阿片/神经肽FF受体的双功能配体BN-9在临床前痛模型
中的镇痛位点和耐受现象研究
中文摘要分层教学
双功能药物的疗效在癌症、糖尿病、抑郁症以及感染性疾病的治疗中已被验证，近年来镇痛类的双功能分子在高效、低副作用的镇痛新药研发中也受到广泛关注。最近，本实验室设计并合成了一类阿片/神经肽FF（NPFF）受体的双功能分子用于镇痛新药研究，其中新型肽类分子BN-9（Tyr-D.Ala-Gly-Phe-Gln-Pro-Gln-Arg-Phe-NH2）能同时激活阿片和NPFF受体，并在急性痛模型中能介导高效、无耐受的镇痛作用。为了进一步验证双功能激动剂BN-9的无耐受镇痛作用，本论文在临床前病理痛模型中全面地评价了其镇痛作用位点、镇痛药效和耐受现象。
首先，本论文选用经典的角叉菜胶炎症痛模型，结合阿片和NPFF受体的拮抗剂，通过不同给药途径来探讨BN-9的镇痛作用位点。研究结果表明小鼠脊髓及脊髓以上水平注射BN-9可通过激活阿片受体而剂量依赖地减缓角叉菜胶炎症痛诱导的机械性异常疼痛，并且该中枢镇痛作用能被NPFF受体拮抗剂RF9增强。相比之下，足跖部、腹腔、皮下和肌肉注射BN-9的抗异常性疼痛作用主要依赖于阿片受体，与NPFF受体无关。此外，侧脑室注射阿片受体拮抗剂甲碘化纳洛酮不能拮抗BN-9（i.p.）的镇痛而腹腔注射拮抗剂时可以拮抗，表明腹腔注射BN-9主要在外周产生镇痛。同时，脚掌注射纳洛酮能够显著阻断腹腔注射B
N-9的镇痛，进一步证明BN-9的外周镇痛特性，而NPFF受体不参与BN-9的外周镇痛。上述研究结果表明在角叉菜胶炎症痛中各个水平（中枢、外周和系统）注射BN-9都能够剂量依赖地缓解机械性痛觉敏感，侧脑室水平的镇痛作用最强，脊髓水平的镇痛作用次之。其中，中枢注射BN-9表现为阿片和NPFF受体的激动活性。而外周和系统注射BN-9主要通过激活外周阿片受体发挥镇痛，并且BN-9不能通过血脑屏障。
鉴于BN-9无法透过血脑屏障且中枢镇痛较强，因此选用临床上可以实现的脊髓注射来研究BN-9在其他临床前实验动物模型的镇痛作用机制以及耐受现象。结果表明BN-9在CFA炎症痛模型、手术后痛模型、醋酸扭体和福尔马林痛中均能够产生剂量依赖的镇痛作用，但是在每个模型中的作用机制各不相同。在CFA炎症痛中，阿片受体拮抗剂可以拮抗BN-9的抗伤害感受作用而
钱万三RF9可以加强其镇痛，进一步证明BN-9的阿片与NPFF受体的双功能激动特性。手术后痛模型中纳洛酮和RF9均能够拮抗BN-9的镇痛。醋酸扭体与福尔马林痛中BN-9的镇痛主要由阿片受体介导，与NPFF受体无关。另外，BN-9在正常大鼠中的耐受研究表明正常大鼠中连续七天鞘内注射BN-9（1nmol）对大鼠基础痛阈值（机械刺激）没有影响，而吗啡（2nmol）引起了明显的痛觉敏感。CFA炎症痛大鼠中连续注射BN-9（1nmol）表现出强效的抗痛敏（光热刺激）活性，且与吗啡（2nmol）没有交叉耐受。总之，BN-9对急性痛和慢性痛都有明显调节作用，且主要通过阿片受体介导镇痛。在慢性痛模型中BN-9可以产生有效的无耐受形成的抗伤害感受作用。
关键词：BN-9；阿片；神经肽FF；双功能激动剂；镇痛
吸烟检讨书Studies of antinociceptive site(s)and tolerance induced by the bifunctional opioid/NPFF receptors agonist BN-9in
preclinical pain models散字组词
Abstract
Bifunctional drugs had been ud in the treatment of cancer,diabetes,depression and infectious dias.In recent years,bifunctional analgesics with high efficiency and low side effects were attracted widespread attention.The novel bifucntional nonapeptide BN-9 (Tyr-D.Ala-Gly-Phe-Gln-Pro-Gln-Arg-Phe-NH2)behaved as a full agonist towardsμ-,δ-,κ-opioid, NPFF1and NPFF2receptors in functional assays.And BN-9produced potent non-tolerance forming antinociception in the tail-flick test.To further validate the non-tolerance forming antinociception of BN-9,the anti-allodynic activities,site(s)of action and tolerance of BN-9are comprehensively evaluated in the preclinical models.
In the prent studies,we initially chon the classic carrageenan inflammatory pain model and inves
tigated the analgesic sites of BN-9by different routes of administration.The results suggest that v.)and )administrations of BN-9do-dependently decread carrageenan-induced mechanical allodynia via opioid receptors,and the antinociceptive actions were augmented by the NPFF receptor antagonist RF9.In contrast,hindpaw（i.paw）, intraperitoneal(i.p.),),and )injection of BN-9produced anti-allodynic effects in an opioid receptor-dependent manner,independent of the NPFF receptor. Furthermore,the anti-allodynia of systemic BN-9was blocked by naloxone methiodide(i.p.),but not v.injection of naloxone methiodide,indicating that anti-allodynic effect of intraperitoneal injection of BN-9mainly mediated by peripheral opioid receptors.In addition, i.paw of naloxone can significantly block the anti-allodynic effect of BN-9(i.p.),it’s also demonstrated the peripherally anti-allodynic properties of BN-9,and the peripherally anti-allodynia of BN-9did not mediated by NPFF receptor.The results suggest that central, peripheral or systemic administrations of BN-9exert potent analgesic activities in the inflammatory pain v.injection of BN-9had the strongest analgesic effect,followed BN-9,and the central injection BN-9showed the opioid and NPFF receptors agonistic character.While the anti-allodynic effect induced by peripheral and systemic injection of BN-9 mainly mediated by peripheral opioid receptors,and BN-9can not cross the blood-brain barrier.
In view of the above-mentioned facts that,BN-9can not cross the blood-brain barrier and the central anti-allodynic effect analgesia is more strong.Therefore,we investigated the analgesic mechanism and tolerance in other experimental models injection of BN-9.The results showed that BN-9could produce do-dependently analgesic effect in CFA inflammatory pain, incisional pain,acetic acid writhing and formalin test via different mechanisms.In the CFA inflammatory pain,opioid receptor antagonist naloxone blocked anti-allodynic effect of BN-9,and were augmented by the NPFF receptor antagonist RF9,which support the bifunctional opioid/NPFF receptors agonism character of BN-9.Both naloxone and RF9can antagonize the anti-allodynic effect of BN-9in the incisional pain.In acetic acid writhing test and formalin test, BN-9produced antinociceptive effects in an opioid receptor-dependent manner,which were not modified by RF9.In addition,BN-9(1nmol)has no effects on the mechanical latency of naive rats after concutively7days treatment.However,morphine(2nmol)induced significant allodynia after chronical administration.Repeated administration of BN-9(1nmol)did not lead to the development of anti-allodynic tolerance in the CFA inflammatory pain and no cross-tolerance to morphine(2nmol).In conclusion,BN-9produced a significant analgesic effect on acute and chronic pain,which is mainly mediated by opioid receptors.And BN-9produced potent non-tolerance forming antinociception in chronic pain models.
Key words:BN-9,Opioid,Neuropeptide FF,bifunctional agonist,Antinociception
目录
双抛桥中文摘要......................................................................................................III 目录. (1)
第一章研究背景 (1)
1.1内源阿片系统 (1)
1.1.1内源阿片受体 (1)
1.1.2内源阿片配体 (2)
1.1.3阿片系统与镇痛 (3)
1.1.4阿片系统与药物成瘾 (4)
1.1.5阿片系统与药物耐受 (5)
1.2神经肽FF系统 (6)
1.2.1神经肽FF受体 (6)
1.2.2神经肽FF的抗阿片活性 (7)四叶草寓意>感动的话
1.2.3神经肽FF的镇痛活性 (7)
1.2.4神经肽FF对阿片耐受和成瘾的调节 (7)
1.3双功能药物 (8)小林龟介
1.3.1阿片类物质与阿片类物质之间的嵌合 (9)
1.3.2阿片类物质与其他镇痛物质之间的嵌合 (11)
1.3.3阿片肽类激动剂与NPFF之间的嵌合 (12)
1.4立题依据与实验设计 (13)
1.4.1小鼠角叉菜胶炎症痛中BN-9的作用位点研究 (13)
