CN1241549C - 两性脂质体及其应用 - Google Patents
两性脂质体及其应用 Download PDFInfo
- Publication number
- CN1241549C CN1241549C CNB028052137A CN02805213A CN1241549C CN 1241549 C CN1241549 C CN 1241549C CN B028052137 A CNB028052137 A CN B028052137A CN 02805213 A CN02805213 A CN 02805213A CN 1241549 C CN1241549 C CN 1241549C
- Authority
- CN
- China
- Prior art keywords
- liposome
- amphoteric lipid
- lipid body
- active component
- charge carrier
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 239000002502 liposome Substances 0.000 title claims abstract description 208
- 239000002800 charge carrier Substances 0.000 claims abstract description 46
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical group CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 claims description 51
- 150000002632 lipids Chemical class 0.000 claims description 50
- 238000002360 preparation method Methods 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 27
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 24
- 108020004414 DNA Proteins 0.000 claims description 20
- 230000007935 neutral effect Effects 0.000 claims description 18
- 239000003925 fat Substances 0.000 claims description 17
- 239000000463 material Substances 0.000 claims description 14
- 102000004169 proteins and genes Human genes 0.000 claims description 13
- RMFHJNQCSIVIEA-UHFFFAOYSA-N 3,4,5-trihydroxyheptane-2,6-dione Chemical compound CC(=O)C(O)C(O)C(O)C(C)=O RMFHJNQCSIVIEA-UHFFFAOYSA-N 0.000 claims description 12
- 235000012000 cholesterol Nutrition 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 claims description 11
- 150000008104 phosphatidylethanolamines Chemical class 0.000 claims description 11
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 claims description 9
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 claims description 9
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- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 claims description 9
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 claims description 9
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- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- LRYZPFWEZHSTHD-HEFFAWAOSA-O 2-[[(e,2s,3r)-2-formamido-3-hydroxyoctadec-4-enoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium Chemical class CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](NC=O)COP(O)(=O)OCC[N+](C)(C)C LRYZPFWEZHSTHD-HEFFAWAOSA-O 0.000 claims description 7
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- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 description 4
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Abstract
本发明涉及两性脂质体及其应用,该两性脂质体同时包含基膜的或构膜的正电荷和负电荷载体。
Description
技术领域
本发明涉及两性脂质体及其应用,该两性脂质体同时包含基膜的或构膜的正电荷和负电荷载体。
背景技术
脂质体这一概念包括三种能从生物膜中分离出的天然产物:磷脂、鞘脂、胆固醇及其衍生物。它还包括具有相同特征的合成物质,提及的代表物质有二乙酰甘油、二烷基甘油、3-氨基-1,2-二羟基丙烷酯或醚以及N,N-二烷基氨。
这些物质对于制备脂质体具有技术上的重要性。脂质体的应用之一是作为药物制剂中活性成分的载体。基于该目的,需要一种能与体液相兼容且可控的、能选择性定位释放药物的一种高效、稳定的包装。
不利之处在于将两种需求结合起来存在困难。包装越紧密、稳定,再一次释放被包裹的活性成分就越困难。因此,人们开发出在外界刺激下改变其性质的脂质体。人们已了解热敏感和PH敏感性的脂质体。PH敏感性脂质体具有特殊的重要性,因为PH这个参数可以随着生理环境的改变而发生变化,例如脂质体在细胞内被内吞(endocytotic)吸收或者通过胃肠道时的PH变化。根据文献情况,PH-敏感性脂质体特别包括胆固醇半琥珀酸酯(CHEMS)。
胆固醇半琥珀酸酯被用于与磷脂酰乙醇胺混合以制备PH-敏感性脂质体(Tachibana et al.(1998);BBRC 251:538-544,U.S.patent 4,891,208))。这样的脂质体能被多个细胞内吞,采取这种方式可将被载分子转运到细胞内,而无需破坏细胞膜的完整性。
CHEMS的阴离子特征使其具有很不利因素。由其制备的脂质体具有完全负电荷,其被细胞吸收的效率则很低。因此,无论上面描述的转运机理怎样,这些脂质体很难将大分子转运到细胞内。
具有可能最高价的、恒定的表面电荷的阳离子脂质体可以用来将活性成分转运至细胞内(转染)。这种带全部正电荷的粒子导致对细胞的静电吸附,从而有效地转运至细胞内。然而,这些化合物和由此产生的脂质体被限制到离体(in vitro)或来自于体外(ex vitro)的使用,因为这样的带正电荷的脂质体与血清中成分形成了无法控制的聚集。
根据文献描述的情况,将PH值限制到少数几个PK值,通常限定到胆固醇半琥珀酸酯的羧基PK值(大约4.5)是目前的PH-敏感性脂质体的弊端之一。该化合物的另一个弊端是对负电荷载体的限制。它们不适合与核酸及蛋白质进行有效的结合。
带正电荷的脂质体显示与核酸和蛋白质能很好的结合,并且能够将这些活性成分带到细胞中,但其缺点是不能在体内使用。
发明内容
基于以上现有技术的状况,使制备以下脂质体结构成为一大目标:
(i)允许包容制剂中的活性成分,
(ii)能将这些活性成分转运到生物细胞中,
(iii)适宜于在体内条件下使用,
(iv)生产简单、成本低。
本发明的目的通过两性脂质体而得以实现,该两性脂质体包括至少一个正电荷载体和一个不同于正电荷载体的负电荷载体,脂质体的等电点在4和8之间。该目的的实现是因为脂质体是在PH-依赖性的、电荷变化的条件下制备而得。
所形成的具有所需性质的脂质体结构,例如,在低PH时,构膜的和基膜的正电荷载体的数量超过负电荷载体的数量,则在高PH时,其比率会反过来。这种情况通常适用于可电离成分的Pka值在4-9之间时。随着介质PH值的降低,所有正电荷载体所带电荷更多,而所有负电荷载体都失去电荷。
以下为本发明所用的缩写形式:
CHEMS 胆固醇半琥珀酸酯
PC 磷脂酰胆碱
PE 磷脂酰乙醇胺
PS 磷脂酰丝氨酸
PG 磷脂酰丙三醇
Hist-Chol 组氨酰胆固醇半琥珀酸酯
构膜的或基膜的带电荷载体具有以下通用的两性结构:
带电荷基团-膜支撑物
已知的天然物或其改造形式都可作为膜支撑物,其包括,尤其是二乙酰甘油、二乙酰磷酸甘油(磷脂)和甾醇,还包括二烷基甘油、二烷基或二乙酰基-1-氨基-2,3-二羟基丙烷、具有8-25个碳原子的长链烷基或酰基、鞘脂、神经酰胺等。这些膜支撑物是本领域共知的。与支撑物结合的带电荷基团可分为以下6组:
强正离子,Pka>9,净正电荷:根据其化学性质,它们是,例如,铵、脒盐、胍盐或吡啶盐基团或有时是仲胺或叔胺官能基。弱正离子,Pka<9,净正电荷:根据其化学性质,它们尤其是含氮的碱,例如,哌嗪、咪唑和吗啉、嘌呤或嘧啶。存在于生物系统的分子片段,优选为,如4-咪唑(组胺)、2-,6-,或9-嘌呤(腺嘌呤、鸟嘌呤、腺苷或鸟苷)、1-,2-或4-嘧啶(尿嘧啶、胸腺嘧啶、胞嘧啶、尿苷、胸苷、胞苷)或吡啶-3-羧酸(烟酯或酰胺)。
具有优选Pka值的含氮碱也是由低分子量的羟基烷烃诸如羟甲基或羟乙基经过一次或多次取代氮原子而形成。例如,氨基二羟基丙烷、三乙醇胺、三-(羟甲基)甲胺、二-(羟甲基)甲胺、三-(羟乙基)甲胺、二-(羟乙基)甲胺或其对应的取代乙胺。
PH从4至9的中性或两性离子:根据其化学性质,这些中性基团是,诸如羟基、酰胺、硫醇或具有一个强正离子和强负离子基团的两性基团,诸如磷脂酰胆碱、氨基羧酸、氨基磺酸、甜菜碱或其它结构。
弱阴离子,Pka>4,净负电荷:根据其化学性质,它们尤其是羧酸。其包括脂肪族的含有至多12个碳原子的,且含0,1或2个烯类不饱和键的线性或支化的单-,双-或三羧酸和。在合适的情况下,羧酸也可以是芳香族的取代物。
其它的阴离子基团是能够存在于抗坏血酸中并能分离出的含羟基化合物或硫醇,N-取代的阿脲、N-取代的巴比妥酸、佛罗那(veronal)、苯酚或为硫醇基。
强阴离子,Pka<4,净负电荷:根据其化学性质,它们是诸如磺酸酯或磷酸酯的官能团。
两性电荷载体,PI在4.5和8.5之间,PI值以下为净正电荷,PI值以上为净负电荷:根据其化学性质,这些电荷载体由两个或更多的上述基团片段组成。实施本发明时,起初带电荷基团无论是在一个相同的膜支撑物上还是在不同的支撑物上都不是实质性问题。实施本发明时,PI值在5至7之间的两性电荷载体为特别优选的载体。
强正电荷化合物是,例如:
DC-Chol 3-β-[N-(N’,N’-二甲基乙烷)氨甲酰基]胆固醇,
TC-Chol 3-β-[N-(N’,N’,N’-三甲氨基乙烷)氨甲酰基]胆固醇
BGSC 双胍盐-亚精胺-胆固醇
BGTC 双-胍盐-三(氨乙基)胺-胆固醇
DOTAP (1,2-二油酰氧丙基)-N,N,N-三甲基铵氯化物
DOSPER (1,3-二油酰氧基-2-(6-羧基-精胺基(spermyl))-丙酰胺
DOTMA 氯化(1,2-二油酰氧丙基)-N,N,N-三甲基铵(Lipofectin)
DORIE 溴化(1,2-二油酰氧丙基)-3-二甲羟乙铵
DOSC (1,2-二油酰基-3-琥珀酰-顺-甘油胆碱酯)
DOGSDSO (1,2-二油酰基-顺-甘油-3-琥珀酰-2-羟乙基二硫化鸟氨酸)
DDAB 溴化二甲基二-十八铵
DOGS ((C18)2GlySper3+) N,N-二-十八氨基-乙二醇-精胺(Transfectam)
(C18)2Gly+ N,N-二-十八氨基甘氨酸
CTAB 溴化鲸蜡基三甲铵
CpyC 氯化鲸蜡基吡啶盐
DOEPC 1,2-二油酰基-顺-甘油-3-乙基磷酸胆碱
或其它O-烷基-磷脂酰胆碱或乙醇胺,赖氨酰胺、精氨酰胺或鸟氨酰胺和磷脂酰乙醇胺。
弱阴离子化合物的例子是:His-Chol 组胺酰-胆固醇半琥珀酸酯、Mo-chol 吗啉-N-乙氨基-胆固醇半琥珀酸酯或组胺酰-PE。
中性化合物的例子是:胆固醇、神经酰胺、磷脂酰胆碱、磷脂酰乙醇胺、四醚脂或二乙酰甘油。
弱阴离子化合物的例子是:CHEMS 胆固醇半琥珀酸酯、具有8至25个碳原子的烷基羧酸或二乙酰甘油半琥珀酸酯。其它的弱阴离子化合物是天冬氨酰胺、谷氨酰胺和PE,还有PS及其与甘氨酸、丙氨酸、谷氨酸盐、天冬酰胺酸、丝氨酸、半胱氨酸、苏氨酸、酪氨酸、谷氨酸、天冬氨酸或其它的氨基酸或氨基二羧酸形成的酰胺。根据相同的原则,羟基羧酸或羟基二羧酸和PS也是弱的阴离子化合物。
强的阴离子化合物是,例如:SDS 十二烷基硫酸钠、胆固醇硫酸酯、胆固醇磷酸酯、胆固醇磷酸胆碱、·磷脂酰甘油、磷脂酸、磷脂酰肌醇、二乙酰甘油磷酸酯、二乙酰甘油硫酸酯、鲸蜡醇磷酸酯或溶血磷脂(lyosophospholipids)。
两性化合物是,例如,His-Chol Nα-组胺酰-胆固醇半琥珀酸酯、EDTA-chol 亚乙基二氨基四乙酸胆固醇酯,Hist-PS Nα-组胺酰-磷脂酰丝氨酸或N-烷基肌肽。
本发明的脂质体包括多种含量的构膜或基膜的两性物质,以使其具有两性特征。这意味着脂质体能够完全改变电荷符号。在介质特定的PH中存在的脂质体的电荷载体数量可以利用以下公式计算出:
z=∑ni((qi-1)+10(PK-PH)/(1+10(PK-PH))
其中,
qi是各离子基团在其PK值以下的绝对电荷(例如,羧基=0,单个含氮碱=1,二级电离的磷酸盐基=-1,等。)
ni是脂质体中这些基团的数目。
在等电点,脂质体的净电荷数为0。对于可选择等电点的结构,其可通过将阴离子和阳离子部分混合而制得。
这种结构特别是能够被构建成,当PH值下降时,分子的电荷总体上从负的转变成正的。当制得的带这些结构的脂质体需要用于生理学中的相互关系上时,这样的电荷逆转尤其有利。只有整体上带负电荷的脂质体与血液和血清中的成分相兼容。正电荷会导致聚集。然而,带正电荷的脂质体具有非常好的融合性(fusogenically),并且能够将活性成分转运至细胞内。PH-依赖性的电荷逆转因此使得被构建的化合物与血清相兼容,原因在于这些化合物具有负电荷;而当它们被内吞吸收后,其电荷发生逆转,在细胞内只成为融合状态。
在本发明实施方式中的一个优选实施方式中,两性脂质体的等电点在5至7之间。
本发明也涉及包括至少一个两性电荷载体的两性脂质体,该两性电荷载体的等电点在4和8之间。
在一种优选的实施方式中,脂质体中两性电荷载体的等电点在5和7之间。
本发明还涉及至少包括一个两性电荷载体和一个阴离子和/或阳离子电荷载体的两性脂质体。
在一种优选的方式中,两性脂质体合适的等电点在5和7之间。
本发明的一个特殊方式是,本发明的脂质体包括磷脂酰胆碱、磷脂酰乙醇胺、二乙酰甘油、胆甾醇、四醚脂、神经酰胺、鞘磷脂、和/或二乙酰甘油。当然,可以用本发明所教导的方法通过很多脂质的结合制备脂质体。例如,脂质体可以利用大量的CHEMS(大约40%)和较少量的DOTAP(大约30%)来合成。在CHEMS中羧基的PK值下,该成分的负电荷总是被抑制着,而由正电荷载体整体支配。另一种制剂是将CHEMS和His-Chol相混合,正电荷载体HisChol的电荷随着CHEMS的负电荷损失得越多而变得越强。
对于本身是两性的His-Chol,如果其与例如磷脂酰胆碱的一种中性膜相结合,则也会形成一等电点与His-Chol相一致的两性脂质体。
本领域技术人员都知道如何通过本发明所教导的多种变化形式来调节各参数:
(i)脂质体电荷逆转时的电荷密度通过所用的电荷载体数量和Pka值调节,
(ii)电荷逆转曲线的斜率通过两种电荷载体的比率,通过载体的绝对数量和两种互补的PH-敏感性脂质体的最佳协同作用调节,
(iii)Z电势通过零值是由于两种电荷载体的比率或其PK值的位置调节。
在本发明所涉及的两性脂质体中,优选以下三类:
在第一类两性脂质体中,该脂质体包括至少一种正电荷载体和至少一种不同于正电荷载体的负电荷载体,该脂质体的等电点位于4和8之间,且该脂质体包括选自磷脂酰胆碱、磷脂酰乙醇胺、胆甾醇、四醚脂、神经酰胺、鞘磷脂和/或二乙酰甘油的中性油脂。
在另一类两性脂质体中,该脂质体包括至少一种两性电荷载体,该两性电荷载体在4到8的pH值范围内可逆转电荷(reversiblyrechargeable),而该脂质体包括一种选自磷脂酰胆碱、磷脂酰乙醇胺、胆甾醇、四醚油脂、神经酰胺、鞘磷脂和/或二乙酰甘油的中性油脂。
在再一类两性脂质体中,该脂质体包括至少一种两性电荷载体和至少一种阴离子和/或阳离子电荷载体,该两性电荷载体在4到8的PH范围内可逆转电荷,而该脂质体包括一种选自磷脂酰胆碱、磷脂酰乙醇胺、胆甾醇、四醚脂、神经酰胺、鞘磷脂和/或二乙酰甘油的中性油脂。
在一种优选的方式中,两性脂质体合适的等电点在5和7之间。
本发明进一步的变化方式中,脂质体的平均大小在50和1000nm之间,优选在70和250nm之间,特别优选在60和130nm之间。两性脂质体可通过本领域已知的方法合成,例如将乙醇注入到缓冲水溶液中的脂质体溶液中,通过水化干燥的油脂膜或通过去垢剂的透析得到。脂质体的大小可以改变,通常在50和10,000nm之间。相同大小的脂质体可通过高压匀化或过滤获得。
在本发明的一个优选变化方式中,脂质体包括一种活性成分。
合适的一种优选方式是活性成分为蛋白质、肽、DNA、RAN、反义核苷酸和/或诱饵(decoy)核苷酸。
本发明进一步优选的方式是脂质体内部具有至少80%的活性成分。
本发明也涉及一种将活性成分装入脂质体的方法,该方法是在一个确定的PH值下进行包囊,再将PH调节至第二个值以分离出未结合的物质。
本发明还涉及将活性成分装入脂质体的方法,该脂质体是可渗透的,并在一确定的PH值下封闭渗透通道。
本发明也涉及脂质体在通过脂质体层上沉积聚合物或聚电解质而制备纳囊中的应用。这样的物质可以一次或多次地沉积在表面上。重复沉积时,也可以在没有交联剂的存在下进行,这种脂质体的纳囊在WO00/28972或WO01/64330中有描述。当使用本发明所描述的物质,有一个好处是其与电解质的静电作用能被避免。众所周知,聚电解质与脂质体膜的电荷载体的相互作用能够导致膜成分的分层并形成油脂聚集体。很多情况下,这种分层与脂质体的渗透性有关。本发明的物质能使这种相互作用在涂层过程后被中止。如果此时PH值升高且膜和聚电解质之间不再存在任何的相互作用时,脂质体只能在纳囊中被无菌包裹,并因此避免了油脂聚集体的形成和与此有关的膜渗透。
本发明还涉及本发明的脂质体在包装和释放活性成分中的应用。此时,脂质体特别引起活性成分如核酸的有效包装。核酸与所述的脂质体在一个较低的PH值(大约3至6)下培养,待脂质体形成后,通过提高PH值(大约7至9)可以将附着在其外表的核酸洗掉。
可以用类似的方法包装蛋白质。有利的做法是将介质的PH值调至脂质体的PI值和蛋白质的PI值之间。已得到证明,特别有利的是二者的PI值相差一个单位以上。
本发明的又一个方面是将脂质体用于制备诊断学中的释放系统。
本发明的再一个优选的方式是将脂质体用于转染系统,即将活性成分带到细胞内。
本发明的另一个方式是利用脂质体通过膜的融合或渗透作用控制其内包含物的释放。例如,一种油脂的脂质体,其本身并不是构膜成分,可以通过与带电荷载体,如PE的结合而得以稳定。当电荷载体被转化成中性的、无电荷的或两性离子的状态,膜的渗透性就增加了。文献中(PE/CHEMS,Tachibana et al.)已知的脂质体状态允许在低PH值下有渗透能力,该PH条件在生理条件下只有在核内体内部或通过胃时才能获得。可以按照上述方法制备两性脂质体,并使其中性点为在pH4到9之间的任意预定值。在这样条件下制备的脂质体是可渗透的并能够将所包含物转运到介质中。
然而,脂质体制剂可以在渗透性更小的条件下制备、加工和储藏。在本发明的一个优选实施方式中,制备的脂质体能使其包含物在生理性PH值条件下被释放,而在低PH值下可安全地包裹该包含物。这样的脂质体尤其适于制备动力学上的缓慢释放制剂,包含物的释放只有在与体液接触时被启动,而在储藏或转运过程中不发生。
因此,本发明中的一个优选实施方式包括用于治疗目的的脂质体的应用,特别是利用有特定靶向作用的脂质体的应用。微弱的非特异性结合是将脂质体转运到靶点的先决条件。相反,强的非特异性结合会抑制脂质体向靶点部位的转运。可以通过文献中的其它方法进行特异性结合,即通过选择脂质体的大小或通过将配体结合到脂质体的表面,该脂质体再结合到细胞表面的靶向受体上。配体可以是诸如,抗体或其片段、糖、激素、维生素、肽如精氨酸-甘氨酸-天冬氨酸(RGD)、生长因子、胆红素或其它成分。
本发明的优选方式还涉及脂质体在体内治疗或诊断中的应用。优选地,这样的脂质体是在生理条件下有微弱的非特异性结合并伴随有微弱的融合趋向,而在变化了的条件下,结合力则强且融合能力也强。这样的脂质体是两性脂质体,其在生理条件下整体上为阴离子粒子,而在PH低于6.5的条件下,阳离子的电荷增加。在脂质体被内吞至细胞时才有这样的PH值。在肿瘤内和皮肤表层也存在这样的PH值。对体外器官进行灌注达一定时间后也会得到这样的低PH值。因此高结合力和融合性仅限于被细胞和特定组织占据的脂质体。结合力和融合能力的增加支持脂质体膜与细胞膜进行融合。这一行为导致了包容物被直接释放到细胞内,而没有释放危及包容物或细胞成分的核内体溶胞成分。
而且,将脂质体应用到持续释放制剂和/或作为循环性的储库也是适合的。脂质体也能够有利地应用在静脉内或腹腔内给药。本发明中的一个特别优选的方式是将脂质体作为体内、离体(in vitro)和体外(ex vivo)细胞转染的载体中的应用。
本发明的脂质体具有几个优点。即使是在中性PH条件下,含40%的HisChol和PC的阳离子脂质体可将核酸,如DAN,结合至其膜上。令人惊异的是,当上述脂质体再加入5%的PG来制备,并具有两性的特点,则这种结合就被完全抑制了。然而,随着PH值的降低,核酸与膜的结合能够再一次被恢复。因此,本发明的脂质体特别适合于核酸的PH-依赖性结合。
而且,我们惊异地发现一系列的蛋白质也具有所描述的核酸的行为表现。例如,抗体与本发明的脂质体膜的结合并不是在中性PH下,而是在弱酸性条件下进行有效的结合。这样的现象不能从中性油脂和CHEMS的PH-敏感性脂质体中观察到,也不能从中性油脂和HisChol的PH-敏感性脂质体中观察到。因此,这是两性脂质体的一种特殊性质。另人惊异的发现是,与已知构成的阳离子脂质体相反,本发明的脂质体与血清相兼容。因此,本发明一种合适的实施方式包括了这样的脂质体在治疗性质上的应用。与已知构成的阳离子脂质体相比,本发明的脂质体的一个优点是其与细胞的非特异性结合明显减少。
也惊异地发现,本发明脂质体的融合能力依赖于介质的PH值。与细胞生物膜相比,融合能力由选择的油脂和脂质体的电荷决定。通常,结合步骤先于真正的融合。然而,正如上面所描述的,脂质体与细胞膜的强结合并不总是需要的和应该发生的,这种情况只有在特定的细胞或组织中的控制条件下才发生。
脂质体也因此能被用于构建脂质体的载体以将活性成分转运至细胞内。所有不形成胶束的物质都在活性成分的考虑范围内。水溶性物质特别适合作为活性成分。它们包括许多蛋白质和肽,特别是抗体或酶或抗原,所有的核酸,而不管它们的分子量,也不管是从RAN或DAN衍生而来的。然而,它们也包括其它的生物大分子,诸如复合糖、天然产物和其它化合物,也有合成的或天然的低分子量活性成分,其用其它方法不能穿透细胞膜屏障。在载体的帮助下,这些物质能够被转运到细胞内并开始发生作用,这是在没有这种转运的情况下所不可能发生的。
因此,在本发明的教导下,制备得到这样的脂质体,其融合和结合的性质在不同的PH值下表现不同。负载大量的活性成分并将其转运到细胞内的与血清兼容的脂质体,也可以用这种方法制备。本领域的技术人员能够根据本发明阐述的内容,制备那些最适用于某特定用途的脂质体。
以下结合实施例对本发明进行详细的阐述,但本发明并不受限于这些例子。
具体实施方式
实施例1
带有电荷载体的两性脂质体的制备及其电荷性质,该电荷载体通常带负电,但也可以转化为正电
将His-chol(5mg)、7.8mg的POPC和2mg的DPPG溶于4ml的1∶1(v/v)的氯仿和甲醇的混合液中,再在旋转蒸发仪上彻底旋干。该油脂膜用4.3ml的适当的缓冲液(10mM Kac,10mM HEPES,150mMNaCl,PH7.5)水化,用超声处理该浓度为5mM的油脂达5分钟。然后,冻结悬浮液,融化后,再经过几次过滤(extrude)(Avestin LiposoFast,用200nm孔径的聚合碳酸酯滤器)。测定Z电势时,需将脂质体的浓度调节至0.2mM。用PH值是7.5或4.2的上述缓冲体系进行稀释。测定的Z电势位于18mV(在PH为7.5时)和+35mV(在PH为4.2时)之间。
实施例2
带电荷载体的两性脂质体的制备及其电荷性质,该电荷载体带恒定的正电荷和可变的负电荷
将POPC、DOTAP和CHEMS以下述所给摩尔比溶于4ml的1∶1(v/v)的氯仿和甲醇的混合液中,再在旋转蒸发仪上彻底旋干。该油脂膜用4.3ml的适当的缓冲液(10mM KAc,10mM HEPES,150mM NaCl,PH7.5)水化,用超声处理该浓度为5mM的油脂达5分钟。然后,冻结悬浮液,融化后,再经过几次过滤(Avestin LiposoFast,用200nm孔径的聚合碳酸酯滤器)。下表显示了Z电势随PH变化而产生的变化。
脂质体组分的摩尔百分比:
脂质体1 POPC 50 DOTAP40 Chems 10
脂质体2 POPC 50 DOTAP30 Chems 20
脂质体3 POPC 50 DOTAP25 Chems 25
脂质体4 POPC 50 DOTAP20 Chems 30
脂质体5 POPC 50 DOTAP40 Chems 10
表1:用mV表示的Z电势
PH | 脂质体1 | 脂质体2 | 脂质体3 | 脂质体4 | 脂质体5 |
4567.5 | 44.239.93729.2 | 38.425.621.41.8 | 34.727.216.4-7.9 | 31.722.12.5-18.9 | 16.23.3-7.3-34.6 |
一种适当组分的Z电势的高度和斜率可以在界限值范围之内选择获得。
实施例3
具有完全可转化性(switchability)化合物的两性脂质体的制备及其电荷性质
将His-chol(5mg)和9.8mg的POPC溶于4ml的1∶1(v/v)的氯仿和甲醇的混合液中,再在旋转蒸发仪上彻底旋干。该油脂膜用4.3ml的适当的缓冲液(10mM KAc,10mM HEPES,150mM Nacl,PH7.5)水化,用超声处理该浓度为5mM的油脂达5分钟。然后,冻结悬浮液,融化后,再经过几次过滤(Avestin LiposoFast,用200nm孔径的聚合碳酸酯滤器)。下表(表2)显示了不同的PH值和离子强度下的Z电势。
表2
PH | 不含盐 | 100mM的Nacl |
45678 | 45.626.9-4.1-31.4-45.7 | 20.22.2-5.2-15.3-25.4 |
实施例4
血清的聚集
油脂膜按照实施例1的方法制备。不含DPPG的油脂混合物作为对照样品。油脂膜在缓冲液(10mM的磷酸盐,150mM的氯化钠,PH为7.4)中被水化并按上述方法过滤。人血清用等量的缓冲液(10mM的磷酸盐,150mM的氯化钠,PH为7.4)稀释,通过离心将特定的成分和脂肪去除(20分钟,13000rpm,4℃);清澈的血清用孔径为0.2μm的过滤器进行无菌过滤。
将上述制备的脂质体以1mM的浓度加入到血清中,37℃下,温育15分钟。温育后,含有DPPG的脂质体悬浮液是均匀的浑浊体;然而,未观察到絮结现象。通过动力学的光散射方法测定脂质体的直径,其比起始样品的直径减小了10%。不含DPPG的脂质体的悬浮液很清楚地显示出絮结现象。
实施例5
膜的血清稳定性
除了血清聚集以外,还进行了活性成分(羧基荧光素,CF)在人血清存在下的沉淀研究。基于该目的,按照实施例2的方法制备了不同分解情况的POPC/DOTAP/CHEMS脂质体:POPC 100%(作为对照),POPC/DOTAP/CHEMS 60∶30∶10,60∶20∶20和60∶10∶30(摩尔百分比)。没有被包囊的CF通过凝胶过滤而被去除。为用于测试,将脂质体稀释到0.1mM,在37℃下,温育15分钟。以适当的次数移出30μL的样品,并将其用100mM的Tris缓冲液稀释至300μL,PH为8.2,测定荧光。用10μL的Tritron X-100(10%的水溶液)将脂质体溶解,得到100%的值。下表显示了被包囊的CF随时间的变化而发生的变化。
脂质体在4个小时的测试期间只在血清中损失了少量的CF。POPC/DOTAP/CHEMS 60∶30∶10,和60∶20∶20仍含有大约75%的CF,POPC和POPC/DOTAP/CHEMS 60∶10∶30甚至还保留最初CF含量的100%(见表3)。
表3
时间(分钟) | POPC | POPC/DOTAP/CHEMS60∶30∶10 | POPC/DOTAP/CHEMS60∶20∶20 | POPC/DOTAP/CHEMS60∶10∶30 |
01560120240 | 100%91%94%96%96% | 100%84%81%80%80% | 100%95%87%76%77% | 100%107%110%105%107% |
实施例6
DNA的结合
含有以下组分(摩尔百分数)的脂质体如实施例1的方法制备(所有数据都是以摩尔百分数表示)。
A:60 POPC 40 HisChol
B:55 POPC 40 HisChol 5 CHEMS
C:60 POPC 20 HisChol 20 CHEMS
将脂质体悬浮在缓冲液(10mM的醋酸钾,10Mmhepes,PH为4.2或7.5)中,浓度为0.2mM。将DNA溶液(45μL,1mgDNA(鲱鱼(Herring)精子,SIGMA D3159)的1mL水溶液)分别加到各种脂质体的样品中至1mL,并快速混合。培养15分钟后,用6mL的适宜的缓冲液装满,测定各种脂质体的Z电势(表4)。
表4
脂质体 | pH4.2 | pH7.5 | ||
ABC | -DNA+47.6+47.8+34.0 | +DNA-32.0-28.1-28.6 | -DNA+2.4+0.1-10.1 | +DNA-44.4-38.4-24.7 |
当正电荷过剩(pH为4.2)时,粒子发生很强的电荷逆转。在中性pH7.5时,高浓度的CHEMS(脂质体C)可以过度代偿HisChol的电荷,且粒子的Z电势为负值。只有少量的DNA与这样的粒子相结合。
实施例7
DNA的结合和分离
按实施例2的方法制备组分为POPC/DPTAP/CHEMS,比率为60∶15∶25和POPC/DCChol/CHEMS,比率为60∶15∶25(摩尔百分比)的脂质体。在PH值为4.2时,采取上述实施例的方法结合DNA并测定Z电势。然后,将样品的pH值调节至7.5,再一次测定Z电势。
混合物 Z电势(mv)
(a)POPC/DCChol/CHEMS 60∶15∶25(pH4.2)(聚集) -43.5
(b)POPC/DPTAP/CHEMS -43.7
(c)POPC/DCChol/CHEMS -18.5
(d)POPC/DPTAP/CHEMS -14.5
在DNA存在下,低PH值下测定的Z电势是负值;而最初的粒子是带正电荷的。在PH变成中性后,由于存在DNA而使电荷减少了。Z电势值接近未处理过的脂质体的Z电势(PH为7.5时,值为-11mV)。
实施例8
DNA的包容及未被包囊的物质的分离
将分别具有组分POPC60/DOTAP15/CHEMS25和POPC85/DOTAP15的两种脂质体制剂,制备成如前述的干燥油脂膜。每一种中油脂的总含量为4μmol。进行水化时,在PH为4.0时,将鲱鱼(Herrings)DNA溶解在10mM的醋酸钾,10mM的HEPES和100mM的氯化钠中。将DNA(4mg)直接加入到油脂膜中。得到的脂质体被冻结后,反复融化,然后通过200nm的滤器过滤。
每500μL的粒子与2.5mL的蔗糖溶液混合(0.8M的上述缓冲液中的蔗糖,PH值为4.2或7.5)。在其上,再加入1.5mL的0.5M蔗糖溶液和0.5mL的缓冲液。
然后,浮在上面的脂质体就与未结合的DNA分离开来。浮出后,将脂质体从缓冲液/0.5M的蔗糖接触面上移出。结合的DNA的量通过加入碘化丙锭进行测定。用Stewart检测法测定油脂的量。只有PC对Stewart检测法有反应。其它的油脂不采取该数值计算。结果显示于下表中(表5)。
表5
脂质体 | PH4.0 | PH7.5 |
POPC/DOTA/CHEMS60/15/25POPC/DOTAP 85/15 | 2μgDNA/μgDOTAP2.3μgDNA/μgDOTAP | 1.2μgDNA/μgDOTAP2.3μgDNA/μgDOTAP |
在改变PH值至7.5后,两性脂质体中只有大约一半的结合DNA浮起来。这样的物质是真正的被包囊的物质。类似的结果通过脱氧核糖核酸酶的消化得到。
DNA不能通过改变PH或通过增加离子强度的方法再一次从构成的阳离子脂质体中分出,而总是保留在外面。
实施例9
融合性质
具有下面组分的脂质体如实施例1的方法进行制备(所有数据以摩尔百分比表示):
(A)POPC 60 HisChol 40
(B)POPC 55 HisChol 40 CHEMS 5
(X)POPC 100
(Y)POPC 60 DPPG 40
兼性的(facultative)阳离子脂质体A或B与中性脂质体X或阴离子脂质体Y在缓冲液中(10mMHEPES,10mM醋酸钾,PH为4.2或7.5)中培育。通过动力学的光散射方法测定大小来分析可能发生的脂质体融合(表6)。
表6
脂质体1 | X | X | Y | Y |
脂质体2 | A | B | A | B |
PH4.2PH7.5 | 161.6nm191.8nm | 191.9nm202.4nm | 1689.3nm250.0nm | 2373.2nm206.3nm |
脂质体的起始大小在PH4.2时是161.8nm,在PH7.5时是165.9nm。
(A)183.2nm
(X)195.2nm
(Y)183.2nm
补充电荷对(YA和YB)的大小完全不同于具有中性脂质体X的混合悬浮液的大小。相互作用的程度取决于兼性的阳离子脂质体的电荷数量。与更大的脂质体融合的程度并不依赖于融合性脂质体PE。
实施例10
大分子的渗透性
将DOPE(13.75μmol),2.5μmol的CHEMS和10μmol的HisChol溶解在异丙醇中,在真空下除去溶剂。将缓冲液(1mg/mL的蛋白酶K,10mM的醋酸钾,10mMHEPES,150mM氯化钠,PH为4.2)中的蛋白酶K溶液(2.5mL)加入到干燥的油脂膜中。待膜被水化后,形成的脂质体通过400nm的膜过滤。没有被包裹的蛋白酶在梯度变化的蔗糖中,通过脂质体的浮出而被分离。在PH4.2和7.2(缓冲液同上,起始PH为4.2和8.0)时,通过在7.5mL的缓冲液中培育如此制备出的脂质体。在结合后,用0.1μm的膜滤除释放出的蛋白酶K。然后,用7.5mLTriton X-100的缓冲液溶液(同上,PH8.0)处理滤液中余下的脂质体。
测定所有滤液中是否含有蛋白酶K。在进行该测试时,使用了偶氮酪蛋白溶液(1M尿素中含6mg/mL的偶氮酪蛋白,200mM的tris硫酸盐,PH8.5)。将该溶液(500μL)与100μL的滤液或缓冲液混合,然后在37℃温育30分钟。通过加入10%的三氯乙酸终止该反应。沉淀的蛋白质通过离心去除。在390nm下测量显色度(表7)。
表7
温育的PH | Triton X-100 | 390nm下的吸收空白 |
4.24.27.27.2 | -+-+ | 0.01920.23450.22100.0307 |
如果脂质体的温育是在PH约为4.2时进行,几乎没有蛋白酶K释放。只有溶解在Triton X-100的脂质体才导致酶的释放。如果脂质体的温育是在PH约为7.2时进行,即使不加入Triton也会释放大量的蛋白酶K,并在第一次滤液中就能发现。即使后来加入Triton,几乎没有额外的酶从脂质体中溶解出来。
实施例11
蛋白质的结合
如前述实施例的制备方法制备组分为POPC50/DOTAP10/CHEMS40(所有数据以摩尔百分比表示)的脂质体。用0.26mg/ml的缓冲液(PH5.0或6.0的10mM MES,或PH7.0或8.0的10mM HEPES)中的溶菌酶水化油脂膜。水化后,将所有的样品反复冻结和融化,然后用超声匀化脂质体,并通过200nm的滤器过滤。
加入乙酸调节PH至4.0,制备脂质体悬浮液。然后从未结合的蛋白质中分离出浮起来的脂质体。蛋白质的包裹率如下表所示(表8)。
表8
包容期间的PH | 被包裹物质的百分数 |
5.06.07.08.0 | 4217580 |
该组成的脂质体显示了PI值为5;溶菌酶是PI值为11.5的碱性蛋白质。因此,在PH为6至8之间,这两种物质带有相反的电荷。一种有效的脂质体的包容方式是由静电吸引引起的。未被包裹的蛋白质在PH为4时被去除。在该PH值下,二物质间无相互作用。
实施例12
转染至细胞内
将HeLa细胞或CHO细胞(3×105)加入到6孔滴定板的每一个孔中,并培养3天。在荧光标记的右旋糖苷(10mg/ml的TRITC右旋糖苷的水化缓冲溶液)的存在下制备脂质体(POPC/DOTAP/CHEMS60/30/10)。未结合的TRITC右旋糖苷通过凝胶过滤除去。如此制得的脂质体加入到细胞中并在37℃培养6个小时。然后,用缓冲液冲洗涤细胞两次。再在显微镜下观察右旋糖苷的吸收情况。结果如图1所示。
实施例13
脂质体的结合和转染
如实施例2法制备组成为POPC/DOTAP/CHEMS/N-戊二酰-DPPE(50∶10∶30∶10(摩尔百分比))的脂质体。同时,在PH为7.5时,用3mg/mL的10mMHEPES和150mM氯化钠中的TRITC-右旋糖苷(分子量大约为4400)溶液进行水化。未包裹的TRITC-右旋糖苷通过Sephadex G-75柱凝胶过滤除去。用EDC(1-乙基-3-(3-二甲氨丙基碳二亚胺)(每400μL脂质体悬浮液中含3.5mgEDC)活化N-戊二酰DEPPs,然后在暗处搅拌5个小时以使环状的肽RCDCRGDCFC结合到脂质体表面。然后加入RGD肽(150μL缓冲液中含250μg)并持续搅拌过夜。通过凝胶过滤将脂质体与未被结合的肽分离。
将人内皮细胞(HUVEC)在特殊的介质中培养。将0.5mM配体修饰的脂质体和无RGD配体的对照脂质体悬浮液加入到细胞中。2个小时后,除去脂质体,用PBS缓冲液冲洗细胞室3次并在荧光显微镜下观察。已用RDG脂质体处理过的细胞中的TRITC荧光比对照脂质体的荧光显得更红。
实施例14
药物代谢动力学研究(PH-可转化的脂质体的血液水平和器官分布)
将POPC/Chol(60∶40),POPC/Hist-Chol/Chol(60∶20∶20)和POPC/DOTAP/Chems(60∶10∶30)(500μL)的脂质体注射到雄性Wistar鼠的尾静脉中。
脂质体悬浮液(50mM)通过水化相应制剂(在PH为7.5时,在10mMHEPES,150nm的氯化钠中加入0.03摩尔的[14]C-DPPC)和2mL含1mg的[3]H-菊粉)进行制备。经过3个冻结和融化循环,悬浮液通过400nm的膜反复过滤(LiposoFast,Avestin)。未包裹的[3]H-菊粉通过G-75Sephadex-柱凝胶过滤除去,然后在CENTRIPREP(微孔)离心机上浓缩。
将每种制剂的脂质体悬浮液(0.5ml)对4个实验动物给药,在5分钟、15分钟、60分钟、3个小时、12个小时和24个小时后抽取血液样品。膜片段和可溶解的包含物的放射活性通过闪烁法测定,以下为所测数值:
从血液获得的半衰期
POPC/Chol 超过120分钟
POPC/DOTAP/Chems 超过120分钟
POPC/Hist-Chol 超过120分钟
本发明的脂质体在血液中具有相对长的半衰期使其具备一个载体系统的基本先决条件。它们没有剧烈毒性,也不被网状内皮系统快速吸收。直到本试验结束,血液中3[H]与14[C]的放射活性比率仍稳定不变。因此,在任何情况下都没有发生通过补充胞溶作用而释放包容物的现象。
Claims (32)
1.两性脂质体,其中,该脂质体包括至少一种正电荷载体和至少一种不同于正电荷载体的负电荷载体,该脂质体的等电点位于4和8之间,且该脂质体包括选自磷脂酰胆碱、磷脂酰乙醇胺、胆甾醇、四醚脂、神经酰胺、鞘磷脂和/或二乙酰甘油的中性油脂。
2.如权利要求1所述的两性脂质体,其中,该脂质体的等电点位于5和7之间。
3.如权利要求1或2所述的两性脂质体,其中,该脂质体的平均尺寸在50和1000nm之间。
4.如权利要求3所述的两性脂质体,其中,该脂质体的平均尺寸在70和250nm之间。
5.如权利要求3所述的两性脂质体,其中,该脂质体的平均尺寸在60和130nm之间。
6.如权利要求1或2所述的两性脂质体,其中,该脂质体包括一种活性成分。
7.如权利要求6所述的两性脂质体,其中,该活性成分为蛋白质、肽、DNA、RNA、反义核苷酸和/或诱饵核苷酸。
8.如权利要求6所述的两性脂质体,其中,该脂质体内含有至少80%的活性成分。
9.两性脂质体,其中,该脂质体包括至少一种两性电荷载体,该两性电荷载体在4到8的pH值范围内可逆转电荷,而该脂质体包括一种选自磷脂酰胆碱、磷脂酰乙醇胺、胆甾醇、四醚油脂、神经酰胺、鞘磷脂和/或二乙酰甘油的中性油脂。
10.如权利要求9所述的两性脂质体,其中,该两性电荷载体的等电点在5和7之间。
11.如权利要求9或10所述的两性脂质体,其中,该脂质体的平均尺寸在50和1000nm之间。
12.如权利要求11所述的两性脂质体,其中,该脂质体的平均尺寸在70和250nm之间。
13.如权利要求11所述的两性脂质体,其中,该脂质体的平均尺寸在60和130nm之间。
14.如权利要求9或10所述的两性脂质体,其中,该脂质体包括一种活性成分。
15.如权利要求14所述的两性脂质体,其中,该活性成分为蛋白质、肽、DNA、RNA、反义核苷酸和/或诱饵核苷酸。
16.如权利要求14所述的两性脂质体,其中,该脂质体内含有至少80%的活性成分。
17.两性脂质体,其中,该脂质体包括至少一种两性电荷载体和至少一种阴离子和/或阳离子电荷载体,该两性电荷载体在4到8的PH范围内可逆转电荷,而该脂质体包括一种选自磷脂酰胆碱、磷脂酰乙醇胺、胆甾醇、四醚脂、神经酰胺、鞘磷脂和/或二乙酰甘油的中性油脂。
18.如权利要求17所述的两性脂质体,其中,该脂质体的等电点位于5和7之间。
19.如权利要求17或18所述的两性脂质体,其中,该脂质体的平均尺寸在50和1000nm之间。
20.如权利要求19所述的两性脂质体,其中,该脂质体的平均尺寸在70和250nm之间。
21.如权利要求19所述的两性脂质体,其中,该脂质体的平均尺寸在60和130nm之间。
22.如权利要求17或18所述的两性脂质体,其中,该脂质体包括一种活性成分。
23.如权利要求22所述的两性脂质体,其中,该活性成分为蛋白质、肽、DNA、RNA、反义核苷酸和/或诱饵核苷酸。
24.如权利要求22所述的两性脂质体,其中,该脂质体内含有至少80%的活性成分。
25.将活性成分装入权利要求1至24任意之一所述的脂质体中的方法,其中,在一个确定的PH值下进行包囊,再在另一个PH值下分离未结合的物质。
26.将活性成分装入权利要求1至24任意之一所述的脂质体中的方法,其中,使该脂质体具有渗透性,然后在一个确定的PH下关闭渗透通道。
27.如权利要求1至24任意之一所述的脂质体在制备纳囊中的应用。
28.如权利要求1至24任意之一所述的脂质体在制备诊断学释放系统中的应用。
29.如权利要求1至24任意之一所述的脂质体在制备用于转运和/或释放活性成分的药物中的应用。
30.如权利要求1至24任意之一所述的脂质体在制备持续释放制剂和/或循环储库中的应用。
31.如权利要求1至24任意之一所述的脂质体在制备静脉内或腹腔内给药剂型的药物中的应用。
32.如权利要求1至24任意之一所述的脂质体在制备体内、离体和体外转染细胞的载体中的应用。
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US20030099697A1 (en) | 2003-05-29 |
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BR0207775A (pt) | 2004-03-30 |
DE50210271D1 (de) | 2007-07-19 |
WO2002066012A3 (de) | 2002-12-19 |
JP2014031383A (ja) | 2014-02-20 |
JP2014218520A (ja) | 2014-11-20 |
DE10109897A1 (de) | 2002-11-07 |
CA2438116A1 (en) | 2002-08-29 |
JP5480764B2 (ja) | 2014-04-23 |
ATE363893T1 (de) | 2007-06-15 |
US20070269504A1 (en) | 2007-11-22 |
JP2011021026A (ja) | 2011-02-03 |
BRPI0207775B1 (pt) | 2015-10-20 |
JP2016104786A (ja) | 2016-06-09 |
EP1363601B1 (de) | 2007-06-06 |
WO2002066012A2 (de) | 2002-08-29 |
AU2002234643B2 (en) | 2007-06-21 |
US20070252295A1 (en) | 2007-11-01 |
JP2004525898A (ja) | 2004-08-26 |
ES2289079T3 (es) | 2008-02-01 |
US20110293695A1 (en) | 2011-12-01 |
CA2438116C (en) | 2011-10-11 |
CN1492756A (zh) | 2004-04-28 |
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