The intercostal NMJ assay - a new alternative to the conventional LD50 assay for the determination of the therapeutic potency of botulinum toxin preparations
The intercostal NMJ assay - a new alternative to the conventional LD50 assay for the determination of the therapeutic potency of botulinum toxin preparations
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DOI：
10.1177/026119290803600205
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发表时间：
2008-05-01
影响因子：
2.7
通讯作者：
Pickett, Andy
中科院分区：
文献类型：
--
作者：
Huber, Alexander;France, Richard M.;Pickett, Andy
已经发现治疗性A型肉毒杆菌神经毒素制剂越来越多地用于多种神经肌肉病症和皮肤病况的临床用途。准确测定肉毒杆菌毒素临床应用中的效力对于确保临床疗效和安全性至关重要，目前通过在小鼠中使用致死剂量（LD 50）测定来实现。与该测定相关的伦理问题和操作限制促使开发可能导致其替代的替代测定系统。作为一种替代方法，我们描述了一种新型离体测定法（肋间神经肌肉连接[NMJ]测定法）的开发和评价，该测定法使用的动物数量大幅减少，并解决了与LD 50测定法相关的伦理问题。该测定记录了电刺激肌肉组织切片对毒素的反应力的衰减，因此结合了毒素分子的受体结合、易位和酶促作用的重要机制。毒素应用导致收缩力的时间相关性和剂量相关性降低。通过统计学方法确定了描述施加剂量与力衰减之间关系的回归模型，并成功进行了测试，结果表明该模型能够正确预测未知样品的效价。发现所用的组织切片具有高度可重复性，如通过神经支配模式和原位NMJ定位所确定的。此外，通过使用分子示踪剂分子严格评估了试验方案成功将供试品递送至细胞靶位点的有效性。
Therapeutic botulinum neurotoxin type A preparations have found an increasing number of clinical uses for a large variety of neuromuscular disorders and dermatological conditions. The accurate determination of potency in the clinical application of botulinum toxins is critical to ensuring clinical efficacy and safety, and is currently achieved by using a lethal dose (LD50) assay in mice. Ethical concerns and operational constraints associated with this assay have prompted the development of alternative assay systems that could potentially lead to its replacement. As one such alternative, we describe the development and evaluation of a novel ex vivo assay (the Intercostal Neuromuscular Junction [NMJ] Assay), which uses substantially fewer animals and addresses ethical concerns associated with the LD50 assay. The assay records the decay of force from electrically-stimulated muscle tissue sections in response to the toxin, and thus combines the important mechanisms of receptor binding, translocation, and the enzymatic action of the toxin molecule. Toxin application leads to a time-related and dose-related reduction in contractile force. A regression model describing the relationship between the applied dose and force decay was determined statistically, and was successfully tested as able to correctly predict the potency of an unknown sample. The tissue sections used were found to be highly reproducible, as determined through the innervation pattern and the localisation of NMJs in situ. Furthermore, the efficacy of the assay protocol to successfully deliver the test sample to the cellular target sites, was critically assessed by using molecular tracer molecules.