Construction and Analysis of BoNT derivatives as Pain Specific Inhibitors

描述（由申请人提供）：肉毒神经毒素（BoNT）利用其抑制作用广泛用于治疗各种神经肌肉疾病 局部注射后运动终板传递的影响，并导致局部肌肉麻痹。很明显，BoNT也可以进入其他神经元细胞，包括感觉和CNS神经元。神经轴初级传入和二级神经元加工的强大阻滞可以通过直接脊髓鞘内递送毒素来实现。我们（和其他人）已经表明，这种鞘内递送BoNT可以在各种疼痛模型中产生有效且持久但可逆的中枢伤害性处理阻断。然而，这种方法的治疗效用受到以下事实的限制：全毒素的靶向和摄取对神经元亚型不是特异性的。因此，由于抑制性中间神经元的阻断而伴随肌肉麻痹和促进感觉处理的可能性是至关重要的。一般效应将导致疼痛状态加剧而不是疼痛缓解，并且事实上曾经在将BoNT/A意外注射到患者的鞘内空间中后描述过。我们建议通过将BoNT轻链（LC）的酶促部分特异性地靶向参与疼痛处理的终末和神经元来克服这种损害。我们假设，稳定的和有针对性的BoNT衍生物可以通过将重组截短版本的BoNT LC偶联到P物质或肽DAMGO来产生，并且这些衍生物将提供对伤害性处理的持久且可逆的阻断。过去产生这种构建体的努力受到BoNT LC不能在不具有BoNT重链区域的情况下进入细胞的假设的阻碍。我们的创新方法在感觉神经元配体与它们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，使得内化不需要重链。我们的初步数据和最近的其他sP证明了这种方法的可行性。我们在此建议使用不同BoNT血清型的轻链（其寿命不同，从而能够调节）来创建一系列构建体 药物作用持续时间）和至少两种不同的配体（P物质和DAMGO），它们分别与NK 1和μ阿片受体结合。这些受体分布在已知在疼痛处理中发挥核心作用的脊髓终末/细胞体上。细胞模型将检查对表达这些受体的神经元的选择性进入，动物模型将评估构建体对伤害感受的影响并建立治疗比率（例如镇痛/副作用剂量）。鞘内毒素的使用已经用诸如sP-皂草素和树脂毒素的药剂进行了验证，这些药剂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，靶向LC可以产生高选择性和不同作用持续时间的阻滞剂。该项目的结果有可能改善慢性疼痛的治疗选择。在这个项目中创建的结构也将为未来的疼痛处理途径的研究提供工具。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