CN101448524A - 脑血管痉挛抑制剂 - Google Patents
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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Abstract
本发明的技术问题在于提供对蛛网膜下腔出血后发生的脑血管痉挛有效且副作用少的脑血管痉挛抑制剂。本发明的脑血管痉挛抑制剂的特征在于以抗HMGB1单克隆抗体为有效成分。
Description
技术领域
本发明涉及用于抑制蛛网膜下腔出血后发生的脑血管痉挛的药剂。
背景技术
蛛网膜下腔出血主要发生在40~60多岁的壮年层,主要指由于动脉瘤的破裂而在包围脑的蛛网膜与脑之间出血的状态。该出血会使头盖骨内压瞬时上升,给脑造成损伤。据统计,约10%的患者在蛛网膜下腔出血发病后立即死亡,约25%的患者危及生命。据说即使初次发病后存活,但约20%患者会在2周内再次出血。作为该蛛网膜下腔出血的治疗,有除去血肿、预防已破裂过的动脉瘤再破裂等。
如上所述,蛛网膜下腔出血本身就是非常恐怖的疾病,但在蛛网膜下腔出血治疗结束后,半数以上的患者会出现脑血管痉挛这一特异的病态。该脑血管痉挛是在蛛网膜下腔出血后3~14天发生并持续1~2周的脑主动脉可逆性狭窄。其会导致脑缺血,从而使约40%的患者死亡,约30%的人留下严重的后遗症,能回归社会的最终只有约30%,已成为严重的问题。
但是,对该脑血管痉挛的研究并未充分展开,其预防方法和治疗方法也尚未确立。例如,蛛网膜下腔出血引发脑血管痉挛的机制尚不明确,但据说自由基、脂质过氧化反应、花生四烯酸级联反应、血管周围神经的损伤、内皮依赖性舒血管效应的损伤、血管壁的结构改变等多种因素复杂地参与其中。因此认为即使阻碍上述因素中的任一个,也很难预防或治疗脑血管痉挛。
现在,作为针对脑血管痉挛的全身药物治疗法,有给予盐酸法舒地尔、奥扎格雷钠。另外,也在蛛网膜下腔出血的手术中向脑池内给予组织型纤溶酶原激活剂。但它们的效果并不充分。
HMGB1是从啮齿类到人类中存在的95%以上的氨基酸序列相同的蛋白质。该HMGB1也虽然存在于正常细胞中,但在败血症(全身性炎症反应症候群)放出的菌体内毒素LPS(脂多糖)的刺激下,血中浓度上升,最终导致组织损伤。因此,在日本专利特表2003-520763号公报所述的技术中,为了治疗以炎症性细胞因子级联的活化为特征的症状,给予HMGB1拮抗剂。但是,虽然在日本专利特表2003-520763号公报中例示了许多介以炎症性细胞因子级联的疾病或症状作为治疗对象,但对脑血管痉挛未作记载和提示。
另外,在日本专利特表2005-537253号公报中,作为用于治疗由坏死组织诱发的副作用的组合物,公开了含有HMGB1抗体等的组合物。但是,作为该副作用,仅例示了邻近活细胞的活化、骨髓细胞的动员及活化、内皮屏障功能的丧失、浮肿,对脑血管痉挛未作记载和提示。
发明内容
如上所述,尽管蛛网膜下腔出血后发生的脑血管痉挛会导致死亡或严重的后遗症,但没有可靠的抑制方法
为此,本发明应解决的技术问题在于提供对蛛网膜下腔出血后发生的脑血管痉挛有效且副作用少的脑血管痉挛抑制剂。
本发明者为了解决上述技术问题,对于对抑制脑血管痉挛有效的药剂进行了各种研究。结果发现,抗HMGB1单克隆抗体具有比过去报道的任何药剂均优异的效果,从而完成了本发明。
本发明的脑血管痉挛抑制剂的特征在于,以抗HMGB1单克隆抗体为有效成分。
在本发明中,为了制造用于抑制脑血管痉挛的药剂,使用抗HMGB1单克隆抗体。
本发明的脑血管痉挛抑制方法的特征在于给予抗HMGB1单克隆抗体。
附图说明
图1是蛛网膜下腔出血模型兔子的无处理组在蛛网膜下腔出血前后的脑基底动脉的造影照片。图中,各组的左侧(前)表示蛛网膜下腔出血前的照片,右侧(后)表示蛛网膜下腔出血前的照片。各组中,蛛网膜下腔出血后(右侧)血管径明显变细,可见强烈的脑血管痉挛。
图2是蛛网膜下腔出血模型兔子的IgG给予组在蛛网膜下腔出血前后的脑基底动脉的造影照片。图中,各组的左侧(前)表示蛛网膜下腔出血前的照片,右侧(后)表示蛛网膜下腔出血前的照片。各组中,蛛网膜下腔出血后(右侧)血管径明显变细,可见强烈的脑血管痉挛。
图3是蛛网膜下腔出血模型兔子的本发明抗体给予组在蛛网膜下腔出血前后的脑基底动脉的造影照片。图中,各组的左侧(前)表示蛛网膜下腔出血前的照片,右侧(后)表示蛛网膜下腔出血前的照片。各组中,蛛网膜下腔出血后(右侧)脑血管痉挛与图1的无处理组及图2的IgG给予组相比得到明显抑制。
图4是用于比较无处理组、IgG给予组以及本发明抗HMGB1单克隆抗体组的血管收缩率的图。本发明的抗HMGB1单克隆抗体的脑血管痉挛抑制作用与无处理时和给予IgG时相比具有显著性优势。
具体实施方式
本发明的脑血管痉挛抑制剂以抗HMGB1单克隆抗体为有效成分。抗HMGB1单克隆抗体只作用于组织损伤因子之一的HMGB1,其作用机制不明,但能抑制蛛网膜下腔出血后发生的脑血管痉挛。另一方面,基本上不作用于其他化合物等。因此认为产生副作用的可能性为零或极少。
抗HMGB1单克隆抗体的制备可采用常规方法。例如,用市售的HMGB1将小鼠或大鼠等免疫,使其抗体产生细胞或脾细胞与骨髓瘤细胞融合得到杂交瘤。将该杂交瘤克隆,筛选产生与HMGB1特异性反应的抗体的克隆。培养该克隆,将分泌的单克隆抗体精制即可。
本发明使用的抗HMGB1单克隆抗体的种类没有特殊限制。例如,可使用人型抗体或完全人抗体。
本发明的脑血管痉挛抑制剂的剂型没有特殊限制,若考虑到有效成分抗HMGB1单克隆抗体为肽,则优选以注射剂给药,制成溶液或乳液制剂等液体制剂。
作为液体制剂的溶剂,可使用经pH调节的生理盐水、葡萄糖水溶液等血浆等渗液。当抗体与盐类等一起冷冻干燥的情况下,也可以使用纯水、蒸馏水、灭菌水等。其浓度可与普通的抗体制剂一样,通常为0.1~1mg/mL左右,用于点滴时可为0.02~0.2mg/mL左右。但是,注射剂的渗透压必须与血浆相等。
本发明中的“抑制”包含抑制脑血管痉挛产生即“预防”、减轻已产生的脑血管痉挛即“治疗”这两方面的概念。因此,本发明的脑血管痉挛抑制剂可在蛛网膜下腔出血后到脑血管痉挛发生前进行预防性给药,也可以在脑血管痉挛发生后进行治疗性给药。
在蛛网膜下腔出血后3~14天,患者中有半数以上会发生脑血管痉挛。但是关于蛛网膜下腔出血引发脑血管痉挛的机制,有人指出有几种要素,但认为多个因素复杂地参与其中,现在并不明确。因此,在蛛网膜下腔出血后或在脑血管痉挛发生后,应该维持本发明的脑血管痉挛抑制剂在脑血管中的血药浓度。因此,本发明的脑血管痉挛抑制剂优选在蛛网膜下腔出血后多次给药或持续给药。
多次给药时的给药频率和给药量可根据是脑血管痉挛发生前还是发生后以及患者的情况等做适当调整。如后述实施例所示,当对体重约3kg的蛛网膜下腔出血模型兔子以每次2mg的抗HMGB1单克隆抗体给药2次时,得到显著的脑血管痉挛抑制效果。由该结果可知,对人的给药量可为每次0.1~2mg/kg抗HMGB1单克隆抗体,更优选为0.2~2mg/kg,每日可给药2次。给药形态没有特殊限制,例如可以静脉注射,在紧急情况下,也可通过在蛛网膜下腔出血手术中设置脑池分流管来给药。
持续给药时的制剂浓度、给药量等也可做适当调整,例如将浓度0.02~0.2mg/mL的液体制剂用2~4小时、每日2次左右进行点滴给药。
当脑血管痉挛患者在蛛网膜下腔出血后14天左右之后存活的情况下,通常脑血管痉挛会自然消失。因此,从蛛网膜下腔出血后经过14天左右,可考虑患者的情况等而逐渐减少本发明的脑血管痉挛抑制剂的给药量等。
本发明的脑血管痉挛抑制剂能有效地抑制在蛛网膜下腔出血后发生的迟发性、会对患者造成严重不良影响的脑血管痉挛。另外,考虑到现在使用的抗体药剂,产生严重副作用的可能性极小。因此,本发明的脑血管痉挛抑制剂能抑制迄今为止无特效治疗方法的脑血管痉挛并防止后遗症、加速患者回归社会,非常有用。
[实施例]
以下,列举实施例更具体地说明本发明,但本发明不限于下述实施例,可在符合前述和后述的主旨的范围内做适当变更后实施,它们均属于本发明的技术范围。
实施例1 抗HMGB1单克隆抗体的制备
(a)大鼠的免疫
取市售的来自牛胸腺的HMGB1和HMGB2的混合物(和光纯药工业公司制、编号:080-070741)1mg/mL于2mL玻璃制注射筒内,借助连接管与置于另一2mL玻璃制注射筒的等容量的弗氏完全佐剂缓慢混合,制成乳液。在被七氟醚麻醉的大鼠的后肢足跖各注射0.1mL所得到的乳液,注射给药共计0.2mL。2周后,从颈静脉采血,证实抗体效价上升。然后,在上述注射给药5周后,无菌取出肿大的肠骨淋巴结。从得到的2个淋巴结可回收约6×107个细胞。
(b)细胞融合和克隆
用聚乙二醇将上述肠骨淋巴结细胞和小鼠骨髓瘤SP2/O-Ag14(SP2)细胞融合,将得到的融合细胞植入96孔微孔板。1周后,进行初次ELISA筛选,对于阳性孔,通过Western杂交进行二次筛选。将显示阳性的孔细胞移置24孔微孔板,培养细胞至几乎汇合的状态(约2×105),用0.5mL的冷冻培养基(向GIT培养基中添加10%胎牛血清和10%二甲基亚砜得到的培养基),在液氮中冷冻保存。将该冷冻保存细胞解冻后,用96孔微孔板进行克隆。
(c)抗体的精制
用旋转培养装置(Vivascience公司制)大量培养上述阳性细胞2周,得到浓度为2~3mg/mL的抗体液。将该抗体液在中性pH下与亲和凝胶(Invitrogen公司制、MEP-HyperCel)混合,使抗HMGB1抗体与凝胶特异性结合。将特异性结合于凝胶的抗体用甘氨酸-盐酸缓冲液(pH4)洗脱。通过超滤装置将洗脱液浓缩后,利用Sepharose CL8B凝胶过滤柱(直径2cm×长97cm)进一步精制。
得到的单克隆抗体是以HMGB1蛋白质的C末端序列208EEEDDDDE215(E表示谷氨酸、D表示天冬氨酸)为表位特异性识别的抗体。例如,作为与HMGB1类似的蛋白质,有HMGB2,HMGB2中不存在211以下的DDDDE序列,因此本发明的单克隆抗体不与HMGB2结合,仅特异性识别HMGB1并结合。
实施例2
在将12只从日本Charles River公司得到的雄兔(体重约3~3.5kg)制成蛛网膜下腔出血模型的前一周,用盐酸氯胺酮(50mg/kg、肌肉给药)和戊巴比妥(20mg/kg、静脉给药)麻醉后,向从右大腿动脉插入至左脊椎骨动脉起始部的管中注入造影剂,通过血管造影拍摄其脑基底动脉。在制作蛛网膜下腔出血模型当天,用盐酸氯胺酮(50mg/kg、肌肉给药)和戊巴比妥(20mg/kg、静脉给药)麻醉后,采集1mL动脉血,将采集的该动脉血注射到该兔子的枕大池(小脑延髓池内)。然后使其低头并保持30分钟,由此制成蛛网膜下腔出血模型。
另外,将实施例1中精制的大鼠的抗牛HMGB1单克隆抗体2mg溶于1mL磷酸缓冲液,得到抗体溶液。在模拟蛛网膜下腔出血后1小时和24小时,向4只蛛网膜下腔出血模型兔子静脉注射该抗体溶液1mL(抗牛HMGB1单克隆抗体2mg)。
在模拟蛛网膜下腔出血后3天,与上述同样地拍摄脑基底动脉。另外,为了比较,对未给予抗牛HMGB1单克隆抗体的兔子4只(无处理组)和按同样操作给予了具有多种抗体活性的大鼠正常多克隆免疫球蛋白G(2mg)的兔子4只(IgG给予组)也进行了同样的拍摄。得到的各4例、共12例的照片如图1~3所示。图1是无处理的例子,图2是给予多克隆免疫球蛋白G的例子,图3是给予本发明的抗HMGB1单克隆抗体的例子。另外,用NIH Image J(美国国家卫生研究所),从得到的照片测定9处脑基底动脉的内径,求出蛛网膜下腔出血后的内径的收缩率,算出各个体的平均值。进而,由各个体的平均值算出各组的收缩率平均值。各组间的显著性差异用t-检验来测定。结果如表1和图4所示。另外,图4中,“**”表示与无处理组相比具有显著性差异,p<0.01,“##”表示与IgG给予组相比有显著性差异,p<0.01。
[表1]
如图1所示,未给予抗体的兔子的脑基底动脉在模拟蛛网膜下腔出血后变细并发生强烈的脑血管痉挛。另外,如图2所示,给予IgG的兔子也同样发生强烈的脑血管痉挛。与此相对,如图3所示,给予本发明的抗HMGB1单克隆抗体的兔子的脑基底动脉的粗细在很大程度上得以维持,显著抑制了脑血管痉挛。此外,本发明的抗HMGB1单克隆抗体的脑血管痉挛抑制作用与无处理时和给予IgG时相比,在统计学上具有显著性优势。从而证实本发明的脑血管痉挛抑制剂能显著抑制脑血管痉挛。
Claims (7)
1.一种脑血管痉挛抑制剂,其特征在于,以抗HMGB1单克隆抗体为有效成分。
2.根据权利要求1所述的脑血管痉挛抑制剂,其中,在蛛网膜下腔出血后被多次给药。
3.根据权利要求2所述的脑血管痉挛抑制剂,其中,以每次0.2~2mg/kg的方式被给予抗HMGB1单克隆抗体。
4.根据权利要求1所述的脑血管痉挛抑制剂,其中,在蛛网膜下腔出血后被持续给药。
5.根据权利要求1~4中任一项所述的脑血管痉挛抑制剂,其为静脉注射给药。
6.抗HMGB1单克隆抗体的使用,其用于制造抑制脑血管痉挛的药剂。
7.一种脑血管痉挛的抑制方法,其特征在于,给予抗HMGB1单克隆抗体。
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