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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

基本信息

批准号：
8213530
负责人：
PING-YEE LAW
金额：
$ 44.32万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-04-01 至 2015-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8213530
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

项目摘要

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(申请人提供)：阿片类药物已经非常成功地用于治疗中到重度的急性和慢性疼痛。不幸的是，它们的使用伴随着许多麻烦的副作用，如恶心、便秘、呼吸抑制、镇静、瘙痒、耐受性和依赖性的形成。许多方法被用来在不降低止痛效果的情况下减轻这些副作用，并取得了不同的成功。值得注意的方法是设计受体选择性配体，激活三个克隆的阿片受体之一，并联合给药以阻断阿片类药物的副作用。虽然这些方法和其他方法是可行的，但它们成功有限的原因之一是克隆的受体之间的氨基酸序列同源性很高。这种同源性减缓了阿片类药物的设计和开发，这种药物将靶向并激活特定的阿片受体。在目前的研究中，我们建议使用另一种方法，即设计突变的阿片受体来控制疼痛。我们的方法是基于偶然发现的一个受体突变，U-阿片受体(MOR)中的S4.45(196)L，该突变导致阿片生物碱拮抗剂在体外和体内都能够激活突变的受体。我们推测，如果这样的突变受体能够传递到伤害性神经元并在其中表达，那么拮抗剂激活突变受体应该会产生镇痛反应，而不会在使用拮抗剂的慢性治疗过程中引发耐受反应。我们的总体目标是开发这种受体突变体，作为疼痛治疗的治疗剂。因此，在目前的建议中，我们将(1)确定阿片类拮抗剂激活受体突变的分子基础；(2)验证和完善S4.54突变的受体模型，以设计一个突变的MOR，其中纳洛酮和纳曲酮表现为完全激动剂；以及(3)开发双链腺相关病毒(DsAAV)，用于在疼痛通路的特定位置递送突变受体，并评估拮抗剂和激动剂在诱导抗伤害感受反应中的效果。我们将检测注射dsAAV后对阿片激动剂和拮抗剂的急性和慢性反应。通过建立受体模型，并通过受体突变研究和突变小鼠系的建立，我们可以设计出一个阿片类拮抗剂表现为完全激动剂的突变型受体，从而证明拮抗剂的结构基础。由于dsAAV已被成功地用于基因治疗，最终通过dsAAV传递并在正常表达MOR的伤害性神经元上表达突变受体，应该会在全身应用阿片类拮抗剂后产生镇痛反应，而不会产生耐受。这种突变的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式。公共卫生相关性：在治疗中度到重度疼痛时，吗啡仍然是首选药物。然而，这些药物的许多副作用，特别是长期治疗中的耐受性和依赖性的形成，降低了使用这种阿片类止痛剂治疗疼痛的可取性。开发药物分子或治疗范例，在没有任何副作用的情况下产生止痛效果，一直是药理学家和药物化学家的圣杯。随着一种突变的u-阿片受体(MOR)的发现，它可以被阿片类拮抗剂激活，而不改变激动剂的性质，我们假设这种突变的受体可以发展成为治疗疼痛的药物。我们已经证明了这种方法的可行性，方法是将这种突变S4.54(196)A“敲入”MOR，并产生一个阿片类拮抗剂、纳洛酮和纳曲酮产生抗伤害感受反应的小鼠系。然而，这种突变只导致了这两种阿片类拮抗剂的部分激动性。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。将确定更多的受体突变，以便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dsAAV2病毒，将突变的受体运送到疼痛通路的不同区域，并研究使用阿片类拮抗剂作为抗伤害感受剂的可行性。外源性引入的突变体MOR的激活，以及内源性阿片受体的失活，将为开发一种不可能产生耐受和依赖的疼痛治疗范例提供独特的机会。