【作者】 孙晓芳；

【导师】 牛建昭； 王丹巧；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2011， 博士

【摘要】 研究背景及目的帕金森病(PD)是一种以黑质、纹状体区多巴胺(DA)能神经元进行性退变、DA含量明显减少为特征的中枢神经系统疾病,临床常用左旋多巴(L-DOPA)替代疗法治疗。L-DOPA在体内转化为DA,可以减轻PD的症状,但随着治疗时间延长,其疗效降低,副作用增强。PD发展导致脑DA储存能力下降,当L-DOPA血药浓度超过治疗阈,而神经元对DA水平波动缓冲能力丧失时,则形成波动性刺激,诱发症状波动、运动障碍等不良反应。L-DOPA自身氧化产生自由基,也可能加速PD患者神经元变性。因此,如何合理地使用L-DOPA,以较小的剂量发挥较大的药效,减少不良反应,是目前面临的重要课题。临床应用中药首乌方与L-DOPA制剂美多芭结合治疗PD,取得了良好效果。首乌方由首乌、鹿茸、天麻等中药组成,是在中医理论指导下针对PD选出的合理中药处方。研究表明,临床使用首乌方可以协同美多芭作用,减少美多芭用量。动物实验显示其可能通过影响兴奋性毒性、氧化损伤等减小、L-DOPA的神经损伤。微透析采样技术可以监测清醒自由活动动物特定组织细胞外液中物质的动态变化。近年来,该技术被应用于药代-药效学结合研究中,以阐明药物在作用靶部位的浓度-效应-时间三维关系。本实验通过建立血-脑双位点同步微透析采样、高效液相色谱-荧光检测(HPLC-FLD)和高效液相色谱-电化学检测(HPLC-ED)法,同步监测清醒自由活动大鼠L-DOPA血药浓度-靶部位(纹状体)药物浓度-靶部位递质水平变化情况。观察首乌方对6-羟基多巴胺(6-OHDA)导致的PD模型大鼠血液和纹状体细胞外液L-DOPA药代动力学的影响,以及由L-DOPA引起的脑内多巴胺及其代谢产物和羟自由基变化的影响,并进行药动-药效相结合的分析研究,进一步探讨首乌方对L-DOPA治疗PD的增效减毒和神经保护作用机制。研究方法1 HPLC-FLD测定L-DOPA的方法色谱柱为MERCK Lichrospher 100 C18 (5μm,4mm×250mm),预柱为Nova-Pak C18,柱温：35℃,激发波长278nm,发射波长325nm,流动相为：乙二胺四乙酸二钠(0.08mmol·L-1),磷酸二氢钾(70mmol·L-1),庚烷磺酸钠(2.08mmol·L-1),甲醇(10%)。流速1.0ml·min-1。2 HPLC-ED同步测定单胺类递质、羟自由基和L-DOPA的方法色谱柱为Antec Leyden BVC18分析柱(3μm, 2.1mm×100mm)。Antec DecadeⅡSDC电化学检测器,玻璃碳电极和Ag/AgCl参考电极,工作电压为0.52v。流动相为：乙二胺四乙酸二钠(0.027mmol·L-1)、辛烷磺酸钠(0.74mmol·L-1)、氯化钾(2mmol·L-1)、磷酸二氢钠(100mmol·L-1)、甲醇(15%)、乙腈(1%)、乙酸(0.05%),用磷酸调pH值至3.32,流速0.2mL·min-1。3大鼠纹状体注射6-OHDA造成PD模型微透析采样前7天,大鼠ip戊巴比妥钠麻醉(40mg·kg-1),埋入探针套管于右侧纹状体内(A：+0.2mm,L：+3mm,V：7.5mm),用牙科水泥固定,纹状体内缓慢注射0.2%的6-OHDA生理盐水溶液(32μg·kg-1 1μL·min-1),造成脑内单点注射6-OHDA所致的PD大鼠模型。对照组注射相应体积生理盐水。4首乌方对大鼠血液-纹状体细胞外液L-DOPA药代动力学及脑内递质的影响动物分组与药物处理SD大鼠随机分为6组：对照组、模型组、高剂量对照组、高剂量模型组、低剂量模型组,首乌方+低剂量模型组。参见上述方法造成PD模型,造模当天首次投药,高剂量对照组和高剂量模型组ig美多芭60mg·kg-1(L-DOPA 48mg·kg-1和苄丝肼12mg·kg-1),低剂量模型组ig美多芭30mg·kg-1(L-DOPA 24mg·kg-1和苄丝肼6mg·kg-1),首乌方+低剂量模型组ig首乌方煎剂(生药量：18g·kg-1)和美多芭30mg·kg-1,对照组和模型组ig等体积蒸馏水,连续6天。手术和微透析第6天以同样方法麻醉大鼠,左侧颈静脉插入血探针,复方氯化钠灌流2μl·min-1过夜。大鼠腹腔埋植导管,从背部引出并固定,用于次日微透析过程中腹腔给药。第7天大鼠清醒自由活动状态下插入脑探针,进行同步血、脑双位点微透析。双位点探针用复方氯化钠灌流(2.5μL·min-1)平衡60min后,脑探针灌流液改为5mmol·L-1的水杨酸钠-复方氯化钠溶液,平衡60min后,开始收集透析液,每15min收集1管。取前4管测定目标检测物,计算均值作为基础水平。之后,各组大鼠经腹腔留置的导管ip L-DOPA和苄丝肼,给药剂量见前。对照组和模型组ip等量生理盐水,收集血、脑透析液至给药后420min。研究结果1 HPLC-FLD测定血透析液中L-DOPA的方法测定0.3125～5mg·L-1 5个浓度的L-DOPA标准液,线性关系良好,相关系数(R2)为0.9987。测定0.3125-5mg·L-1的L-DOPA标准,日内、日间相对变异系数(RSD)≤4.9%,最低检测限19μg·L-1。2 HPLC-ED同时测定脑透析液中单胺类递质、羟自由基和L-DOPA的方法配置含L-DOPA、DA、3,4-二羟基苯乙酸(DOPAC)、5-羟吲哚乙酸(5-HIAA)、5-羟色胺(5-HT)、2,3-二羟基苯甲酸(2,3-DHBA).2,5-二羟基苯甲酸(2,5-DHBA)、肾上腺素(EP)、去甲肾上腺素(NE)、高香草酸(HVA)的10mix标准液。测定5个不同浓度的10mix标准液,线性关系良好,各物质R2≥0.9991。各检测物在3.125～250μg·L-1浓度的日内、日间RSD≤8.7%,检测限≤1.0μg·L-1。3 PD模型大鼠血液和纹状体细胞外液L-DOPA药代动力学同步研究PD大鼠腹腔注射L-DOPA后,药物迅速吸收入血,并通过血脑屏障进入纹状体。血液和纹状体细胞外液的药物浓度-时间曲线符合一室模型,纹状体细胞外液药物达峰时间(Tmax)晚于血液,药-时曲线下面积(AUC(o-∞))和达峰浓度(Cmax)小于血液。4 PD模型大鼠L-DOPA血液药动学-靶部位药动学-靶部位药效学指标的相关性研究L-DOPA药动学指标选择大鼠血液、纹状体细胞外液的药物浓度,药效学指标选择大鼠纹状体细胞外液DA变化率(△DA=(测定浓度-基础值)/基础值)。腹腔注射L-DOPA后,随着血液中L-DOPA浓度的增加,纹状体细胞外液L-DOPA、ΔDA水平也逐渐增加。血液浓度与纹状体细胞外液药物浓度、血药浓度与纹状体细胞外液△DA、纹状体细胞外液药物浓度与纹状体细胞外液△DA均呈现较好的相关性。血药浓度-效应曲线、纹状体细胞外液药物浓度-效应曲线均呈典型的逆时针滞后环。当血液、纹状体细胞外液LDOPA浓度达到最大时,纹状体DA水平并未达到最大值。5首乌方对PD模型大鼠血液和纹状体细胞外液L-DOPA药代动力学的影响与低剂量模型组相比,首乌方+低剂量模型组L-DOPA血药浓度在6个时间点升高,纹状体细胞外液药物浓度未见升高,而在15min时降低。首乌方使L-DOPA血液AUC(0-∞)增加,Tmax延后,平均驻留时间(MRT(0-∞)延长；使纹状体细胞外液Tmax滞后,MRT(0-t)延长。6首乌方对PD大鼠纹状体细胞外液DA及其代谢产物的影响6-OHDA造模使模型组纹状体细胞外液DA水平较对照组降低,DOPAC/DA、HVA/DA比率升高。与模型组相比,低剂量模型组、首乌方+低剂量模型组DA基础水平升高,DOPAC/DA、HVA/DA比率基础值降低。腹腔注射L-DOPA后,两组DA水平均迅速升高,然后缓慢回落。与低剂量模型组相比,首乌方+低剂量模型组DA水平升高更显著,而△DA升高幅度较低。两组间DOPAC/DA、HVA/DA比率基础值及变化无差异。7首乌方对PD大鼠纹状体细胞外液羟自由基的影响以总DHBA (2,3-DHBA与2,5-DHBA之和)代表羟自由基水平。模型组纹状体细胞外液总DHBA较对照组升高。与模型组相比,低剂量模型组、首乌方+低剂量模型组总DHBA基础水平降低。腹腔注射L-DOPA后,总DHBA逐渐升高,275min后缓慢回落。其中高剂量模型组升高最为显著,低剂量模型组居中,首乌方+低剂量模型组总DHBA最低,且有9个时间点低于低剂量模型组。结论1本实验建立了大鼠血-脑双位点微透析采样、HPLC-ED同时检测脑透析液中单胺类递质、羟自由基和L-DOPA的方法。成功地对清醒自由活动大鼠L-DOPA血药浓度-纹状体细胞外液药物浓度-纹状体细胞外液多种递质水平进行了相同生物个体的同步动态研究。2清醒PD模型大鼠L-DOPA血药浓度、纹状体细胞外液药物浓度、纹状体细胞外液△DA三者间具有明显的相关性,血药浓度可以在一定程度上反映靶部位药物浓度及效应指标的变化趋势。3首乌方可减慢L-DOPA消除,增加血液L-DOPA的吸收,减少纹状体L-DOPA药物浓度波动。4首乌方协同L-DOPA治疗PD,可使纹状体细胞外液DA浓度更长时间维持在较高水平,同时可以降低△DA,减少DA水平的波动。此协同作用与首乌方对L-DOPA药动学的影响密切相关。5低剂量L-DOPA可以降低纹状体细胞外液羟自由基基础水平,与首乌方合用降低作用更为显著。首乌方具有抗氧化作用。更多还原

【Abstract】 Background and ObjectiveParkinson’s disease (PD) is a central nervous system diseases, featured mainly by progressive neurodegeneration and reduce of DA content in the system of substantia nigral and striatum. Generally replacement therapy with L-DOPA, it conversion to DA, and abate the symptoms. But long-time administration can weaken L-DOPA curative effects and caused side effects. There a reduction of DA storage ability in neuron with the development of PD, when blood concentration of L-DOPA over the response threshold, and no buffer effect of neuron to DA fluctuation. L-DOPA oxidation and releases free radical, may improve process of PD. Thus rational administration of L-DOPA is important. Shouwu Fang is a clinical effective Chinese herbal formula, it consists of radix polygoni multiflori preparata, pilose Antler, Gastrodia elata, et al. It has been found that the treatment with integration of Shouwu Fang and western medicine can not only increase therapeutic action, but also reduce toxic and side-effects.Microdialysis is an in vivo sampling technique permitting the continuous determination of test substances. Recently, this technique has been used in pharmacokinetics-pharmacodynamics study to determine the relationship between drug’s concentration, effects and time.The experiment combined dual blood-brain sites microdialysis technique with HPLC-FLD and HPLC-ED to synchronous monitor the level of L-DOPA in blood and striatum, and neurotransmitter in striatum. To observe the effects of Shouwu Fang on pharmacokinetics of Levodopa in blood and extracellular fluids of striatum, and the effects on neurotransmitter, such as monoamine transmitters and hydroxyl free radical, in normal and PD rats. And then, further analysis the data with method of pharmacokinetics-pharmacodynamics study, to investigate the mechanism of Shouwu Fang on effects of L-DOPA and treatment of PD.Methods1 The method of measuring the L-DOPA with HPLC-FLD. Aglient 1200 system was adopted with a fluorescence detector (λex=278nm,λem=325nm), a MERCK Lichrospher 100 C18 column (5μm,4mm×250mm), a Nova-Pak C18 pre-column, a mobile phase consisted of 90% solution (containing EDTA 0.08mmol·L-1, KH2PO4 70mmol·L-1, solium heptanesulfonate 2.08mmol·L-1) and 10% methanol, and using a flow rate of 1.0ml·min-1, temperature with 35℃.2 The method of synchronous measuring the neurotransmitter, hydroxyl free radical and L-DOPA with HPLC-ED. We used a Antec Leyden BV C18 column (3μm, 2.1mm×100mm), a Antec DecadeⅡSDC electrochemical detector was set at 0.52v with a glass carbon electrode and a Ag/AgCl reference electrode. The mobile phase (pH 3.32, adjusted with phosphoric acid)consisted of EDTA (0.027mmol·L-1), sodium octane sulfonate (0.74mmol·L-1), KCL (2mmol·L-1), KH2PO4(100mmol·L-1), methanol(15%), acetonitrile (1%), acetic acid (0.05%), using a flow rate of O.2mL·min-13 To establish the PD rats model by injecting 6-OHDA into striatum. In this study, seven days before sampling, rats were anesthetized with sodium pentobarbital (40mg·kg-1) and placed on a stereotaxic frame. Unilateral injection of 6-OHDA (32μg·kg-1 1μL·min-1)was performed in striatum (coordinates with respect to bregma:A:+0.2mm, L:+3mm, V:7.5mm). The same volume normal saline was injected into the striatum of the control group rats.4 Effects of Shouwu Fang on pharmacokinetics of L-DOPA in blood and extracellular fluids of striatum and neurotransmitter in normal and PD rats.Rats grouping and dosage. Rats were randomly divided into six groups:control group, model group, control high dose group(L-DOPA 48mg-kg-1 and benserazide 12mg·kg-1), model high dose group(L-DOPA 48mg-kg-1 and benserazide 12mg·kg-1), model low dose group(L-DOPA 24mg·kg-1 and benserazide 6mg-kg-1), SWF+model low dose (L-DOPA 24mg·kg-1 and benserazide 6mg·kg-1, and Shouwu Fang 18g·kg-1 equivalent to herbs). The model groups establish the PD rats model by method above. The drugs or water were ig to rats for 6 days.Operation and blood-brain microdialysis. The rats were anesthetized with same method on the 6th day, the blood microdialysis probe was positioned within the jugular vein toward the right atrium and then was perfused with compound sodium chloride solution at a rate of 2.5μL·min-1. A piece of tubing was looped subcutaneously on the back of the rats to the surface of the neck from the abdominal space for the administration of drugs.On the 7th day of the experiment, a probe was inserted into the striatal guide, in freely moving rats. The blood and brain microdialysis probes were perfused with compound sodium chloride solution at a flow rate of 2.5μL·min-1 for 60 min, then the brain microdialysis probe was perfused with compound sodium chloride solution containing 5mmol·L-1 salicylic acid sodium salt. After a 60-min stabilization period, L-DOPA and benserazide were ip to rats at same dosage above. Brain and blood dialysates were collected in 15 min intervals for 420 min. Result1 The method of measuring the concentration of L-DOPA in blood with HPLC-FLD. Intra-day and inter-day RSD for L-DOPA(0.3125-5mg·L-1) were≤4.9%. correlation coefficients of linear regression equations from 0.3125 mg·L-1 to 5mg·L-1was 0.9987. the minimum detection limit of L-DOPA was 19μg·L-2 The method of synchronous measuring the neurotransmitter, hydroxyl free radical and L-DOPA with HPLC-ED. The 10mix standard liquid were consisted of L-DOPA, DA, DOPAC,5-HIAA,5-HT,2,3-DHBA,2,5-DHBA, EP, NE and HVA. Intra-day and inter-day RSD for 5 different concentrations of 10mix were≤8.7%. Correlation coefficients of linear regression equations of the each substance in 10 mix were≥0.9991。3 Synchronous pharmacokinetics study on Levodopa in PD rats blood and extracellular fluids of striatum. Both in normal and PD rats, drugs were absorbed in blood rapidly after administration through intraperitoneal injection, then entered the striatum through blood brain barrier. Both the blood and striatum concentration-time profiles of L-DOPA were described by a one compartment model. Tmax, AUC(o-∞) and Cmax in striatum were significantly different from that in blood.4 The relativity between the concentration of L-DOPA in blood, concentration of L-DOPA in target organ, and effects in target organ in PD rats. The target of pharmacokinetics is concentrations of L-DOPA in rat’s blood or extracellular fluids of striatum. The target of pharmacodynamics is the level of DA in extracellular fluids of striatum, witch expressed asΔDA((determination value -basic value)/basic value). After administration of L-DOPA, ip, the L-DOPA concentration andΔDA value in extracellular fluids of striatum were increased, with the blood L-DOPA concentration was increased. The RSD showed strong correlation between L-DOPA concentration in blood and in extracellular fluids of striatum, between L-DOPA concentration in blood andΔDA value in extracellular fluids of striatum, between L-DOPA concentration andΔDA value in extracellular fluids of striatum.5 Synchronous pharmacokinetics study on effects of Shouwu Fang on L-DOPA in PD rats blood and extracellular fluids of striatum. Conmpared with model low dose group, the blood L-DOPA concentration of SWF+model low dose group were increased significantly at 6 time points, the concentration in striatum was decreased at 15 min after a administration. the blood AUC(o-∞) of SWF+model low dose group was increased, blood Tmax and MRT(o-t) were delayed, and striatum Tmax and MRT(o-t) were delayed too.6 Effects of Shouwu Fang on DA and its metabolic products in extracellular fluids of striatum in PD rats. After one week injection of 6-OHDA into stratum, compared with the control group, the DA in extracellular fluids of striatum significantly was decreased, the DOPAC/DA and HVA/DA were risen of model group. Compared with the model group, model low dose group and SWF +model low dose group DA basic value increased, DOPAC/DA and HVA/DA basic value decreased. After administration of L-DOPA, ip, DA level increased rapidly and then fell slowly. Compared with the model low dose group, the SWF+model low dose group DA increased more significant, andΔDA increased less.7 Effects of Shouwu Fang on hydroxyl free radical in extracellular fluids of striatum in PD rats. The target of hydroxyl free radical is total DHBA(2,3-DHBA +2,5-DHBA). The total DHBA of model group is above that of control group. Compared with model group, model low dose group and SWF +model low dose group total DHBA basic level decreased. After administration of L-DOPA, ip, total DHBA increased gradually, then fell slowly after 275min. The SWF +model low dose group DHBA increase is lowest, and lower than model low dose group significantly at 9 time points.Conclusion1 Through the combination of blood-brain microdialysis technique and HPLC-FLD and HPLC-ED, make it possible to simultaneous detection of the L-DOPA concentration in blood, L-DOPA concentration and DA and its metabolic products and hydroxyl free radical in extracellular fluids of striatum, in dialysates of freely moving rats.2 There are strong correlation between L-DOPA concentration in blood, L-DOPA concentration in extracellular fluids of striatum, andΔDA value in extracellular fluids of striatum in awake PD rats. The blood concentration of L-DOPA can reflected the concentration of L-DOPA and effects in target organ partly.3 Shouwu Fang delayed L-DOPA elimination, increased its absorption in blood, and reduced fluctuation of concentration in striarum.4 Shouwu Fang and L-DOPA synergy therapy PD. SWF can retain high level of DA in extracellular fluids of striatum for a longer time, and decreasesΔDA at the same time, and reduced the DA fluctuation. This synergy effect was closely related to SWF effects on L-DOPA pharmacokinetics.5 The hydroxyl free radical basic level in extracellular fluids of striatum was decreased when administration of low dose L-DOPA than model group. SWF can promote the decrease, have an antioxidant effect.更多还原