Role of CB2 in Analgesic Mechanisms

 描述(申请人提供)：癌症化疗经常导致疼痛的周围神经病变，这是剂量有限的，可以是不可逆转的。加巴喷丁在临床上用于治疗各种形式的神经病理性疼痛。然而，其抗伤害性作用的机制(S)仍然知之甚少。我们小组未发表的工作表明，加巴喷丁通过与CB2大麻素受体相互作用，抑制化疗药物紫杉醇在啮齿动物中诱导的神经病理性疼痛。在细胞水平上，加巴喷丁选择性地增加内源性大麻素2-花生四烯酸甘油(2-AG)向CB2受体募集β-arrestin的能力。这些观察表明，CB2受体、β-arrestin/ERK1/2信号和加巴喷丁诱导的抗伤害作用之间存在先前未知的相互作用。我们推测加巴喷丁的止痛效果是由于(至少部分)CB2特异的机制，该机制涉及小胶质细胞或神经元中β-arrestin信号的增加。我们将通过完成三个特定的目标来彻底检验这一假设：目标1将通过转基因细胞系、天然表达CB2受体的细胞系和表达CB2受体的细胞的原代培养来表征加巴喷丁对CB2受体信号转导的影响。此外，还将探讨加巴喷丁和CB2之间潜在的变构相互作用。目的2利用从神经元、小胶质细胞和星形胶质细胞中条件性缺失CB2来确定在紫杉醇神经病的发展和维持阶段，哪种细胞类型(S)表达介导加巴喷丁抗伤害感受的CB2受体。由于CB2激动剂能有效缓解紫杉醇引起的痛觉过敏和痛觉过敏，因此该方法也将被用于确定介导直接作用CB2激动剂抗伤害效应的细胞类型(S)。目的3将扩展前两个AIMS的发现，以确定加巴喷丁在其他神经损伤和炎症性疼痛模型中是否也由CB2介导。相关细胞类型(S)将根据需要和第二个具体目标中所述，使用有条件的删除CB2来确定。这一目标还将使用上述条件缺失方法来研究在这些疼痛模型中直接作用的CB2激动剂的机制。我们的研究团队结合了(1)CB2受体结合、信号传递、运输和调节，(2)大麻素药理和抗伤害感受机制，以及(3)小鼠临床前疼痛模型的专业知识。我们的研究表明，CB2受体、内源性大麻素和arrestin信号之间存在高度新颖的交叉，这是加巴喷丁治疗效果的基础。了解这些通路之间的相互作用对于阐明加巴喷丁的作用机制、开发和优化其治疗效果以及为药物开发寻找新的治疗靶点、消除传统疗法的不良副作用都是至关重要的。最后，我们的研究还将确定CB2激动剂缓解各种病理性疼痛状态的细胞靶点。

 DESCRIPTION (provided by applicant): Cancer chemotherapy frequently causes a painful peripheral neuropathy that is dose-limiting and can be irreversible. Gabapentin is clinically used to treat diverse forms of neuropathic pain. However, the mechanism(s) responsible for its antinociceptive effects remain poorly understood. Unpublished work from our groups suggests that gabapentin suppresses neuropathic pain induced by the chemotherapeutic agent paclitaxel in rodents through interactions with CB2 cannabinoid receptors. At the cellular level, gabapentin selectively increases ability of the endocannabinoid 2-arachidonoylglycerol (2-AG) to recruit β-arrestin to CB2 receptors. These observations suggest a previously unrecognized interaction between CB2 receptors, β- arrestin/ERK1/2 signaling and gabapentin-induced antinociception. We postulate that gabapentin analgesic efficacy is due (at least in part) to a CB2-specific mechanism that involves increased β-arrestin signaling in microglia or neurons. We will thoroughly test this hypothesis by completing three Specific Aims: Aim 1 will characterize the impact of gabapentin on CB2 receptor signaling using transfected cells lines, cell lines natively expressing CB2 receptors and primary cultures of CB2-expressing cells. In addition, potential allosteric interactions between gabapentin and CB2 will be probed. Aim 2 will use conditional deletion of CB2 from neurons, microglia, and astrocytes to determine which cell type(s) express the CB2 receptors mediating gabapentin antinociception during the development and maintenance phases of paclitaxel neuropathy. Since CB2 agonists efficaciously relieve paclitaxel-induced allodynia and hyperalgesia, this approach will also be used to determine the cell type(s) mediating antinociception elicited by direct acting CB2-agonists. Aim 3 will extend the findings of the first two aims to determine if gabapentin efficacy is also CB2-mediated in other nerve injury and inflammatory pain models. The relevant cell type(s) will be determined using conditional deletion of CB2 as warranted and as described in the second specific aim. This aim will also investigate the mechanism of direct-acting CB2 agonists in these pain models using the above conditional deletion approach. Our research team combines expertise in (1) CB2 receptor binding, signaling, trafficking, and regulation, (2) cannabinoid pharmacology and antinociceptive mechanisms, and (3) mouse preclinical models of pain. Our studies suggest a highly novel and previously unrecognized intersection between CB2 receptors, endocannabinoids, and arrestin signaling that underlies the therapeutic efficacy of gabapentin. Understanding the cross-talk between these pathways is critical both for elucidating the mechanism of action of gabapentin to exploit and optimize its therapeutic efficacy and for identifying novel therapeutic targets for drug development that lack unwanted side effects of conventional treatments. Lastly, our studies will also identify the cellular targets of CB2 agonist as they relieve a variety of pathological pain states.