Role of ceramide in morphine hyperalgesia and tolerance

描述（由申请人提供）：以硫酸吗啡为代表的阿片类/麻醉性镇痛药是治疗急性和慢性剧烈疼痛最有效的镇痛药，但其临床应用往往受到对无害和有害刺激的镇痛耐受性和疼痛超敏反应的发展的阻碍。慢性阿片类药物暴露诱导痛觉过敏和抗痛觉耐受性的机制尚不清楚，但已经提出了脊髓的神经免疫激活、细胞凋亡和氧化/硝化应激。神经酰胺是一种鞘脂信号分子，具有强大的促凋亡和促炎特性，也可能参与氧化/硝化应激。神经酰胺是由丝氨酸棕榈酰基转移酶和神经酰胺合成酶协同重新合成和/或由鞘磷脂酶（SMases）酶解鞘磷脂产生的。通过建立良好的小鼠模型，我们的初步实验表明，反复给药吗啡会增加脊髓腰椎段背角的神经酰胺水平，而神经酰胺合成酶抑制剂伏马菌素B1对其的抑制作用会减弱抗伤害性耐受的发展。这些事件与抑制背角组织的细胞凋亡和氧化/硝化应激有关。此外，D609和肉豆蔻素对神经酰胺合成的抑制、SMAse/鞘磷脂合成酶和丝氨酸棕榈酰转移酶的抑制剂分别阻断了抗伤性耐受。总之，这些发现支持了这一探索性建议的中心论点：脊髓中神经酰胺形成的增加是吗啡诱导的痛觉过敏和抗痛觉耐受性发展的重要途径。为了解决这一新的假设，我们提出了一个综合的实验策略，采用分子，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Specific Aim 1中，我们将通过药理学和遗传学方法证明，抑制神经酰胺形成的增加可阻断吗啡诱导的痛觉过敏和抗痛觉耐受性的发展，从而确定其生物合成的主要酶途径。在特异性目标2中，我们将阐明神经酰胺调节痛觉过敏和抗痛觉耐受性的分子和生化机制。具体来说，我们将确定神经酰胺对脊柱组织内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。我们假设的成功验证将首次确定神经酰胺在吗啡诱导的痛觉过敏和抗痛觉耐受性中的重要作用，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。阿片类药物（如吗啡）是治疗严重慢性疼痛最有效的镇痛药，但在长期给药过程中，其镇痛作用往往减弱，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神经酰胺生物合成的抑制，用新的药物，恢复吗啡的镇痛作用。我们的研究结果可能会为慢性疼痛管理开辟一个新的前沿，从而减轻其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.