CN102985102A - 引起局部肌麻痹的非扩散型肉毒杆菌毒素及其精制方法 - Google Patents
引起局部肌麻痹的非扩散型肉毒杆菌毒素及其精制方法 Download PDFInfo
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Abstract
本发明涉及引起局部肌麻痹的非扩散型肉毒杆菌毒素的精制方法以及由此精制的非扩散型肉毒杆菌毒素,更具体地,本发明涉及包括如下步骤的非扩散型肉毒杆菌毒素的精制方法:用阴离子色谱处理精制后的肉毒杆菌毒素A型制剂以分离亚组分,在该阴离子色谱中添加了受控量的氯化钠(NaCl);以及从所述亚组分中获得具有预定范围的A260/A280值的组分;以及涉及由此精制的非扩散型肉毒杆菌毒素。
Description
技术领域
本发明涉及一种引起局部肌麻痹的非扩散型肉毒杆菌毒素的精制方法以及由此获得的非扩散型肉毒杆菌毒素。
背景技术
美国FDA声称已经报道有除注射有肉毒杆菌毒素的部位以外的其他部位也出现肌麻痹副作用,并且有注射肉毒杆菌毒素的人住院或者死亡。
FDA认为当扩散到除腿部以外的部位时会影响呼吸肌肉的功能,由此导致副作用[FDA NEWS RELEASE FOR IMMEDIATE RELEASE April 30,2009,FDA Patient Safty News:Show#74,April 2008]。关于此点,FDA声称患者和医生需要注意在注射后是否出现呼吸频率降低或吞咽困难[FDA-approved Patient Labeling 7/31/09 APPENDIX 1:MEDICATION GUIDEBOTOX,BOTOX Cosmetic(on a botulinum toxin A)for Injection]。
美国消费者协会要求FDA加强警示:在1997年至2006年间向FDA报告有180件有关的副作用的病例,其中16件导致死亡的病例,以及使用可能导致畸形。
在现有技术中,已经通过进行酸沉淀(USP 7,354,740,其题目为“Animalproduct free system and process for purifying a botulinum toxin”,爱力根公司(Allergan Inc.))或进行酸沉淀后进行色谱(USP 7,452,697,其题目为“Chromatographic method and system for purifying a botulinm toxin”,爱力根公司(Allergan Inc.);韩国专利申请第10-2008-001680,其题目为“Method ofpurifying botulinum toxin from Clostridium botulinum”,明傣克斯金公司(Medexgen Inc);韩国专利申请第10-2002-000685,其题目为“Method forpurifying Clostridium botulinum type A toxin”,明傣克斯金公司(Medexgen Inc))来精制肉毒杆菌毒素。
然而,众所周知的是通过上述方法精制的肉毒杆菌毒素会显著地扩散到除了被注射的部位以外的身体部位,从而麻痹周围器官或呼吸肌肉;以及在情况严重时会导致死亡的严重副作用。
发明内容
技术问题
因此,鉴于上述问题进行了本发明,本发明人发现:当使用氯化钠将通过酸沉淀或酸沉淀后再进行色谱而精制得到的已有的A型肉毒杆菌毒素进行离子交换色谱时,获得三种异质的亚组分,其中一种亚组分具有肌麻痹活性,且同时不会在体内扩散。
因此,本发明的目的之一是提供一种在体内不扩散且同时使肌麻痹活性限于局部的非扩散型肉毒杆菌毒素以及精制该非扩散型肉毒杆菌毒素的方法。
技术方案
为了实现上述目的,根据本发明的一个方面,提供一种非扩散型肉毒杆菌毒素精制方法,该方法包括如下步骤:通过采用pH为4.5~6.5的缓冲液和0.02~0.2M的氯化钠(NaCl)进行离子交换色谱,将A型肉毒杆菌毒素产品分离成亚组分;以及收集A260/A280值为0.4~0.6的非扩散型肉毒杆菌毒素亚组分。
根据本发明的另一个方面,提供通过上述方法精制的非扩散型肉毒杆菌毒素制品,其包含离子浓度至少分别为每100U/ml有150ppb、80ppb和140ppb的Zn、Fe和Mg离子。
根据本发明的又一个方面,提供一种用于测定非扩散型肉毒杆菌毒素的方法,其包括:将权利要求2所述的非扩散型肉毒杆菌毒素以相当于该毒素LD50的1.5~3倍的量注射至小鼠(4~6周龄,重18~22g)的左侧或右侧后肢肌肉;以及判断所述小鼠的右侧后肢肌肉和呼吸肌肉是否麻痹或小鼠是否死亡。
根据本发明的再一个方面,提供所述方法,其中,所述非扩散型肉毒杆菌毒素制品显示出在小鼠(4~6周龄,重18~22g)的左侧或右侧后肢肌肉注射50μL体积中2U的毒素制品后96小时的存活率至少为80%。
有益效果
根据本发明,可以获得一种引起局部肌麻痹的非扩散型肉毒杆菌毒素,该非扩散型肉毒杆菌毒素在所希望的部位引起肌麻痹,而不会扩散到除给药部位以外的其他部位,并且具有快速且持续时间长的肌麻痹效果。
并且,本发明可以获得与传统的肉毒杆菌毒素产品相比具有高的毒素效价的非扩散性肉毒杆菌毒素。
此外,根据本发明,可以通过控制阴离子色谱条件来大量获得不与扩散性毒素混合的非扩散性肉毒杆菌毒素。
附图说明
图1表示在实施例1中通过离子交换色谱将肉毒杆菌毒素分离成亚组分的结果。
图2是表示亚组分的峰面积%随着缓冲溶液的pH值的改变而变化的曲线图。
图3是表示每个亚组分的峰面积%随着试样的投药量的改变而变化的曲线图。
图4表示存活率在实施例1中分离的亚组分中的活性亚组分(pI和pII)的剂量(A)与给药后的时间(B)的改变而变化的示意图[小鼠:ICR,雌性,4周龄,18-22g,n=10,IM注射入右后腿]。
图5是表示将在实施例1中分离的亚组分中的非扩散型活性组分(pII)和市售产品给药到小鼠的右后肢后12小时测量的从给药部位扩散至体内的比较的一组照片。
图6是表示将在实施例1中分离的亚组分中的非扩散型活性组分(pII)和市售产品给药到小鼠的右后肢后测量的剂量(A)和给药后时间(B)与存活率的关系的示意图。
图7表示在实施例1中分离的亚组分中的非扩散型活性组分(pII)和市售产品各以1U的量给药至小鼠的右后肢后测量的、肌麻痹开始的时间的比较图。
图8表示在实施例1中分离的亚组分中的非扩散型活性组分(pII)和市售产品各以0.5U的量给药到小鼠的右后肢后测量的肌麻痹持续的时间的比较图。
具体实施方式
下面进一步详细描述根据本发明的非扩散型肉毒杆菌毒素的精制方法。
根据本发明的非扩散型肉毒杆菌毒素的精制方法包括如下步骤:通过采用pH为4.5~6.5的缓冲液和0.02~0.2M的氯化钠(NaCl)进行离子交换色谱,将A型肉毒杆菌毒素产品分离成亚组分;以及收集A260/A280值为约0.4~0.6的非扩散型肉毒杆菌毒素亚组分。
用于精制非扩散型肉毒杆菌毒素的本发明方法是如下的方法:其中将通过进行酸沉淀或者进行酸沉淀后进行离子交换色谱获得的已有的肉毒杆菌毒素进行由本发明人开发的新颖离子交换色谱法,由此得到非扩散型肉毒杆菌毒素组分。
因此,用于精制非扩散型肉毒杆菌毒素的本发明方法是使用从A型肉毒梭状芽胞杆菌株的培养液中酸沉淀或者在酸沉淀后色谱精制获得的A型肉毒杆菌毒素进行非扩散型肉毒杆菌毒素的精制。
当使精制后的A型肉毒杆菌毒素进行使用氯化钠的离子交换色谱时,可以从离子交换树脂得到三种亚组分。为了进行离子交换色谱,使用醋酸钠缓冲液,并且依次增加添加到缓冲溶液中的氯化钠的浓度以获得亚组分,收集分离的各组分。发现当不使用氯化钠(0M的NaCl)时,分离的组分是在体内扩散的活性组分(peak Ⅰ,pI);当采用浓度为0.02~0.2M的氯化钠时,分离的组分是在体内不扩散的活性组分(peak Ⅱ,pII);以及当采用浓度为1.0M的氯化钠时,分离的组分是在体内扩散的活性组分(peakⅢ,pIII)。
具体地,用于获得组分pII的氯化钠浓度在0.02~0.2M范围内,且当不使用氯化钠或者氯化钠的浓度超出上述范围时pII组分含有pI或pIII组分时,这表明很难获得受本发明保护的具有局部肌麻痹效果的非扩散型活性组分。
同时,当使用在具有4.5至5.5范围内pH值的缓冲液中的氯化钠进行离子交换色谱时,降低了亚组分中的pI或pIII组分的量,并且增加了pII组分的纯度。这可以通过比较A260/A280值或者测量在注射小鼠的右后肢肌肉后的麻痹程度来证实。
此外,当使用氯化钠进行离子交换色谱使得精制后的A型肉毒杆菌毒素试样的量为所使用的离子交换色谱法柱的容量的1/5~1倍时,pII组分的量增加。具体地,当色谱柱的容量为1ml,且精制的A型肉毒杆菌毒素试样的量为0.2~1ml时,所获得的pII组分的量在20%至50%范围内。
同时,发现通过用于精制非扩散型A型肉毒杆菌毒素的本发明方法所获得的pII组分具有如使用小鼠测量的1×105-5×105U/ml的总LD50。此外,可以看出pII组分在注射时并未扩散到除了所希望的部位外的其他部位,并且与市售产品相比,其在显著短的时间内开始肌麻痹以及长时间维持肌麻痹。
另外,通过用于精制非扩散型肉毒杆菌毒素的本发明方法获得的pII组分包含每100U/ml至少150ppb的Zn离子、至少80ppb的Fe离子和至少140ppb的Mg离子。
用于精制非扩散型肉毒杆菌毒素的本发明方法的特征在于,Mg、Fe和Zn的含量明显高于由传统方法精制的A型肉毒杆菌毒素的那些离子的含量。
发现将由本发明的精制方法获得的非扩散型肉毒杆菌毒素以相当于该毒素的LD50的1.5~3倍的量注射到小鼠的右后肢时,右后肢肌肉麻痹,而没有麻痹呼吸肌肉且无致命活性,这表明本发明的非扩散型肉毒杆菌毒素没有在体内扩散。
实施例
下面结合具体实施例和附图对本发明做进一步详细说明,但本发明的范围并不局限于以下实施例及附图。
用于本发明实施例的市售产品是用由USP7,354,740[题目为“Animalproduct free system and process for purifying a botulinum toxin”]所公开的方法制备的A型肉毒毒素制剂产品(爱力根公司(Allergan,Inc))。但是,本发明不仅适用于上述市售产品或者由上述方法精制而得的A型肉毒杆菌毒素,而且适用于由酸沉淀方法精制的任何A型肉毒杆菌毒素。
实施例1:离子交换色谱分离的组分
将通过酸沉淀由肉毒杆菌毒素培养基精制的试料进行离子交换色谱分离,由此将该试料分离成3个亚组分,色谱分离的结果如图1所示[pI、peak I(在体内扩散的活性组分);pII、peak II(在体内不扩散的组分);pIII、peak III(非活性组分)];
使用AKTA FPLC仪器(GE Healthcare)、Hitrap DEAE FF色谱柱(GEHealthcare)和作为实验用缓冲液的醋酸钠缓冲液(pH为5.5)(洗提缓冲溶液),进行离子交换色谱分离。
具体地,采用醋酸钠中0M的NaCl(pH为5.5)来洗提该样品,以获得非结合的洗涤组分(pI、在体内扩散的活性组分);然后采用0.05M的NaCl洗提以获得一种组分(pII、在体内不扩散的组分);然后采用1M的NaCl洗提以获得一种组分(pIII、惰性组分)。对该试料进行流速为1ml/min的酸沉淀,然后进行脱盐。
实施例2:作为缓冲溶液的pH值变化的函数的峰值和特性的变化
图2表示根据离子交换色谱中使用的缓冲溶液的pH值变化,亚组分的峰面积%。由图2可以看出,当缓冲溶液的pH值在4.5~5.5范围时,pII组分的峰面积%增加。下表1显示了作为缓冲溶液的pH值的函数,所述亚组分的A260/A280值;
下表2显示了所述亚组分的总LD50(unit)
[表1]
由上表1可以看出,当缓冲溶液的pH值在4.5~5.5范围内时,pII组分的A260/A280值为0.45~0.6,这表明pII组分适于给药。
因此,优选将缓冲液的pH值调整为4.5~5.5以获得在体内不扩散的精致活性组分(pII)。
[表2]
由上表2可以看出,当缓冲溶液的pH值为5.0~5.5时,pII组分亚组分的总LD50为0.8×105~3.5×105U/ml,这表明p Ⅱ的回收率相当高。
因此,优选将缓冲液的pH值调整为4.5~5.5以得到在体内不扩散的安全活性组分(pII)。
实施例3.作为试氧装载量的变化的函数峰值和特性的变化
按照实施例1相同的方式获得亚组分。
图3显示了作为用于获得亚组分的试样的装载量的变化的函数各亚组分(pI、pII、pIII)的峰面积的变化。另外,表3显示了作为试样装载量的函数所述亚组分的A260/A280值变化,以及下表4显示了作为试样装载量的函数所述亚组分的总LD50(unit)的变化。
[表3]
由上表3可以看出,当试样的装载量在0.25~2ml时,pII组分的A260/A280为0.4~0.6,这表明pII组分是用于给药的合适适试样。
[表4]
由上表4可以看出,当试样装载量在0.25~1ml时,p Ⅱ组分的总LD50为0.8×105~3.5×105U/ml,这表明p Ⅱ的回收率相当高。
因此,优选控制试样装载量为色谱柱容量的1/5~2倍以获得在体内不扩散的安全活性组分(p Ⅱ)。
测试例1:肌麻痹效果的比较
1)注射到右后肢肌肉
为了测试由实施例1得到的亚组分的肌麻痹效果,对小鼠右后肢肌肉进行注射。
将ICR小鼠(雌性,4周龄,18~22g)分为3组,每组各有10只。精确测量和记录每只小鼠的重量。在没有空气下将每个亚组分以浓度为1U/20μL填满50μL的汉密尔顿注射器(Hamilton syringe),然后将注射器针头扎入每只ICT小鼠的右后肢脚踝至3cm深度,并把每个亚组分注射到每只小鼠的肌肉。在注射以后,按照表5的标准在各时间点评价每只小鼠的肌麻痹指数以及测量小鼠的存活率。测量的结果如图4所示。
[表5]
得分 | 标准。 |
0 | 正常小鼠的肢体外观和行走 |
1 | 小鼠腿拖地行走,但是脚趾不是集中在一起 |
2 | 与标准1一样,脚趾集中在一起 |
3 | 与标准2一样,脚关节向内弯曲 |
4 | 与标准3一样,脚与腿部肌肉接触且瘸腿 |
[表6]
组分 | 在注射到小鼠右后肢时麻痹的部位 |
体内扩散的活性组分 | 体内各部位,包括右后肢、膈膜。 |
体内不扩散的活性组分 | 右后肢。 |
体内扩散的非活性组分 | - |
由图4可以看出,在实施例1中所获得的亚组分中,在体内不扩散的活性组分(pII)与在体内扩散的活性组分(pI)相比显示了显著增加的剂量和存活率(图4A),以及在剂量为2U时p Ⅱ组分的存活率为100%,而p Ⅰ组分的存活率为0%,相互之间完全不同。
测试例2:市售产品与在体内不扩散的活性组分在体内扩散性的比较
为了测试市售产品(Allergan’s Botox)和由实施例1得到的亚组分p Ⅱ的肌麻痹效果,注射到小鼠的右后肢肌肉。
ICR小鼠(雌性,4周龄,18~22g)分为两组,每组各有10只小鼠。精确测量和记录每只小鼠的重量。在没有空气下将市售产品和组亚组分pII都以浓度为1U/20μL填满50μL的汉密尔顿注射器(Hamilton syringe),然后将注射器针头扎入每只ICT小鼠的右后肢脚踝约3cm深度,将市售产品和亚组分注射到每只小鼠的肌肉。在注射到小鼠后,肉眼观察小鼠的麻痹程度以测定市售产品和亚组分在活的有机体内的扩散程度。图5显示了在注射后12小时拍照的小鼠状态;图6表示作为剂量和给药时间的函数的小鼠存活率;图7表示肌麻痹效果的起始时间;图8表示肌麻痹效果的持续时间。
如图5所看到的,亚组分p Ⅱ仅麻痹肌内注射的右后肢,但是市售产品没有扩散至腰部和对侧肢体以及麻痹腰部和对侧肢体。
如图6所看到的,与市售产品相比,亚组分p Ⅱ显示了在某剂量下存活率明显增加(图6A);在剂量为2U下pⅡ亚组分的存活率为100%,而使用市售产品的存活率为0%,相互之间有极大的差异。
如图7所看到的,与市售产品相比,pⅡ亚组分在剂量为1U时在相当短的时间内开始肌麻痹效果。
如图8所看到的,与市售产品相比,pⅡ亚组分在0.5U剂量下肌麻痹持续时间长80天以上。
试验例3:分析冷冻干燥的肉毒杆菌毒素的离子含量
将市售产品(Allergan’s Botox)和在实施例1中获得的亚组分p Ⅱ冷冻干燥,并且分析其离子含量。
为了与市售产品比较,稀释的p Ⅱ组分和同等量的肉毒杆菌毒素(100U)与添加剂进行冷冻干燥;采用小鼠致死试验证实市售产品冷冻干燥物与pⅡ亚组分的冷冻干燥产品具有相同的活性;然后分析了冷冻干燥产品的离子含量。具体地,将两种冷冻干燥的产品每个都溶于10ml的蒸馏水中,然后采用电感耦合等离子体质谱分析测量了离子的含量。测量结果示于下表7。
[表7]
由上表7可以看出,由本发明精制的非扩散型肉毒杆菌毒素的冷冻干燥物中的Mg、Fe和Zn的含量明显高于市售产品的冷冻干燥物中的Mg、Fe和Zn的含量。
尽管为了说明目的描述了本发明的优选实施例,但是本域技术人员在没有偏离如所附的权利要求书公开的本发明的范围和精神下可以对本发明进行各种修改、添加或者替换。
工业实用性
根据本发明,可以大量获得引起局部肌麻痹的非扩散型肉毒杆菌毒素。该非扩散型肉毒杆菌毒素在所希望的部位引起肌麻痹,不会扩散到除所希望的部位以外的部位,且具有短和持续时间长的肌麻痹效果。
权利要求书(按照条约第19条的修改)
1.一种非扩散型肉毒杆菌毒素的精制方法,包括以下步骤:
通过采用pH为4.5~6.5的缓冲液和0.02~0.2M的氯化钠(NaCl)进行离子交换色谱,将A型肉毒杆菌毒素产品分离成亚组分;以及
从所述分离后的亚组分中收集具有0.4~0.6的A260/A280值的非扩散型肉毒杆菌毒素亚组分。
2.一种非扩散型肉毒杆菌毒素制品,其通过上述方法进行精制,且包含每100U/ml分别至少150ppb、80ppb和140ppb浓度的Zn、Fe和Mg离子。
3.一种测定非扩散型肉毒杆菌毒素的方法,其包括:将权利要求2所述的非扩散型肉毒杆菌毒素制品以相当于在20-50μL体积范围该毒素LD50的1.5~3倍的量注射到小鼠(4~6周龄,重18~22g)的左侧或右侧后肢肌肉;以及测定注射后的小鼠后肢肌肉和呼吸肌肉是否麻痹或小鼠是否死亡。
4.根据权利要求3所述的方法,其中,非扩散型肉毒杆菌毒素制品显示出在小鼠(4~6周龄,重18~22g)的左侧或右侧后肢肌肉注射50μL体积中2U毒素制品后96小时的存活率至少为80%。
Claims (3)
1.一种非扩散型肉毒杆菌毒素的精制方法,包括以下步骤:
通过采用pH为4.5~6.5的缓冲液和0.02~0.2M的氯化钠(NaCl)进行离子交换色谱,将A型肉毒杆菌毒素产品分离成亚组分;以及
从所述分离后的亚组分中收集具有0.4~0.6的A260/A280值的非扩散型肉毒杆菌毒素亚组分。
2.一种非扩散型肉毒杆菌毒素制备品,其通过上述的方法进行精制,且包含每100U/ml分别至少150ppb、80ppb和140ppb浓度的Zn、Fe和Mg离子。
3.一种用于测定非扩散型肉毒杆菌毒素的方法,其包括:将权利要求2所述的非扩散型肉毒杆菌毒素以相当于该毒素LD50的1.5~3倍的量注射到小鼠(4~6周龄,重18~22g)的左侧或右侧后肢肌肉;以及测定所述小鼠的右侧后肢肌肉和呼吸肌肉是否麻痹或小鼠是否死亡。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1491205A1 (en) * | 2002-03-29 | 2004-12-29 | Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute | Remedy for hypermyotonia |
US20060022780A1 (en) * | 2004-07-30 | 2006-02-02 | Delta Systems, Inc. | Electromagnetic latching switch |
US7160699B2 (en) * | 2003-09-25 | 2007-01-09 | Allergan, Inc. | Media for clostridium bacterium and processes for obtaining a clostridial toxin |
CN101204577A (zh) * | 2006-12-21 | 2008-06-25 | 中国人民解放军军事医学科学院微生物流行病研究所 | 一种a型肉毒毒素结晶复合物的制备方法 |
Family Cites Families (34)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
SE303567B (zh) * | 1959-02-17 | 1968-09-02 | Behringwerke Ag | |
US3409605A (en) * | 1965-06-15 | 1968-11-05 | American Cyanamid Co | Concentration and purification of growth factor-placental origin (human) |
AU2907695A (en) * | 1994-08-08 | 1996-03-07 | Wisconsin Alumni Research Foundation | Purification and pharmaceutical compositions containing type g botulinum neurotoxin |
US5939070A (en) * | 1996-10-28 | 1999-08-17 | Wisconsin Alumni Research Foundation | Hybrid botulinal neurotoxins |
AU2340299A (en) * | 1998-01-26 | 1999-08-09 | University Of Massachusetts | Biologically active hemagglutinin from type a (clostridium botulinum) and methods of use |
DE19925739A1 (de) * | 1999-06-07 | 2000-12-21 | Biotecon Ges Fuer Biotechnologische Entwicklung & Consulting Mbh | Therapeutikum mit einem Botulinum-Neurotoxin |
GB9921592D0 (en) * | 1999-09-13 | 1999-11-17 | Microbiological Res Authority | Preparation of highly pure toxin fragments |
US20030118598A1 (en) * | 2000-02-08 | 2003-06-26 | Allergan, Inc. | Clostridial toxin pharmaceutical compositions |
US6670322B2 (en) * | 2000-06-01 | 2003-12-30 | Wisconsin Alumni Research Foundation | Method of targeting pharmaceuticals to motor neurons |
KR100455280B1 (ko) | 2000-06-28 | 2004-11-08 | 삼성코닝 주식회사 | 인듐 틴 옥사이드(ito)의 제조방법 |
DE10035156A1 (de) * | 2000-07-19 | 2002-02-07 | Biotecon Ges Fuer Biotechnologische Entwicklung & Consulting Mbh | Proteinkomplex als Vehikel für oral verfügbare Protein-Arzneimittel |
JP2003009897A (ja) * | 2001-07-03 | 2003-01-14 | Keiji Oguma | ボツリヌス毒素の分離・精製法 |
KR20030060150A (ko) * | 2002-01-07 | 2003-07-16 | (주)메디톡스 | 클로스트리디움 보툴리눔 a형 독소를 정제하는 방법 |
US6673598B1 (en) * | 2002-10-29 | 2004-01-06 | Synthecon, Inc. | Disposable culture bag |
US7148041B2 (en) * | 2003-09-25 | 2006-12-12 | Allergan, Inc. | Animal product free media and processes for obtaining a botulinum toxin |
US7452697B2 (en) * | 2003-09-25 | 2008-11-18 | Allergan, Inc. | Chromatographic method and system for purifying a botulinum toxin |
KR20050074806A (ko) * | 2004-01-14 | 2005-07-19 | 팜텍(주) | 결정형 보툴리늄 독소의 제조방법 |
US7514088B2 (en) * | 2005-03-15 | 2009-04-07 | Allergan, Inc. | Multivalent Clostridial toxin derivatives and methods of their use |
BRPI0508299A (pt) | 2005-03-03 | 2007-07-31 | Allergan Inc | sistema livre de produto animal e processo para purificação de uma toxina botulina |
US7465457B2 (en) * | 2005-04-14 | 2008-12-16 | Wisconsin Alumni Research Foundation | Method for preparing botulinum neurotoxin type A light chain |
KR101234969B1 (ko) | 2005-06-03 | 2013-02-20 | 라이온 가부시키가이샤 | 지방산 저급 알킬에스테르의 제조 방법 및 지방산 저급알킬에스테르 및 경유 대체연료 |
US8949033B2 (en) * | 2006-04-28 | 2015-02-03 | The Chemo-Sero-Therapeutic Research Institute | Method for quantification of neurotoxin |
AR061669A1 (es) * | 2006-06-29 | 2008-09-10 | Merz Pharma Gmbh & Co Kgaa | Aplicacion de alta frecuencia de terapia con toxina botulinica |
ES2537579T3 (es) * | 2006-10-27 | 2015-06-09 | The Chemo-Sero-Therapeutic Research Institute | Preparación que contiene toxina botulínica de tipo A muy purificada derivada de patógeno de botulismo del lactante |
KR101025617B1 (ko) * | 2008-02-25 | 2011-03-30 | 메덱스젠 주식회사 | 클로스트리디움 보툴리눔으로부터 보툴리눔 독소를정제하는 방법 |
KR20090120222A (ko) * | 2008-05-19 | 2009-11-24 | (주)메디톡스 | 식물 유래 성분 함유 배지 및 가요성 폐쇄 용기를 이용하여클로스트리디움 보툴리눔 독소를 생산하는 방법 |
SG172812A1 (en) * | 2008-12-31 | 2011-08-29 | Revance Therapeutics Inc | Injectable botulinum toxin formulations |
US8440204B2 (en) * | 2009-04-30 | 2013-05-14 | Wisconsin Alumni Research Foundation | Subtype of Closteridium botulinum neurotoxin type A and uses thereof |
US8129139B2 (en) * | 2009-07-13 | 2012-03-06 | Allergan, Inc. | Process for obtaining botulinum neurotoxin |
US20120196349A1 (en) * | 2009-10-21 | 2012-08-02 | Revance Therapeutics, Inc. | Methods and Systems for Purifying Non-Complexed Botulinum Neurotoxin |
EP2512519A2 (en) * | 2009-12-18 | 2012-10-24 | Allergan, Inc. | Clostridium botulinum carrier complex for the administration of therapeutic agents |
HUE029962T2 (hu) * | 2010-01-25 | 2017-04-28 | Allergan Inc | Egyláncú fehérjék kétláncú formájúvá történõ intracelluláris konverziójának módszerei |
KR101134146B1 (ko) * | 2010-05-31 | 2012-04-19 | 메덱스젠 주식회사 | 국소 근마비 효과를 갖는 비확산형 보툴리눔 독소와 그의 정제방법 |
MX342920B (es) * | 2010-07-30 | 2016-10-19 | Medimmune Llc | Metodo para purificar polipeptidos o inmunoconjugados activos. |
-
2010
- 2010-05-31 KR KR1020100051076A patent/KR101134146B1/ko active IP Right Grant
-
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- 2011-05-13 CN CN201180028634.2A patent/CN102985102B/zh not_active Expired - Fee Related
- 2011-05-13 ES ES11789976.5T patent/ES2659820T3/es active Active
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- 2011-05-13 EP EP11789976.5A patent/EP2578228B1/en not_active Revoked
- 2011-05-13 WO PCT/KR2011/003547 patent/WO2011152618A2/ko active Application Filing
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1491205A1 (en) * | 2002-03-29 | 2004-12-29 | Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute | Remedy for hypermyotonia |
US7160699B2 (en) * | 2003-09-25 | 2007-01-09 | Allergan, Inc. | Media for clostridium bacterium and processes for obtaining a clostridial toxin |
US20060022780A1 (en) * | 2004-07-30 | 2006-02-02 | Delta Systems, Inc. | Electromagnetic latching switch |
CN101204577A (zh) * | 2006-12-21 | 2008-06-25 | 中国人民解放军军事医学科学院微生物流行病研究所 | 一种a型肉毒毒素结晶复合物的制备方法 |
Non-Patent Citations (1)
Title |
---|
CARL J.MALIZIO等: "Purification of Clostridium botulinum type A neurotoxin", 《METHODS IN MOLECULAR BIOLOGY》, vol. 145, 31 December 2000 (2000-12-31), pages 27 - 39 * |
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CN102985102B (zh) | 2016-06-08 |
WO2011152618A4 (ko) | 2012-07-12 |
US10369235B2 (en) | 2019-08-06 |
WO2011152618A3 (ko) | 2012-05-24 |
JP2013533856A (ja) | 2013-08-29 |
JP5998419B2 (ja) | 2016-09-28 |
AU2011262499B2 (en) | 2015-01-22 |
WO2011152618A2 (ko) | 2011-12-08 |
WO2011152618A9 (ko) | 2012-03-22 |
EP2578228A2 (en) | 2013-04-10 |
KR101134146B1 (ko) | 2012-04-19 |
US20130071331A1 (en) | 2013-03-21 |
AU2011262499A1 (en) | 2013-01-24 |
EP2578228B1 (en) | 2017-11-15 |
JP2015200659A (ja) | 2015-11-12 |
EP2578228A4 (en) | 2013-10-02 |
US20170145399A1 (en) | 2017-05-25 |
JP5919608B2 (ja) | 2016-05-18 |
ES2659820T3 (es) | 2018-03-19 |
US9598683B2 (en) | 2017-03-21 |
US20170143849A1 (en) | 2017-05-25 |
KR20110131572A (ko) | 2011-12-07 |
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