Development of Anti-OLAM Aptamers as Novel Analgesics

描述(由申请人提供)：作为新型镇痛剂的抗Olam适体的开发由于对疼痛机制的不完全理解，疼痛的管理仍然是一个主要的卫生保健问题。TRPV1是配体门控离子通道瞬时受体电位(Trp)家族中的重要成员，检测周围组织中的有害化学和物理刺激。药理学和基因缺失研究都表明，TRPV1在炎症、热、痛觉过敏和其他疼痛条件下发挥着关键作用。氧化亚油酸代谢物(OLAM)最近被证明包含一个新的内源性TRPV1激动剂家族，有助于急性和炎症性疼痛。因此，阻断OLAM系统的化合物很可能构成一个新的止痛药家族。为了直接支持这一预测，这里提供的初步数据表明，注射针对两种主要OLAM的抗体，9-HODE和13-HODE，在两种疼痛模型中产生显著的镇痛作用。尽管这些数据为概念验证提供了证据，但由于与免疫原性相关的不良反应，兔多克隆抗体的临床开发是不可行的。因此，运营技术公司(Optech)建议使用组合适配子化学来发现与这些OLAM特异结合的DNA适配子，并中和它们产生疼痛的活性。这将允许用更特异、更便宜、也许亲和力更高的DNA适配子来取代抗体。在第一阶段，Optech预计将完成两个总体具体目标。目的1将开发、克隆和测序几种高度特异的DNA适配子，这些适配子与9-羟基十碳二烯酸(HODE)和13-HODE结合，而不与前体脂质亚油酸结合(图1A)。目的2利用几种临床前大鼠体内疼痛模型评价适配子的镇痛活性。在第二阶段，Optech将提炼、优化并开始 其抗奥兰适体化合物的商业化。第二阶段的优化过程将包括对推测的适体结合口袋与OLAM的相互作用进行3-D建模。基于3-D模拟结果，Optech预计添加具有各种官能团(例如伯胺、甲基、硫醇等)的改性碱基。现在可以在商业上用于在化学合成时添加到寡核苷酸中。这些额外的官能团对适体-OLAM结合亲和力的影响有望更好地模拟氨基酸侧链，并将通过类似ELISA的平板分析和表面等离子共振(SPR)来研究。最高亲和力和最特异的未修饰或修饰的抗Olam适配子将进入动物研究。在动物疼痛研究中最有效的适配子将被修改为更长的体内寿命(3‘-胆固醇添加和脂质体中的包合物，聚乙二醇化等)。 或时间释放的配方，并进入FDA的批准流程。 公共卫生意义：作为新型止痛剂的抗奥兰适体的开发数百万患者遭受疼痛，许多现有的止痛药(“止痛药”)遭受不完全止痛或不可接受的副作用。该提案将开发一种新型的止痛药，通过阻断组织损伤期间释放的内源性辣椒素类物质来发挥作用。

DESCRIPTION (provided by applicant): Development of Anti-OLAM Aptamers as Novel Analgesics The management of pain remains a major health care problem due to an incomplete understanding of pain mechanisms. TRPV1, a prominent member of the transient receptor potential (TRP) family of ligand-gated ion channel, detects noxious chemical and physical stimuli in peripheral tissues. Both pharmacological and gene deletion studies have demonstrated a pivotal role for TRPV1 in inflammatory heat hyperalgesia and other pain conditions. Oxidized linoleic acid metabolites (OLAMs) have been recently demonstrated to comprise a novel family of endogenous TRPV1 agonists that contributes to acute and inflammatory pain conditions. Therefore, compounds that block the OLAM system are likely to constitute a novel family of analgesics. In direct support of this prediction, preliminary data provided herein demonstrate that injection of antibodies against two of the major OLAMs, 9-HODE and 13-HODE, produce significant analgesia in two pain models. Although these data provide evidence for proof-of-concept, the clinical development of rabbit polyclonal antibodies is not feasible due to adverse effects related to immunogenicity. Accordingly, Operational Technologies Corporation (OpTech) proposes to use combinatorial aptamer chemistry to discover DNA aptamers that specifically bind to these OLAMs and neutralize their pain-producing activities. This would permit replacing antibodies with more specific, less expensive and perhaps higher affinity DNA aptamers. In Phase 1, OpTech expects to complete two overall Specific Aims. Aim 1 will develop, clone, and sequence several highly specific DNA aptamers that bind 9-hydroxydecadienoic acid (HODE) and 13-HODE without binding to the precursor lipid, linoleic acid (Fig 1A). Aim 2 will evaluate the analgesic activity of the aptamers using several in vivo preclinical rat models of pain. In Phase 2, OpTech will refine, optimize, and begin commercialization of its anti-OLAM aptamer compounds. The Phase 2 optimization process will include 3-D modeling of putative aptamer binding pocket interactions with the OLAMs. Based on 3-D modeling findings, OpTech anticipates adding modified bases having various functional groups (e.g., primary amines, methyl, thiol groups, etc.) that are now commercially available for addition to oligonucleotides at the point of chemical synthesis. The effects of these additional functional groups on aptamer-OLAM binding affinity are expected to better emulate amino acid side chains and will be studied by ELISA-like plate assays and surface plasmon resonance (SPR). The highest affinity and most specific unmodified or modified anti-OLAM aptamers will move into animal studies. The most effective aptamers in animal pain studies will be modified for longer in vivo lifetimes (3'-cholesterol addition and inclusion in liposomes, PEGylation, etc.) or time-released formulation and enter the FDA approval pipeline. PUBLIC HEALTH RELEVANCE: Development of Anti-OLAM Aptamers as Novel Analgesics Millions of patients suffer from pain and many available analgesic drugs ("pain killers") suffer from either incomplete analgesia or unacceptable side-effects. The proposal will develop a novel class of analgesics that work by blocking the endogenous capsaicin-like substances that are released during tissue injury.