)、坐骨神经痛、特发性脊柱侧凸和/或脊髓病。盘退变等级可以通过手术前MRI的分析来分级。
50为了本发明的目的,术语″透盘地(transdiscally)″包括但不限于注射入椎间盘、特别是椎间盘的髓核(NP),其包括完整的盘、不同阶段的退变性盘、突出的盘、破裂的盘、分层的盘(delaminateddisc)或开裂的盘。如果要注射的体积可能引起NP的压力,可以在注射或实施脊柱的灌输应用之前除去至少部分NP。在某些情况下,除去材料的体积为约要施用的体积的量±20%。对于NP,术语“透盘地”还包括注射入如上所述的退变性或完整盘的纤维环(AF)。在应用较大尺寸载体材料的实例中,在应用根据本发明的药物组合物之前部分或全部除去盘可能是必要的。其还包括提供灌输入AF壁的外部但紧邻该壁的位置,或相邻的椎体的终板(endplate)的位置,这可能避免AF的刺穿以及由此在盘上潜在的负荷。
51当用于本文时,术语“脂质”,旨在指定任何可用于制备脂质双层的物质。典型的脂质包括糖脂、卵磷脂、磷脂、神经酰胺及其混合物。
52根据本发明的单独或以混合物存在的合适的脂质,无论氢化与否,包括中性或带正电的脂质,例如天然卵磷脂或磷脂。脂质的实例为磷脂酰胆碱(PC)、卵黄磷脂酰胆碱(EPC)、磷脂酰丝氨酸(PS)、胆固醇(Chol)、二硬脂酰磷脂酰胆碱(DSPC)、鞘磷脂(SM)、二油酰磷脂酰胆碱(DOPC)、二油酰磷脂酰甘油(DOPG)、二月桂酰磷脂酰胆碱(DLPC)、磷脂酰甘油(PG)、二肉豆蔻酰磷脂酰胆碱(DMPC)、二棕榈酰磷脂酰胆碱(DPPC)、神经节苷脂、神经酰胺、磷脂酰肌醇(PI)、磷脂酸(PA)、磷酸二鲸蜡脂(DcP)、二豆蔻酰磷脂酰胆碱(DMPC)、神经节苷脂和其它糖脂、硬脂酰胺、二棕榈酰磷脂酰甘油脂(DPPG)、以及其它合成或半合成脂质。磷脂可以为衍生自蛋黄、大豆或其它动物或植物的天然脂质例如蛋黄卵磷脂、大豆卵磷脂等。脂质体制剂典型地为至少一种、优选至少两种脂质例如胆固醇和磷脂酰胆碱以及更优选三种或以上脂质的混合物。
53在进一步优选的实施方式中,脂质包括低于20、15、10、8、5、3、1重量百分比(wt%)的不饱和脂质(基于总脂质的量)。优选脂质为饱和中性脂质。
54为了说明书内所述的本发明的目的,不饱和和/或中性脂质为根据本发明的优选脂质,以增加形成的脂质体的稳定性和以改进药物应用的缓释。不饱和脂质具有非常低的转变温度。由于在生产的所有阶段脂质将处于液晶相,处理例如分散或复水成脂质体将容易和快速的多。如果周围的温度接近或超过脂质的相变温度,结果将是引入的化合物的极大损失。
55优选地,如上所述的肠胃外用药物组合物包括两种脂质、优选两种中性脂质、更优选磷脂酰胆碱(PC)和胆固醇(Chol)。优选地,基于总脂质的量的两种脂质(例如PC∶Chol)的重量百分比为从约2至约7、约2至约6、约2至约5、约3至约7、约3至约6、约3至约5、约2,7至约5,4、约2,8至约5,2、约2,8至约4,2、约2,8至约3,2(例如3、4或5)。
56虽然描述了中性脂质经常产生MLVs的聚集,但对于本发明的MLVs不能观察到聚集。
57优选脂质混合物是带电的。阳离子脂质的实例包括dioctadecyldemethylammonium chloride(双十八烷基脱甲基氯化铵(DOPAC))、N-(2,3-二油酰氧基)丙基-N,N,N-三甲基铵(DOTMA)、双十二烷基溴化铵(didodecylammonium bromide)(DDAB)、1,2-二油酰基-3-三甲基铵丙烷(DOTAP)、3-N-(N′,N′,-二甲基氨基乙烷)-氨基甲酰基(carbamol)胆固醇(DC-Chol)、1,2-二肉豆蔻酰氧基丙基-3-二甲基羟乙基铵(DMRIE)、2,3-二油酰氧基-N-[2(精胺甲酰氨基)乙基]-N,N-二甲基-1-丙铵三氟乙酸酯(DOSPA)等。
58阴离子脂质的实例对本领域技术人员是已知的,并且包括但不限于心磷脂、抗坏血酸棕榈酸酯、二硬脂酰磷脂酰甘油(DSPG)、磷脂酸和磷脂酰丝氨酸(PS)。其它阴离子脂质包括磷脂酰乙醇胺(例如花生四烯酸乙醇胺(anandamides)和甲基花生四烯酸乙醇胺(methanandamides))的酰胺、磷脂酰丝氨酸、磷脂酰肌醇和其脂肪酸酯、磷脂酰乙二醇、酸性溶脂质(acidic lysolipids)、棕榈酸、硬脂酸、花生四烯酸、油酸、亚麻酸、亚油酸、肉豆蔻酸、脑硫脂和硫苷酯、饱和和不饱和的游离脂肪酸,及其带负电衍生物。更优选地,阴离子脂质为磷脂酸、磷脂酰甘油、磷脂酰甘油脂肪酸酯、磷脂酰乙醇胺花生四烯酸乙醇胺、磷脂酰乙醇胺甲基花生四烯酸乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇、磷脂酰肌醇脂肪酸酯、心磷脂、磷脂酰乙二醇、酸性溶脂质、脑硫脂和硫苷酯、饱和游离脂肪酸、不饱和游离脂肪酸、棕榈酸、硬脂酸、花生四烯酸、油酸、亚麻酸、亚油酸或肉豆蔻酸。此处所述的任何阴离子脂质都可以通过用氟原子取代至少一个氢原子来氟化。
59为了提高水溶性物质例如CD-RAP的包封,优选选择脂质组分以使至少一种脂质带电,以增加活性剂的包封率。因此,在另一实施方式中,肠胃外用药物组合物包括达20%、15%、10%、5%、2%、1%,、约10%至1%、约5%至1%、0,1%至0,5%(w/w)带电的脂质,基于总脂质的量。
60优选带电的脂质为心磷脂或抗坏血酸棕榈酸酯,优选为总脂质的0,1%至5%(w/w)、总脂质的0,1%至3%(w/w)、0,1%至1,5%(w/w)、0,1%至1%(w/w)的心磷脂或抗坏血酸棕榈酸酯。
61合适的脂质混合物的优选实例为磷脂酰胆碱(PC)、胆固醇(Chol)和抗坏血酸棕榈酸酯,优选PC∶Chol∶抗坏血酸棕榈酸酯的比例为总脂质组分的60%-1%∶0%-40%∶0%-5%,更优选比例为70%-90%∶7%-30%∶0,1%-3%,最优选比例为70%-80%∶20%-28%∶0.1%-1,5%(w/w)。
62现有技术教导带电的磷脂物质对降低脂质体的尺寸可能是重要的(Liposomes第二版,A Practical Approach,edited byVladimir P.Torchilin and Volkmar Weissig,Oxford UniversityPress(2002),第7页第1段),并且可能对冷冻干燥/复水后的物质保持(substance retention)有负面影响,与现有技术相反,发明人令人惊奇地发现带电脂质的添加增加了重建后的MLVs的尺寸(参见图3)。
63脂质体可以进一步包括用亲水性聚合物衍生以形成脂质聚合物的脂质。此类脂质聚合物优选包括在其头部基团用聚合物通过共价或非共价键改性的脂质。脂质聚合物可以通过以下引入脂质体:向形成脂质体的脂质混合物添加脂质聚合物,或通过首先制备脂质体,然后向预成形的脂质体的外层引入脂质聚合物。脂质聚合物例如描述于WO2006/027786,在此引入以作参考。
64根据本发明的蛋白质包括例如亲水性蛋白质。
65优选地,亲水性蛋白质为软骨或骨再生剂例如软骨促进剂或骨形成蛋白。此类试剂,具体地包括以下家族的成员:TGF-β家族(转化生长因子,Roberts和Sporn,Handbook of ExperimentalPharmacology 95(1990),第419-472页);DVR-族(等,Biochem.Biophys.Res.Comm.206(1995),第608-613页,及其进一步引用的文献),包括BMPs(骨形成蛋白,Rosen和Thies,GrowthFactors in Perinatal Development(1993),第39-58页)和GDFs(生长分化因子);抑制素/激活素(activin)(Vale等,The Physiology ofReproduction,第二版(1994),第1861-1878页);GDNF、SOX、IGF和EGF蛋白家族。
66TGF-β超家族或其活性变体的目的成员包括TGF-β蛋白例如TGF-β1、TGF-β2、TGF-β3、TGF-β4、TGF-β5(U.S.5,284,763:EP 0376785;U.S.4,886,747:DNA 7(1988),第1-8页),EMBOJ.7(1988),第3737-3743页),Mol.Endo.2(1988),第1186-1195页),J.Biol.Chem.265(1990),第1089-1093页);OP1,OP2和OP3蛋白(U.S.5,011,691.U.S.5,652,337.WO91/05802)以及BMP-2,BMP-3,BMP-4(WO88/00205,U.S.5,013,649和WO89/10409,Science242(1988),第1528-1534页)、BMP-5,BMP-6和BMP-7(OP1)(Proc.Natl.Acad.Sci.87(1990),第9841-9847页,″WO90/11366)、BMP-8(OP2)(WO91/18098)、BMP-9(WO93/00432)、BMP-10(WO94/26893)、BMP-11(WO94/26892)、BMP-12(WO95/16035)、BMP-13(WO95/16035)、BMP-15(WO96/36710)、BMP-16(WO98/12322)、BMP-3b(Biochem.Biophys.Res.Comm.219(1996),第656-662页)、GDF-1(WO92/00382和Proc.Natl.Acad.Sci.88(1991),第4250-4254页)、GDF-8(WO94/21681)、GDF-10(WO95/10539)、GDF-11(WO96/01845)、GDF-5(CDMP1,MP52)(WO95/04819;WO96/01316;WO94/15949,WO96/14335和WO93/16099and Nature 368(1994),第639-643页)、GDF-6(CDMP2,BMP-13)(WO95/01801,WO96/14335和WO95/16035)、GDF-7(CDMP3,BMP-12)(WO95/01802和WO95/10635)、GDF-14(WO97/36926)、GFD-15(WO99/06445)、GDF-16(WO99/06556)、60A(Proc.Natl.Acad.Sci.88(1991),第9214-9218页)、DPP(Nature 325(1987),第81-84页)、Vgr-1(Proc.Natl.Acad.Sci.86(1989),第4554-4558页)、Vg-1,(Cell51(1987),第861-867页),背蛋白(dorsalin)(Cell73(1993),第687-702页)、MIS(Cell45(1986),第685-698页),pCL13(WO97/00958)、BIP(WO94/01557)、抑制素a、激活素βA和激活素βB(EP 0222491),激活素βC(MP121)(WO96/01316)、激活素βE和GDF-12(WO96/02559和WO98/22492)、激活素βD(Biochem.Biophys.Res.Comm.210(1995),page 581-588)、GDNF(Science 260(1993),第1130-1132页,WO93/06116)、神经营养因子(neurturin)(Nature 384(1996),第467-470页)、Parsephin(Neuron 20(1998),第245-253页,WO97/33911)、Artemin(Neuron 21(1998),第1291-1302页),Mic-1(Proc.Natl.Acad.Sci USA 94(1997),第11514-11519页),Univin(Dev.Biol.166(1994),第149-158页),ADMP(Development 121(1995),第4293-4301页),Nodal(Nature 361(1993),第543-547页),Screw(Genes Dev.8(1994),第2588-2601页)或其组合。其它可用的蛋白质包括生物活性的生物合成构建体,包括使用来自两种或以上已知的形态形成蛋白的序列设计的生物合成的蛋白质。生物合成的构建体的实例公开于U.S.5,011,691(例如COP-1,COP-3,COP-4,COP-5,COP-7和COP-16)。可用的SOX蛋白家族成员的实例(例如SOX-9)公开于WO96/17057。在此将包括专利或专利申请的引用出版物的内容引入以作参考。
67在一个实施方式中,软骨或骨再生剂选自具有SH3结构域或具有采用类SH3结构域折叠的结构域的蛋白质,例如CD-RAP。SH3结构域或类SH3结构域例如公开于Stoll等(Stoll,R.等(2003)Backbone dynamics of the human MIA protein studied by(15)N NMRrelaxation:implications for extended interactions of SH3domains.Protein Sci.12:510-519;Stoll,R.et al.(2001)Theextracellular human melanoma inhibitory activity(MIA)proteinadopts an SH3 domain-like fold.Embo J 20:340-349),并且可以通过Kelley等公开的3D-PSSM网络服务器(Kelley,L.A.等.(2000)Enhanced genome annotation using structural profiles inthe program 3D-PSSM.J Mol.Biol 299:499-520)通过预测SH3折叠来确定。SH3结构域,也被称为Src同源结构域,为在许多细胞内蛋白中发现的蛋白质分子。迄今为止,还没有描述SH3结构域用来治疗脊柱疾病。
68在另一实施方式中,软骨或骨再生剂为这样的蛋白,其能够特异地结合到如文献(Stoll,R.等.(2001)The extracellularhuman melanoma inhibitory activity(MIA)protein adopts an SH3domain-like fold.Embo J 20:340-349;Homandberg,G.A.and Hui,F.(1996)Association of proteoglycan degradation withcatabolic cytokine and stromelysin release from cartilagecultured with fibronectin fragments.Arch.Biochem.Biophys.334:325-331;Homandberg,G.A.等(1997)Fibronectin-fragment-induced cartilage chondrolysis isassociated with release of catabolic cytokines.Biochem.J 321(Pt 3):751-757)中所述的纤连蛋白、纤连蛋白片段和/或富脯氨酸序列。
69在一个实施方式中,软骨或骨再生剂包括纤连蛋白或整联蛋白结合结构域。可以通过例如ELISA确定软骨分化和维持因子(cartilage differentiation and maintenance factor)至细胞外蛋白例如纤连蛋白或纤连蛋白片段以及整联蛋白的结合。纤连蛋白、其片段或整联蛋白可以涂布在塑料表面,并暴露于软骨分化和维持因子。结合的量可以通过抗软骨分化和维持因子的过氧化酶连接的单克隆抗体来确定。整联蛋白结合还可以如Bauer等所述(Bauer,R.等.(2006)Regulation of integrin activity by MIA.J Biol Chem 281:11669-11677)来确定,在此引入以作参考。
70优选地,将软骨或骨再生剂定义为:a)软骨细胞蛋白,包括或具有CD-RAP(SEQ ID No 1)的成熟序列和其功能片段或变体,b)与CD-RAP的C末端的4个半胱氨酸骨架(skeleton)、SEQ ID No.1的12至107氨基酸具有至少63%、优选80%,更优选90%氨基酸序列同源性的蛋白质;或c)具有任何此处定义的通用序列1至3(SEQ ID No2,3和4)的蛋白质。
71与CD-RAP具有相同生物功能的功能性片段优选具有SEQ IDNO:1所示的序列的至少20、特别至少40、更优选至少50、最优选80个连续氨基酸的长度。优选地,功能性片段包括从SEQ ID No 1的位置1至50、1至70、1至80、20至80、20至107的氨基酸。
72成熟CD-RAP序列(SEQ ID No 1)
GPMPKLADRKLCADQECSHPISMAVALQDYMAPDCRFLTIHRGQVVYVFS
KLKGRGRLFWGGSVQGDYYGDLAARLGYFPSSIVREDQTLKPGKVDVKTD
KWDFYCQ
73通用序列1(SEQ ID No 02)
CX4CX17CX12VX11-13WX7-18FX4VX21CX
74通用序列2(SEQ ID No 03)
KXCXDXECX11DX3PDCX12VX2KLX7-9WXGSX5-13GYFPX3
VX18DFXCX
75通用序列3(SEQ ID No 04)
KXCXDX2CX8AX2DX3PDCRFX5GXVX5KLX7WXGSVX12
GYFPX22DFXCQ
76其中每次出现的″X″独立地表示任何氨基酸,小写的数字表示任何氨基酸的数目。优选地,″X″独立地表示天然发生的氨基酸,特别是A,R,N,D,B,C,Q,E,Z,G,H,I,L,K,M,F,P,S,T,W,Y或V。
77特别优选地,软骨或骨再生剂为CD-RAP(软骨源性视黄酸敏感蛋白),也称为MIA(黑素瘤抑制性活性因子)、OTOR(纤维细胞衍生蛋白(fibrocyte derived protein)、FDP、类MIA、MIAL)和TANGO130,所述TANGO 130属于分泌蛋白的类别(Bosserhoff,A.K.等(2004)Characterization and expression pattern of the novel MIAhomolog TANGO.Gene Expr.Patterns.4:473-479;Bosserhoff,A.K.和Buettner,R.(2003)Establishing the protein MIA(melanoma inhibitory activity)as a marker for chondrocytedifferentiation.Biomaterials 24:3229-3234;Bosserhoff,A.K.等.(1997)Mouse CD-RAP/MIA gene:structure,chromosomallocalization,and expression in cartilage and chondrosarcoma.Dev.Dyn.208:516-525;WO00/12762)。CD-RAP或MIA为130个氨基酸的蛋白质(EP 0710248,EP1146897,在此全部引入以作参考),其对于软骨分化是高度特异性的标记。
78优选地,根据本发明的蛋白质包括CD/RAP(MIA)、BMP-2、BMP-7、BMP-12、BMP-13、GDF-5(MP-52)、TGF-β1、TGF-β2、TGF-β3、TGF-α或其活性片段或其组合,最优选CD/RAP(MIA)。
79此处预期的蛋白质可以从完整或截短的基因组DNA或cDNA或从合成DNA在原核或真核宿主细胞中表达。蛋白质可以从培养基或包涵体中分离和/或再折叠以形成生物活性的组成。参见例如EP0710248和Lougheed等(Lougheed,J.C.等.(2001)Structure ofmelanoma inhibitory activity protein,a member of a recentlyidentified family of secreted proteins.Proc.Natl.Acad.Sci.U.S.A 98:5515-5520)以用于CD-RAP的重建蛋白纯化的示例性方案。如何测试此类分离蛋白的活性(例如软骨形成)的详细描述描述于Tscheudschilsuren等和Stoll等(Tscheudschilsuren,G.等.(2005)Regulation of mesenchymal stem cell and chondrocytedifferentiation by MIA.Experimental Cell Research 1-10;Stoll,R.等.(2003)Backbone dynamics of the human MIA protein studiedby(15)N NMR relaxation:implications for extended interactionsof SH3 domains.Protein Sci.12:510-519),在此将其内容引入以作参考。软骨诱导的生物测定描述于EP 1146897的实施例2至5,在此引入以作参考。
80优选地,融合促进剂以足以避免生理环境中的渗透胁迫和/或维持肠胃外应用的等渗条件的量使用。优选地,融合促进剂以低于8%(m/v)、优选低于5%、优选2%至5%基于干燥前颗粒材料的量使用。
81在优选的实施方式中,包括包含冷冻干燥蛋白质的囊泡的肠胃外用药物组合物以用水溶液复水后足以形成等渗脂质体分散体的量包括融合促进剂。优选地,复水的脂质体分散体的pH为pH4至pH9、pH5至pH8,优选pH6至pH7.5。
82合适的无机或有机阴离子为例如琥珀酸根、富马酸根、柠檬酸根、苹果酸根、磷酸根、乙酸根、盐酸根,优选磷酸根。
83优选地,融合促进剂为磷酸精氨酸,冻干的脂质体重建后优选100-800mM、更优选100-600mM或最优选280-400mM磷酸精氨酸。
84其它添加剂或添加物质,如抗氧化剂例如蛋氨酸、抗坏血酸、维生素E、丁基羟基甲苯(butylhydroxytoluol)(BTH)、丁基羟基苯甲醚、没食子酸丙酯;带电的物质例如硬脂胺、油胺、双十六烷基磷酸酯等可以适当地调整。优选的添加剂是优选为总脂质的0,1至5%(w/w)丁基羟基甲苯(BTH)、丁基羟基苯甲醚和/或蛋氨酸、总脂质的0,1至3%(w/w)、0,1至1,5%(w/w)、0,1至1%(w/w)的抗氧化剂例如丁基羟基甲苯和/或5至100mM复水脂质体的蛋氨酸终浓度、更优选5至50mM、最优选10至25mM复水脂质体的蛋氨酸终浓度。其它的物质可以为用来改进缓释、增加脂质体的半衰期或将脂质体从而将药物靶向针对特定组织或细胞类型那些。
85除了活性剂,本发明的药物脂质体组合物可以包括另外的治疗或生物活性的试剂。例如,可以存在用于治疗特定适应症例如骨关节炎的治疗因子,例如涉及破坏关节软骨或滑液组分的一种或多种抑制剂,不限于抗金属蛋白酶、周期蛋白化合物(cycline compounds)、细胞因子拮抗剂、糖皮质激素、TNF抑制剂、IL抑制剂、抗血管形成物质、聚蛋白多糖酶抑制剂、p38激酶抑制剂、细胞凋亡抑制剂、透明质酸抑制剂和蛋白裂解酶抑制剂。可以控制炎症的因子也可为组合物的一部分,所述因子包括英夫利西(infliximab)、依那西普(etanercerpt)、阿达木单抗(adalimulab)、nerelimonmab、来那西普(lenercerpt)等,或其组合。还设想药物脂质体组合物可以包括细胞外基质组分例如透明质酸或其衍生物,所述衍生物包括其盐、酯、内酯和硫酸衍生物,优选透明质酸的偏酯。
86本发明还设想涉及根据本发明任何实施方式的肠胃外用药物组合物或其制备方法,其中,用水溶液复水时形成的包封活性剂的脂质体的至少70%、80%、90%、95%、98%为多层脂质体。更具体地,上述百分比是指复水时由上述比例形成的脂质体占形成的总脂质体的部分。
87在一个实施方式中,水溶液是缓冲或无缓冲的,优选无缓冲的,最优选注射用水。
88在优选的实施方式中,多层脂质体,其优选为脂质体分散体,具有200至400mosmol、优选250至350mosmol的渗透压。
89在一个实施方式中,根据本发明的肠胃外药物组合物包含至少0.125pmol蛋白质,优选1.25pmolCD-RAP每μmol脂质体脂质。
90在一个实施方式中,根据本发明的多层脂质体通过如上所述的脱水和复水方法制备,其包含在溶液、优选等渗溶液中的内部和外部脂质体活性剂。
91另一优选的实施方案涉及包括包含冷冻干燥蛋白质的囊泡的药物组合物,所述药物组合物包含以下:a)磷脂酰胆碱、胆固醇和抗坏血酸棕榈酸酯,比例为总脂质的约78-80%:19.5%-28%:0.5-2%,b)骨和/或软骨诱导剂,优选CD-RAP,和c)融合促进剂,优选具有以下pH的磷酸精氨酸:pH4至pH9,优选pH5至pH 8,最优选pH6至pH7.5,其中在用包封活性剂的水溶液使干燥组合物复水时形成具有包含骨和/或软骨诱导剂的内部水性空间、平均脂质体直径大于1μm、优选1μm至2,5μm的多层脂质体。
92本发明的方法提供干燥脂质体组合物以及重建脂质体组合物。干燥脂质体组合物优选为冷冻干燥和/或无菌的。重建脂质体组合物优选为可肠胃外给药并且也是无菌的。特别地,该方法允许在水性介质中复水并且形成多层脂质体后高的包封率和高的脂质体内蛋白浓度。
93这些方法包括将脂质、脂质混合物或脂膜在不存在有机溶剂下脱水的步骤,由此形成大的MLVs,并且随后产生小单层囊泡或脂质体,所述小单层囊泡或脂质体优选具有50至200nm、50至150nm、50至120nm、70至120nm的平均直径。
94根据本发明,脂质或脂质混合物例如脂质粉末可以通过添加水溶液来水合。水溶液可以为缓冲或无缓冲的(例如缓冲或无缓冲的蛋白质本体溶液)。优选地,水溶液包含至少50%(w/w)、更优选至少90%(w/w)、以及最优选至少99%(w/w)的水。脂质膜可以通过在有机溶剂例如叔丁醇中溶解脂质,然后在氮气流下干燥或冻干来制备。
95可获得几种产生小单层脂质体和估量脂质体的技术。这些方法包括施加足以减少脂质体尺寸的力的各种技术,并且产生较小单层囊泡。此类方法包括匀质化,其将大的脂质体通过剪切破碎成较小的脂质体。在典型的匀质化方法中,脂质体通过乳液匀化器循环直至达到所期望的尺寸,例如平均直径50至200nm、50至150nm、50至120nm或70至120nm。用于脂质体制造的高压匀化器例如描述于Liposomes第二版,A Practical Approach,Vladimir P.Torchilinand Volkmar Weissig,编辑,Oxford University Press page 17(2002)。其它方法包括使脂质体在加压下通过多孔的聚碳酸酯膜挤出。通常,脂质体分散体循环几次通过膜。连续的较小孔的膜可以用于逐步减少尺寸。优选的过滤器或膜具有小于或等于250nm、200nm、150nm、100nm、80nm、50nm或15nm的尺寸。优选的循环数目为1、更优选2、最优选3以上。另外的方法为超声处理、微射流处理(microfluidization)或机械剪切以及不同方法的组合。可以使用传统方法例如光散射监视脂质体的尺寸。
96形成小单层脂质体后,添加水性蛋白溶液以产生小单层脂质体。在此步骤之后、之前或一起添加融合促进剂。
97在形成SUVs之后和/或添加水性蛋白溶液之后可以包括灭菌步骤。灭菌步骤例如无菌过滤(sterilfiltration)可以由通过孔径0.22μm的无菌过滤器过滤来进行。此类无菌过滤器的实例为UltiporN66(PALL),Acrodisc 4455T(PALL)或Millex GV SLGV025LS(Millipore)。
98在优选的实施方式中,蛋白质的浓度为25μg/ml至10mg/ml,但更典型地为250g/ml至2.5mg/ml重建多层脂质体制剂。
99优选地,融合促进剂以足以用水溶液复水后形成等渗的脂质体分散体的量包含于本发明的产品和方法中,优选浓度为50mM至800mM、200mM至600mM以及最优选250mM至400mM。优选地,复水的多层脂质体和/或脂质体分散体的pH为pH 4至pH 9、更优选pH 5至pH 8、最优选pH 6至pH 7.5。
100使所述脂质分散体脱水包括冻干或冷冻干燥。可以意识到除了冻干之外的干燥方法可以用于本发明,例如真空干燥、氮气流下干燥、喷射干燥、盘式干燥和滚筒干燥。在其它实施方式中,使所述脂质分散体脱水为通过脱水例如通过冻干或冷冻干燥来破碎、破裂或打开小单层囊泡。
101任选地,可以在如上所述任一制造步骤之后包括另外的步骤,例如通过膜(例如聚碳酸酯膜)的过滤步骤,以消除例如冻干物质的晶体。
102可以在本发明的任一工艺步骤包括如上所述的那些添加剂(例如抗氧化剂、稳定剂)。
103以脱水,即干燥特别是冷冻干燥的形式,组合物可以长时间地稳定贮存。
104然后可以通过添加蒸馏水、水性溶液或其它合适的溶液,无论缓冲还是无缓冲的,重建脱水产品(例如冻干的″块(cake)″)。可以通过轻轻地旋转溶液使脂质体在水性溶液中再悬浮或复水。可以在室温下或在其它适合脂质体和它们的内含物的的组合物的温度下进行复水。冻干制剂的复水形成多层脂质体的悬浮液或分散体,与干燥前的起始脂质体悬浮液相比,其具有增加的尺寸分布和形态。
105在另一优选的实施方式中,包封蛋白质的多层囊泡实质上在冷冻干燥脂质体组合物的复水期间(步骤e)形成,更优选地,其中多层脂质体在所述脂质分散体脱水期间(步骤d)不形成。
106优选地,至少一种生物活性化合物的包封率或高引入率为大于活性剂的40%、55%、大于60%、大于70%、大于80%。
107本发明涉及包括干燥重建囊泡的药物组合物以及其生产方法,所述干燥重建囊泡包括包含冷冻干燥蛋白的囊泡,它们具有可忽略的或不可检测的活性剂的聚集或降解。
108在优选的实施方式中,大于60%、80%、90%、95%或约100%的复水的MLV脂质体维持或具有大于1μm、更优选1,0μm至5μm、1,5μm至5μm、1,0μm至3μm、最优选1,2μm至2,5μm的尺寸分布。平均囊泡直径可以通过以下方法确定:电子显微检测、光子相关显微检测(photon correlation microscopy)、激光散射、激光衍射(例如MastersizerTM)或通过遮光技术(例如AccusizerTM)或描述于Liposomes第二版,A Practical Approach,Vladimir P.Torchilin和Volkmar Weissig编辑,Oxford University Press(2002)的另一方法,在此引入以作参考。优选的方法为光子相关显微检测。
109本发明的另一方面包括可通过本发明方法获得的药物冷冻干燥组合物,以及通过本文所述方法获得的重建药物组合物。
110包封活性剂例如骨或软骨再生剂(例如CD/RAP)的多层脂质体能够在许多治疗领域例如在以下疾病的软骨再生中提供益处:骨软骨缺损、全层组织缺损(full-thickness defect)、部分组织缺损(partial-thickness effect),关节炎例如骨关节炎、类风湿性关节炎、银屑病关节炎、幼年慢性关节炎、肢根假性多关节炎(rhizomelicpseudoarthritis)、类风湿性多关节炎,滑膜炎或绒毛结节性滑膜炎,脊柱疾病、退行性盘疾病(degenerative disk disease)、腱和/或韧带诱导(ligament induction)、腱炎、半月板撕裂和/或前交叉韧带(ACL)损伤。活性剂的此类脂质体递送的优点为至所期望的周围组织的更有效的、局部的递送。可以设计脂质体以在目的位置提供缓释库(Sustained release depot),以及缓慢释放包封的药物。
111与例如SUVs相反,本发明MLVs的另一优点在于在通过注射入滑液治疗骨关节炎的情况下,包含MLVs的药物由于它们的大尺寸,被限制在施用的位置,并且缓慢地释放包封的活性剂。
112因此,本发明的另一方面为本发明的无菌药物在制备用于治疗受试者中的骨或软骨缺陷、免疫疾病优选骨关节炎、类风湿性关节炎、以及脊柱疾病例如退行性盘疾病的药物组合物中的用途,其中所述治疗优先在用水溶液使冷冻干燥组合物复水后,通过一次或重复注射进行。
113在优选的实施方式中,脊柱疾病为先天性下背痛、盘突出、盘内破裂或盘开裂、神经根病、椎管狭窄、髓核突出诱导的坐骨神经痛、坐骨神经痛、特发性脊柱侧凸或脊髓病。
114在优选的实施方式中,注射为局部或非全身注射(non-systemic injection),优选盘内地或透盘地注射入滑液、滑液腔(synovia space)、髓核、髓核腔(nucleus pulposus space)。
115给药方案可以从每周多次至每月多次,优选不超过每三天一次。总疗程优选每周至少一次,更优选每月至少一次。
116药物组合物也适用于长期给药至少3个月、至少6个月、至少12个月、高达18个月、高达24个月或甚至更长。
117本发明的药用组合物优选以约0.25mg至高达约25mg、特别约0.5mg至约15mg、更优选5mg至高达15mg脂质体蛋白的单次剂量给药。
118另外的优选的治疗策略包括给药本发明所述的药物组合物:
a)第一周内至少一次,特别1至3次,然后1至5个周的间隔不给药,并且任选地重复1次或更多次该给药策略;
b)每周一次或每连续几个周一次,或;
c)每月一次或每连续几个月一次,
其中每月的剂量优选为约1mg至高达约100mg、特别约2mg至约60mg、更优选约20mg至50mg脂质体蛋白。
119脂质体的贮存稳定性为至少3个月,优选至少6个月,更优选至少1年。
120特别将本文披露的所有参考文献的全部内容引入以作参考。
121虽然已经举例说明和描述了优选的实施方式,应该理解本领域技术人员可以在权利要求限定的较宽范围形成改进而不背离本发明。
122通过附图和以下实施例进一步描述本发明。
附图说明
123图1显示与根据本发明实施例1的方法(B)相比,通过根据实施例3的冷冻和解冻方法(A)制造的脂质体的形态。
124图2举例说明通过在双偏振光下的光学显微镜分析的复水的脂质体的形态。
125图3显示依赖于使用的抗坏血酸棕榈酸酯的量,复水后根据实施例6B制造的MLVs的平均尺寸分布±标准偏差。
具体实施方式
实施例1:具有融合促进剂的冷冻干燥重建脂质体(DRVs)的制备
126A:在圆底烧瓶中将750mg磷脂酰胆碱(Lipoid S100)、250mg胆固醇、10mg抗坏血酸棕榈酸酯(有或没有)溶于20ml乙醇。在旋转蒸发器中定量地除去溶剂。产生的薄脂质膜在室温下通过平缓搅拌在10ml水中复水以得到脂质体(10%(w/v)脂质)。通过随后的超声处理制备直径约100nm的单层囊泡(SUV)。将300μl的SUV与250μl CD-RAP溶液(在420mM精氨酸/H3PO4中3mg/ml,pH7.5)混合并冻干。在用300μl蒸馏水使冻干块复水和温和旋转期间发生包封。根据HPLC和酶联免疫分析(ELISA)确定,这导致超过40%的包封率以及平均直径1.5μm的MLV,而没有包埋药物的化学变化。
127B:将111.4g磷脂酰胆碱(Lipoid S100)、37.1g胆固醇和1.5g抗坏血酸棕榈酸酯溶于800ml叔丁醇以获得分子分散的混合物。通过冷冻干燥定量地除去溶剂。通过用1.5L水使干燥脂质水合和剧烈摇动,形成直径几微米的多层脂质体。使用高压匀化器使脂质分散体匀质化,随后挤出通过100nm的膜以获得单分散的小单层囊泡(SUV)。将3ml SUV加至2.5ml CD-RAP溶液(在420mM精氨酸/H3PO4中3mg/ml,pH7.5)并且冷冻干燥。用3ml蒸馏水重建稳定均匀的冻干块,随后的摇晃产生多层脂质体(DRV)的均匀分散体,所述多层脂质体直径约1.5μm,包封率大于40%。
实施例2具有不同赋形剂例如融合促进剂的DRVs的制备
128分析不同赋形剂或添加剂例如融合促进剂对形成干燥重建囊泡(DRVs)的影响,所述干燥重建囊泡包括用于治疗以下例如各种疾病的亲水性蛋白质例如CD-RAP:软骨和骨疾病例如骨关节炎、软骨缺损或退行性盘疾病。测试其它添加剂和/或缓冲系统来代替使用根据实施例1的作为添加剂和/或融合促进剂的pH 7.5的精氨酸/H3PO4,并总结于表1。分析以下参数:通过光子相关光谱(PCS)的尺寸分布、通过渗压计的渗透压(osmolarity)、通过HPLC的蛋白质稳定性、重建囊泡的外观和包封率。
129表1具有不同融合促进剂的DRVs的特征
添加剂 | 蛋白质稳定性 | 平均尺寸 | 分散性 | EE[%] | 生理渗透压(physiol.Osmol.) |
海藻糖5%(w/v) | + | - | + | nd | + |
海藻糖3%(w/v)/甘露醇2%(w/v) | + | - | + | nd | + |
海藻糖2%(w/v)/甘露醇3%(w/v) | + | - | + | nd | + |
甘露醇5%(w/v) | + | - | + | nd | + |
PEG4000 5%(w/v) | + | + | - | nd | + |
甘氨酸1.1%(w/v) | - | + | + | - | + |
甘氨酸1.1%(w/v)/20mM KCl/150mMKH2PO4 | + | + | + | - | - |
350mM精氨酸/H3PO4/pH 7.5 | + | + | + | + | + |
350mM组氨酸/H3PO4/pH 7.5 | + | + | + | + | + |
350mM L-赖氨酸/H3PO4/pH7.5 | + | + | + | + | + |
磷酸缓冲盐水pH7.4 | - | + | + | nd | + |
130在使干燥脂质体复水后确定蛋白质稳定性、脂质体的平均尺寸、均匀分散性和包封率(EE)。nd:未确定,如果未满足其它要求,参数未确定。
131通过使用海藻糖、甘露醇和其混合物作为防冻剂,获得蛋白质和脂质体膜的稳定效果,导致未受影响的小单层囊泡而不是大的MLVs。其它制剂包括聚乙二醇4000作为防冻剂,其不能在冻干块复水成脂质体后确定均匀的分散体。作为公知的制剂缓冲系统的磷酸缓冲盐水的应用,在干燥过程中只添加pH6.0和pH 4.2的乙酸不能稳定蛋白质,导致蛋白质的强烈破坏。然而,本发明人发现包括氨基酸的制剂导致不同的令人惊奇的结果。作为添加剂的甘氨酸需要添加盐以维持蛋白质稳定性,但是导致在非生理介质中的重建脂质体制剂。然而,令人惊奇地是,添加碱性氨基酸例如但不限于精氨酸、组氨酸和赖氨酸满足获得均匀的脂质体溶液或包封在大多层脂质体(≥1.5μm)的CD-RAP的分散体的所有的要求,所述大多层脂质体具有高的包封率(≥40%),在制造过程中没有蛋白质的化学变化。
实施例3:使用″冷冻和解冻″方法制造脂质体
132在圆底烧瓶中使742mg磷脂酰胆碱(Lipoid S100)、248mg胆固醇和10mg抗坏血酸棕榈酸酯溶于20ml乙醇。在旋转蒸发器中定量地除去溶剂。产生的薄脂质膜在室温下通过平缓搅拌在7.8ml水中复水以得到脂质体(12.8%(w/v)脂质)。通过随后的超声处理制备直径约100nm的单层囊泡(SUV)。将234.8μl的SUV溶液与65.2μlCD-RAP溶液(在420mM精氨酸/H3PO4中1.15mg/ml,pH7.5)混合。3个冷冻和解冻循环(在液N2中冷却,随后在室温下解冻)后由于脂质膜的融合和脂质体形成,脂质体分散体变成乳状。随着冷冻和解冻循环次数的增加例如5个循环保持脂质体的形态。产物为粘性乳状的悬浮液,具有大于40%的包封率(根据实施例5方法A测定)。然而,与根据本发明方法制备的MLVs相反,通过冷冻和解冻方法产生的大多数脂质体仅是单层的。通过在双偏振光下的光学显微镜的脂双层(lamellarity)的检测显示低于5%的马耳他十字(maltesercrosses)(在多层脂质体中的检测参数),而根据本发明(例如实施例1)冷冻干燥制备的脂质体产生≥95%的多层脂质体(图1)。在冷冻和解冻方法的情况下形成单层脂质体而不是MLVs被文献(Liposomes,APractical Approach由R.R.C.New编辑,IRL Press(1990),58页最后一段)进一步支持。
实施例4:在脂质粉末重建时制造的脂质体
133在圆底烧瓶中使750mg磷脂酰胆碱(Lipoid S100)、250mg胆固醇和10mg抗坏血酸棕榈酸酯溶于20ml乙醇。在旋转蒸发器中定量地除去溶剂。产生的薄脂质膜在室温下通过平缓搅拌在10ml pH7.5的200mM精氨酸/H3PO4中复水以得到脂质体(10%(w/v)脂质)。通过随后的超声处理制备直径约100nm的单层囊泡(SUV)。将3000μl的SUV与2500μl的蒸馏水混合并冻干。在用3000μl CD-RAP溶液(在150mM Arg/PO4中0.3mg/ml,pH 7.5)使脂质冻干块复水和温和旋转期间发生包封。
134将这样产生的脂质体与根据实施例1A制造的脂质体对比。根据实施例5A确定包封率。
135令人惊奇地是,用CD-RAP溶液使冻干的脂质块复水导致非常差的20%±4%CD-RAP的包封率(n=4),而使根据实施例1A的CD-RAP和脂质的共冻干物水合导致3倍增加的60%±10%的包封率(n=10)。
136脂质和要被包埋的蛋白质的均匀紧密接触对于高的包封率而言是关键的,其通过组分的共冻干来最好地实现。
实施例5:CD-RAP的包封率的确定
137使用三种不同的方法确定根据上述实施例制造的脂质体中的CD-RAP的包封率。方法A用于通过离心从未包封的CD-RAP中分离脂质体内部的包封的CD-RAP。方法B使用透析步骤确定包封。方法C为通过离心/超滤的改进确定。
方法A:通过离心步骤确定
138用300μl注射用水非常仔细和缓慢地稀释100μl复水的脂质体溶液,以获得脂质体间的密度差异。较快的稀释导致脂质体的破坏以及引入的蛋白质的损失。稀释步骤对于形成脂质体和周围溶剂间的差异是必需的,其对于成功地使脂质体旋转下降也是必要条件。将稀释的脂质体溶液在室温下在16.000rcf下离心15分钟,并产生相当多的小球(pellet)和澄清的上清液。仔细地除去上清液,用磷酸缓冲盐水和0.01%(v/v)Tween 80稀释之后通过反相HPLC确定蛋白质水平(=未包封的蛋白质)。
139将小球用300μl 20%(w/v)Triton X-100溶液溶解,然后剧烈摇晃。令人惊奇地是,本发明人发现添加聚L-赖氨酸例如50μl 2%(w/v)的聚L-赖氨酸溶液对于离解通过离子相互作用结合至脂质的蛋白质而言是必须的。用550μl 50%(v/v)乙腈/0.1%(v/v)三氟乙酸稀释后,通过反相HPLC确定蛋白质浓度。
方法B:通过透析确定
140将1ml重建脂质体转移入硝酸纤维素酯膜的软管(hose)中。随后将溶液相对于30ml的350mM精氨酸/H3PO4 pH 7.5/0.1%(w/v)胎牛血清白蛋白透析。在4℃下孵育4小时并温和地摇晃后完成脂质体溶液的纯化。直接通过反相HPLC确定透析入接收介质(acceptormedium)的未包封的蛋白质的量。
方法C:通过离心/超滤确定
141 125μl复水的脂质体溶液在超滤装置(Amicon MicroconUltracel YM-100units,Millipore,CAT.No.:42413,100′000Dacut-off)中以13′200rcf离心60分钟。该步骤导致脂质体从周围溶液中的分离。在过滤中,使用标准曲线和渗透的体积通过RP-HPLC测量BMP-2的浓度来确定未包封的BMP-2的量。
实施例6:使用各种脂质的DRVs的制备
142不使用磷脂酰胆碱和胆固醇作为用于制备脂质体的组分,而是测试几种其它脂质以全部取代上述脂质组分或部分加至上述脂质组分上。
A.完全饱和的脂质
1431g完全饱和的大豆卵磷脂(LIPOID SPC-3)溶于20ml乙醇,在根据实施例1A的制备后可以获得10%SUV的溶液。将300μl的SUV溶液与250μl的CD-RAP溶液(在420mM Arg/PO4中0.3mg/ml,pH7.5)混合并在玻璃反应器中冻干,产生非常稳定的冻干块。添加300μl蒸馏水后,以非常慢的方式不在20分钟之下发生冻干块的复水,其不满足快速注射产品的要求。
B.添加带负电的脂质
144除了在大豆卵磷脂和胆固醇的脂质组合物中引起带负电的脂质外,根据实施例1A进行在DRV中CD-RAP的脂质体配制。
1.添加心磷脂
145向375mg磷脂酰胆碱(Lipoid S100)和125mg胆固醇中添加0mg、5mg、10mg、15mg、25mg和100mg心磷脂,并随后在10ml乙醇中溶解。根据实施例1A完成进一步的处理例如制备SUV、配方设计(formulation)、冷冻干燥。用0.3ml蒸馏水使脂质/蛋白质块复水和摇晃后,在包含0mg-25mg心磷脂的显示快速增加粘度的样品中获得多层囊泡。包含100mg心磷脂的样品产生具有高粘度的仅具有非常小囊泡的凝胶状溶液(通过光子相关广谱测量为400nm)。
146通过光子相关光谱确定脂质体的直径,随着添加25mg的心磷脂,其产生从平均直径1500nm开始至最终2500nm的增加。
2.添加抗坏血酸棕榈酸酯
147向于750mg磷脂酰胆碱(Lipoid S100)和250mg胆固醇中添加0mg、2.5mg、5mg、10mg、25mg、50mg和100mg抗坏血酸棕榈酸酯,并在20ml乙醇中溶解。根据实施例1A完成进一步的处理例如制备SUV、配方设计、冷冻干燥。用0.3ml蒸馏水使脂质/蛋白质块复水和摇晃后,在包含0mg-50mg抗坏血酸棕榈酸酯的显示粘度根据心磷脂的添加而快速增加的样品中获得多层囊泡。包含100mg抗坏血酸棕榈酸酯的样品产生具有非常高粘度的凝胶状溶液。
148然而,令人惊奇地是,与文献(Liposomes第二版,APractical Approach,Vladimir P.Torchilin和Volkmar Weissig编辑,Oxford University Press(2002),第7页)中所述的相反,使用高量例如1%以上抗坏血酸棕榈酸酯(图2),多层脂质体显示强烈的聚集的趋势,以及随着抗坏血酸棕榈酸酯的量的增加复水后显示较大的直径(图3),将此文献在此引入以作参考。
实施例7:在重建DRVs中的rhBMP-2的包封
149使用750mg磷脂酰胆碱(Lipoid S100)和250mg胆固醇,根据实施例1A制造冷冻干燥脂质体。将300μl的SUVs与250μl的rh-BMP-2溶液(在420mM精氨酸/H3PO4中0.50mg/ml,pH7.5和5.0),并且随后冻干。使用两种不同的rh-BMP-2制剂,coli源rhBMP-2(Ruppert,R.等.(1996)Human bone morphogenetic protein2contains a heparin-binding site which modifies its biologicalactivity.Eur.J Biochem.237:295-302)和CHO源rhBMP-2(InductOs,Wyeth Pharma GmbH)。使用300μl的复水产生大于80%进入直径约1.5μm的MLV的包封率。根据方法C-离心/超滤确定包封率。所有测量一式两份的完成。结果示于表2。
150表2不同pH值下在精氨酸/H3PO4中的rhBMP-2的脂质体包封的物料平衡
变量 | 未包封的 | 包封的 |
rhBMP-2,未糖基化,pH7.4 | 18% | 82% |
rhBMP-2,未糖基化,pH5.0 | 17% | 83% |
实施例8:兔前交叉韧带横断模型(transection model)
151为了评价MIA/CD-RAP是否减轻或预防在骨关节炎的动物模型中的软骨退变,使用兔前交叉韧带横断模型。在通常的麻醉和无菌条件下,通过内侧髌骨旁(medial parapatellar)切口(在内侧副韧带和髌韧带之间)接近(approach)兔的膝关节。使髌骨侧向移位,移动髌骨下脂肪垫并使其缩回以暴露整个前交叉韧带。将切口在层内缝合,缝合后应用绷带。在荧光检查和镇静下进行关节内注射。手术和注射方案如下:每组使用6个动物(组1:假手术(ACL未横切),组2:脂质体,组3:脂质体加低剂量MIA/CD-RAP,组4:脂质体加中剂量MIA/CD-RAP,组5:脂质体加高剂量MIA/CD-RAP,n=30)。使除了假手术组中的动物的动物进行5次注射。每10天完成注射。最后的注射之后10天杀死动物,获得X-射线。
152随后,样品固定在10%中性缓冲的甲醛中,在EDTA中脱钙并包埋入石蜡。切口用藏红O-快绿、苏木精和曙红染色,并进行组织学分析。
153在此模型中,递送CD-RAP的多层脂质体降低软骨破坏和进展。组织学分析证明了以中剂量和高剂量CD-RAP将包封入脂质体的CD-RAP注射入关节腔内(intrarticular space)的功效,其降低了OA的发展(p<0.05)。与假手术组相比,如保持在中剂量组的藏红O染色的密度所证明的,看来以中剂量递送的CD-RAP对于预防OA的发展是最有力的(p<0.05)。放射性同位素分析显示在所有治疗组(CD-RAP低、中和高剂量组)中OA的进展的预防(p<0.05)。
实施例9:纤维环刺伤模型(Annulus fibrosus puncture model)
154在该实施例中,在兔纤维环刺伤模型中在部分复原盘高度中CD-RAP的注射是有效的。
155可以通过使用规定的针规(needle gauges)使纤维环刺入盘来在青年新西兰白兔中诱导盘退变(Singh,K.等.(2005)Animalmodels for human disc degeneration.Spine J 5:267S-279S)。通过注射利多卡因至盘的背部区域提供局部麻醉之后,获得侧向的平放射图(lateral plain radiographs)以确定对于IVD高度的预注射基线值。随后将兔以侧躺(lateral prone position)姿势放置,使用后外侧腹膜后方法以暴露腰部IVDs。在每一兔中,用18G的针刺伤AF。四周后动物接受缓冲盐水(在PBS中)或作为对照的囊泡脂质体或2.5mg/ml CD-RAP(在PBS中)或脂质体包封的CD/RAP(2.5mg/ml)注射入髓核,然后经过12个周。通过核磁共振成像(MR I)扫描腰椎分析临床前结果,计算通过放射性观察监视的IVD高度,所述IVD高度使用成像软件和%DHI(手术后的DHI/手术前的DHIx100)的常规程序测量。对于IVDs的组织学分析,截面用苏木精曙红和藏红O染色。通过单向方差分析(ANOVA)评定组间的差异以用于统计学分析。
156每只兔子将用盐水溶液中的CD-RAP处理一盘,其余的用盐水溶液或脂质体或脂质体包封的CD-RAP处理。
序列表
<110>Scil Technology GmbH
<120>药物应用的干燥重建囊泡组合物
<130>41512PWO
<150>EP 06021093.7
<151>2006-10-06
<160>1
<170>PatentIn version 3.3
<210>1
<211>107
<212>PRT
<213>智人
<400>1
Gly Pro Met Pro Lys Leu Ala Asp Arg Lys Leu Cys Ala Asp Gln Glu
1 5 10 15
Cys Ser His Pro Ile Ser Met Ala Val Ala Leu Gln Asp Tyr Met Ala
20 25 30
Pro Asp Cys Arg Phe Leu Thr Ile His Arg Gly Gln Val Val Tyr Val
35 40 45
Phe Ser Lys Leu Lys Gly Arg Gly Arg Leu Phe Trp Gly Gly Ser Val
50 55 60
Gln Gly Asp Tyr Tyr Gly Asp Leu Ala Ala Arg Leu Gly Tyr Phe Pro
65 70 75 80
Ser Ser Ile Val Arg Glu Asp Gln Thr Leu Lys Pro Gly Lys Val Asp
85 90 95
Val Lys Thr Asp Lys Trp Asp Phe Tyr Cys Gln
100 105
Claims (21)
1.干燥药物组合物,其包括包含冷冻干燥活性剂的囊泡,所述干燥药物组合物包括:
a)至少一种脂质,
b)至少一种活性剂,
c)融合促进剂,和
d)不含膜稳定剂,
其中用水溶液使干燥药物组合物复水导致平均脂质体直径大于1μm的多层脂质体的形成,所述脂质体包封活性剂。
2.根据权利要求1所述的干燥药物组合物,其中至少一种活性剂为蛋白质,特别是亲水性蛋白质或其活性片段。
3.根据权利要求1或2所述的干燥药物组合物,其中融合促进剂为选自精氨酸、组氨酸、赖氨酸或瓜氨酸的碱性氨基酸。
4.根据上述权利要求任意一项所述的干燥药物组合物,其中不存在保护性的糖、糖醇或糖苷。
5.根据上述权利要求任意一项所述的干燥药物组合物,进一步包括无机或有机阴离子,具体地,选自琥珀酸根、富马酸根、柠檬酸根、苹果酸根、磷酸根、乙酸根和盐酸根。
6.根据权利要求2所述的肠胃外的药物组合物,其中亲水性蛋白质为骨和/或软骨再生剂。
7.干燥脂质体组合物的制备方法,所述干燥脂质体组合物用水溶液复水后形成平均脂质体直径大于1μm的包封活性剂的多层脂质体,该方法包括以下步骤:
a)在不存在有机溶剂下将脂质、脂质混合物或脂质膜脱水,
b)形成优选具有50至200nm平均直径的小单层囊泡,
c)添加活性剂的水溶液,
d)在步骤c)之后、之前或与c)一起,添加融合促进剂和任选的无机或有机阴离子,和
e)不添加膜稳定剂,将所述脂质分散体脱水。
8.根据权利要求7所述的方法,其中在步骤b)和/或步骤c)之后进行无菌过滤步骤。
9.可给药的脂质体组合物的制备方法,所述可给药的脂质体组合物包括平均脂质体直径大于1μm的包封活性剂的多层脂质体,该方法包括以下步骤:
a)在不存在有机溶剂下将脂质、脂质混合物或脂质膜脱水,
f)形成优选具有50至200nm平均直径的小单层囊泡,
g)添加活性剂的水溶液,
h)在步骤c)之后、之前或与c)一起,添加融合促进剂和任选的无机或有机阴离子,和
i)不添加膜稳定剂,将所述脂质分散体脱水,
j)用水溶液复水并且形成平均脂质体直径大于1μm的包封活性剂的多层囊泡,
其中在步骤b)和/或c)之后进行灭菌过滤步骤。
10.根据权利要求7至9任一项所述的方法,其中所述脂质包括至少一种中性脂质,优选至少两种中性脂质,更优选磷脂酰胆碱(PC)和胆固醇(Chol)。
11.根据权利要求7至10任一项所述的方法,其中融合促进剂为选自精氨酸、组氨酸、赖氨酸或瓜氨酸的氨基酸。
12.根据权利要求7至11任一项所述的方法,其中无机或有机阴离子选自琥珀酸根、富马酸根、柠檬酸根、苹果酸根、磷酸根、乙酸根和盐酸根。
13.根据权利要求7至12任一项所述的方法,其中所得的多层脂质体在冷冻干燥脂质体组合物的复水期间形成。
14.根据权利要求7至13任一项所述的方法,其中活性剂为蛋白质,优选亲水性蛋白质。
15.根据权利要求14所述的方法,其中活性剂为骨和/或软骨再生剂,优选CD-RAP或其活性片段。
16.根据权利要求7至15任一项所述的方法,其中活性剂的包封率大于40%。
17.通过权利要求7至8任意一项方法可获得的药物冷冻干燥组合物。
18.通过权利要求9至16任意一项方法可获得的药物组合物。
19.权利要求1至6或17任意一项所述的药物冷冻干燥组合物在制备药物组合物中的用途,所述药物组合物用于通过在用水溶液使冷冻干燥组合物复水后注射,治疗受试者中骨和/或软骨缺损、免疫性疾病优选骨关节炎、类风湿性关节炎和脊柱疾病。
20.权利要求10所述的药物冷冻干燥组合物的用途,其中注射是局部或非全身注射,优选盘内地或透盘地(transdiscally)注射入滑液、滑液腔、髓核、髓核腔。
21.权利要求19或20所述的药物冷冻干燥组合物的用途,其中至少每周一次进行注射。
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EP06021093 | 2006-10-06 | ||
EP06021093.7 | 2006-10-06 | ||
PCT/EP2007/008659 WO2008040556A1 (en) | 2006-10-06 | 2007-10-05 | Dried reconstituted vesicle formation for pharmaceutical application |
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CN2012104557068A Pending CN102940878A (zh) | 2006-10-06 | 2007-10-05 | 脊柱髓核植入物 |
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JP (2) | JP5404402B2 (zh) |
KR (1) | KR101333279B1 (zh) |
CN (3) | CN101528197B (zh) |
CA (1) | CA2664637C (zh) |
CY (1) | CY1113934T1 (zh) |
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HR (1) | HRP20130328T1 (zh) |
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CN102869679B (zh) * | 2010-04-27 | 2015-11-25 | Scil技术股份有限公司 | 稳定的水性mia/cd-rap制剂 |
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