CN100389820C - 含肉毒神经毒素的治疗剂 - Google Patents

含肉毒神经毒素的治疗剂 Download PDF

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CN100389820C
CN100389820C CNB008086419A CN00808641A CN100389820C CN 100389820 C CN100389820 C CN 100389820C CN B008086419 A CNB008086419 A CN B008086419A CN 00808641 A CN00808641 A CN 00808641A CN 100389820 C CN100389820 C CN 100389820C
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J·夫莱沃特
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Abstract

一种含有A，B，C，D，E，F或G型肉毒杆菌属肉毒神经毒素之一或两种或多种此类神经毒素的混合物，其特征为，神经毒素或神经毒素的混合物不含自然与神经毒素一起构成肉毒神经毒素复合物的复合蛋白质。

Description

含肉毒神经毒素的治疗剂
本发明涉及药物制剂，其含有肉毒杆菌属肉毒神经毒素，其中神经毒素不含自然存在于复合物中的复合化蛋白质。由此直接的结果是本发明基于的认识，自由神经毒素相对于病人体内的复合体而言没有导致或只导致产生显著降低的中和抗体诱导。本发明还涉及肉毒杆菌的肉毒神经毒素在制备治疗神经系统疾病的药物方面的用途。本发明的另一方面涉及肉毒杆菌属肉毒神经毒素在美容处理方面的用途。
肉毒杆菌属毒素复合体A型(Mr 900,000)自从多年以来即用来治疗不同种的张力障碍。现在许可用于治疗睑痉挛，血方面痉挛和痉挛性斜颈的两种不同的含有此复合体的制剂：
Figure C0080864100031
和
Figure C0080864100032
。其它神经系统疾病(例如痉挛，偏头痛，腰偏，颈部综合症，唾液过多)的治疗目前还在临床试验。此外，制剂还用于化妆适应症如盗汗和明显的皱纹生成。其余的肉毒杆菌属毒素复体(B，C，D，E，F，G型)也适于这种治疗。当然，目前还没有允许含有投寄毒素B-G型的产品进入市场。
肉毒毒素复合物由肉毒杆菌蛋白质的混合物组成。其是具有不同分子量的血凝集素，一种非毒性的非凝血蛋白质(Mr约120,000)和神经毒素(Mr约150,000)。它们形成一种酸稳定的复合物，其是食物中毒方面导致口腔毒性的原因。与纯神经毒素相比，这种复合物抵制胃肠道中的攻击性环境，实现神经毒素的肠吸收，其通过血液循环或淋巴系统到达目标细胞并在此解开传输器释放的阻滞。对此还有横纹光滑肌的瘫痪和不同植物性神经功能的枯竭。中毒的病人死于呼吸肌不足。因为纯的神经毒素在胃肠管道中分解，以此并不为肠吸收，那么消化后没有毒。肠外服用时，神经毒素和复合物的治疗作用不同，因为复合物在组织内分解为其组分，且只有神经毒素在目标细胞中吸收。
为治疗使用，根据目前的技术状况是将复合物直接注射入张力障碍或痉挛的肌肉，在那里，神经毒素在生理pH条件下从复合物中释放，引起希望的药理作用。虽然复合物只以十分少的剂量服用(1-25ng，根据适应症和涉及肌肉的大小)，但是在对病人重复注射相当大量之后会生成特殊的，中和抗体，它也抵抗神经毒素。直接的结果是抗体阳性的病人不再适应此复合物。但是可以用其它类型毒素处理，其中自然没有一种允许用于治疗。当病人试验了所有类型的毒素，抵抗它们的抗体已生成时，再服用肉毒毒素复合物(任意类型)也不再有用。这里要考虑，每剂复合物对提高抗体滴定度有贡献，直至继续服用复合物不再有意义，因为不再达到任何效果。到抗体滴定度显著降低经常已过了数年，以致于这些病人很长时间(可以)不用(用肉毒神经毒素)治疗。
特殊抗体的生成由两个因素促进。一个是，固定在复合体中的神经毒素，长时间保留于组织中，可激活游移至组织中的免疫细胞产生抗体。但是，长时间停留导致目标细胞的吸收不再升高，因为中毒的目标细胞可能不再吸收毒素。这种缓慢地由复合体中解离出的神经毒素也只还是免疫机理上有效。另一个是，复合体中含有的蛋白质增强免疫反应。血凝集素是卵膦脂，也是蛋白质，其特征是高度亲合某些糖。根据其在糖结构上的结合，将卵膦脂有激发免疫的作用。这可指出的有，卵膦脂刀豆球蛋白(Concanavalin A)，植物性血凝集素和美洲商陆有丝分裂原激活了T-和B-淋巴细胞。同样结合到膜上糖的肉毒毒素复合物的血凝集素也可以按类似方式作为免疫辅药起作用，用于生成抗体，并借此引起无效治疗。
由此，对于本发明的发明者提出一个目标，即发展一种对上述疾病和障碍的其它治疗路径。特别是，本发明者推荐一种可治疗已经产生中和抗体的病人的合适的活性物质。
作为上面提及的目的的解决方法，作为A型肉毒毒素复合体组成的两种商业上的制剂
Figure C0080864100041
和
Figure C0080864100042
的替代品，也作为技术状况中所述的其余类型(B，C，D，E，F，G)的复合物的替代品，发展一种新型只含有纯的神经毒素(类型A或B，C，D，E，F，G)，不含血凝聚素和其它异体蛋白质的药物。由于很小的分子量，它很快扩散到目标细胞，在其中被吸收，然后免疫细胞被血凝集素吸引，被激活。抗原性研究中发现，所有类型的纯神经毒素，不同于商业上的A型制剂和B至G型复合物，没有或任何情况下都很少引起抗体的生成。在治疗使用中，这种新发展的药物(纯的类型A，B，C，D，E，F，G的神经毒素)在重复服用后不出现抗体决定的治疗失效。此外，还可表明，纯神经毒素由于其立即的可生物支配性还适于治疗在服用肉毒毒素复合体之后，例如用
Figure C0080864100051
或
Figure C0080864100052
治疗后，已经开发了对抗相应类型的抗体滴定度的病人(所谓次级非应答者)，以及继续用或
Figure C0080864100054
治疗不再可行，因为商业上毒素的服用不再减轻病人的痛苦。
按本发明制备好的药物作为治疗剂，特别适合具有对肉毒毒素，特别是对A型肉毒毒素有抗体滴定度的病人。特别合适的是，按本发明的治疗剂(纯神经毒素或多种纯神经毒素的混合物)用于抗体滴定度不大于50，优选不大于30，更优选不大于20，特别优选不大于10，更特别优选不大于5mU/ml的病人。这里，1mU抗体是中和10U毒素的抗体用量。
另外，按本发明的药剂可特别有利地用于以前从未，或很长时间没再用肉毒神经毒素治疗的病人，因为其抗体滴定度从开始就低或等于0。本发明的优点还在于，通过用按本发明的纯毒素治疗的病人的滴定度没有或都是很微不足道地提高。换句话说，按本发明的治疗剂可长时间服用，不损失效力。
在用肉毒杆菌属毒素治疗时抗体的诱导也受到阻止，通过不用高分子量有毒复合体，而服用纯神经毒素来实现。这种与复合蛋白质完全分开的神经毒素是立即可生物支配的，可以直接结合到运动尾板的神经末梢上。
由此本发明的一方面涉及药物制剂，其含有至少一种A，B，C，D，E，F或G型肉毒杆菌属肉毒神经毒素(或两种或多种这类神经毒素的混合物)，其中，所有的神经毒素不含在复合体中自然存在的复合蛋白质。
按优选的实施方案，其涉及一种药物制剂，其特征为，神经毒素或神经毒素的混合物在病人体内不会引起或，与复合物比较，只引起降低的中和抗体诱导。
另一个优选的实施方案是，含有作为神经毒素或作为神经毒素的混合物的一种天然神经毒素或天然神经毒素的混合物的药物制剂。
另一个优选的实施方案是，含有作为神经毒素或作为神经毒素的混合物的一种重组神经毒素或重组神经毒素的混合物的药物制剂。
此外，本发明的药物制剂的优选的实施方案是，制剂中作为神经毒素的是类型A或B的肉毒杆菌属神经毒素，或作为神经毒素的混合物的是由类型A和B的肉毒杆菌属神经毒素的混合物。
本发明的另一方面还涉及将A，B，C，D，E，F或G型肉毒杆菌属肉毒神经毒素或两种或多种此类神经毒素的混合物用于制备治疗神经系统疾病或张力障碍的药物。神经系统疾病或张力障碍，按一种优选的实施方案，是斜颈痉挛和睑痉挛，是马蹄足样痉挛，血方面痉挛，偏头痛，腰偏，颈部综合症或唾液过多。
本发明的另一方面还涉及将A，B，C，D，E，F或G型肉毒杆菌属肉毒神经毒素或两种或多种此类神经毒素的混合物用于美容处理，其中特别优选用于治疗盗汗和皱纹生成，特别是面部的美容治疗。
本发明意义中更特别优选将神经毒素单独或以混合物用于制备治疗上述神经疾病的药物，涉及的病人(优选人，但也指动物)是已经具有对肉毒神经毒素复合物，特别是对类型A或B的肉毒杆菌属复合物，或对于更多的复合物，特别是类型A和B的肉毒杆菌属复合物的中和抗体的病人(所谓的次级非应答者)。
神经毒素、其混合物或按本发明的药物制剂可以作为水溶液，特别是含水的注射液，但也作为冻干产品存在。
已知的A-G型纯神经毒素的制备，按文献中提及的公开内容包括的记录来进行。典型地，下面的实施例中说明两种神经毒素(A和B型)的提纯。
实施例1：纯神经毒素的分离
A型肉毒杆菌属纯神经毒素按DasGupta和Sathyamoorthy的方法获得。A型肉毒杆菌在20l发酵器中培养，其中的培养基包括2％蛋白胨，1％酵母提取物，1％葡萄糖和0.05％硫甘醇酸钠。生长72小时后，加入3N H2SO4(最后pH＝3.5)沉析毒素。沉淀和离心的生物量用0.2M pH 6.0的磷酸钠缓冲液萃取。
用硫酸精蛋白沉淀分离出核酸后，加入硫酸铵沉淀毒素。溶解后用50mM pH 6.0的磷酸钠渗析的沉淀与同样pH值的DEAE-Sephadex柱结合，用150mM NaCl溶解。然后，通过用50mM Tris/HCl-缓冲液pH 7.9平衡的QAE-Sephadex柱进行色层析。毒素通过NaCl梯度分级。最后，毒素通过SPSephadex在pH 7.0色层析。结合的毒素在此借助NaCl梯度(0-300mM)与柱分离。纯化的毒素在SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)中分析，具有纯度95±5％。生物活性在鼠LD50-试验中测定：LD50单元相应于4.8pg蛋白质。
实施例2：含肉毒毒素的成药的制备
用实施例1中纯化的神经毒素制备每ml中含200个鼠LD50单元，10mg蔗糖和2mg人血清白蛋白的溶液。0.5ml此溶液装入小玻璃瓶中，冷冻干燥。冻干物用生理盐水重新组成，测定生物活性。小瓶中含100±30LD50单元。
实施例3：纯神经毒素B的分离
B型肉毒杆菌属在与A型相同的培养基和相同的条件下培养，直至制成硫酸铵沉淀。然后，再次在pH 6.0进行DEAE-Sephadex色层析。用150mM NaCl从柱中洗脱的级分进行合并，用磷酸钠pH 7.0渗析，接着通过QAE-Sephadex色层析。含毒素的级分再通过DEAE-Sephadex-色层析在pH 8.5(50mM Tris/HCl pH 8.5)进行色层析。
最后通过用10mM磷酸钠pH 8.0平衡的羟基磷灰石色层析得到高纯度B型肉毒毒素。结合均匀的毒素用80mM磷酸钠pH 8.0洗脱，然后在LD50试验中测试生物活性(2-4×107LD50-单元/mg蛋白质)。
实施例4：抗体的检测
20只家兔皮内注射25U
Figure C0080864100081
，持续12周，间隔14天(5次注射)。3周后，间隔14天取血清。
肉毒杆菌属神经毒素A的抗体用酶免疫试验检测，通过将均匀的神经毒素固定到微滴定板上。神经毒素结合的抗体借助第2种酶标记的抗体定量。
结果列于表1中。第1次服药后已5周时可在5只家兔中证明有抗体。11周后，17只家兔，也是所用动物的85％的血清中，显示出对神经毒素的抗体。生理活性实验中表明，17个血清中的12个含有中和抗体(表2)。
表1用酶免疫试验测定用
Figure C0080864100091
处理的家兔的血清样品(1∶100稀释)。提供OD490nm＞0.1。所有的OD值用预先免疫血清的OD值(OD约0.150)来校正。
  家兔序号   3周   5周   7周   9周   11周
  1   -   -   -   0,11   0,36
  2   -   -   -   2,36   2,23
  3   -   -   0,57   1,43   1,44
  4   -   -   0,68   1,68   0,93
  5   -   0,97   3,52   3,49   3,44
  6   -   -   1,34   2,32   2,70
  7   -   -   2,13   3,09   3,00
  8*   -   0,53   1,47   2,75   2,75
  9   -   0,43   2,44   2,85
  10   -   2,99   3,15   2,73
  11   -   0,10   2,42   2,45   1,93
  12   -   -   -   1,13   1,95
  13   -   -   -   -   1,89
  14   -   -   -   -   -
  15   -   -   -   -   -
  16   -   -   -   -   -
  17   -   2,93   3,62   3,72   3,44
  18   -   -   1,18   2,28   2,62
  19   -   -   0,43   0,43   0,81
  20   -   1,65   3,20   2,97   2,88
＊此值不较正，因为不存在预先免疫血清
“-”意指光密度(OD490)＜0.1
表2通过在鼠半隔膜试验中用
Figure C0080864100101
(第一次免疫后11周)的家兔血清中和(检测极限：0.35mU/ml抗体)
  家兔   中和mU/ml
  1   2,0
  2   n.d.
  3   n.d.
  4   ＞10
  5   ＞100
  6   n.d.
  7   ＞10
  8   ＞10
  9   n.d.
  10   n.d.
  11   n.d.
  12   ＞10
  13   n.d.
  14   n.d.
  15   ＜0,35
  16   0,4
  17   ＞10
  18   ＞10
  19   2,0
  20   ＞10
n.d.＝未测定
实施例5：用市场产品和纯神经毒素的抗原性试验
在表明，由神经毒素和血凝集素和非毒性，非血清凝集的蛋白质组成的复合物引起中性抗体生成之后，试验纯神经毒素(A型)的免疫作用。对此，8只家兔用毒素复合物处理，12只家兔用纯毒素处理。按上述方法(参见实施例1)皮内注射25U的各制剂。神经毒素的量，测为重量，在两种制剂中相等(200pg/剂)，如ELISA中所检测。
Figure C0080864100111
额外还含有一种复合蛋白质(约800pg/剂)。
8只用
Figure C0080864100112
处理的动物中有4只在ELISA中显示出抗体滴定度，而12只用纯神经毒素处理的动物证明没有对纯神经毒素有任何抗体。此结果在生物活性实验中确认。所有4只家兔血清含有阻止毒素作用的中性抗体(表3)。
表3通过在鼠半隔膜试验中用
Figure C0080864100113
(第一次免疫后11周)的家兔的血清(1∶3稀释)来中和(检测极限：1mU/ml抗体)
  家兔   中和mU/ml
  1   12mU
  2   ＞30mU
  3   4.5mU
  8   ＞30mU
实施例6：(比较实施例)
本试验中比较通过
Figure C0080864100114
与通过
Figure C0080864100115
的抗体生成。对此，各10只家兔或用
Figure C0080864100116
(组1)，用(组2)或用纯神经毒素(组3)按下列流程处理。
组1和2中多于50％的动物产生中性抗体滴定度，而组3动物的血清中无抗体。
实施例7：临床试验
因为斜颈痉挛而用
Figure C0080864100118
治疗5年的病人(年龄：45岁)已经产生抗体滴定度为3mU/ml血清。
Figure C0080864100119
或对本病人有治疗效果。用纯肉毒神经毒素以与最后注射的
Figure C00808641001111
的剂量相当的145U剂量进行的治疗试验，72小时内导致肌肉松驰，头部支撑正常，肌肉疼痛消除。没有产生不希望的效果。
实施例8：临床试验
病人(年龄：52岁)因为脑溢血麻痹而用
Figure C0080864100121
治疗3年。他已经出现抗体滴定度为1mU/ml血清，因此治疗必须中断。注射200U纯神经毒素实现显著治疗。
文献
DasGupta，B.R.& Sathyamoorthy，V.(1984)，Purification and Amino Acid Composition ofType A Botulinum Neurotoxin；Toxicon 22(3)，p.415-424
De Jongh，K.S.，Schwartzkoff，C.L.&Howden，M.E.H.(1989)，Clostridium botulinum TypeD Neurotoxin Purification and Detection；Toxicon 27(2)，p.221-228
Schmidt，J.J.& Siegel，L.S.(1986)，Purification of Type E Botulinum Neurotoxin by High-Performance Ion Exchange Chromatography；Analyt.Biochemistry 156，p.213-219
Nukina，M.，Mochida，Y.，Sakaguchi，S.&Sakaguchi，G.(1988)，Purification of Clostridiumbotulinum Type G Progenitor Toxin；Zbl.Bakt.Hyg.A 268，p.220-227
Terajima，J.，Syuto，B.，Ochanda，J.O.&Kubo，S.(1985)，Purification and Characterization ofNeurotoxin Produced by Clostridium botulinum Type C 6813；Infection and Immunity48(2)，p.312-317
Wadsworth，J.D.F.，Desai，M.，Tranter，H.S.et al.(1990)，Botulinum type F neurotoxin：Large-scale Purification and Characterization of its Binding to Rat Cerebrocortical Synaptosomes；Biochem.J.268，p.123-128

Claims (6)

1.一种A型肉毒杆菌属的肉毒神经毒素的用途，其特征为，神经毒素不含自然与肉毒神经毒素构成复合物的复合蛋白质，用于制备已经形成抗这种肉毒神经毒素的中和抗体的动物或病人中美容处理用或治疗张力障碍或治疗神经系统疾病的治疗剂。
2.权利要求1的用途，其特征为，待治疗的对象已经具有对A型肉毒杆菌属复合物的中和抗体。
3.权利要求1或2的用途，其特征为，美容处理用于治疗盗汗。
4.权利要求1或2的用途，其特征为，美容处理用于治疗皱纹形成。
5.权利要求4的用途，其特征为，美容处理用于治疗面部皱纹形成。
6.权利要求1或2的用途，其特征为，神经系统疾病或张力障碍为斜颈痉挛，睑痉挛，马蹄足样痉挛，血方面痉挛，偏头痛，腰偏，颈部综合症或唾液过多。
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