P450 Epoxygenase Mechanisms of Opioid Analgesia

描述(申请人提供)：吗啡作用于大脑和脊髓中的u阿片受体，产生显著的止痛效果，但u相关的副作用(包括耐受性和药物滥用)限制了它的使用。尽管有大量关于Mu信号的信息，但吗啡激活脑干止痛回路的机制仍然不清楚。这项建议的目的是验证吗啡镇痛的新机制，并研究可能模拟吗啡作用但没有副作用的新止痛药。细胞色素P450是一类药物代谢酶家族，在大脑中也执行内源性代谢氧化。许多P450具有花生四烯酸(AA)环氧酶活性，即它们将AA转化为环氧二十碳三烯酸(EET)。基于三项新发现和一篇文献综述，提出了脑干阿片类止痛作用的环氧合酶假说： 吗啡受体？AA-&gt；(P450)-&gt；？S-&gt；？K&gt；？止痛回路 验证和利用这一假说的实验包括：1)初步研究表明，新开发的脑P450缺陷转基因小鼠(BCPRN)存在吗啡抗伤害性缺陷。为了验证这种阿片类止痛表型，用BCPRN和对照小鼠进行的吗啡实验将研究吗啡剂量、给药途径、伤害性测试和性别的重要性。2)P450环氧合酶抑制剂可阻断吗啡的抗伤害作用。由于吗啡作用于中脑导水管周围灰质腹外侧区、延髓头端腹内侧区和脊髓背角，将P450抑制剂和吗啡微量注射到大鼠中枢这些区域将识别P450相关部位。在BCPRN和对照组小鼠体内注射类似的吗啡，将证实大鼠的结果。3)吗啡介导的副作用包括对呼吸、运动、旋转和体温的调节。在大鼠中使用吗啡和P450抑制剂，在BCPRN小鼠中使用吗啡，将发现这些副作用中的哪些利用了P450机制。4)在大鼠身上使用P450抑制剂和EET代谢抑制剂的额外实验将进一步证实和表征P450环氧合酶在吗啡镇痛中的作用。5)中试结果表明，AA环氧化产物EETs具有抗伤害作用。通过实验验证EET的镇痛作用，寻找EET的副作用，并对EET的作用机制进行研究，可能会使EET成为一类新的实验止痛药。拟议中的研究将增加对吗啡缓解疼痛和产生有害副作用的机制的理解。环氧合酶假说的证实可能会导致开发非阿片类、非成瘾止痛药的新方法。 公共卫生相关性： 人们迫切需要发现新型止痛药，这种止痛药缺乏令人上瘾的特性。这项提案将揭示新的止痛生化机制，并测试一组新的止痛特性化学物质。这项研究可能会导致开发新的、非成瘾的止痛药。

DESCRIPTION (provided by applicant): Morphine acts on mu opioid receptors in the brain and spinal cord to produce dramatic pain relief, but mu-associated side effects (including tolerance and substance abuse) limit its use. Despite substantial information on mu signaling, the mechanisms by which morphine activates brain stem analgesic circuits remain unknown. The goal of this proposal is to validate a new mechanism for morphine analgesia and to investigate new analgesic agents which may mimic morphine actions but lack side effects. Cytochrome P450s are a family of drug-metabolizing enzymes that also perform endogenous metabolic oxidations in the brain. Many P450s have arachidonic acid (AA) epoxygenase activity, i.e. they convert AA to epoxyeicosatrienoic acids (EETs). Based on three new findings and a literature review, an epoxygenase hypothesis is proposed for opioid analgesic action in the brain stem: Morphine -> ¿ receptor -> ? AA -> (P450) -> ? EET s -> ? K+ -> ? Analgesic Circuits Experiments to validate and exploit this hypothesis include: 1) Pilot studies show that newly-developed brain P450-deficient transgenic mice (BCPRN) have defective morphine antinociception. To validate this opioid analgesic phenotype, morphine experiments with BCPRN and control mice will investigate the importance of morphine dose, routes of administration, nociceptive tests and gender. 2) P450 epoxygenase inhibitors block morphine antinociception. Since morphine acts in the ventrolateral periaqueductal gray, the rostral ventromedial medulla, and the spinal dorsal horn, microinjections of P450 inhibitors and morphine into these regions of the rat CNS will identify the P450-relevant sites. Similar microinjections of morphine into BCPRN and control mice will confirm results from rats. 3) Morphine's mu-mediated side effects include modulation of respiration, locomotor activity, rotorod performance, and body temperature. Experiments with morphine and P450 inhibitors in rats and with morphine in BCPRN mice will discover which of these side effects utilize P450 mechanisms. 4) Additional experiments in rats with P450 inhibitors and with inhibitors of EET metabolism will further confirm and characterize the role of P450 expoxygenases in morphine analgesia. 5) Pilot results show that EETs, the products of AA epoxidation, have antinociceptive properties. Experiments to validate EET analgesia, to search for EET side effects, and to characterize EET mechanisms may reveal EETs to be a new class of experimental analgesic agents. The proposed studies will increase understanding of the mechanisms by which morphine relieves pain and produces unwanted side effects. Confirmation of the epoxygenase hypothesis may lead to new approaches for developing non-opioid, non-addicting pain relievers. PUBLIC HEALTH RELEVANCE: There is an urgent need to discover new kinds of pain-relievers that lack addictive properties. This proposal will uncover new biochemical mechanisms for pain relief, and test a new group of chemicals for pain-relieving properties. This research could lead to the development of new, non-addicting pain relievers.