CN1122718C - 能修饰外周感觉传入功能的肉毒毒素衍生物 - Google Patents
能修饰外周感觉传入功能的肉毒毒素衍生物 Download PDFInfo
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Abstract
本发明涉及对外周感觉传入特异性的试剂。该试剂经过控制从第一感觉传入释放的至少一种神经递质或神经调制物可抑制第一感觉传入与触突神经元之间的信号传递。该试剂可用于或作为药品用于治疗疼痛，特别是慢性疼痛。
Description
技术领域：
本发明涉及能修饰外周传入功能的新试剂。该试剂可抑制从神经元的分离的群体释放神经递质，从而减轻或优选阻止从外周将传入痛觉信号传递到中央痛觉纤维。该试剂可用于或作为药品用于治疗疼痛，特别是慢性疼痛。
背景：
在传统上将触觉认为是5个标准的感觉之一，但实际上它是高度复杂的，传导许多不同的感觉。经过各种各样的特化神经末梢和相关结构在外周检测到这些感觉。其中一些对各种机械刺激具有特异性，如接触，压迫，振动及毛发或胡须的变形。另一类神经能检测温度，由热和冷激活不同的纤维。还有一个类群的神经末梢在正常情况下不被轻微的刺激激发，只被强刺激激发。这类感觉神经常相应于一种以上的刺激，称为高阈值多形纤维。它们可用于感觉潜在的损伤状态或物体。这种多形纤维也传导化学信号，如由酸性引起的“灼烧”感觉。因此，触觉可传递对物体非常详细的描述及用于提示和警告偶然事件。
感觉信号从外周向感觉本身的传导是经过多神经元途经及大脑中枢的信息加工来实现的。涉及感觉刺激传递的第一神经细胞途经称为第一感觉传入。从头部和一些肠道器官发出的第一感觉传入的细胞体存在于各种与脑神经相关的神经节中，特别是三叉核和单束核中。身体其它部分的第一感觉传入的细胞体位于脊柱的后根神经节中。第一感觉传入和其加工按组织学进行分类；细胞体分为两类：A型较大(直径60-120μm)，而B型较小(14-30μm)且数量较多。同样，加工分成2类：C-纤维缺乏A纤维所具有的髓鞘。A纤维可进一步细分成直径较大且充分形成髓鞘的Aβ纤维，和较细且形成髓鞘不太充分的Aδ-纤维。一般相信从A型细胞体产生Aβ-纤维且从B型细胞体产生Aδ-和C-纤维。经过研究分子标记范围的选择性表达可将这些分类进一步延伸和细分。
功能性分析表明，在正常情况下Aβ-纤维传递触觉并调节温度辨别力，而C纤维主要相当于上面提到的多形高阈值纤维。Aδ纤维的作用不太清楚，因为它们似乎具有各种各样的反应方式，较高和较低阈值均有。
激活第一感觉传入后，感觉信号传导中的下一步是激活触突神经元，它携带信号到中央神经系统的更高部分，如丘脑核。这些神经元的细胞体(不同于与脑神经相关的那些细胞体)位于脊髓的背角。这也是第一传入和触突神经元之间的突触所在的位置。背角组织成一系列堆积的神经板，神经板I在最后部，接着是神经板II等，第一传入的不同类型在不同神经板上形成突触。对于皮肤第一传入，C纤维在神经板I和II形成触突，Aδ纤维在神经板I，II和V，Aβ纤维在神经板III，IV和V。认为越深层的神经板(V-VII，X)与越深层的组织，如肌肉和内脏的感觉途径到达有关。
在第一传入和触突神经元之间的触突处主要的神经递质是谷氨酸，尽管较重要的是C纤维含有一些诸如P物质和降钙素基因相关性肽(CGRP)的一些神经肽。A纤维在某些情况下也可表达诸如神经肽Y的神经肽。
这些触突的传递效率可经过下行途径和经过脊髓中的局部中间神经元来改变。这些调节神经元释放许多抑制的(例如，阿片样肽，甘氨酸)或兴奋的(例如，氧化氦，胰酶分泌素)调节剂以提供增强或减轻感觉意识的机制。
需要这类生理调节的一类感觉是痛觉。痛觉是能警告损伤或疾病的一种感觉，且它是日常生活中必不可少的。然而有时需要能够忽视它，在生理学上，这是诸如阿片样肽的一种功能。不幸的是，尽管有这些生理学机制，但在疾病期间或损伤后其效用过去较长时间后疼痛仍会继续。在这些情况下，疼痛变成一个疾病的症状，最好能使其减轻。
在临床上，疼痛可分成三类：(1)急性疼痛，通常由损伤或手术引起，当伤口愈合时预期疼痛消失。(2)由恶性疾病引起的慢性疼痛，具有转移癌的大多数人具有中等至严重的疼痛，经过成功地台疗该疾病或经过病人死亡来解决这一问题。(3)不是由恶性疾病引起的慢性疼痛；这是一种异源性疾病，由各种疾病引起，包括关节炎和外周神经痛，通常不威胁生命但可能持续几十年且疼痛水平不断增加。
对由组织损伤引起的疼痛的生理学比对由中央神经系统缺陷引起的疼痛的生理学有更好的了解。在正常情况下，引起疼痛的感觉首先由携带高阈值信号的Aδ-和C纤维传导。因此，在神经板I和II中的突触涉及疼痛信号的传递，使用由C纤维释放的谷氨酸和肽以产生合适突触神经元的激活。然而，有证据表明在某些慢性疼痛症状中，其它A纤维(包括Aβ-纤维)可携带疼痛信号，从而充当第一伤害性传入，例如在与神经病理性疼痛相关的痛觉过敏和allody-nia中。已经将这些改变与诸如A纤维中神经肽Y的肽的表达联系起来。在各种慢性疼痛状态期间，各种感觉传入与突触神经元间的触突可以几种方式修饰：在形态学上可能发生改变，导致触突数目增多，不同肽的水平和比率可改变，突触神经元的敏感性可改变。
由于疼痛引起的临床问题巨大，为发现减轻疼痛的方法花费了相当大的努力。用于减轻疼痛的最常用的药品分成2类：(1)非甾类抗发炎药(NSAID)，包括阿司匹灵和布洛芬；(2)阿片样物质，包括吗啡。
NSAID经过抑制损伤组织生产前列腺素而在外周具有其主要止痛作用。已显示前列腺素是疼痛和发炎的外周介质，其浓度的减小使病人疼痛减轻。在轻度关节炎疾病中特别会出现这种情况，其中发炎是主要的疼痛病因。已有人建议前列腺素涉及脊髓和大脑中的痛觉介导，这可解释为什么在不涉及发炎或外周组织损伤的一些疼痛症状中NSAID具有止痛效应。然而，由于前列腺素仅是疼痛的一些介质之一，所以单独的NSAID仅在将一些轻度疼痛的类型减轻到可接受的水平中有效。认为它们具有最高限度的活性，增加超过这限度不会引起疼痛减轻的程度增加。而且，在慢性疾病中它们具有限制其效用的副作用。NSAID的使用与胃肠道的刺激相关，延长使用可导致形成消化道的广泛溃疡。这在老年病人中特别现实，他们形成了具有例如关节炎的病人的最大类群。
阿片样物质在脊髓水平上发生作用以抑制第一伤害性纤维(原则上是C纤维)和触突神经元之间的神经传递效力。它们经过引起这些触突两个成份的超极化延长而达到这一效果。使用阿片样物质在减轻大多数类型的急性疼痛和慢性恶性疼痛中有效。然而，有许多慢性恶性疼痛症状部分或完全对阿片样物质的止痛具有不应性，特别是那些由诸如肿瘤形成引起的神经压迫的症状。不幸的是阿片样物质也具有不需要的全身性副作用，包括：(1)在脑干呼吸中枢水平抑制呼吸系统；(2)经过对胃肠道平滑肌系统的各种效应诱导便秘；和(3)包括镇静和诱导欣快症的精神性效应。这些副作用在相似于产生止痛的剂量下出现，因而限制了给病人用药的剂量。
在脊髓水平传递阿片样物质可减小副作用情况，但需要经常重复脊髓注射或安置导管，两者都会增加带给病人的危险。安置导管需要将病人基本上限制于床上，从而进一步限制了他们的生活质量。
阿片样物质在治疗一些其它类型的慢性疼痛中的应用一般是无效或不需要的。例如包括与类风湿性关节炎相关的疼痛及神经损伤后形成的神经瘤。在这些病人中阿片样物质治疗所不希望的特性不仅与已提到的副作用和疾病的可能持续时间相关，而且与阿片状物质的第4种主要的副作用：依赖性相关。诸如吗啡和海洛因的阿片样物质是熟知的导致身体依赖性的滥用毒品，这最后一个副作用涉及形成耐受性：产生相同止痛效应所需的药物剂量随时间而增加。这可能导致出现由于前3种副作用，减轻疼痛所需的剂量是有生命危险的情况。
尽管NSAID和阿片样物质在治疗疼痛中有用，但一般认同它们通常不适合于适当治疗疼痛，特别是慢性和严重疼痛。
也使用其它治疗方法，特别是用于治疗慢性严重疼痛，包括在外周神经的一些水平从背根切片和脊髓前侧柱切断术到垂体破坏来手术损伤疼痛途径。
由此可见，仍然明显地需要形成用于治疗许多类型的疼痛的新类型的药物。这类新疗法的所需特征可简单地表达如下：(1)提供明显地减轻疼痛，包括严重疼痛的能力；(2)缺乏明显地损伤病人生活质量的全身性副作用；(3)不需经常注射或对病人进行长期插管的长效作用；(4)提供不导致耐受性和相关依赖性的试剂。
本发明的陈述
本发明涉及一种能减少且优选防止疼痛信号从外周向中央神经系统传递，从而减轻痛觉的试剂。具体地说，本发明可提供一种能减少且优选防止疼痛信号从伤害性传入神经向触突神经元传递的试剂。更具体地说，本发明可提供一种能抑制至少一种神经递质或神经调制物从至少一类伤害性传入神经的胞吐作用的试剂。
在本发明的第一个方面，提供了一种能全身性用药且能特异性地靶向伤害性传入神经的限定群体以抑制至少一种神经递质或神经调制物从神经触突末端释放的试剂。
在本发明的第二个方面，提供了一种能在外周局部用药的试剂，且它能抑制至少一种神经递质或神经调制剂从经外周传递痛觉信号的伤害性传入的触突末端的释放。
在本发明的第三个方面，提供了一种能施用到脊髓的试剂，且它能抑制至少一种神经递质或神经调制物从在脊髓区终止的伤害性传入的触突末端的释放。
在本发明的第四个方面，提供了一种试剂，它能特异性地靶向限定群体的传入神经元，使该试剂的影响局限于该细胞类型。
在本发明的第五个方面，提供了一种治疗疼痛的方法，包括施用有效剂量的根据本发明的试剂。
在本发明的第六个方面，该试剂可作为包括该试剂所需成份的融合蛋白质以重组的方式表达。
本发明提供下面技术方案：
1.一种对外周感觉传入表现出特异性的试剂，它包括一个偶联到修饰的梭菌神经毒素上的靶向部分TM，其中TM包含第一感觉传入上细胞表面结合位点的配体且在功能上能与结合位点相互作用引起该试剂与第一感觉传入的表面之间的有形联系；且梭菌神经毒素的重链H链经化学修饰，突变或蛋白水解去掉或修饰以减小或去掉其对运动神经元的天然结合亲合力；梭菌神经毒素轻链L-链或其片段保留对神经分泌机制的成份特异性的蛋白酶活性；试剂形成的分子将L链或其片段导入第一感觉传入的细胞溶质，从而经过控制至少一种神经递质或神经调制物从第一感觉传入的释放而抑制第一感觉传入和触突神经元之间的信号传递。
根据方案1的试剂，它包括偶联到梭菌神经毒素上的靶向部分TM，其中H链的Hc部分被去掉或修饰。
根据方案1的试剂，其中修饰的H链是梭菌神经毒素的HN片段。
根据上述方案1的试剂，其中的梭菌神经毒素成份从肉毒神经毒素中获得。
根据方案1的试剂，其中的梭菌神经毒素成份从肉毒神经毒素A型中获得。
根据上述方案的试剂，它经过将TM偶联到肉毒神经毒素A型LHN片段上而形成。
根据上述方案的试剂，它经过将TM偶联到肉毒神经毒素B型LHN片段上而形成。
根据上述方案的试剂，它经过将TM偶联到肉毒神经毒素C1型的LHN片段上而形成。
根据方案1的试剂，其中的梭菌神经毒素成份从肉毒神经毒素B型中获得。
根据方案1的试剂，其中的梭菌神经毒素成份从肉毒神经毒素C1型中获得。
根据方案1的试剂，其中的H链从与获得L链不同的梭菌神经毒素中获得。
根据上述方案的试剂，其中的H-链从肉毒神经毒素A型中获得，L链从肉毒神经毒素B型中获得。
根据上述方案的试剂，它由连接于肉毒神经毒素A型的HN片段和肉毒神经毒素B型L链上的TM组成。
根据上述方案1的试剂，其中的L链或L链片段以直接共价链连接于H链上。
根据方案1的试剂，其中的L链或L链片段以包括一个或多个间隔区的共价键连接于H链上。
根据上述方案1的试剂，其中的TM能将L链或L链片段传递进独立的第一感觉传入的细胞溶质中。
根据上述方案1的试剂，其中传递L-链或L-链片段进入第一感觉传入的细胞溶质的能力完全包含于TM内。
根据上述方案1的试剂，其中TM结合于其特征在于第一感觉传入的特定群体的结合位点上。
根据上述方案1的试剂，其中的TM结合于其特征在于第一伤害性传入的特定群体的结合位点上。
根据上述方案1的试剂，其中TM结合到在第一感觉传入内进行逆向运输的结合位点上。
根据上述方案1的试剂，其中TM结合到在第一伤害性传入内进行逆向运输的结合位点上。
根据上述方案1的试剂，其中的TM包含一种第一感觉传入上的细胞表面受体的配体。
根据上述方案1的试剂，其中的TM包含一种第一感觉传入上的生长因子受体的配体。
根据上述方案的试剂，其中TM包含一种神经生长因子受体的配体。
根据上述方案的试剂，其中的TM包含神经生长因子。
根据上述方案的试剂，它包含连接于肉毒神经毒素A型LHN片段上的神经生长因子。
根据方案1的试剂，其中的TM包含一种第一感觉传入上的神经肽受体的配体。
根据方案1的试剂，其中TM包含一种第一感觉传入上的细胞因子受体的配体。
根据方案1的试剂，其中TM包含一种第一感觉传入上的激素受体的配体。
根据上述方案1的试剂，其中TM以直接共价键连接到来自梭菌神经毒素的成份上。
根据上述方案1的试剂，其中TM以包括一个或多个间隔区的共价键连接到来自梭菌神经毒素的成份上。
根据上述方案1的试剂，它防止神经递质或神经调制物从第一感觉传入的释放。
根据上述方案1的试剂，它抑制神经递质或神经调制物从第一伤害性传入的释放。
2.一种获得根据上述方案1的试剂的方法，它包含将TM共价附着于修饰的梭菌神经毒素或梭菌神经毒素的片段上。
一种获得根据方案1的试剂的方法，它包含用一个或多个间隔区将TM共价附着于修饰的梭菌神经毒素或梭菌神经毒素的片段上。
一种获得根据方案1的试剂的方法，包含构建编码修饰的梭菌神经毒素或梭菌神经毒素的片段的遗传构建体，将所说的构建体掺入宿主生物，表达该构建体以产生修饰的梭菌神经毒素或梭菌神经毒素的片段，将修饰的梭菌神经毒素或梭菌神经毒素的片段共价附着于TM上。
一种获得根据方案1的试剂的方法，包含构建编码修饰的梭菌神经毒素或梭菌神经毒素的片段的遗传构建体，将所说的构建体掺入宿主生物，表达构建体以产生修饰的梭菌神经毒素或梭菌神经毒素的片段，用一个或多个间隔子区将修饰的梭菌神经毒素或梭菌神经毒素的片段共价附着于TM上。
一种获得根据方案1的试剂的方法，包含构建编码该试剂的遗传构建体，将所说的构建体掺入宿主生物中，表达该构建体以生产该试剂。
3.根据方案1的试剂在生产用于减轻疼痛的药物中的应用。
根据方案1的试剂在生产用于防止疼痛的药物中的应用。
定义：
不希望以下列定义来进行限制，它意指在本说明书中，下列术语具有下面的意义：
轻链指形成梭菌神经毒素的两条多肽链中较小的一条，它具有大约50KDa的分子量且一般称为L-链或简单称为L。
重链指形成梭菌神经毒素的两条多肽链中较大的一条；它具有大约100KDa的分子量，通常称为H-链或简单称为H。
Hc片段指来自梭菌神经毒素H链的一个片段，它大约相当于H链的羧基末端一半或相应于在完整H链中的该片段的功能域。它含有涉及结合运动神经元的天然毒素功能域。
HN片段指来自梭菌神经毒素H链的片段，它大约相当于H链的氨基末端一半，或相应于在完整H链中该片段的功能域。它含有涉及将L链转移通过内体膜的区域。
LHN指来自梭菌神经毒素的片段，它含有L链，或偶联到HN片段上的其功能性片段。它通常来自蛋白水解的完整神经毒素。
靶向部分(TM)指在功能上与结合位点相互作用引起试剂与第一感觉传入表面之间的有形联系的试剂的任何化学结构。
结合位点(BS)指在细胞表面的一种结构，外源分子能与它相互作用以致引起与细胞的有形联系。
第一感觉传入指能从外周携带感觉信息到中央神经系统的神经细胞。
第一伤害性传入是能从外周携带感觉信息到中央神经系统的神经细胞，其中该信息可导致痛觉。
附图的简要描述
图1显示了衍生的神经生长因子(NGE)和来自BoNT/A(指肉毒杆菌血清型A的神经毒素)的衍生的LHN之间偶联反应产物的大小排阻色谱的馏分的SDS-PAGE分析的考马斯染色。
图2显示了在还原和非还原条件下NGF和LHN的连接物的SDS-PAGE分析的考马斯染色。
图3显示了用NGF和LHN连接物处理的PC12细胞提取物的Western印迹，用识别SNAP-25蛋白水解产物的抗体以BoNT/AL链进行探测。
图4显示了用NGF和LHN连接物处理的大鼠背根神经节神经元的提取物的Western印迹，用识别SNAP-25的蛋白水解产物的抗体以BONT/AL链进行探测。
本发明的详细描述：
可以发现用于减轻或防止疼痛信号从外周伤害性传入神经元向触突神经元传递的试剂在减轻痛觉、特别是严重的慢性疼痛中具有许多潜在的应用。
根据本发明，提供了一种能抑制至少一种神经递质或神经调制物或两者从伤害性传入的触突末端释放的试剂。
该试剂具有许多独立的功能：1)它结合表面结构(结合位点[BS])，它是伤害性传入神经元的特征且对伤害性传入神经元有一定程度的特异性。2)它进入神经元。经过内吞过程分子可进入细胞。仅在某些细胞表面BS进行内吞，优选该试剂结合的BS是其中之一。在本发明的一个方面，BS存在于外周伤害性传入神经元的感觉纤维上，同化后，逆行运输到细胞体和神经元的中央突起使该试剂也传递到神经元的这些区域。在本发明的另一个方面，该试剂所结合的BS存在于伤害性传入神经元的中央突起或细胞体上。3)该试剂进入细胞溶质。4)该试剂修饰存在于这些神经元中央突起触突末端上的胞吐机制的成份，使触突末端释放的至少一种神经递质或神经调制物减少或优选被阻止。
令人惊奇的是，本发明的试剂可经过修饰梭菌神经毒素或其片段来产生。
梭菌神经毒素是具有150KDa级分子量的蛋白质。它们由梭状芽胞杆菌属的不同种类产生，最重要的是C.tetani和一些肉毒梭菌的菌株。目前有8类不同的已知神经毒素：破伤风毒素和在其血清型A，B，C1，D，E，F和G中的肉毒神经毒素，它们都具有相似的结构和作用方式。梭菌神经毒素由细菌以单个多肽的形式来合成，该多肤进行翻译后修饰，形成以二硫键连接起来的2条多肽链。这两条链称为分子量约为100KDa的重链(H)和分子量约为50KDa的轻链。梭菌神经毒素结合到神经肌肉连接点的运动神经元细胞膜的受体位点上并经内吞机制被同化。同化的梭菌神经毒素具有高特异性的锌依赖性内肽酶活性，它水解三种蛋白质小突触小泡蛋白，突触融合蛋白或SNAP-25中至少一种的特异性肽键，这3种蛋白质是神经分泌机制的决定性成分，梭菌毒素的这一活性导致肌肉麻痹延长。发现梭菌神经毒素的锌依赖性内肽酶活性位于L链中。由于这些神经元上受体位点的具体特性，梭菌神经毒素对运动神经元具有高度选择性。已知梭菌神经毒素的特异性神经肌肉连接点结合活性位于双链神经毒素分子重链成份的羧基末端部分，该区域称为HC。
令人惊奇的是，经过将梭菌神经毒素或H链HC区已被去掉或修饰的两个梭菌神经毒素的杂交体共价连接到结合于感觉神经元表面BS上的一个新分子或部分，即靶向部分(TM)上时，产生一种能抑制至少一种神经递质或神经调制物从伤害性传入释放的新试剂。本发明更令人惊奇的一个方面是如果梭菌神经毒素L链或含内肽酶活性的L-链的片段共价连接到也能影响L-链或其片段同化进感觉神经元胞质的TM上，也产生能抑制至少一种神经递质或神经调制物释放的新试剂。用于偶联试剂的组份部分的共价键可包括合适的间隔区。
TM提供了对伤害性传入神经元上的BS的特异性。该试剂的TM成份可包括许多细胞结合分子之一，包括但不限于抗体，单克隆抗体，抗体片段(Fab，F(ab)2′，Fv，ScFv，等)，植物凝血素和激素细胞因子，生长因子或神经肽的受体的配体。可能的TM目录在表1中给出。该目录是举例性的且不打算用于限制能满足本发明的需要的TM的范围。在本发明的一个实施方案中，TM结合于BS上进行逆行运输。
在本领域中已知神经毒素分子的HC部分可从称为HN的重链其它部分中去掉，使HN片段保留二硫键连接到神经毒素分子轻链(L-链)上以提供称为LHN的片段。因此，在本发明的一个实施方案中，梭菌神经毒素的LHN片段使用可包括一个或多个间隔区的连接物共价连接到TM上。
在本发明的另一实施方案中，梭菌神经毒素的HC区例如经过化学修饰进行突变或修饰以减小或优选破坏其将神经毒素结合到神经肌肉连接处的受体上的能力。然后使用可包括一个或多个间隔子区的连接物将修饰的梭菌神经毒素共价连接到TM上。
在本发明的另一实施方案中，例如经化学修饰突变或修饰HC区以减小或优选破坏其将神经毒素结合到神经肌肉连接点的受体上的能力的梭菌神经毒素重链与不同梭菌神经毒素的L-链组合。然后使用可包括一个或多个间隔区的连接物将杂合的，修饰的梭菌神经毒素共价连接到TM上。
在本发明的另一实施方案中，梭菌神经毒素的HN部分与不同梭菌神经毒素的L链组合。然后使用可包括一个或多个间隔子区的连接物将杂合的LHN共价连接到TM上。
在本发明的另一实施方案中，梭菌神经毒素的轻链或含内肽酶活性的轻链片段使用可包括一个或多个间隔子区的连接物连接到TM上，它也影响轻链或其含内肽酶活性的片段同化进细胞质。
在本发明的另一实施方案中，该试剂以融合蛋白质的形式经重组表达，它除了所需的间隔子区外还包括靶向部分的合适片段。该重组表达的试剂可完全来自编码一种神经毒素血清型的基因或是来自编码两种不同血清型的基因的嵌合体。
在本发明的另一实施方案中，可以是来自不同梭菌毒素型的L和HN的杂合体的所需LHN作为具有TM的融合蛋白的形式经重组表达，且可以包括一种或多种间隔子区域。
在本发明的另一实施方案中，梭菌神经毒素的轻链或含内肽酶活性的轻链片段作为具有TM的融合蛋白以重组形式表达，它也能影响轻链或含内肽酶活性的其片段同化进细胞质。表达的融合蛋白质也可包括一种或多种间隔区域。
本说明书的基础是产生了对限制的且限定类的神经元(第一感觉传入)具有非常特异性及限定活性的新试剂，因此该试剂可认为代表一种形式的神经毒素。天然肉毒神经毒素的治疗应用是现有技术中熟知的。然而如现有技术所述，肉毒神经毒素的作用方式的机制是抑制乙酰胆碱分泌，且针对一类靶神经元，即传出运动神经元，明显不同于本文所述的试剂。背景技术未教导所公开的试剂的活性或化学结构。因此，尽管如本申请所讨论的，背景技术教导了许多关于天然梭菌神经毒素，但天然未修饰的梭菌神经毒素不是本说明书的主题。本发明的试剂需要修饰梭菌神经毒素以便去掉背景技术所教导的靶向特征。然后修饰的神经毒素偶联到新的靶向功能(TM)上以产生具有区别于天然梭菌神经毒素且现有技术未教导的新的生物学特征的新试剂。本说明书的主题是具有新特性的该新的试剂。工业利用：
本发明所述的试剂可直接或作为药用上可接受的盐用于体内治疗疼痛。
例如，根据本发明的试剂可全身性地用于治疗严重的慢性疼痛。其具体例子是用于治疗与影响多处关节的类风湿性关节炎相关的临床疼痛。
在另一个例子中，根据本发明的试剂可局部应用于治疗疼痛。其具体例子是经过局部注射进被发炎疼痛所感染的关节来进行治疗。
在另一个例子中根据本发明的试剂可经过脊髓注射(硬膜或内膜)在涉及神经支配感染器官的脊髓段的水平用药以治疗疼痛。例如它可应用于治疗深层组织疼痛，如慢性恶性疼痛。
现在本发明将参考下面非限制性的实施例进行描述：实施例1 NGF和BoNT/A的LHN片段连接物的合成
经过加水将冻干的鼠2.5S NGF溶解并在MES缓中液(0.1MMES，0.1M氯化钠，pH5.0)中透析。向该溶液(浓度为大约0.3mg/ml)中加入PDPH(以100mg/ml溶于DMF中)至终浓度为1mg/ml。混合后，加入固体EDAC使产生大约0.2mg/ml的终浓度。反应在室温下进行至少30分钟。然后经过在预先用MES缓中液平衡的PD-10柱(Phamacia)上脱盐去掉过剩的PDPH。
基本上以Shone C.C.，Hambleton，P.，和Melling.J.1987，欧洲生化杂志167，175-180的方法产生BoNT/A的LHN片段。相当于所用的NGF一半重量的LHN量溶于三乙醇胺缓冲液(0.02M三乙醇胺/HCl，0.1M氯化钠，pH7.8)中得到的大约1mg/ml的浓度的溶液与2mM终浓度的Traut’s试剂(100mM贮液溶液溶于1M三乙醇胺/HCl，pH8.0中)反应。1小时后，使用PD-10柱(Pharmaci-a)将LHN脱盐进入PBSE(具有1mM EDTA的磷酸盐缓冲溶液)中。使用Microcon 50(Amicon)将柱洗脱物的蛋白质峰浓缩到大约2mg/ml的浓度。
对衍生的NGF进行最终浓缩步骤，使体积减少到不超过起始体积10％的体积，然后在室温下与衍生的LHN混合过夜。在十二烷基磺酸钠存在下经聚丙烯酰胺凝胶电泳(SDS-PAGE)分析反应产物。
在Bio-Gel P-100(Bio-Rad)上经大小排阻色谱部分纯化从上面反应所得的连接物。经过在280nm下测光密度和SDS-PAGE分析馏份得到洗脱图。这允许从自由NGF和反应副产物中分离连接物。
图1显示了对一个这种Bio-Gel P-100柱的馏分进行SDS-PAGE分析。从大多数的游离NGF(Mr 13KDa)中清楚地分离出游离的LHN和连接物(Mr100KDa及以上)。由于2.5S NGF是由非共价相互作用形成的同型二聚体，用SDS处理使其解离。因此通过一个亚基与LHN形成共价交联的分子在SDS-PAGE分析期间分离且产生在馏分4-6可见的游离NGF带。结果表明修饰后NGF的同型二聚体结构保持完整。在这些馏分中可见游离的LHN代表未偶联NGF的少量成份。合并馏分4-6，进行进一步的分析。
图2显示了在还原和非还原条件下以SDS-PAGE分析连接物。泳道1是在非还原条件下的游离LHN，泳道2是用50mM二硫苏糖醇还原的相同LHN量。泳道3和4显示了未还原(泳道3)或用二硫苏糖醇还原的大小排阻色谱后的连接物。同样，泳道5和6分别显示未还原或还原的NGF。结果清楚地表明泳道5中具有大于100KDa的表观分子量的物质在还原条件下仅产生LHN和HGF的组份带。而且还原后该带的强度是很可能来自不同于在未还原的样品中观察到的少量自由LHN和NGF的物质。这一额外产物的唯一可能的来源是具有表观分子量＞100KDa的物质。因此大小排阻色谱后获得的馏份中的连接物代表由可还原的二硫键共价连接的NGF和LHN。
含连接物的馏分贮存于4℃备用。实施例2 PC-12细胞中NGF和LHN连接物的活性
PC12细胞是常用作神经功能研究的模型系统的神经外胚层衍生物的细胞系。作为试验NGF和LHN连接物的功能的模型系统，它们具有2个必要的特征：首先它们已知具有NGF的细胞表面受体，且显示出涉及与低浓度的NGF反应的分化过程。其次已知它们含有神经递质释放，包括重要的例子是SNAP-25的胞吐机制。
将PC12细胞以每孔大约5×105个细胞的密度涂布到已用MATRIGEL基底膜基质(Collaborative生物医药产品)覆盖的24孔平板上。在培养基(具有2mM谷氨酰胺，10％马血清和5％胎牛血清的RPMI1640，37℃，5％CO2)培养几天后，用含加有连接物(按实施例1所述制备)或LHN或未加的新鲜培养基来代替旧培养基。在培养基中保持过夜后去掉培养基并用新鲜培养基洗涤细胞一次。然后经过加入0.45ml氢氧化钠(0.2M)30分钟裂解细胞。此后，经过加入0.45ml盐酸(0.2M)中和该溶液，接着加入0.1ml HEPES/NaOH(1M，pH7.4)。为了从这些混合物中提取膜蛋白，加入Triton-X-114(10％，v/v)并在4℃培养60分钟，经离心去掉不溶物，然后将上清于37℃保温30分钟。经离心分离所得的两相并弃去上层。用氯仿/甲醇沉淀下层中的蛋白质用于Western印迹分析。
经SDS-PAGE分离样品并转移到硝酸纤维上。然后经过用识别新揭示的裂解的SNAP-25的羧基末端的抗体(在专利申请PCT/GB95/01279)进行探测来检测SNAP-25的蛋白水解产物。一种神经分泌过程的关键成份和BoNT/A的锌依赖性内肽酶活性的底物。图3显示了该Western印迹的一个实施例。在来自对照细胞的样品中(泳道1和2)观察不到明显的免疫反应性，而在用10mg/mlHN培养的样品中(泳道5和6)观察到微弱地相应于29KD分子量的带，在用10mg/ml NGF和LHN连接物培养的样品中(泳道3和4)观察到较强的带。因此，用连接物培养PC12细胞导致SNAP-25的明显水解表明连接物将BONT/AL链的锌依赖性蛋白水解活性导入了细胞质。用连接物的组成成份可见很小或没有该活性。
在过剩的自由NGF存在下将细胞与连接物培养导致比仅用连接物的培养所产生的SNAP-25蛋白水解产物减小。这表明连接物的作用借助于NGF靶向部分与NGF细胞表面受体之间的相互作用发生。实施例3在背根神经节神经元的原代培养物中NGF和LHN连接物
的活性
背根神经节含有第一伤害性传入的细胞体。已建立了在该组织的原代体外培养物中该神经元可保留伤害性传入的许多特征。这些特征包括与已知引起体内疼痛的化学刺激(如，辣椒碱)反应释放诸如P物质的神经肽的能力。
从大鼠胚胎(胚龄12-15天)解剖分离的神经节后建立背根神经节神经元的原代培养物。以3×105个细胞/孔的起始密度将细胞涂布到含NGF(100ng/ml)的培养基的12孔平板中。在培养基中培养1天后，加入含阿糖胞苷(10mM)的新鲜培养基以杀死非神经元细胞。2-4天后去掉阿糖胞苷。在培养基中再培养几天后在不含NGF的条件下用含连接物或LHN的新鲜培养基代替旧培养基。37℃培养过夜后去掉培养基，裂解细胞并使用实施例2所述的Tri-ton-X-114提取疏水蛋白质。
使用识别SNAP-25的BoNT/A蛋白水解产物的抗体按实施例2所述经Western印迹分析样品。在来自对照细胞的样品中(泳道4)观察不到免疫反应性，而在用10mg/ml LHN培养的样品中(泳道3)观察到微弱的相应于29KD分子量的带，在用10mg/ml NGF和LHN连接物培养的样品中(泳道1和2)观察到较强的带。
结果表明该连接物可将BoNT/A的蛋白水解活性L链传递给体内形成第一伤害性传入的神经元细胞的细胞质。实施例4 L链来自BoNT/B且HN片段来自BoNT/A的嵌合LHN
的生产
根据Shone.C.C.，Hambleton，P.，和Melling.J.(1987，欧洲生化杂志，167，175-180)所述的方法生产BoNT/A的HN片段，且根据Sathyamoorthy，V.和DasGupta，B.R.(1985，生物化学杂志，260，10461-10466)的方法生产BoNT/B的L链。然后经过加入超过10倍摩尔数的双吡啶二硫化物接着在4℃培养过夜改变BoNT/A HN片段上的自由半胱氨酸。然后经过将蛋白质在PD10柱(Pharmacia)上脱盐进PBS去掉过剩的双吡啶二硫化物和硫代吡啶酮副产物。
然后在与等摩尔的来自BoNT/B的L链部分(21mg/ml，溶于PBS)混合前将修饰的HN浓缩到蛋白浓度超过1mg/ml。在室温下培养过夜后，经过在Superose6(Pharmacia)上进行大小排阻色谱分离混合物，以SDS-PAGE分析馏份。然后可对嵌合LHN进行修饰以产生实施例1所述的靶向连接物。
上述实施例仅仅是对本发明的说明。在合成的试剂中，TM与修饰的梭菌神经毒素或其片段的偶联是经过使用本领域的技术人员已知的试剂和技术经化学偶联实现的。因此，尽管所给的实施例专一性地使用了PDPH/EDAC和Traut试剂的化学，但是能共价附着该试剂的TM成份到来自梭菌神经毒素的成份上的且本领域的技术人员已知的其它偶联化学也被本申请的范围所覆盖。同样，本领域的技术人员明白编码任一完整试剂或该试剂的片段的DNA易于构建，且当在合适的生物中表达时，可用于重组生产该试剂或该试剂的片段。以本领域的技术人员已知的技术获得的本发明的试剂的遗传构建体也在本发明的范围内。
表1-可能的靶向部分(TM)
生长因子：
1.神经生长因子(NGF)；
2.白细胞瘤抑制因子(LIF)；
3.碱性成纤维细胞生长因子(bFGF)；
4.来自大脑的神经营养因子(BDNF)；
5.神经营养因子-3(NT-3)
6.水螅头部激活肽(HHAP)
7.转化生长因子1(TGF-1)
8.转化生长因子2(TGF-2)；
9.转化生长因子(TGF-)
10.表皮生长因子(EGF)；
11.睫状神经营养因子(CNTF)。
细胞因子：
1.肿瘤坏死因子(TNF-)；
2.白细胞介素-1(IL-1)；
3.白细胞介素-1(IL-1)；
4.白细胞介素-8(IL-8)。
肽：
1.内啡呔
2.甲硫氨酸-脑啡肽
3.D-Ala2-D-Leu5-脑啡肽
4.缓激肽抗体：
1.抗在背根神经节神经元表面发现的乳糖类糖类抗原决定簇的抗体(例如，单克隆抗体IB2和LA4)；
2.抗上面给出的配体的任意受体的抗体。
3.抗表面表达的抗原Thy1的抗体(例如，单克隆抗体MRCOX7)。
Claims (40)
1.一种对外周感觉传入表现出特异性的试剂，它包括一个偶联到修饰的梭菌神经毒素上的靶向部分TM，其中TM包含第一感觉传入上细胞表面结合位点的配体且在功能上能与结合位点相互作用引起该试剂与第一感觉传入的表面之间的有形联系；且梭菌神经毒素的重链H链经化学修饰，突变或蛋白水解去掉或修饰以减小或去掉其对运动神经元的天然结合亲合力；梭菌神经毒素轻链L-链或其片段保留对神经分泌机制的成份特异性的蛋白酶活性；试剂形成的分子将L链或其片段导入第一感觉传入的细胞溶质，从而经过控制至少一种神经递质或神经调制物从第一感觉传入的释放而抑制第一感觉传入和触突神经元之间的信号传递。
2.根据权利要求1的试剂，它包括偶联到梭菌神经毒素上的靶向部分TM，其中H链的Hc部分被去掉或修饰。
3.根据权利要求1的试剂，其中修饰的H链是梭菌神经毒素的HN片段。
4.根据上述权利要求1的试剂，其中的梭菌神经毒素成份从肉毒神经毒素中获得。
5.根据权利要求1的试剂，其中的梭菌神经毒素成份从肉毒神经毒素A型中获得。
6.根据权利要求1的试剂，其中的梭菌神经毒素成份从肉毒神经毒素B型中获得。
7.根据权利要求1的试剂，其中的梭菌神经毒素成份从肉毒神经毒素C1型中获得。
8.根据权利要求5的试剂，它经过将TM偶联到肉毒神经毒素A型LHN片段上而形成。
9.根据权利要求6的试剂，它经过将TM偶联到肉毒神经毒素B型LHN片段上而形成。
10.根据权利要求7的试剂，它经过将TM偶联到肉毒神经毒素C1型的LHN片段上而形成。
11.根据权利要求1的试剂，其中的H链从与获得L链不同的梭菌神经毒素中获得。
12.根据权利要求11的试剂，其中的H-链从肉毒神经毒素A型中获得，L链从肉毒神经毒素B型中获得。
13.根据权利要求12的试剂，它由连接于肉毒神经毒素A型的HN片段和肉毒神经毒素B型L链上的TM组成。
14.根据上述权利要求1的试剂，其中的L链或L链片段以直接共价链连接于H链上。
15.根据权利要求1的试剂，其中的L链或L链片段以包括一个或多个间隔区的共价键连接于H链上。
16.根据上述权利要求1的试剂，其中的TM能将L链或L链片段传递进独立的第一感觉传入的细胞溶质中。
17.根据上述权利要求1的试剂，其中传递L-链或L-链片段进入第一感觉传入的细胞溶质的能力完全包含于TM内。
18.根据上述权利要求1的试剂，其中TM结合于其特征在于第一感觉传入的特定群体的结合位点上。
19.根据上述权利要求1的试剂，其中的TM结合于其特征在于第一伤害性传入的特定群体的结合位点上。
20.根据上述权利要求1的试剂，其中TM结合到在第一感觉传入内进行逆向运输的结合位点上。
21.根据上述权利要求1的试剂，其中TM结合到在第一伤害性传入内进行逆向运输的结合位点上。
22.根据上述权利要求1的试剂，其中的TM包含一种第一感觉传入上的细胞表面受体的配体。
23.根据上述权利要求1的试剂，其中的TM包含一种第一感觉传入上的生长因子受体的配体。
24.根据权利要求1的试剂，其中的TM包含一种第一感觉传入上的神经肽受体的配体。
25.根据权利要求1的试剂，其中TM包含一种第一感觉传入上的细胞因子受体的配体。
26.根据权利要求1的试剂，其中TM包含一种第一感觉传入上的激素受体的配体。
27.根据权利要求23的试剂，其中TM包含一种神经生长因子受体的配体。
28.根据权利要求27的试剂，其中的TM包含神经生长因子。
29.根据权利要求28的试剂，它包含连接于肉毒神经毒素A型LHN片段上的神经生长因子。
30.根据上述权利要求1的试剂，其中TM以直接共价键连接到来自梭菌神经毒素的成份上。
31.根据上述权利要求1的试剂，其中TM以包括一个或多个间隔区的共价键连接到来自梭菌神经毒素的成份上。
32.根据上述权利要求1的试剂，它防止神经递质或神经调制物从第一感觉传入的释放。
33.根据上述权利要求1的试剂，它抑制神经递质或神经调制物从第一伤害性传入的释放。
34.一种获得根据上述权利要求1的试剂的方法，它包含将TM共价附着于修饰的梭菌神经毒素或梭菌神经毒素的片段上。
35.一种获得根据权利要求1的试剂的方法，它包含用一个或多个间隔区将TM共价附着于修饰的梭菌神经毒素或梭菌神经毒素的片段上。
36.一种获得根据权利要求1的试剂的方法，包含构建编码修饰的梭菌神经毒素或梭菌神经毒素的片段的遗传构建体，将所说的构建体掺入宿主生物，表达该构建体以产生修饰的梭菌神经毒素或梭菌神经毒素的片段，将修饰的梭菌神经毒素或梭菌神经毒素的片段共价附着于TM上。
37.一种获得根据权利要求1的试剂的方法，包含构建编码修饰的梭菌神经毒素或梭菌神经毒素的片段的遗传构建体，将所说的构建体掺入宿主生物，表达构建体以产生修饰的梭菌神经毒素或梭菌神经毒素的片段，用一个或多个间隔子区将修饰的梭菌神经毒素或梭菌神经毒素的片段共价附着于TM上。
38.一种获得根据权利要求1的试剂的方法，包含构建编码该试剂的遗传构建体，将所说的构建体掺入宿主生物中，表达该构建体以生产该试剂。
39.根据权利要求1的试剂在生产用于减轻疼痛的药物中的应用。
40.根据权利要求1的试剂在生产用于防止疼痛的药物中的应用。
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| GB9508204.6 | 1995-04-21 | ||
| GBGB9508204.6A GB9508204D0 (en) | 1995-04-21 | 1995-04-21 | A novel agent able to modify peripheral afferent function |
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| CN1187217A CN1187217A (zh) | 1998-07-08 |
| CN1122718C true CN1122718C (zh) | 2003-10-01 |
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| CZ (1) | CZ332297A3 (zh) |
| DE (1) | DE69633228T2 (zh) |
| ES (1) | ES2225876T3 (zh) |
| GB (1) | GB9508204D0 (zh) |
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| SK (1) | SK143597A3 (zh) |
| TR (1) | TR199701215T1 (zh) |
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1995
- 1995-04-21 GB GBGB9508204.6A patent/GB9508204D0/en active Pending
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1996
- 1996-03-18 SG SG1996006595A patent/SG52602A1/en unknown
- 1996-04-16 AU AU53398/96A patent/AU705924B2/en not_active Ceased
- 1996-04-16 WO PCT/GB1996/000916 patent/WO1996033273A1/en active IP Right Grant
- 1996-04-16 ES ES96910091T patent/ES2225876T3/es not_active Expired - Lifetime
- 1996-04-16 BR BR9609870A patent/BR9609870A/pt not_active Application Discontinuation
- 1996-04-16 SK SK1435-97A patent/SK143597A3/sk unknown
- 1996-04-16 CA CA2218857A patent/CA2218857C/en not_active Expired - Fee Related
- 1996-04-16 EP EP96910091A patent/EP0826051B1/en not_active Expired - Lifetime
- 1996-04-16 PT PT96910091T patent/PT826051E/pt unknown
- 1996-04-16 KR KR1019970707466A patent/KR19990007943A/ko not_active Application Discontinuation
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- 1996-04-16 DE DE69633228T patent/DE69633228T2/de not_active Expired - Lifetime
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- 1996-04-16 RU RU97119181/13A patent/RU2165976C2/ru active
- 1996-04-16 AT AT96910091T patent/ATE274581T1/de active
- 1996-04-16 HU HU9802392A patent/HUP9802392A3/hu unknown
- 1996-04-19 ZA ZA963129A patent/ZA963129B/xx unknown
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1997
- 1997-10-20 NO NO974845A patent/NO974845L/no not_active Application Discontinuation
- 1997-10-21 BG BG101984A patent/BG101984A/xx unknown
-
1999
- 1999-11-22 US US09/447,356 patent/US6395513B1/en not_active Expired - Lifetime
-
2002
- 2002-05-20 US US10/150,262 patent/US6962703B2/en not_active Expired - Fee Related
-
2005
- 2005-09-26 US US11/234,250 patent/US8158132B2/en not_active Expired - Fee Related
-
2007
- 2007-06-28 US US11/819,650 patent/US7887810B2/en not_active Expired - Fee Related
- 2007-06-28 US US11/819,648 patent/US7892560B2/en not_active Expired - Fee Related
-
2011
- 2011-10-11 US US13/270,662 patent/US20120141511A1/en not_active Abandoned
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102422158A (zh) * | 2009-03-13 | 2012-04-18 | 阿勒根公司 | 基于免疫的重靶向内肽酶活性测定 |
| CN102422158B (zh) * | 2009-03-13 | 2015-04-08 | 阿勒根公司 | 基于免疫的重靶向内肽酶活性测定 |
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