THERAPEUTIC NA+ CHANNEL BLOCKERS: RECEPTOR & DRUG DESIGN

这项续期申请的长期目标是开发 与疼痛管理相关的治疗性钠通道阻滞剂。传统型 局麻药通常不适合治疗慢性或顽固性疾病。 癌症疼痛，因为他们的神经阻滞持续时间不足。有4个 具体目标：1.研究各种化合物的构效关系 有效的体外钠通道阻滞剂；2.设计和合成其 两亲性衍生物；3.筛选出适合长期使用的阻滞剂 体内神经阻断；4.定位其在钠通道内的受体位置 阿尔法亚单位。第一个被测试的药物是三环类抗抑郁药 阿米替林，这是一种有效的钠通道阻滞剂，除了 它对其他受体的作用。以布比卡因为比较标准， 各种三环化合物与其他强效钠通道的结合亲和力 阻滞剂将在HEK细胞的电压钳研究中瞬时确定 转染大鼠骨骼肌和人心脏钠通道克隆； 也将使用大鼠垂体GH3细胞上的天然神经元钠通道。 要表征的元素包括数据块的使用依赖性和IC50 静息和失活的通道阻滞剂。这一阶段的工作假设是 研究表明，体内阻滞的持续时间与 对停用的通道状态使用依赖和负IC50。入内 活体研究将使用行为终点来检查感觉和运动 一次注射每种药物后坐骨神经的神经阻滞 大鼠；在大鼠身上有效的药物也将在#年在马尾神经间隙进行测试。 绵羊来模拟脊椎治疗路线。药物设计和合成将 最初使用的是Amitritiline衍生品。黄曲霉毒素受体结合部位的研究 钠通道将探测两个疏水结构域的位置 HEK细胞突变通道的点突变及药效研究 细胞，特别强调可能负责的残基 三环的高亲和力结合。最终，这些研究将是 扩展到其他类别的药物，包括苯乙酰胺、钙通道 阻滞剂和钾通道阻滞剂也能有效地阻断钠 频道。与三环类抗抑郁药一样，这些药物也含有多个苯基 环，但它们被分成两个大的疏水结构域，而不是 一。

The long term objective of this renewal application is to develop therapeutic Na+ channel blockers pertinent to pain management. Traditional local anesthetics are often unsuited for treatment of chronic or intractable cancer pain because of their insufficient duration of nerve block. There are 4 specific aims: 1. To study the structure-activity relationship of various potent Na+ channel blockers in vitro; 2. To design and synthesize their amphipathic derivatives; 3. To test selected blockers suitable for prolonged nerve block in vivo; and 4. To map their receptor site within the Na+ channel alpha subunit. The first agent to be tested is the tricyclic antidepressant amitryptyline, which is a potent sodium channel blocking agent in addition to its actions at other receptors. With bupivacaine as a standard for comparison, the binding affinities of various tricyclics and other potent sodium channel blockers will be determined in voltage clamp studies on HEK cells transiently transfected with rat skeletal muscle and human heart sodium channel clones; native neuronal sodium channels in rat pituitary GH3 cells will also be used. Elements to be characterized include use-dependence of block and IC50 for resting and inactivated channel block. The working hypothesis for this phase of the studies is that duration of block in vivo will correlate positively with use dependence and negatively with IC50 for inactivated channel states. The in vivo studies will employ behavioral endpoints to examine both sensory and motor nerve block of sciatic nerve following a single injection of each agent in rats; drugs effective in rats will also be tested in the cauda equina space in sheep to model spinal routes of therapy. Drug design and synthesis will initially employ amitryptiline derivatives. Studies of the receptor site in the sodium channel will probe for the locations of two hydrophobic domains using point mutations and studies of drug potency on the mutated channels in HEK cells, with a special emphasis on residues which may be responsible for high-affinity binding of the tricyclic ring. Eventually the studies will be extended to other classes of drugs including phenylacetamides, calcium channel blockers, and a potassium channel blocker that also potently blocks sodium channels. Like tricyclic antidepressants, these agents have multiple phenyl rings but they are separated into two large hydrophobic domains rather than one.