Construction and Analysis of BoNT derivatives as Pain Specific Inhibitors

作为疼痛特异性抑制剂的 BoNT 衍生物的构建和分析

• 批准号: 8906736
• 负责人: Eric A. Johnson
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-07 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8906736
• 关键词: Adverse effects; Afferent Neurons; Analgesics; Animal Model; Binding; Biological Assay; Biological Products; Bontoxilysin; Botulinum Toxin Type A; Botulism; C-terminal; Cell Culture Techniques; Cell Line; Cell model; Cells; Cervical Dystonia; Cisplatin; Cleaved cell; Clinical Trials; Coupling; Cysteine; Cytosol; Data; Dependence; Disease; Disulfide Linkage; Dose; Drug effect disorder; Elements; Endocytosis; Endopeptidases; Escherichia coli; Esthetics; Formalin; Future; G-Protein-Coupled Receptors; Goals; Health; In Vitro; Injection of therapeutic agent; Interneurons; Intrathecal Injections; Intrathecal Space; Laboratories; Ligands; Ligation; Light; Link; Longevity; Lysosomes; Measures; Medicine; Membrane; Modeling; Mono-S; Morphine; Motor; Motor Endplate; Muscle; Nerve; Neuromuscular Diseases; Neurons; Neuropathy; Nociception; Opioid Peptide; Pain; Pain management; Paralysed; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Play; Process; Proteins; Recombinant Fusion Proteins; Recombinants; Regulation; Resiniferatoxin; Role; S-nitro-N-acetylpenicillamine; SNAP receptor; Sensory; Sensory Process; Series; Serotyping; Site; Specificity; Spinal; Spinal Ganglia; Spinal nerve root structure; Substance P; Substance P Receptor; Synaptic Vesicles; System; Testing; Therapeutic; Toxin; Western Blotting; Zinc; base; chronic pain; design; disulfide bond; dorsal horn; ganglion cell; holotoxins; improved; in vivo; inhibitor/antagonist; innovation; mouse model; mu opioid receptors; neuronal cell body; neurotransmitter release; novel; pain behavior; peptide A; prototype; receptor; response; substance P-saporin; tool; transmission process; uptake

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

描述（由申请人提供）：肉毒杆菌神经毒素（BONTS）在医学中广泛用于治疗各种神经肌肉疾病，利用其抑制作用 在局部注射后的运动式板传输上，并导致局部肌肉麻痹。显而易见的是，BONTS还可以进入其他神经元细胞，包括感觉和CNS神经元。通过直接的脊髓鞘内递送毒素，可以实现神经末期传入和二阶神经元加工的强大块。我们（和其他人）表明，这种胸腔递送的骨会产生一种有效的，持久但可逆的中央伤害性处理的块。然而，这种方法的治疗效用受到以下事实的限制：靶向和摄取Holotoxin并非特定于神经元亚型。因此，由于抑制性中间神经元的阻滞而导致的肌肉瘫痪和促进的感觉处理的可能性至关重要。一般影响会导致疼痛状态增强，而不是缓解疼痛，实际上，在意外注入BONT/A进入患者鞘内空间后曾经描述过。我们建议通过针对专门针对参与疼痛处理的终末和神经元的酶促部分来克服这种损害。我们假设可以通过将Bont LC的重组截短版本与物质P或肽Damgo耦合来创建稳定和靶向的BONT衍生物，并且这些衍生物将提供持久且可逆的伤害性处理块。假设Bont LC不能没有拥有重链的区域的假设无法进入细胞。我们的创新方法使用G蛋白偶联受体驱动的内在化，在感觉神经元配体与其各自受体的特定结合之后，因此不需要重链来进行内在化。我们的初步数据以及其他具有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的Bont血清型的轻链创建一系列构造（它们的寿命有所不同，从而使调节能力 药物作用的持续时间和至少两个不同的配体（物质P和Damgo），它们分别与NK1和MU阿片受体结合。这些受体分布在已知在疼痛加工中起核心作用的脊柱末端 /细胞体上。细胞模型将检查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）。鞘内毒素的使用已通过SP-saporin和Insiniferatoxin等药物进行了验证，这些药物正在针对慢性疼痛患者的临床试验中。但是，与这些代理不同，他针对的LC可以产生高选择性和不同作用持续时间的块。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疼痛处理途径的未来研究提供工具。 1

项目成果

Current status and future directions of botulinum neurotoxins for targeting pain processing.

• DOI: 10.3390/toxins7114519
• 发表时间: 2015-11-04
• 期刊: Toxins
• 影响因子: 4.2
• 作者: Pellett S;Yaksh TL;Ramachandran R
• 通讯作者: Ramachandran R