Role of CB2 in Analgesic Mechanisms

 描述（由申请人提供）：癌症化疗经常导致疼痛性周围神经病变，这是剂量限制性的，可能是不可逆的。加巴喷丁在临床上用于治疗各种形式的神经性疼痛。然而，其抗伤害作用的机制仍然知之甚少。我们小组未发表的工作表明，加巴喷丁通过与CB 2大麻素受体的相互作用抑制啮齿动物中化疗药物紫杉醇诱导的神经性疼痛。在细胞水平，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）募集β-抑制蛋白到CB 2受体的能力。这些观察结果表明CB 2受体，β- arrestin/ERK 1/2信号和加巴喷丁诱导的抗伤害感受之间的先前未被认识的相互作用。我们推测加巴喷丁的镇痛效果（至少部分）是由于CB 2特异性机制，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具体目标来彻底检验这一假设：目标1将使用转染细胞系、天然表达CB 2受体的细胞系和表达CB 2的细胞的原代培养物来表征加巴喷丁对CB 2受体信号传导的影响。此外，加巴喷丁和CB 2之间的潜在变构相互作用将被探测。目的2将使用从神经元、小胶质细胞和星形胶质细胞中条件性缺失CB 2来确定哪种细胞类型在紫杉醇神经病的发展和维持阶段表达介导加巴喷丁抗伤害感受的CB 2受体。由于CB 2激动剂有效地缓解紫杉醇诱导的异常性疼痛和痛觉过敏，该方法也将用于确定介导由直接作用的CB 2激动剂引起的抗伤害感受的细胞类型。目的3将扩展前两个目的的发现，以确定加巴喷丁的疗效是否也是CB 2介导的其他神经损伤和炎性疼痛模型。将根据第二个具体目标中的要求和描述，使用CB 2的条件性缺失确定相关细胞类型。该目的还将使用上述条件性缺失方法研究直接作用的CB 2激动剂在这些疼痛模型中的机制。我们的研究团队结合了以下方面的专业知识：（1）CB 2受体结合，信号传导，贩运和调节，（2）大麻素药理学和抗伤害机制，以及（3）疼痛的小鼠临床前模型。我们的研究表明，CB 2受体，内源性大麻素和arrestin信号之间存在一种高度新颖且先前未被认识的交叉点，这是加巴喷丁治疗效果的基础。了解这些途径之间的串扰对于阐明加巴喷丁的作用机制以利用和优化其治疗功效以及确定用于药物开发的新治疗靶点都是至关重要的，这些靶点没有常规治疗的不必要的副作用。最后，我们的研究还将确定CB 2激动剂的细胞靶点，因为它们缓解了各种病理性疼痛状态。

 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 Unpublished work from our groups suggests that gabapentin suppresses neuropathic pain induced by the chemotherapeutic agent paclitaxel in rodents through interactions with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.