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1、1,Tat Leang LEE Department of Anaesthesia Yong Loo Lin School of Medicine National University of Singapore,Advances in Amino Acids and Neuropeptides Research and Pain 关于氨基酸和神经肽与疼痛关系的研究,2,Rational treatment of pain requires identification of pain mechanisms as targets of drug therapy,致病因素,临床实体,疾病机制,症
2、状体征,疼痛机制,基于疼痛机制的治疗,与疼痛相关的症状体征,特定机制结果的评测,3,Rational treatment of pain requires identification of pain mechanisms as targets of drug therapy,致病因素,临床实体,疾病机制,症状体征,疼痛机制,与疼痛相关的症状体征,基于疼痛机制的治疗,特定机制结果的评测,4,Key molecular elements in the transduction, transmission and signal processing of nociceptive input in
3、the peripheral and central nervous system represent potential targets for the development of new analgesics.,Mechanism-based pain diagnosis: issues for analgesic drug development Woolf CJ, Max MB. Anesthesiology 2001;95: 241-9,Pain: moving from symptom control toward mechanism-specific pharmacologic
4、 management Woolf CJ. Ann Intern Med. 2004;140:441-5,Can we conquer pain? Schol J, Woolf CJ. Nature neuroscience suppl. 2002;5:1062-7,基于病理机制的疼痛诊断: 镇痛药发展的问题,我们能否征服疼痛?,疼痛: 从症状控制向特异机制的药理学治疗转移,5,Background 背景,Cerebral spinal fluid (CSF) reflects metabolic state of brain 脑脊液反映疼痛的新陈代谢状态 Analysis of CSF am
5、ino acids and peptides highly relevant in pain perception and also neurological diseases 脑脊液中氨基酸和缩氨酸与疼痛感知和神经系统疾病高度相关 Chronic Pain inflammatory, neuropathy and cancer 慢性疼痛分类发炎，神经系病，癌症 Mechanisms very challenging and not yet understood 对于疼痛机制，目前还没有被足够的了解，非常具有挑战性 Lack of effective therapeutics 缺乏有效的治疗法
6、,6,Background,Excitatory and inhibitory amino acids and also nitric oxide (NO) are involved in pain modulation. 兴奋性，抑制性氨基酸和一氧化氮涉及到对疼痛的调节 Neuropeptides are implicated in pain modulation. 神经肽影响对疼痛的调节,7,Objectives,A two pronged study on CSF 对脑脊液的两次研究 To quantitatively analyze physiological amino acids
7、and pain related compounds in CSF samples from pain patients 对疼痛病人脑脊液中生理氨基酸和疼痛相关的化合物定量研究 To find new pain-related peptides (molecular mass 15,000 Da) from CSF samples of chronic pain patients 从慢性疼痛病人的脑脊液中寻找与疼痛相关的肽（分子量小于15000） Results should lead to better understanding of pain mechanisms and new tre
8、atment strategy 实验结果帮助我们更深的了解疼痛机制和寻找新的治疗方法,8,Amino acids in CSF and Pain脑脊液中氨基酸和疼痛关系的研究,9,液相色谱法对生理氨基酸在未被预处理的脑脊液中的定量测量,10,Typical Chromatogram典型色谱层析图,STD AA 氨基酸分离标准图,1-aspartate天(门)冬氨酸盐 2-glutamate 谷氨酸 3-asparagine天冬酰胺酸 4-glutamine谷酰胺 5-citrulline瓜氨酸 6-serine丝氨酸 7-threonine苏氨酸 8-glycine甘氨酸 9-alanine丙
9、胺酸 10-arginine精氨酸 11-taurine牛磺酸 12-GABA伽马氨基丁酸 13-proline脯氨酸 14-valine缬氨酸 15-methionine蛋氨酸 16-isoleucine异亮氨酸 17-leucine亮氨酸 18-tryptophan色氨酸 19-phenylalanine苯基丙氨酸 20-cystine胱氨酸 21-Ammonia氨 22-lysine赖氨酸 23-histidine组氨酸 24-tyrosine酪氨酸,11,1-aspartate天(门)冬氨酸盐 2-glutamate 谷氨酸 3-asparagine天冬酰胺酸 4-glutamine谷
10、酰胺 5-citrulline瓜氨酸 6-serine丝氨酸 7-threonine苏氨酸 8-glycine甘氨酸 9-alanine丙胺酸 10-arginine精氨酸 11-taurine牛磺酸 12-GABA伽马氨基丁酸 13-proline脯氨酸 14-valine缬氨酸 15-methionine蛋氨酸 16-isoleucine异亮氨酸 17-leucine亮氨酸 18-tryptophan色氨酸 19-phenylalanine苯基丙氨酸 20-cystine胱氨酸 21-Ammonia氨 22-lysine赖氨酸 23-histidine组氨酸 24-tyrosine酪氨酸,
11、CSF sample,12,Salient features of the method 方法特色,Use of Triton X-100 （ Triton X-100 的应用） No pre-treatment of CSF 脑脊液不被预处理 Enhanced recovery of hydrophilic amino acids 提高亲水性氨基酸的恢复 Detection of citrulline improved 提高对瓜氨酸的检测 Requires small volume of CSF - 10-20 L 使用较少量的脑脊液 20 L of CSF sample yields 14
12、0 L of dabsylated sample. 20微升脑脊液样本经过dabsylation产生140微升样本 Injection volume 20 L 一次样本液相色谱分析只需20微升 6 repeated analysis of same sample once dabsylated is possible 能够对同一 Dabsylate 过的样本进行6次重复分析 Q effectively separated - 100 times higher concentration in CSF 谷酰胺被有效分离在脑脊液中浓度是其它氨基酸的100倍 Stability of dabsyl
13、derivates Dabsyl衍生物稳定性 Dabsylated sample stable for one week in 4C and for 48 hrs at room temperature 经过Dabsylation的样本能够在4度保持一周，室温下保持48小时,13,Analysis of amino acids and nitric oxide in labor pain对产前疼痛病人脑脊液中氨基酸和一氧化氮的分析,Differential amino acid profile in the cerebrospinal fluid of parturients with and
14、 without labor pain 对产前疼痛和非疼痛产妇脑脊液中氨基酸量不同的分析 Lee TL, Sethuraman R, Chui JW, Tachibana S Annual meeting of the American Society of Anesthesiologists, 23-27 Oct 2004, Las Vegas, USA,14,Concentrations of pain related amino acids in CSF of pregnant women 怀孕妇女脑脊液中和疼痛相关的氨基酸浓度,Values are expressed as mean
15、SEM; a Pregnant women with no labor pain; b Pregnant women in acute labor pain (P 0.05) * (P 0.01) * (P 0.001) Statistically significant when compared to the control group. 产前急性疼痛b与产前无疼痛a相比具有统计价值,15,Statistical correlation between Pain intensity (PI) and the concentration of pain related amino acids
16、 in cerebrospinal fluid of the labor pain group as compared against the no pain control. 疼痛强度和与疼痛相关氨基酸在产妇脑脊液中浓度的统计相关性,PI Pain intensity graded as Mild轻度 (n = 11), Moderate 中度(n = 18), Severe严重 (n = 9) * (P 0.05) * (P 0.01) * (P 0.001) Statistically significant correlation relative to the control gro
17、up.,16,CSF concentration of other amino acids not related to pain 与疼痛不相关的氨基酸在脑脊液中浓度,Values are expressed as mean SEM a Pregnant women with no labor pain b Pregnant women in acute labor pain * (P 0.05) * (P 0.01) * (P 0.001) Statistically significant when compared to the control group.,17,Results结果,A
18、spartate is involved in labor pain but not glutamate 天冬氨酸盐与产前疼痛相关，谷氨酸不相关 Inhibitory amino acids glycine, GABA, taurine involved in labor pain 抑制性氨基酸如甘氨酸，伽马氨基丁酸，牛黄酸与产前疼痛相关 NO might be involved in labor pain may have differential roles 一氧化氮可能涉及至产前疼痛，但可能有不同作用,18,A comparative study on the roles of amin
19、o acids and nitric oxide in chronic and acute pain 氨基酸和一氧化氮在慢性和急性疼痛中的作用对比研究,Relationship between physiological amino acids in cerebrospinal fluid and pain. 脑脊液中生理氨基酸和疼痛的联系 Tachibana S, Sethuraman R and Lee TL In: Anna Capasso (Ed) Recent Developments in Pain Research, Research Signpost; 2005, pp ISB
20、N 81-308-0012-8.,19,Concentration of pain-related amino acids in CSF Chronic pain Vs Control group and Chronic pain Vs Acute pain group 和疼痛相关的氨基酸在脑脊液中浓度慢性疼痛组和标准组，慢性疼痛组和急性疼痛组,Values are expressed as mean SEM; *, (P 0.05) *, (P 0.01) *, (P 0.001) Statistically significant when compared to the chronic
21、pain group 与慢性疼痛组相比有统计. Citrulline detected in only 3 out of 35 control CSF, 17 out of 29 chronic pain CSF and 22 out of 46 acute pain CSF. 在标准组（35样本）中，仅有3个样本中含瓜氨酸；29个慢性疼痛病人中17个含有瓜氨酸；46个急性疼痛病人中有22个含瓜氨酸,20,Comparison between Pain related amino acids in CSF of Chronic and Acute pain patients - Differe
22、ntial roles for these amino acids in the two types of Pain states 和疼痛相关的氨基酸在慢性和急性病人脑脊液浓度对比这些氨基酸在两种不同疼痛状态中的作用 *(P 0.05) * (P 0.01) * (P 0.001) Statistically significant differences between the two groups compared Glutamine (Gln) concentrations are expressed as x10-2 mol/L,21,Results,Chronic pain Vs N
23、o pain group 慢性疼痛和无疼痛 Aspartate, glutamate, glycine and NO involved in chronic pain perception 天门冬氨酸盐，谷氨酸，甘氨酸和一氧化氮与疼痛的感知有关 Chronic pain Vs Acute pain Differential roles of amino acids in chronic and acute pain states 慢性疼痛和急性疼痛氨基酸在慢性和急性疼痛状态的不同作用 Aspartate - a key player in pathogenesis of chronic pai
24、n 天门冬氨酸盐导致慢性疼痛的重要因素,22,Future Scope前景,This simple CSF amino acid analysis method can be applied to study various other pain states and CNS diseases. 这种简易的氨基酸分析方法可以用于其它疼痛状态和神经系疾病分析 This method of CSF analysis without pretreatment in the presence of Triton can also be applied to other analysis procedu
25、res. 这种含有Triton，无预处理脑脊液分析方法可以用于其它分析方法中 With more number of age matched subjects (increase “n”) and with well defined pain conditions potential markers can be identified for each type of pain. 同样年龄组中如果有足够样本，在有明确的对疼痛定义的前提下，对各种疼痛类型，可以鉴别出潜在的生物标记,23,Purification and characterization of peptides from CSF
26、of chronic pain patients慢性疼痛病人脑脊液中肽的提纯和特性研究,24,Peptide Purification Strategy肽的提纯方法,25,Identification鉴别,Characterisation特性,Quantification量化,Localisation 位置,CNS effects 在中枢神经中的影响,Antagonists 对抗剂,Pain models疼痛原型,26,The Story ,27,1973 morphine binding sites / opiate receptors L Terenius, S Snyder, E Sim
27、on,1975 enkephalin J Hughes, H Kosterlitz,Papaver somniferum pain, diarrhoea 377: 532-35,Reinscheid et al. Science 1995; 270: 792-94,NOCICEPTIN (Noc),ORPHANIN FQ (OFQ),阿片孤儿受体（ORL-1）的内源性配体,孤啡肽,孤啡肽,Rat brain nociceptin Mice (i.c.v.) hyperalgesia,Porcine brain Orphanin FQ Mice (i.c.v.) hyperalgesia (ta
28、il- flick assay) decrease locomotor activity,大鼠脑,猪脑,痛觉过敏 (甩尾试验) 减少运动的活动性,33,34,Noc / OFQ,Heptadecapeptide Structurally similar to dynorphin A i.t. or i.c.v. administration : - hyperalgesia (hot plate 100 m (c, d).,Okuda-Ashitaka E et al. 2000,Spinal trigeminal tract,paramedian raphe nucleus,Ventrome
29、dial nucleus of hypothalamus,47,NST receptor (?),Does not act on the classic opiate receptors,Does not act on ORL1 receptor,痛稳素受体 (?),不作用于典型的阿片受体,不作用于阿片孤儿受体(ORL1 )受体,48,The role of the ORL1 receptor in the modulation of spinal Neurotransmission by nociceptin / orphanin FQ and nocistatin Ahmadi S, Li
30、ebel JT, Zeilhofer HU. Eur. J. Pharmacl.2001;412:39-44,Modulation of synaptic transmission by nociceptin / orphanin FQ and nocistatin in the spinal cord dorsal horn of mutant mice lacking the nociceptin / orphanin FQ receptor Ahmadi S, Kotalla C, Guhring H, Takeshima H, Pahl A, Zeilhofer HU Molecula
31、r Pharmacology 2001;59:612-8,49,Possible pathways involved in Noc/OFQ-induced hyperalgesia and allodynia in the spinal cord,Ito S et al. 2000,50,51,Clinical Significance,Anti-nociceptive Responses Produced by Human Putative Counterpart of Nocistatin,Minami T et al Br J Pharmacol 1998; 124: 1016-18,I
32、dentification of Nocistatin in Human, Rat and Mouse Brain and Human Cerebrospinal Fluid,Lee TL et al. NeuroReport 1999; 10: 1537,临床意义,对抗由人们公认的痛稳素类似体所引起的伤害性反应,人,大鼠,小鼠大脑及人脑脊液中痛稳素的鉴别,52,53,Identification of Nocistatin in Human, Rat and Mouse Brain and Human Cerebrospinal Fluid,Lee TL, Fung F, Tachibana
33、 S et al. NeuroReport 1999; 10: 1537,Present in human nervous system Different NST forms Animal models,人,大鼠,小鼠大脑及人脑脊液中痛稳素的鉴别,存在于人类的神经系统中,痛稳素的不同结构,动物模型,54,Lee TL et al. 1999,55,Pathophysiological roles of the two isoforms of nocistatin in pain perception:comparison of bioactivities between nocistatin
34、-17 and nocistatin-30,Meng MJ. 2001; Thesis, Master of Clinical Science. National University of Singapore,Tested against NCP induced allodynia 8:497-500,Endogenous orphanin FQ: evidence for a role in the modulation of electroacupuncture analgesia and the development of tolerance to analgesia produce
35、d by morphine and electroacupuncture Tian JH, Zhang W, Fang Y et al. Br J Pharmacol 1998; 124:21-6,Functional studies using antibodies against orphanin FQ / nociceptin Tian JH, Han JS. Peptides. 2000; 21:1047-50,59,60,NST (future),NST receptor(s) Antagonists / agonists Correlate with specific disease state,痛稳素 (前景),痛稳素受体,拮抗剂 /激动剂,与特异疾病的相关性,61,Thank You,62,