Engineered Opioid Receptors as Therapeutic Agents for Pain Control

工程阿片受体作为控制疼痛的治疗剂

• 批准号: 8013897
• 负责人: PING-YEE LAW
• 金额: $ 43.71万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8013897
• 关键词: Acute; Acute Pain; Adverse drug effect; Adverse effects; Affect; Agonist; Alkaloids; Amino Acid Sequence Homology; Amino Acids; Analgesics; Area; Biological Assay; Brain; Cell model; Chimera organism; Chronic; Complement; Complementary DNA; Constipation; Data; Dependence; Dependovirus; Development; Docking; Drug usage; Electroporation; Elements; Engineering; Environment; Exhibits; Gene Delivery; Gene Transfer; Generations; Goals; Health; In Vitro; Infection; Injection of therapeutic agent; Intrathecal Injections; Knock-in Mouse; Ligands; Lipids; Medical; Membrane Lipids; Methods; Modeling; Molecular; Morphine; Mus; Mutagenesis; Mutant Strains Mice; Mutate; Mutation; Naloxone; Naltrexone; Narcotic Antagonists; Nature; Nausea; Neurons; Nociception; Opioid; Opioid Analgesics; Opioid Receptor; Pain; Pain management; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Physicians; Property; Pruritus; Receptor Activation; Receptor Gene; Research Personnel; Rodent Model; Second Messenger Systems; Sedation procedure; Sequence Homology; Site; Spinal Cord; Spinal cord posterior horn; Structure; Testing; Therapeutic Agents; Transmembrane Domain; Ventilatory Depression; Virus; Wild Type Mouse; addiction; base; chronic pain; design; dorsal horn; drug development; endogenous opioids; engineering design; extracellular; gene therapy; in vitro Assay; in vivo; midbrain central gray substance; mutant; receptor; receptor binding; response; second messenger; social stigma; success

DESCRIPTION (provided by applicant): Opioids have been used very successfully for the treatment of moderate to severe acute and chronic pain. Unfortunately, their uses have been associated with many troublesome side effects such as nausea, constipation, respiratory depression, sedation, pruritus, tolerance and dependence development. Many approaches have been used to alleviate these side effects without diminishing the analgesic effects, with variable success. Notable approaches have been the design of receptor selective ligands that would activate one of the three cloned opioid receptors and co-administration of pharmaceutical agents to block the opioid side effects. Although these approaches and others are viable ones, one of the reasons for their limited success is the high amino acid sequence homology among the cloned receptors. Such homology has slowed the design and development of an opioid drug that will target and activate a specific opioid receptor. In the current studies, we propose to use an alternative approach, i.e., to engineer mutant opioid receptors for pain management. Our approach is based on an accidentally discovered receptor mutation, S4.45(196)L in the u-opioid receptor (MOR), that resulted in the ability of opioid alkaloid antagonists to activate the mutant receptor, both in vitro and in vivo. We hypothesize that, if such a mutant receptor could be delivered to and expressed in the nociceptive neurons, then activation of the mutant receptors by antagonists should produce analgesic responses without eliciting the tolerance responses during chronic treatment with the antagonists. Our overall goal is to develop such receptor mutants as therapeutic agents for pain management. Thus, in the current proposal, we will (1) determine the molecular bases for the activation of receptor mutants by opioid antagonists; (2) validate and refine the receptor model for S4.54 mutation so as to engineer a mutant MOR in which naloxone and naltrexone behave like full agonists; and (3) develop a double stranded adenoassociated virus (dsAAV) for the delivery of the mutant receptor at specific sites of the pain pathway and evaluate the antagonist and agonist efficacies in eliciting antinociceptive responses. We will examine both the acute and chronic responses to opioid agonists and antagonists after dsAAV injection. By developing the receptor model and demonstrating the structural bases for the antagonist activities via receptor mutagenesis studies and generation of mutant mouse lines, we could engineer a mutant receptor in which opioid antagonists behave like full agonists. Since dsAAV has been used successfully in gene therapy, the eventual delivery by dsAAV and expression of the mutant receptor at the nociceptive neurons that normally express MOR should result in analgesic responses after systemic administration of opioid antagonists without tolerance development. Such a mutant opioid receptor gene therapy approach could be a new paradigm for the eventual treatment of chronic pain. PUBLIC HEALTH RELEVANCE: In the treatment of moderate to severe pain, morphine remains the drug of choice. However, the many side-effects of the drugs, notably tolerance and dependence development in prolonged treatment, have reduced the desirability in the use of this opioid analgesic for pain management. It has been the Holy Grail of pharmacologists and pharmaceutical chemists to develop drug molecules or treatment paradigms that elicit the pain relief effects without any side effects. With the discovery of a mutant u-opioid receptor (MOR) that could be activated by opioid antagonists without altering the agonists' properties, we hypothesize that such a mutant receptor could be developed into therapeutic agents for the purpose of pain management. We have demonstrated the feasibility of such approach by "knocking-in" this mutation, S4.54(196)A, into MOR, and generating a mouse line in which opioid antagonists, naloxone and naltrexone produced antinociceptive responses. However, this mutation only resulted in partial agonistic properties observed with these two opioid antagonists. Therefore, in the proposed studies, we will investigate the molecular bases for such antagonistic activities using modeling and mutational analysis. Additional receptor mutations will be identified in order to convert the antagonists into full agonists. We will develop a gene therapy vehicle, initially using dsAAV2 virus, to deliver the mutant receptors into various regions of the pain pathway and to examine the feasibility of using opioid antagonists as antinociceptive agents. The activation of the exogenously introduced mutant MOR, and the inactivation of the endogenous opioid receptors by the antagonists, will provide a unique opportunity to develop a pain treatment paradigm without possible development of tolerance and dependence.

描述（由申请人提供）：阿片类药物已非常成功地用于治疗中度至重度急性和慢性疼痛。不幸的是，它们的使用与许多令人烦恼的副作用有关，例如恶心、便秘、呼吸抑制、镇静、瘙痒、耐受性和依赖性发展。许多方法已被用来减轻这些副作用，而不减少止痛效果，具有可变的成功。值得注意的方法是设计受体选择性配体，其将激活三种克隆的阿片样物质受体之一，并共同施用药剂以阻断阿片样物质副作用。虽然这些方法和其他方法是可行的，但它们有限成功的原因之一是克隆受体之间的高度氨基酸序列同源性。这种同源性减缓了靶向和激活特定阿片受体的阿片类药物的设计和开发。在目前的研究中，我们建议使用另一种方法，即，来设计用于疼痛控制的变异阿片受体。我们的方法是基于一个偶然发现的受体突变，S 4.45（196）L在u-阿片受体（莫尔），这导致阿片类生物碱拮抗剂的能力，激活突变的受体，在体外和体内。我们假设，如果这样的突变体受体可以被传递到伤害性神经元并在伤害性神经元中表达，那么拮抗剂对突变体受体的激活应该产生镇痛反应，而不会在用拮抗剂长期治疗期间引起耐受反应。我们的总体目标是开发这种受体突变体作为疼痛管理的治疗剂。因此，在目前的建议中，我们将（1）确定阿片受体拮抗剂激活受体突变体的分子基础;（2）验证和完善S4.54突变的受体模型，以便工程改造纳洛酮和纳洛酮表现为完全激动剂的突变型莫尔;和（3）开发双链腺相关病毒（dsAAV）用于在疼痛通路的特定位点递送突变受体，并评价拮抗剂和激动剂在引发抗伤害感受性反应中的功效。我们将检查dsAAV注射后对阿片样物质激动剂和拮抗剂的急性和慢性反应。通过开发受体模型，并通过受体诱变研究和突变小鼠系的产生来证明拮抗剂活性的结构基础，我们可以设计一种突变受体，其中阿片拮抗剂的行为类似于完全激动剂。由于dsAAV已成功地用于基因治疗，dsAAV的最终递送和突变受体在通常表达莫尔的伤害感受神经元处的表达应在全身施用阿片样物质拮抗剂后产生镇痛反应而不产生耐受性。这种突变型阿片受体基因治疗方法可能成为最终治疗慢性疼痛的新范例。公共卫生相关性：在治疗中度至重度疼痛时，吗啡仍然是首选药物。然而，药物的许多副作用，特别是长期治疗中的耐受性和依赖性发展，降低了使用这种阿片类镇痛剂进行疼痛管理的可取性。药理学家和药物化学家的圣杯是开发药物分子或治疗范例，引起疼痛缓解效果而没有任何副作用。随着突变型μ-阿片受体（莫尔）的发现，其可以被阿片拮抗剂激活而不改变激动剂的性质，我们假设这样的突变型受体可以开发成用于疼痛管理的治疗剂。我们已经证明了这种方法的可行性，通过“敲入”这种突变，S4.54（196）A，进入莫尔，并产生一个小鼠系，其中阿片拮抗剂，纳洛酮和纳洛酮产生抗伤害性反应。然而，该突变仅导致在这两种阿片类拮抗剂中观察到的部分激动性质。因此，在拟议的研究中，我们将使用建模和突变分析来研究这种拮抗活性的分子基础。将鉴定其他受体突变，以将拮抗剂转化为完全激动剂。我们将开发一种基因治疗载体，最初使用dsAAV 2病毒，将突变受体传递到疼痛通路的各个区域，并研究使用阿片类拮抗剂作为镇痛剂的可行性。外源引入的突变体莫尔的激活和拮抗剂对内源性阿片样物质受体的失活将提供独特的机会来开发疼痛治疗范例，而不可能产生耐受性和依赖性。