Therapeutic and Mechanistic Evaluation of Cannabis sativa Terpenes in Neuropathic Pain

摘要 慢性疼痛在美国和全世界都是一种严重且不断恶化的流行病， 降低患者的生活质量。吗啡等阿片类药物是治疗中度至重度抑郁症的“黄金标准”。 严重的慢性疼痛，然而，他们负担着重大的副作用，特别是成瘾倾向， 导致了阿片类药物成瘾、滥用和过量的流行。此外，阿片类药物对 一些疼痛类型，最明显的是神经性疼痛。在寻找替代品的过程中，来自大麻的植物大麻素 Sativa已经被大量研究。然而，大麻素通常被证明具有中等至差的 疗效，并有自己的副作用，特别是精神副作用与Δ9-四氢大麻酚 治疗这再次导致了对改善大麻素治疗方法的研究。因此，研究 主要集中在大麻中发现的约150种萜烯化合物，这些化合物赋予植物风味和香气。 有限的证据表明，萜烯本身就能缓解疼痛，也有人提出， 调节并可能改善大麻素如THC的作用，称为“随行效应”假说。 然而，萜烯功效的证据质量一般较差，受到定义不清和复杂的限制， 提取物，很少的机制研究。因此，我们对大麻萜类化合物α- 蛇麻烯、β-蒎烯、香叶醇和芳樟醇。我们发现，所有4个萜烯产生抗伤害性，在一个 化疗诱导的周围神经病变（CIPN）的小鼠模型与 吗啡同时，香叶醇和芳樟醇不产生奖赏或厌恶，表明不会成瘾 或厌恶性责任。寻求机制的见解，我们发现，所有4个萜烯产生甩尾抗- 通过大麻素受体1型（CB 1）机制的伤害感受，并进一步与 cannabinoid WIN 55，212，为随行效应假说提供了证据。我们进一步鉴定了CB 2， 腺苷A2 a和抗炎活性作为潜在的作用机制。在本提案中，我们将 扩展这些研究，以评估这些萜烯在神经病变中的治疗潜力和作用机制， 疼痛，为这些配体作为改善的非阿片样物质疼痛治疗剂的用途提供了潜在的支持。在目标1中， 我们将在CIPN的小鼠模型中全面测试萜烯，包括剂量/反应，交替神经病变模型， 副作用如耐受性和奖赏/厌恶，与其他镇痛药如阿片类和大麻素的协同作用， 和萜烯对这些其他镇痛药的副作用的影响（特别是阿片样物质的奖励）。在目标2中，我们将确定 CIPN中萜烯作用的分子机制，重点是1）CB 1/2，2）A2 a和3）抗炎 活动我们将使用选择性拮抗剂和CRISPR基因编辑，确定作用位点（例如大脑，脊髓）， 脊髓、外周），测量组织对萜烯的反应（例如细胞因子产生），并使用体外模型来 确认这些机制。这些研究将一起提供一个严格的评估的潜在用途， 萜类化合物作为神经性疼痛的有效和低副作用的治疗剂。

ABSTRACT Chronic pain is a serious and worsening epidemic in the United States and worldwide, seriously degrading patient quality of life. Opioid drugs like morphine are the “gold standard” for treating moderate to severe chronic pain, however, they are burdened by major side effects, especially addiction liability, which has contributed to a paralell epidemic of opioid addiction, abuse, and overdose. In addition, opioids are ineffective in some pain types, most notably neuropathic pain. In the search for alternatives, phytocannabinoids from Cannabis sativa have been heavily studied. However, cannabinoids have generally been shown to have modest to poor efficacy, and have their own side effects, especially psychoactive side effects with Δ9-tetrahydrocannabinol treatment. This has led again to a search for methods to improve cannabinoid therapy. For this reason, research has focused on the ~150 terpene compounds found in Cannabis, which impart flavor and aroma to the plant. Limited evidence suggests that terpenes produce pain relief on their own, and they have also been proposed to modulate and potentially improve the effects of cannabinoids like THC, termed the “entourage effect” hypothesis. However the quality of evidence on terpene efficacy is in general poor, limited by poorly-defined and complex extracts, and few mechanistic studies. We thus performed a preliminary study on the Cannabis terpenes α- humulene, β-pinene, geraniol, and linalool. We found that all 4 terpenes produced anti-nociception in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN) comparable or better than morphine. At the same time, geraniol and linalool produced no reward or aversion, suggesting no addictive or aversive liability. Seeking mechanistic insight, we found that all 4 terpenes produced tail flick anti- nociception by a cannabinoid receptor type 1 (CB1) mechanism, and further synergized with the cannabinoid WIN55,212, providing evidence for the entourage effect hypothesis. We further identified CB2, Adenosine A2a, and anti-inflammatory activity as potential mechanisms of action. In this proposal, we will extend these studies to evaluate therapeutic potential and mechanisms of action of these terpenes in neuropathic pain, providing potential support to the use of these ligands as improved non-opioid pain therapeutics. In Aim 1, we will fully test the terpenes in a mouse model of CIPN, including dose/response, alternate neuropathy models, side effects like tolerance and reward/aversion, synergy with other analgesics such as opioids and cannabinoids, and terpene impact on side effects of these other analgesics (especially opioid reward). In Aim 2, we will identify molecular mechanisms for terpene action in CIPN, focusing on 1) CB1/2, 2) A2a, and 3) anti-inflammatory activity. We will use selective antagonists and CRISPR gene editing, identify sites of action (e.g. brain, spinal cord, periphery), measure tissue response to terpene (e.g. cytokine production), and use in vitro models to confirm these mechanisms. Together these studies will provide a rigorous evaluation of the potential use of terpenes as efficacious and low side-effect therapeutics for neuropathic pain.