Therapeutic and Mechanistic Evaluation of Cannabis sativa Terpenes in Neuropathic Pain

摘要 慢性疼痛是一种严重且日益恶化的流行病，在美国和全世界都是如此 降低病人的生活质量。像吗啡这样的阿片类药物是治疗中度到中度到 严重的慢性疼痛，然而，他们有主要的副作用，特别是成瘾倾向，这有 导致阿片成瘾、滥用和过量的平行流行。此外，阿片类药物在 一些疼痛类型，最明显的神经病理性疼痛。在寻找替代品的过程中，大麻中的植物大麻素 人们对紫花苜蓿进行了大量研究。然而，大麻类物质通常被证明具有中等到较差的活性。 有疗效，并有自己的副作用，特别是Δ9-四氢大麻酚的精神活性副作用 治疗。这再次促使人们寻找改进大麻类药物治疗的方法。出于这个原因，研究 专注于大麻中发现的~150种萜类化合物，这些化合物给大麻带来了风味和芳香。 有限的证据表明，萜类化合物本身就能缓解疼痛，而且它们也被提出 调节和潜在地改善类似THC的大麻素的影响，这被称为“环境效应”假说。 然而，关于萜类化合物功效的证据质量总体上很差，受到定义不明确和复杂的限制。 提取，很少有机械学研究。因此，我们对大麻萜烯类化合物α进行了初步研究。 胡萝卜烯、β-蒎烯、香叶醇和芳樟醇。我们发现，所有4个萜类化合物都能产生抗伤害性作用。 化疗所致周围神经病(CIPN)小鼠模型相当或优于 吗啡。同时，香叶醇和芳樟醇不会产生奖励或厌恶，这表明它们不会上瘾。 或厌恶责任。在寻求机理洞察时，我们发现4个萜类化合物都产生了尾部甩尾现象。 通过大麻素1型受体(CB1)机制产生伤害性，并进一步与 大麻素WIN55,212，为环境效应假说提供了证据。我们进一步鉴定了CB2， 腺苷A2a和抗炎活性作为潜在的作用机制。在这项提案中，我们将 扩大这些研究以评估这些萜类化合物在神经病中的治疗潜力和作用机制 疼痛，为将这些配体用作改进的非阿片类止痛药提供了潜在的支持。在目标1中， 我们将在CIPN小鼠模型中全面测试这些萜类化合物，包括剂量/反应、替代神经病变模型、 副作用，如耐受性和奖赏/厌恶，与其他止痛剂如阿片类药物和大麻类药物的协同作用， 以及萜类对这些其他镇痛剂的副作用(特别是阿片类奖赏)的影响。在目标2中，我们将确定 CIPN中萜烯作用的分子机制，主要集中在1)CB1/2，2)A2a和3)抗炎 活动。我们将使用选择性拮抗剂和CRISPR基因编辑，确定作用部位(例如大脑、脊髓 脐带、外周)，测量组织对萜烯的反应(例如细胞因子的产生)，并使用体外模型 确认这些机制。这些研究将一起提供对潜在用途的严格评估 萜类化合物是治疗神经病理性疼痛的有效且副作用低的药物。

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.