INTEGRATIVE AND MOLECULAR STUDIES OF PAIN AND PAIN CONTROL

我们的研究项目致力于中枢神经系统伤害性信息传递的基本分子和生理过程。分子研究是在动物和体外细胞模型中进行的。我们主要研究初级传入痛觉神经元和脊髓背侧的连接。脊髓背侧是伤害性信息处理的第一个突触加工部位，我们的研究发现它是持续性疼痛状态下神经元可塑性和基因表达改变的场所。使用慢性疼痛患者和正常志愿者的活体脑功能成像，对人类进行了与较高中枢神经系统疼痛处理相关的问题。该股还研究控制伤害性传播的新方法，并有三种治疗严重顽固性疼痛的新疗法正在研究中。目前正在解决两个主要的基础科学问题。第一部分通过对香草素受体1的研究，重点研究了痛觉传导的分子机制。香草素受体1是一种热敏性钙/钠离子载体，通过去极化皮肤上的痛觉神经末梢，将痛热转化为神经动作电位。辣椒素是一种典型的香草类化合物，它与辣椒中的活性成分结合，从而刺激了离子载体的活性。在分子的羧基末端的一个独特区域内的点突变表明，热激活和化学激活存在不同的分子基序。我们把它们称为“热区”和“香草区”。这一计划直接导致了床边应用的试验台，其中香草类激动剂被用来通过配体诱导的钙细胞毒性来杀死疼痛神经元，从而提供疼痛控制。第二个项目的中心是脊髓中的基因发现。消减克隆、测序和差异杂交发现了富含在脊髓中的、受疼痛刺激诱导的新基因。我们正在描述新基因的特征，并将已知基因放入上下文中。这些研究揭示了大量的新信息。例如，我们观察到一个重要的抑制性递质的特定磷酸化亚基的背部富集物，Rho/RAC细胞形状信号转导通路中选定的刺激和抑制调节剂的背部富集物，以及在持续性实验性疼痛过程中两个新基因的表达诱导。这个项目是一个长期的、高风险的努力，它为分子疼痛研究设定了新的方向。翻译计划使用我们的基础科学发现来创造治疗慢性疼痛的新方法。从我们的板凳研究中发现了三个目标领域。(1)体内基因转移。我们正在进行腺病毒介导的分泌型β-内啡肽基因转移，准备进行临床试验。这涉及与当地基因治疗公司GenVec的CRADA，将治疗盒放置到他们的腺病毒载体中，并进行临床前毒理学。(2)我们开发了一种称为P物质-外毒素-35(SP-PE)的重组细胞毒性毒素-配体融合蛋白(SP-PE)，作为一种杀死参与疼痛传递的脊髓神经元的手段。鞘内注射15微微克分子的SP-PE即可完全控制疼痛。我们目前正在为临床前毒理学和临床试验准备大量的结合物。(3)我们正在测试一种超强的香草素激动剂--树脂毒素，作为一种用于去除初级传入痛觉神经元的止痛剂。我们开发了一种神经节内注射的方法。节内RTX的结果表明，它将是一种非常有效的方法来控制某些类型的慢性疼痛。

Our research program addresses basic molecular and physiological processes of nociceptive transmission in the central nervous system. The molecular research is performed in animal and in vitro cell-based models. We concentrate on the primary affernt pain sensing neurons and the connections in the dorsal spinal cord. The dorsal spinal cord is the first site of synaptic processing for nociceptive information processing and our research has identified it as a locus of neuronal plasticity and altered gene expression in persistent pain states. Questions related to higher CNS pain processing are performed with humans using in vivo functional brain imaging of chronic pain patients and normal volunteers. The Unit also investigates novel methods for controlling nociceptive transmission and has three novel treatments for sever intractable pain under investigation. Two main basic science issues are being addressed. The first centers on the molecular mechanisms of pain transduction through an investigation of the vanilloid receptor 1. This molecule is a heat-sensitive calcium/sodium ionophore and converts painful heat into nerve action potentials by depolarizing the pain sensing nerve terminals in the skin. Ionophore activity is stimulated by binding of capsaicin a prototypical vanilloid compound and the active ingredient in hot pepper. Point mutations within a unique region in the carboxy end of the molecule suggest the occurrence of separate molecular motifs for thermal and chemical activation. We refer to these as the "heat domain" and "vanilloid domain". This program has directly led to a bench to bedside application in which vanilloid agonists are used to kill pain neurons via ligand-induced calcium cytotoxicity and thereby provide pain control. The second program is centered on gene discovery in spinal cord. Subtraction cloning, sequencing and differential hybridization has revealed new genes enriched in spinal cord and induced by pain stimuli. We are in the process of characterizing the novel genes and placing into context the known genes. These studies have revealed large amounts of new information. For example, we observe dorsal enrichment for a specific phosphorylated subunit of an important inhibitory transmitter, a dorsal enrichment of selected stimulatory and inhibitory modulators of the Rho/Rac cell shape signal transduction pathway, and the induction of expression of two novel genes during persistent experimental pain. This project is a long term, high-risk endeavor, which is setting new directions for molecular pain research. The translational program uses our basic science findings to create new treatments for chronic pain. Three target areas emerged from our bench research. (1) In vivo gene transfer. We are getting the adenoviral-mediated gene transfer of secreted beta-endorphin ready for clinical trial. This involves a CRADA with GenVec, a local gene therapy company, in which the therapeutic cassette is placed into their adenoviral vector and preclinical toxicology is performed. (2) We developed a recombinant cytotoxic toxin-ligand fusion protein called substance P-Pseudomonas exotoxin-35 (SP-PE) as a means to kill spinal cord neurons involved in pain transmission. Complete pain control is achieved by intrathecal administration of as little as15 picomoles of SP-PE. We are currently preparing a large amount of conjugate for preclinical toxicology and clinical trial. (3) We are testing a ultrapotent vanilloid agonist, resiniferatoxin, as a pain control agent for used in removal of primary afferent pain sensing neurons. We developed an intraganglionic injection method. The results of intraganglionic RTX suggest that it will be a very effective approach to control of certain types of chronic pain.