CN101022825B - 预防或治疗阿尔茨海默病的联合疗法及其试剂盒 - Google Patents
预防或治疗阿尔茨海默病的联合疗法及其试剂盒 Download PDFInfo
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Abstract
本发明涉及预防或治疗阿尔茨海默病(AD)的方法。根据所述方法,对人施用诱导血浆中淀粉状蛋白β(Aβ)被捕捉的手段,并用包含固定的载体的单采血液成份术装置处理该人,该固定载体可与血液或血浆流接触,并包含结合淀粉状蛋白β前体蛋白(APP)的载体,通过该单采血液份术装置将APP从该人的血液中去除。本发明还涉及用于实施该方法的试剂盒。
Description
本发明涉及预防或治疗阿尔茨海默病的联合疗法,以及用于实现该疗法的试剂盒。
淀粉状蛋白-β肽(Aβ)在阿尔茨海默病(AD)的神经病理学方面起着重要作用(Roher等,1993:“β-Amyloid-(1-42)is a major component ofcere-brovascular amyloid deposits:Implications for the pathology of Alzheimerdisease”PNAS 90:10836)。该病的家族性类型与淀粉状蛋白前体蛋白(APP)和早老蛋白(presenilin)基因的突变有关。这些基因中与疾病连锁的突变导致这种肽的42个氨基酸形式(Aβ42)的产生增加,它是在阿尔茨海默病的淀粉状蛋白斑(amyloid plaque)中发现的主要形式。该病的动物模型在商业上是可得的。过表达突变的人类基因APP(其中717位上的氨基酸是F而不是V)的PDAPP转基因小鼠,以年龄或脑依赖的方式逐渐显示出很多阿尔茨海默病的神经病理学标志(Games等,1995:“Alzheimer-typeneuropathology in transgenic mice overexpressing V717F β-amyloid precursorprotein”Nature 373:523)。
已经进行了用“正常的”而非以模拟表位(mimotope)为基础的疫苗作的接种研究。用聚集的Aβ42免疫转基因动物,免疫或者在AD型神经病理发作之前(6周),或在更大的年龄(11个月)进行:对年幼动物的免疫阻止了斑形成的发展、神经炎性营养不良(neuritic dystrophy)和astrogliosis。对年龄更大的动物的治疗显著降低了AD样神经病理。这种实验性接种方法诱导抗Aβ42抗体的产生,该抗体可以穿过血-脑屏障,攻击淀粉状蛋白斑(Schenk等,1999:“Immunization with amyloid-β attenuatesAlzheimer-disease-like pathology in the PDAPP mouse”Nature 400:l73)。随后,斑通过数种机制被去除,包括Fc受体介导的吞噬作用(Bard等,2000:“Peripherally administered antibodies against amyloid β-peptide enter the centralnervous system and reduce pathology in a mouse model of Alzheimerdisease”Nature Med 6:916)。这种疫苗也能延缓记忆缺陷(memory deficit)(Janus等2000:“Aβpeptide immunization reduces behavioural impairment andplaques in a model of Alzheimer’s disease”Nature 408:979)。
从1999年晚期起,一种很有前途的AD免疫治疗法已经进入临床试验。人们认为,免疫作用可引发免疫系统进攻斑,从受感染的人脑中清除这些沉积物,虽然潜在的精确机制尚需更详细地被表征。
这些临床试验是由Elan制药公司和它的合作者American Home Products联合实施的(治疗性疫苗AN-1792,QS21为辅剂)。一期试验于2000年成功地完成。二期试验于2001年晚期开始,以在一组轻度到中度AD的病人中检测功效。
现在这些二期试验已经永久性地终止了,因为在一些病人中发生了神经炎症(neuroinflammation)(2002年社论“Insoluble problem?”Nature Med8:191)。症状包括无菌性脑膜脑炎(aseptic meningoencephalitis),使这些世界范围的试验立刻停止。在最坏的病例中,受影响的患者显示出发生了自身免疫反应——自身免疫反应是很多免疫疗法中的内在风险。自身免疫并发症本来是可以预见的,考虑到APP是普遍存在的,它理所当然地与它的蛋白水解产物具有共同的抗原决定簇。最近,更多的研究集中在聚集的Aβ42免疫所诱导的抗体(在人和鼠中)的性质,这些研究揭示大多数抗体识别Aβ42的第4和10位氨基酸之间(Aβ4-10)的小的区域。小鼠抗体阻滞Aβ原纤维生成,破坏已存在的Aβ纤维(McLaurin et al 2002:“Therapeutically effective antibodies against amyloid-β peptide target amyloid-βresidues 4-10 and inhibit cytotoxicity and fibrillogenesis”Nature Med 8:1263)。值得注意的是,人抗体不与暴露在细胞表面的APP发生反应,也不与该前体的任何其他的非聚集蛋白水解产物发生反应(Hock et al 2002:“Generationof antibodies specific for β-amyloid by vaccination of patients with Alzheimerdisease”Nature Med 8:1270)。在人和小鼠血清中,观察到清楚的差别:与人抗体相反,小鼠抗体检测单体的、寡聚的和纤维状的Aβ。这是很重要的,可能是治疗效能的先决条件,因为表明不被人抗Aβ识别的Aβ的小寡聚体是这种病的主要毒性因素的证据正在越来越多,(toxic players)(Walsh etal 2002:“Naturally secreted olijgomers of amyloid β protein potently inhibithippocampal longterm potentiation in vivo”Nature416:535)。因此,一种可能的新策略是用包含β-淀粉状蛋白氨基酸4-10(而不是用聚集的Aβ42)的疫苗免疫。尽管功效未知,该策略也面临着自身免疫问题,因为病人将直接用(线性B细胞(linear B cell))“自身”表位免疫。
尽管最近在AD疫苗接种策略方面的发展令人失望,人们仍然认为Aβ疫苗是抗击AD的最有前途的方法。然而,AD疫苗接种迫切需要改进和新的策略。特别是,这种疫苗应该不诱导自身反应性的T和/或B细胞。
不过,越来越多的其他治疗法正在发展,这些治疗法应该能阻止淀粉状蛋白-β产生、淀粉状蛋白β聚集或由上述聚集体引发的神经毒事件。在Wolfe的综述文章(Nature Reviews Drug Discovery 1(2002 859-866))中总结了迄今为止研究过的针对AD的治疗法。
形成淀粉状蛋白-β斑的基础是所谓的淀粉状蛋白-β前体蛋白(APP),它是一种内在跨膜蛋白(关于它的生理功能尚未清楚地证明;然而,大多数最近的研究结果提示,APP作为所谓的驱动蛋白I的膜运货受体(membrane cargo receptor)而起作用)。APP在所谓的分泌酶作用下发生蛋白酶切,具体地,生理性地形成40个氨基酸长度的肽(Aβ40)。Aβ的其他更短或更长的片段也有形成,特别是一种42氨基酸的有高度的聚集潜力的形式(Aβ42)。因此,上述的Aβ42形式是在淀粉状蛋白斑中出现最多的形式。正因为此,一种可能的AD治疗策略主要集中在进攻负责上述不同切割的分泌酶(α-、特别是β-和γ-分泌酶)。因此,在AD治疗中,针对上述酶,现已尝试分别使用调节剂和抑制剂(例如,苯并二氮、磺胺、苯并己内酰胺)。
另一个与AD有关的基因是载脂蛋白E,其有3个等位变体存在(APOE2,APOE3和APOE4)。和总的人群相比,具有一个或两个APOE4拷贝的人比携带APOE2的人患AD的风险更大。研究还显示,服用抑制素,即抑制胆固醇生物合成的药物的人,患AD的风险显著下降。正因为此,进一步治疗AD的策略集中在抑制胆固醇生物合成,例如使用抑制素。
治疗AD的另一方面是抑制脑斑中淀粉状蛋白的聚合,这也可能通过分泌酶抑制剂等手段来实现。还有人提出要降低锌含量,因为如果锌以生理上有意义的浓度存在,就可能诱导Aβ的聚合。
在现有技术中已经提出的其它的治疗AD的策略涉及阻止APP的表达和增加Aβ的清除,其中为了实现所述阻止,寻找与APP启动子区相互作用的物质。对于Aβ的清除,有人提出增加特定的蛋白酶,例如胰岛素降解酶和中性溶酶(neprolysin)的活性,或外周使用抗Aβ抗体(De Mattos et al.,PNAS 98(15)(2001),8850-8855)。然而,这些测试,在小鼠模型中产生了矛盾的结果(Wolfe,(2002))。最后,人们尝试重新溶解已经存在的淀粉状蛋白斑,例如通过降低AD病人血浆中的淀粉状蛋白-β水平来达到这个目的。在这种背景下,还提出了利用单采血液成分术(apheresis)方法来降低脑中的β淀粉状蛋白的斑沉积物(US 6,551,226,其提出通过单采血液成分术去除分子量大于500kD的大分子),但未在AD中演示该方法。无论如何,在脑细胞中已存在的斑不可能由单采血液成分术直接溶解掉(大于500kD的斑或分子不能穿过血脑屏障)。
正如已经提到的,β-淀粉状蛋白(Aβ40和Aβ42)斑的存在是AD最显著的病理特征。因此Aβ的减少被认为是AD防治中的主要药理学目标。尽管有通过自动免疫诱导抗Aβ抗体导致淀粉状蛋白的移除的描述,在临床试验中,迄今为止这种免疫都因为严重的副作用而失败,这导致这种治疗的停止。后来的临床前结果显示,抗体可能(也可能)引起外周的Aβ减少,可能因此而改变Aβ外周脑动力学。
现已进一步显示,用对Aβ有高度亲和力的试剂(例如凝溶胶蛋白(gelsolin)或GM1)进行外周治疗,可使脑中Aβ的量下降(Masouka et al.,Journal ofNeuroscience 2003:29-33)。因此,有人提出将能够降低血浆中Aβ的含量并减少或阻止脑中淀粉样变(amyloidose)的发生的化合物作为一般手段。在此基础上,可以发展活性不依赖于穿越血/脑屏障的新治疗剂。
由所述血浆Aβ被捕捉(sequestration)所导致的Aβ从脑中流出(efflux),已经显示了对血浆中Aβ的含量具有方法依赖性的效果:血浆中Aβ的含量不被凝溶胶蛋白减少;相反,施用凝溶胶蛋白和用抗Aβ单克隆抗体进行被动免疫导致血浆中Aβ含量的升高。然而,在实验中,只有当使用相对年轻的APP转基因小鼠时,脑中Aβ的载量才下降;当使用超过6月龄的小鼠时,治疗证明无效。这可能由于在较老的小鼠的脑中,Aβ的不溶性增加。另一方面,长期的治疗可能会成功,然而,凝溶胶蛋白或GM1的施用和被动免疫都不适合长期治疗使用。
因此,本发明的目的是提供一种防治阿尔茨海默病的新策略,特别是一种还基于成功的免疫(immunization)的策略。
因此,本发明提供一种联合治疗方案,其包括诱导Aβ流出(efflux)的试剂和Aβ肽特异性的单采血液成分术。根据本发明,诱导Aβ流出(例如利用凝溶胶蛋白、GM1、Aβ-特异性的自动或被动疫苗等试剂来诱导),且通过Aβ单采血液成分术来维持该流出。在本文中,即使用疫苗进行一次或两次自动免疫,也足够诱导IgM和/或IgG介导的血浆Aβ被捕捉,所述疫苗包含Aβ、Aβ衍生物或Aβ模拟表位。
因此本发明的一个特别优选的方面涉及用于预防或治疗阿尔茨海默病(AD)的试剂盒,包括:
-诱导血浆中淀粉状蛋白β(Aβ)被捕捉的试剂,和
-单采血液成分术装置,包括能与血液或血浆流接触、并具有能结合淀粉状蛋白-β前体蛋白(APP)的受体的固体载体。
本发明的试剂盒中,APP结合受体优选选自抗APP抗体,(可溶的)Aβ结合受体,例如抗Aβ40抗体或抗Aβ42抗体,APP结合蛋白,特别是凝溶胶蛋白,apoJ或apoE,APP结合多肽,APP结合神经节苷脂,特别是GM1,或APP结合核酸,特别是适体(aptamers),或所述受体的混合物。
本试剂盒中,优选使用无菌的无热原的柱子作为单采血液成分术的载体。
本试剂盒中,诱导血浆中淀粉状蛋白β(Aβ)被捕捉的试剂优选选自对Aβ有高度亲和力的试剂,特别是凝溶胶蛋白或GM1、Aβ特异性的肽配体或核酸配体、Aβ特异性的自动或被动疫苗或Aβ特异性的人源化单克隆抗体。
Aβ特异性的自动疫苗优选是Aβ衍生物或Aβ模拟表位。
特别优选的Aβ衍生物选自部分或全部由D-氨基酸组成,和/或不由天然氨基酸组成的多肽。
Aβ模拟表位优选由下式的肽组成或包含下式的肽:
X1X2X3X4X5X6,
其中
X1是氨基酸,除了C以外,
X2是氨基酸,除了C以外,
X3是氨基酸,除了C以外,
X4是氨基酸,除了C以外,
X5是氨基酸,除了C以外,
X6是氨基酸,除了C以外,
其中,X1X2X3X4X5X6不是DAEFRH,所述肽与对天然的N-端Aβ42序列DAEFRH具有特异性的抗体有结合能力,并且其5聚体(5-mer)与对天然的N-端Aβ42序列DAEFRH具有特异性的抗体有结合能力。
特别优选下式的肽:X1X2X3X4X5X6,其中:
X1是G,或有羟基基团的氨基酸,或带负电荷的氨基酸,优选E、Y、S或D,
X2是疏水性的氨基酸或带正电荷的氨基酸,优选I、L、V、K、W、R、Y、F或A,
X3是带负电荷的氨基酸,优选D或E,
X4是芳香族氨基酸或L,优选Y、F或L,
X5是H、K、Y、F或R,优选H、F或R,
X6是S、T、N、Q、D、E、R、I、K、Y或G,优选T、N、D、R、I或G。
本文中,在蛋白质中天然存在的20种氨基酸可以被化学类似物或D-氨基酸代替;如,L、I和V可以被Nle、Nva、Cha或有其它线性或环状脂肪族侧链的α氨基酸所代替,W和F可以被芳香族氨基酸所代替,R和K可以被碱性氨基酸如鸟氨酸或高精氨酸所代替。丝氨酸和苏氨酸适合被有脂肪族和/或芳香族侧链,且该侧链终端有OH基团的氨基酸取代。这种变换的效率和有效性在例如PCT/EP04/00162中描述的实验模型能够很容易地进行检验。另外,对于抗体与肽的结合,也可能考虑空间因素(借助于计算机模型)。
特别合适的表位是选自下列的至少一种表位:EIDYHR、ELDYHR、EVDYHR、DIDYHR、DLDYHR、DVDYHR、DIDYRR、DLDYRR、DVDYRR、DKELRI、DWELRI、YREFFI、YREFRI、YAEFRG、EAEFRG、DYEFRG、ELEFRG、DRELRI、DKELKI、DRELKI、GREFRN、EYEFRG、DWEFRDA、SWEFRT、DKELR、SFEFRG、DAEFRWP、DNEFRSP、GSEFRDY、GAEFRFT、SAEFRTQ、SAEFRAT、SWEFRNP、SWEFRLY、SWELRQA、SVEFRYH、SYEFRHH、SQEFRTP、SSEFRVS、DWEFRD、DAELRY、DWELRQ、SLEFRF、GPEFRW、GKEFRT、AYEFRH、DKE(Nle)R、DKE(Nva)R或DKE(Cha)R。
依照本发明,Aβ42模拟表位用于抗AD接种:这种模拟表位诱导产生抗Aβ42但不抗天然APP的抗体,这种模拟表位可以用(单克隆)抗体和(商业上可获得的)肽文库(例如,按照Reineke et al.2002:“Identification ofdistinct antibody epitopes and mimotopes from a peptide array of 5520 randomlygenerated sequences”JImmunol Methods 267:37)来鉴定。使用这样的(单克隆)抗体,其不识别APP,而只检测带有氨基末端天冬氨酸的不同Aβ种类(这种抗体的实例在Johnson-Wood et al 1997中有描述:“Amyloid precursorprotein processing and Aβ42 deposition in atransgenic mouse model ofAlzheimer disease”PNAS 94:1550)。在本发明的过程中,这样的抗体已被证明是鉴定适用于疫苗的模拟表位的理想工具。虽然这样的单克隆抗体在AD的小鼠模型中直接对小鼠给药时显示了有益的作用(Bard et al 2000:“Peripherally administered antibodies against amyloid β-peptide enter the centralnervous system and reduce pathology in a mouse model of Alzheimer disease”Nature Med 6:916),但是这些抗体还从未被提出用作分离AD疫苗化合物的模拟表位搜索工具。
在现有技术中,所有努力都集中在天然存在的Aβ肽。如上所述,Aβ肽疫苗的临床试验,由于发生神经炎症事件而停止了。事实上,T细胞表位预测程序(BIMAS,用于限于I类MHC的表位;TEPITOPE,用于限于II类MHC的表位)提出在该序列内部存在高分值的(自身)表位。这可能意味着神经炎症事件是由于自身免疫反应,这使得这种疫苗不适合普遍使用。
与现有技术提出的这种Aβ疫苗相反,在使用包含本发明的模拟表位的疫苗的治疗过程中,预期不会发生自身免疫反应,因为用于根据本发明的模拟表位鉴定的(单克隆)抗体不识别APP,而且模拟表位序列不同于Aβ42衍生的自身序列,所述Aβ42衍生的自身序列先前已经被用于试验或将被用于未来的试验。
用于按照本发明模拟表位鉴定的抗体检测Aβ衍生的、带有游离氨基末端的天冬氨酸的氨基酸序列DAEFRH(=原始表位),因此不识别天然的APP。抗体是单克隆或多克隆抗体制品,或任何抗体的部分或其衍生物,唯一的先决条件是抗体分子特异性地识别DAEFRH表位,也就是说,它不与天然的N-端延长形式的淀粉状蛋白前体蛋白结合,这意味着,它对DAEFRH表位的结合能力比对APP分子的结合能力(binding capacity)至少高100倍,优选至少1000倍,更优选至少高105倍。抗体可以是和Johnson-Wood等1997所描述的抗体相比对DAEFRH序列有同样的或较高的结合能力的抗体。当然,结合能力较低的抗体也可能使用(>10%、>50%或>80%Johnson-Wood等的抗体的结合能力),虽然更优选较高的结合能力。
按照本发明的化合物与那些抗体结合的特异性与DAEFRH序列相比具有可比性。
按照本发明要使用的模拟表位优选长度是5-15个氨基酸。所述的化合物存在于疫苗中的形式可以是分离的(肽)的形式,或是与其它分子偶联,或者形成复合物(complexed),如药用载体物质或多肽、脂类或糖类结构。按照本发明的模拟表位优选长度是(最少)5-15、6-12个氨基酸残基,特别是9-11个。然而,模拟表位能够与非特异性的接头(linker)或载体(共价或非共价地)偶联,特别是与肽接头或蛋白载体偶联。而且,肽接头或蛋白载体可以由T细胞辅助表位(T cell helper epitopes)组成或者包含T细胞辅助表位。
药用载体优选为KLH、破伤风类毒素、白蛋白结合蛋白、牛血清白蛋白、树状聚体(dendrimer)(MAP;Biol.Chem,358:581)、以及由Singh等所描述的佐剂物质(Nat.Biotech.17(1999),1075-1081)(特别是那些在该文件中表1指明的物质)和O’Hagan等Nature review,Drug Discovery 2(9)(2003),727-735(特别是其中描述的内源免疫增效化合物和分配系统)或其混合物。而且,疫苗组合物可以包括氢氧化铝。
包括本化合物(模拟表位)和药用载体的疫苗可以通过任何适合施用的方式给药,例如,静脉注射、腹腔注射、肌肉注射、鼻内、口服、皮下,等,以及以任何适当的分配装置使用(O’Hagan et al.,Nature Reviews,DrugDiscovery 2(9)(2003),727-735)。疫苗一般含有本发明的化合物的量为0.1ng至10mg,优选10ng至1mg,特别是100ng至100μg,或者,作为选择,在100fMol和10μMol之间,优选10pMol至1μMol,特别是100pMol至100nMol。疫苗还包含典型的佐剂,如缓冲剂、稳定剂等。
按照本发明,单采血液成分术装置在联合治疗过程中的启动之后用来保持Aβ流出,所述装置包括能与血液或血浆流接触的固体载体,所述载体包括与淀粉状蛋白-β前体蛋白(APP)结合的受体。借助本单采血液成分术装置,通过单采血液成分术,可以特异性地清除AD患者和有患AD风险的人的APP或APP分解产物,特别是Aβ40或Aβ42,这样Aβ被捕捉的效果可以在第一步中保持。众所周知,Aβ42在中枢神经系统(CNS)和血浆之间存在动态平衡。如上所述,在小鼠模型中可以看到(DeMattos PNAS 2001,如上),外周使用抗Aβ抗体影响CNS和血浆中Aβ42的清除,降低Aβ42在脑中的载量,而没有抗Aβ抗体穿过血脑屏障。Matsuoka等(Journal ofNeuroscience 2003:29-33)通过外周施用其它的Aβ42结合分子(凝溶胶蛋白)证实了所述结果。由此,斑形成的过程可以在脑中一个非常好的易接近的位点被阻止,即已经在血液中,也就是说所述蛋白和分解的肽各自不能再回到脑中,也不能在那里聚集。脑中斑形成的过程也可以通过捕获血液中的Aβ42而被阻止。在此过程中,在单采血液成分术装置中与患者的血液或血浆接触的受体对Aβ42或APP的其它分解形式是否具有特异性并不重要,唯一重要的是APP和它的(蛋白水解的)分解产物特别是Aβ42由所述特异吸附作用从血液中被除去,从而没有“错误的”蛋白分解作用(即生成Aβ42)发生或没有斑的形成。因此,本发明是基于与US 6,551,266完全不同的单采血液成分术的使用方法,即基于排除已经潜在的结构性的斑组分而不是斑本身。另外,对于借助单采血液成分术治疗AD而言,可以推知通过单采血液成分术消除斑是无效的,因而将其排除在外,原因是血液单采血液成分术不能到达脑中斑形成的区域。
另一方面,和其它的导致Aβ在体内自我损耗(例如,DeMattos等,PNAS98(15)(2001),8850-8855,用外周抗Aβ抗体)且进行较长时间的方法相比,本发明的联合治疗方案具有决定性的优势,即不会引发自身免疫反应。而且,按照本发明,不必给患者提供只能在体内(可能只有在被运到特定位点后才)起作用的物质,而致病因素被选择性地去除,也就是,疾病的起因以体外的方式被特异性地去除,从而不必排除体内反应产物。
按照本发明,所有实施方式的现存的和已知的单采血液成分术装置可很容易地适用于本发明。特别是,当选择固体载体(和单采血液成分术装置)的时候,它的/它们的医疗适用性是应该考虑的。这样的载体、方法或装置在US 5,476,715,US 6,036,614,US 5,817,528或US 6,551,266中有描述。相应的商业的单采血液成分术装置由以下公司销售,Fresenius,Plasmaselect,ASAHI,Kaneka,Braun等,它们提供例如LDL-Therasorb,Immusorba,Prosorba,Globafin,Ig-Therasorb,Immusorba,Liposorba,HELP,DALI,Bilirubin-Bile-Acid-Absorber BR-350,Prometheusdetoxication,MARS,ADAsorb of Medicap或Plasma FLO等系统。虽然所有这些系统的商业上可获得的形式并不总是主要针对单种蛋白的特异性排除,但是单采血液成分术领域的技术人员能够容易地将其适用于本发明,例如作为免疫单采血液成分术(irmmuno aphrensis)和/或通过在单采血液成分术装置中安装发明的固体载体(例如柱子)。
因此,按照本发明,“APP结合受体”理解为对配体APP及其生物副产物(biological by-product)特别是Aβ42有亲和力,并且能够从AD患者及有患AD的风险的人的血液或血浆中去除所述多肽的所有物质。所述APP和Aβ42受体各自优选(单或多克隆)抗体、蛋白、肽、神经节苷脂或核酸。
特别优选的是抗APP抗体、抗Aβ40抗体或抗Aβ42抗体、APP结合蛋白特别是凝溶胶蛋白、apoJ或apoE、APP结合肽、APP结合神经节苷脂特别是GM1、或APP结合核酸特别是适体、或所述受体的混合物。
这样的抗体的例子有,3D6(Aβ1-5)、2H3(Aβ1-12)、2G3(Aβ33-40)、21F12(Aβ3342)、12H7(Aβ33-42)(Johnson-Wood et al.,PNAS 1997:1550-1555)、10D5、16C11(Bard et al.,Nature Medicine 2000:916-919)、DeMattos等(2001)描述的抗体(m266,m243),以及有同样特异性的抗体。这样的抗体可以通过例如用包含APP、Aβ42或其片段或变体的疫苗制剂免疫哺乳动物,然后任选地进行细胞融合和克隆选择(用单克隆抗体)过程而获得。
APP-结合蛋白受体的更多例子有凝溶胶蛋白(Matsuoka et al.2003,见上述)、apoJ或apoE(DeMattos et al.,2001,见上述)。GM1是APP结合神经节苷脂受体的例子(Matsuoka et al.2003,见上述)。
在本文中,作为APP-结合受体的肽可以由D型或L型氨基酸,或由D型和L型氨基酸的组合组成,并任选地通过进一步的修饰、成环或衍生化而被改变。合适的肽受体,例如Aβ42的受体,可由商业上可得到的肽文库提供。这些肽的长度优选至少5个,优选6个氨基酸,特别是至少8个氨基酸,其中优选的长度为最多10个、优选最多14个或20个氨基酸。然而,按照本发明,更长的肽用作APP-结合受体也没有问题。而且,寡聚物(象聚乙烯亚胺(polyethylenimine)和聚赖氨酸)是合适的受体。
当然,噬菌体文库、肽文库(见上述),或例如由组合化学或对不同结构的高通量筛选技术获得的结构文库,也适合生产这样的APP-结合受体。
而且,基于核酸的APP-结合受体(“适体”;还有“诱饵”(decoy)寡脱氧核苷酸(就它们的序列而言组成转录因子的结合位点的双链寡核苷酸)),其中所述的核酸可以被各种(寡核苷酸)文库(例如有2-160个核酸残基)检测(例如,Burgstaller et al.,Curr.Opin.Drug Discov.Dev.5(5)(2000),690-700;Famulok et al.,Acc.Chem.Res.33(2000),591-599;Mayer et al.,PNAS 98(2001),4961-4965;及很多其它的)。核酸骨架可以例如通过天然磷酸二酯(phosphor diester)化合物和硫代磷酸酯(phosphorothioate)或组合或化学变体(如作为PNA)来检测,其中按照本发明,主要是U、T、A、C、G、H和mC能作为碱基使用。按照本发明可用的核苷酸的2’残基优选是H、OH或其它的2’位置保护性基团或修饰基团,其中核酸也能被修饰,例如,被提供给保护性基团,如它们经常用于寡核苷酸的合成那样。“保护性基团”理解为氧原子的醚化,而-OH-基团被与2’位置修饰不同的物质代替。在现有技术中描述了二者的很多不同的可能性;甲基、烯丙基、丙基和类似的保护性基团(即,例如,2’OCH3,2’-O-CH=CH3,等)是特别优选的;特别优选的修饰是2’-脱氧、2’-氨基、2’-氟、2’-溴、2’-叠氮基(azido),还有金属,例如硒等。另外,按照本发明,为反义技术(核酶(ribozymes)、RNAi等)而发展的寡核苷酸稳定化处理方法可用于提供核酸(对比,例如,在这个领域领先的ISIS和Ribozyme Pharmaceuticals公司,特别是它们的专利文件和主页)。
因此在本发明的范围中APP-结合的适体(按照本发明和上面定义的,也包括Aβ42结合的适体)也优选作为APP-结合受体。
因此,优选由肽、抗体或核酸组成的APP-结合受体被用作载体材料,用于在阿尔茨海默病病人(或具有患该病倾向的人)中体外地消除APP及其蛋白分解产物。
在利用该发明进行常规的医疗时,载体要求是无菌和无热原的,因此按照本发明,满足该特性的任何载体物质和任何受体/载体组合都是优选的(参考例如US 6,030,614或US 5,476,715)。合适的例子包括含有乙烯基的单体(如丙烯酸,例如TSK Toyopearl,Fractogel TSK)的多孔的同聚物、共聚物或三元共聚物,被含有环氧乙烷的化合物(如环氧氯丙烷)修饰(活化)或可选地进一步与含有NH3、氨基或羧基、CNBr或CNCl吸附剂(如在EP110,409 A和DE 3,617,672A中所描述的)反应的载体。优选用于治疗目的的吸附材料适合于避免血细胞损失,不(或很少)激活补体系统,并在体外循环中最大程度上延迟聚集物形成。所用的载体材料优选地应该在与受体偶联的形式下对灭菌手段也是稳定的,特别是对环氧乙烷饱和、戊二醛饱和、γ射线、蒸汽处理、UV、溶剂或去污剂等稳定。也可以使用基于sepharose、琼脂糖、丙烯酸、乙烯基和葡聚糖的产品,它们的优选适合结合APP-结合受体的功能基团已经是商业上可获得的。一些整体料(monoliths)(如通过交联的缩水甘油甲基丙烯酸酯-乙二醇二甲基丙烯酸酯共聚物的载体)也可以用来做载体。
可以使用本领域技术人员已知的化学反应把受体偶联到合适的载体上(如Bioconjugate Techniques,Greg T Hermanson主编Academic Press,Inc.San Diego,CA,1995,PP 785)
本发明的另一个方面涉及本发明的设备的如下用途:通过调节该设备使之适用于治疗各个患者,在本发明的联合疗法的范围内,为阿尔茨海默病提供治疗或治疗设备,或预防该疾病。进行治疗时,患者与单采血液成分术装置连接足够长时间,以有效的去除APP多肽,其中患者的血液或血浆流和含有APP结合受体的固体载体相接触,APP和/或APP的蛋白水解物(特别是Aβ42)被结合在该载体上面。在单采血液成分术治疗过程中,当然必须保证外周或中枢静脉通路及动静脉瘘,及充分地防止凝血,记录所述的数值和测量的数据。而且,大多数单采血液成分术方法要求在正式的血浆处理之前初步分离血浆和血细胞。需要这种预防性措施的特殊人群包括有家族因素的老人(>50、>60或>70岁)或者有另外的AD危险性因素特别是遗传因素的人。
根据另一个重要的方面,本发明与涉及一种预防或治疗阿尔茨海默病(AD)的方法有关,其中,
-对人施用诱导血浆中淀粉状蛋白β(Aβ)被捕捉的试剂,并用单采血液成分术装置处理该人,该装置包括能与血液或血浆流接触的固体载体,该载体有与淀粉状蛋白-β前体蛋白(APP)结合的受体,其中利用该单采血液成分术装置将APP从该人的血液中去除。
优选用本发明的试剂盒进行所述方法。
因此,本发明也涉及如上面定义的Aβ模拟表位在生产本发明的预防或治疗AD的联合治疗中使用的试剂中的用途。
下面将通过实施例更详细地解释本发明,当然,本发明不受这些实施例的限制。
1.携带APP受体的载体的生产
1.1.整体柱(monolithic column)
按照产品说明书,用0.5 M pH8.0的磷酸钠缓冲液平衡CIM环氧整体柱(Epoxy Monolithic Column)(BIA Separations,SI),同时,按照产品说明书,将抗Aβ肽的单克隆抗体激活,并与CIM柱偶联。柱子用磷酸盐缓冲液(+1M NaCl)洗几次,多余的环氧基团任选被封闭。
通过控制洗涤和平衡洗出液来保证质量;只有当洗出液中没有活性环氧基团和没有抗体泄漏时,柱子才可用于后面的过程并安装在单采血液成分术装置中。
1.2.Sepharose柱
将散装琼脂糖材料(sepharose CL4B)在无菌条件下装入无菌的且无热原的容器中,材料在无菌条件下洗涤,每步洗涤之间,凝胶材料都在真空中完全干燥。然后,将sepharose在高压灭菌锅中,在115℃条件下蒸汽灭菌30分钟。
灭菌之后,将sepharose装入盛有60%丙酮/水的无菌容器中,用CNBr和三乙胺(每96ml丙酮加14g CNBr;66.2ml 87%丙酮加30ml三乙胺)活化。然后,加入丙酮/HCl溶液(392ml消过毒的且无热原的水;16.3ml 5N HCl,408ml丙酮)。活化的琼脂糖在2h内洗涤并用于偶联反应,以防止已活化的基团水解。
将过滤除菌过的抗体溶液(分别是m266或m243)导入反应容器中,至少搅拌90min。最后,(用等渗磷酸盐缓冲液)彻底洗涤反应溶液,直到在洗出液中不能检测到反应产物,将抗体偶联的琼脂糖装入无菌且去热原的有玻璃熔渣的玻璃柱中,进行最后的质量检查(针对反应产物、重金属等的洗脱液分析;颗粒分析,热原性;无菌性)。
2.阿尔茨海默病患者的单采血液成分术治疗的动物模型
在过去的几年中,德国Karlsburg的“Gerhard Katsch”糖尿病研究所已经建立了一种用于在自由活动的小动物身上进行实验性单采血液成分术的特殊体外系统。这种单采血液成分术疗法可以在同一个动物身上重复进行。而且,所用的动物也能被用于后续的研究,以对单采血液成分术疗法作长期评估。在数种大鼠株系中已经成功地使用了上述的实验性单采血液成分术系统。当大鼠的体重超过250g时,有I型糖尿病的大鼠和胶原蛋白II型诱导的关节炎的大鼠能够很好地忍受重复的单采血液成分术治疗。
在实验性单采血液成分术治疗开始前,给动物提供动脉和静脉导管。在单采血液成分术的第一步,用血浆过滤器分离血细胞和血浆。当血细胞被直接重新注入动物体时(通过静脉导管),引导分离的血浆通过在实例1中生产的吸附试剂(其中由于与固定化的受体结合,配体被从血浆中分离出来),然后将血浆重新供应给动物。
作为选择,也可以使用全血的单采血液成分术,与对LDL的DALI单采血液成分术类似。
3.在动物模型中的本发明的联合疗法:
基本上,在动物模型中进行的联合疗法时,Aβ流出可在单采血液成分术之前、过程中或之后发生。此外,两种疗法使用的频率相对彼此可以变化。
Claims (4)
1.在预防或治疗阿尔茨海默病(AD)中应用的试剂盒,包括:
诱导血浆中淀粉状蛋白β(Aβ)被捕捉的手段(means),其中所述诱导血浆中Aβ被捕捉的手段选自凝溶胶蛋白(gelsolin),GM1,或Aβ-特异性抗体,和
单采血液成分术装置,其包括能与血液或血浆流接触的固体载体,该载体具有与淀粉状蛋白-β-前体蛋白(APP)结合的受体,
其中所述APP结合受体选自抗APP抗体,抗-Aβ抗体,APP-结合蛋白凝溶胶蛋白,apoJ或apoE,APP-结合神经节苷脂,或所述受体的混合物,
其中诱导血浆中淀粉状蛋白β(Aβ)被捕捉的手段被施用于人和所述人在体外循环中用单采血液成分术装置治疗,其包括能与血液或血浆流接触的固体载体,该载体具有与淀粉状蛋白-β-前体蛋白(APP)结合的受体,
其中通过单采血液成分术装置从人的血液中去除APP。
2.根据权利要求1的试剂盒,其特征在于所述抗-Aβ抗体是抗Aβ40抗体或抗Aβ42抗体。
3.根据权利要求1的试剂盒,其特征在于所述APP-结合神经节苷脂是GM1和APP结合适体。
4.根据权利要求1-3中任一项的试剂盒,其特征在于所述载体是无菌的和无热原的柱子。
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TWI350173B (zh) | 2011-10-11 |
EP1765388B1 (de) | 2013-04-17 |
KR101158600B1 (ko) | 2012-07-09 |
CA2577332C (en) | 2014-12-09 |
JP2008506665A (ja) | 2008-03-06 |
US20110201987A1 (en) | 2011-08-18 |
CN101022825A (zh) | 2007-08-22 |
AU2005261687B2 (en) | 2011-02-17 |
DK1765388T3 (da) | 2013-07-22 |
AT500483A4 (de) | 2006-01-15 |
AT500483B1 (de) | 2006-01-15 |
HK1103354A1 (zh) | 2007-12-21 |
WO2006005706A3 (de) | 2006-07-20 |
PT1765388E (pt) | 2013-05-24 |
TW200602072A (en) | 2006-01-16 |
ES2414872T3 (es) | 2013-07-23 |
JP2013116903A (ja) | 2013-06-13 |
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