Construction and Analysis of BoNT derivatives as Pain Specific Inhibitors

描述(申请人提供)：肉毒杆菌神经毒素(BoNTs)被广泛用于治疗各种神经肌肉疾病，利用其抑制作用。 局部注射后对运动终板传递的影响并导致局限性肌肉瘫痪。很明显，BoNTs还可以进入其他神经细胞，包括感觉神经元和中枢神经系统神经元。通过脊髓鞘内直接注射毒素，可以实现对神经轴、初级传入神经元和二级神经元的强大阻断。我们(和其他人)已经证明，这种鞘内注射BoNTs可以在各种疼痛模型中产生有效和持久的、但可逆的中枢伤害性处理障碍。然而，全毒素的靶向和摄取并不是神经元亚型特有的，这一事实限制了这种方法的治疗效果。因此，由于抑制的中间神经元的阻断，伴随肌肉瘫痪和促进感觉处理的可能性是至关重要的。一般效应会导致高度疼痛状态，而不是缓解疼痛，事实上，曾经有过在意外将BONT/A注射到患者鞘内间隙后的描述。我们建议通过靶向BONT轻链(LC)的酶部分来克服这种损害，该部分专门针对参与疼痛处理的终末和神经元。我们假设，通过将重组截短版本的BONT LC与P物质或多肽DAMGO偶联，可以创建稳定和靶向的BONT衍生物，并且这些衍生物将提供长期且可逆的伤害性加工。过去创造这种结构的努力一直受到这样一个假设的阻碍，即BoNT LC在没有BoNT重链区域的情况下无法进入细胞。我们的创新方法是在感觉神经元配体与各自的受体特异性结合后，使用G蛋白偶联受体驱动的内化，这样内化不需要重链。我们的初步数据和最近其他SP的数据证明了这种方法的可行性。我们在这里建议使用不同BONT血清型(它们的寿命不同而允许调节)的轻链来创建一系列结构 至少两种不同的配体(P物质和DAMGO)分别与NK1和MU阿片受体结合。这些受体分布在脊髓终末/细胞体上，已知它们在疼痛处理中发挥核心作用。细胞模型将检测表达这些受体的神经元的选择性进入，动物模型将评估这些结构对伤害性感受的影响并建立治疗比率(例如，止痛剂/副作用剂量)。鞘内毒素的使用已经通过SP-Saporin和Resiniferatoxin等药物的验证，这些药物正在对慢性疼痛患者进行临床试验。但是，与这些药物不同的是，他针对的LCS可以产生高选择性和不同作用时间的块。该项目的结果有可能改善慢性疼痛的治疗选择。该项目中创建的结构还将为未来疼痛处理路径的研究提供工具。1

DESCRIPTION (provided by applicant): Botulinum neurotoxins (BoNTs) are widely used in medicine to treat a variety of neuromuscular disorders, taking advantage of their inhibitory effect on motor-endplate transmission after local injection and leading to localized muscle paralysis. It has become apparent that BoNTs can also enter other neuronal cells including sensory and CNS neurons. A robust block of neuraxial primary afferent and second order neurons processing can be achieved by direct spinal intrathecal delivery of the toxin. We (and others) have shown that such intrathecal delivery of BoNTs can produce a potent and persistent, but reversible, block of central nociceptive processing in a variety of pain models. However, the therapeutic utility of this approach is limited by the fact that the targeting and uptake of holotoxins is not specific to neuronal subtype. Thus the possibility of accompanying muscle paralysis and facilitated sensory processing due to block of inhibitory interneurons is of paramount importance. General effects would result in a heightened pain state rather than relief from pain, and has in fact once been described after an accidental injection of BoNT/A into the intrathecal space of a patient. We are proposing to overcome this detriment by targeting the enzymatic portion of the BoNT light chain (LC) specifically to terminals and neurons involved in pain processing. We hypothesize that stable and targeted BoNT derivatives can be created by coupling of recombinant truncated version of the BoNT LC to substance P or the peptide DAMGO, and that these derivatives will provide a long lasting and yet reversible block of nociceptive processing. Past efforts to create such constructs have been hindered by the postulate that BoNT LC cannot enter cells without possessing regions of BoNT heavy chain. Our innovative approach uses G- protein coupled receptor driven internalization after specific binding of the sensory neuron ligands to their respective receptors, such that the heavy chain is not needed for internalization. Our preliminary data and recently that of others with sP demonstrates the feasibility of this approach. We propose here to create a series of constructs using light chains of different BoNT serotypes (which differ in their longevity enabling regulation of duration of drug action) and at least two different ligands (substance P and DAMGO), which bind to the NK1and mu opioid receptors, respectively. These receptors are distributed on spinal terminals /cell bodies known to play a central role in pain processing. Cell models will examine selective entry into neurons expressing these receptors, and animal models will assess effects of the constructs on nociception and establish therapeutic ratios (e.g. analgesic /side effect doses). The use of intrathecal toxins has been validated with agents such as sP-saporin and resiniferatoxin, which are in clinical trials for chronic pain patients. But, unlike these agents, he targeted LCs can produce blocks of high selectivity and of varying durations of action. The results from this project have the potential to improve treatment options for chronic pain. The constructs created in this project will also provide tools for future studies of pain processing pathways. 1