Role of ceramide in morphine hyperalgesia and tolerance

描述(申请人提供)：以硫酸吗啡为代表的阿片类/麻醉性镇痛剂是治疗急性和慢性剧烈疼痛的最有效的镇痛剂，但其临床应用往往受到止痛耐受性和对无害和有害刺激的疼痛过敏的影响。慢性阿片类药物暴露引起痛觉过敏和抗伤害性耐受的机制尚不清楚，但脊髓神经免疫激活、细胞凋亡和氧化/硝化应激已被提出，神经酰胺是一种神经鞘磷脂信号分子，具有强大的促凋亡和促炎作用，也可能参与氧化/硝化应激。神经酰胺是由丝氨酸棕榈糖基转移酶和神经酰胺合成酶协调的从头合成和/或鞘磷脂酶(SMase)对神经鞘蛋白的酶解而产生的。利用已建立的小鼠模型，我们的初步实验表明，反复给药吗啡增加了脊髓腰段背角神经酰胺的水平，并且神经酰胺合成酶抑制剂伏马尼辛B1对其的抑制减弱了抗伤害耐受性的发展。这些事件与抑制背角组织中的细胞凋亡和氧化/硝化应激有关。此外，sMase/鞘磷脂合成酶和丝氨酸棕榈酰基转移酶的抑制剂D609和myriocin对神经酰胺合成的抑制分别阻断了抗伤害性耐受。综上所述，这些发现支持了这一探索性建议的中心论点：脊髓中神经酰胺的增加是吗啡诱导的痛敏和抗伤害耐受性发展的重要途径。为了解决这一新的假设，我们提出了一种综合的实验策略，使用了分子、生物分析、生化、药理学和遗传学方法。两个具体目标将检验我们的假设。在特定的目标1中，我们将通过药理学和遗传学的方法证明，抑制神经酰胺的增加形成阻止了吗啡诱导的痛敏和抗伤害耐受性的发展，从而确定了负责其生物合成的主要酶途径。在具体目标2中，我们将阐明神经酰胺调节痛觉过敏和抗伤害性耐受的分子和生化机制。具体地说，我们将确定神经酰胺对脊髓组织中三种生化途径的影响：(A)神经免疫激活，(B)氧化/硝化应激和(C)细胞凋亡。我们的假说的成功验证将首次确定神经酰胺在吗啡诱导的痛敏和抗伤害耐受中的重要作用，为开发神经酰胺生物合成抑制剂作为阿片类药物的辅助药物治疗慢性疼痛提供科学基础，特别是对于那些需要长期阿片类药物治疗以缓解疼痛的患者。与公共卫生相关的阿片类药物，如吗啡，是治疗严重慢性疼痛最有效的镇痛剂，但在长期给药期间，它们的止痛作用往往会减弱，需要增加剂量，从而降低患者的生活质量。我们的研究将首次确定，用新型药物抑制神经酰胺的生物合成，可以恢复吗啡的止痛作用。我们发现的更广泛的影响可能会在慢性疼痛管理方面开辟一个新的前沿，从而减轻它所造成的社会经济后果。

DESCRIPTION (provided by applicant): Opiate/narcotic analgesics, typified by morphine sulfate, are the most effective analgesics for treating acute and chronic severe pain, but their clinical utility is often hampered by the development of analgesic tolerance and painful hypersensitivity to both innocuous and noxious stimuli. The mechanisms by which chronic opiate exposure induce hyperalgesia and antinociceptive tolerance are unclear but neuroimmune activation, cellular apoptosis and oxidative/nitrative stress in the spinal cord have been proposed, Ceramide is a sphingolipid signaling molecule with powerful proapoptotic and proinflammatory properties and may also contribute to oxidative/nitrative stress. Ceramide is generated from de novo synthesis coordinated by serine palmitosyltransferase and ceramide synthase and/or by enzymatic hydrolysis of sphingomyelin by sphingomyelinases (SMases). Using a well established murine model, our preliminary experiments revealed that repeated administration of morphine increased the levels of ceramide in the dorsal horn of the lumbar segment of the spinal cord and that its inhibition by fumonisin B1, an inhibitor of ceramide synthase, attenuated the development of antinociceptive tolerance. These events were associated with inhibition of apoptosis and oxidative/nitrative stress in dorsal horn tissues. Furthermore, inhibition of ceramide synthesis by D609 and myriocin, inhibitors of SMAse/sphingomyelin synthase and serine palmitoyltransferase respectively blocked antinociceptive tolerance. Together, these findings support the central thesis of this exploratory proposal: increased formation of ceramide in the spinal cord is an important pathway in the development of morphine-induced hyperalgesia and antinociceptive tolerance. To address this novel hypothesis, we propose a comprehensive experimental strategy employing molecular, bio-analytical, biochemical, pharmacological and genetic approaches. Two Specific Aims will test our hypothesis. In Specific Aim 1, we will demonstrate by pharmacologic and genetic approaches that inhibition of the increased formation of ceramide blocks the development of morphine- induced hyperalgesia and antinociceptive tolerance thus identifying the predominant enzymatic pathway responsible for its biosynthesis. In Specific Aim 2, we will elucidate the molecular and biochemical mechanisms whereby ceramide modulates hyperalgesia and antinociceptive tolerance. Specifically, we will determine ceramide's effects on three biochemical pathways within spinal tissue: (a) neuroimmune activation, (b) oxidative/nitrative stress and (c) apoptosis. Successful validation of our hypothesis will define for the first time the important role of ceramide in morphine-induced hyperalgesia and antinociceptive tolerance providing the scientific foundation towards the development of inhibitors of ceramide biosynthesis as adjunct to opiates for the management of chronic pain, in particular for those patients who require long-term opioid treatment for pain relief. PUBLIC HEALTH RELEVANCE Opioid drugs such as morphine are the most effective analgesics for treating severe chronic pain, but their pain-relieving action is often diminished during chronic administration, necessitating dose escalation that reduces quality of life for the patient. Our studies will determine for the first time that inhibition of ceramide biosynthesis, with novel agents, restores the pain-relieving action of morphine. The broader implications of our findings may open a new frontier in chronic pain management thus alleviating the socioeconomic consequences it causes.