【作者】 刘宏伟；

【导师】 周莉玲；

【作者基本信息】 广州中医药大学 ， 中药学， 2010， 博士

【摘要】 本课题执行国家自然科学基金项目(No.30672669,30801551)的部分研究内容。本课题研究是在导师以往工作基础上研制一种在鼻腔中滞留时间较长、疗效持久的磷酸川芎嗪(TMPP)新型制剂——-TMPP鼻用pH敏感型原位凝胶,在正常大鼠体内进行整体药物动力学和局部药物动力学研究,并在急性脑缺血模型大鼠体内进行脑局部PK/PD结合模型研究。实验采用星点设计-效应面法优化处方工艺；应用微透析采样技术,将探针分别植入大鼠颈静脉和脑部纹状体区,考察静脉注射、鼻腔给药后TMPP在动物血液和脑部的释药特征和浓度的动态变化规律；建立大鼠急性脑缺血模型,并在模型动物体内对静脉注射TMPP溶液、鼻腔给予TMPP溶液和鼻腔给予TMPP凝胶剂等三种给药方式进行脑局部PK／PD结合模型研究,利用同一份脑部微透析样品分析其药物动力学指标和药效动力学指标,建立时间-药物浓度-药效之间的定量关系,并建立脑局部PK／PD结合模型的评价方法,为TMPP的临床给药方案提供更为科学的依据。本实验研究主要分为以下三个部分：1 TMPP鼻用pH敏感型原位凝胶剂的制备1.1 TMPP鼻用pH敏感型原位凝胶的制备工艺研究以Carbopol和HPMC的用量为考察因素,黏度和5 h的药物释放量为效应指标,采用星点设计-效应面法优化了TMPP鼻用pH敏感型原位凝胶的制备工艺。采用SAS 9.0统计软件对数据进行了处理,经二次多项式拟合,得到了优化方程,并绘制出了效应面图和等高线图,从中可以读出考察因素的较优取值,据此制备出的原位凝胶,测定其黏度和5 h的药物释放量,得到实测值,与预测值相比较,偏差较小,说明采用星点设计-效应面法优化原位凝胶制备工艺具有较好的预测效果,确定了TMPP鼻用pH敏感型原位凝胶的最佳制备工艺：取0.5%的Carbopol和1.4%的HPMC分别配成不同浓度的溶液,放置24小时使其充分溶胀,均匀分散,再将两者混合得到空白凝胶；将TMPP溶于蒸馏水后混入空白基质中,搅拌均匀,再用三乙醇胺调节pH值至5.5左右,超声5min,即得到含药凝胶。1.2 TMPP鼻用pH敏感型原位凝胶中TMPP的含量测定用HPLC考察了TMPP的线性范围和精密度,结果表明TMPP的浓度在8.024～206.0μg/mL之间时与其峰面积呈良好的线性关系,得到回归方程为：Y=17450X+34866,R2=0.999。精密度试验RSD%为1.2%,此外根据3批样品的测定结果,并考虑到保存期等因素,规定本品每1g含TMPP不得少于25mg。1.3凝胶剂的质量评价及初步稳定性考察本节对TMPP鼻用pH敏感型原位凝胶的质量进行了评价：原位凝胶制备后为乳白色不透明的液体制剂,药物和基质混合均匀；pH值在5.5～6.0范围；黏度为3.1±0.2Pa·S；稳定性考察无异常现象；制剂中TMPP的平均含量为31.00±1.38 mg/g。将TMPP原位凝胶置于密封容器中,分别于室温和40±2℃条件下放置三个月,对其稳定性进行了初步探索性研究。结果表明：原位凝胶中TMPP的含量和pH值基本没有变化；随着温度的升高,时间的延长,制剂的黏度呈现下降的趋势；40±2℃条件下放置,第三个月开始外观颜色略带淡黄色,且有少许分层现象,搅拌后无分层现象,表明其稳定性出现下降趋势,说明该制剂不适于高温贮存。2 TMPP制剂的药物动力学研究2.1微透析探针的选择及植入方法的建立根据预实验结果对探针的类型、分子截留量及透析膜的长度进行了筛选,最终确定用于血液微透析的探针为CMA／20,活性透析膜长度为10mm,分子截留量为20000Dalton;用于脑部的微透析探针为CMA／12,活性透析膜长度为4mm,分子截留量为20000 Dalton,并检查了探针植入脑部的位置,结果表明探针透析膜部位处于大脑纹状体区,位置准确,方法可靠。2.2微透析样品中TMPP含量测定方法的建立用HPLC法考察了TMPP的线性范围和精密度,结果表明TMPP的浓度在45.2ng/mL～7.23μg/mL范围内线性关系良好,浓度与峰面积的回归方程为：Y=15.668X+2.991,R2=0.9998,精密度试验RSD为1.0%。TMPP的最低定量限为17pg。2.3微透析探针的体内回收率及其稳定性考察在流速对体内回收率的影响、浓度对体内回收率的影响、体内回收率的稳定性以及采样时间间隔和采样量等多方面进行了研究,考察微透析采样技术在TMPP药动学和PK/PD研究中的可行性,最终确定血液微透析探针和脑部微透析探针灌流的速度均为2.0μL/min,用浓度为1.0μg/mL左右的含药灌流液测定体内回收率,采样的时间间隔为10min,采样量为20μL,且当灌流液的流速为2.0μL/min时,血液微透析探针和脑部微透析探针的TMPP体内回收率在10小时内基本保持稳定。2.4 TMPP制剂的整体药物动力学和局部药物动力学研究实验采用微透析采样技术,同步开展静脉注射TMPP溶液、鼻腔给予TMPP溶液和鼻腔给予TMPP凝胶剂后皿液和脑脊液中TMPP的药物动力学研究,采用Kinetica4.4药动学软件对所得到的药物动力学数据进行了房室模型和非房室模型分析,结果表明：各给药组血液和脑中的药物动力学数据均符合二室开放模型,且房室模型和非房室模型得到的血液和脑中的AUC分别都比较接近。从静脉注射TMPP溶液和鼻腔给予TMPP溶液两种给药途径来看,非房室模型的数据显示,鼻腔给药后脑中的MRT大于静脉注射给药后脑中的MRT,且鼻腔给药后脑中的MRT大于血液中的MRT；此外鼻腔给药和静脉注射给药后两者脑中的AUC均大于血液中的AUC,且鼻腔给药后脑中的AUC明显大于静脉注射给药后脑中的AUC。这种结果表明,鼻腔给药后TMPP在脑中的消除速度比血液中消除速度慢,且鼻腔给药后TMPP在脑中的消除速度比静脉注射给药后消除的更缓慢,证明药物TMPP更易从鼻腔到达脑靶部位,驻留时间更长,更有利于药物在病灶部位发挥疗效。TMPP容液和TMPP凝胶剂均通过鼻腔给药,从药物动力学模型分析结果来看,两种剂型给药后脑中TMPP的浓度均高于血液中TMPP的浓度,证明药物通过鼻腔给药后容易在脑中富集,这对于缺血性脑血管疾病的治疗有很大帮助；此外,从房室和非房室模型分析得到的药物动力学参数来看,TMPP凝胶剂的消除半衰期长于TMPP溶液,TMPP凝胶剂组的Tmax为35min长于TMPP溶液的15min,且前者的AUC也大于后者,TMPP凝胶剂鼻腔给药后TMPP在脑中的滞留时间明显长于TMPP溶液,表明TMPP凝胶剂可明显延长药物在大鼠脑和血液中的滞留时间,起到缓释效应。3 TMPP制剂脑局部PK／PD结合模型的研究3.1大鼠急性脑缺血模型的建立及评价指标选择改良的Zea-longa线栓法建立大鼠急性脑缺血模型,将鱼线插入颈总动脉经颈内动脉送至颅内,造成大鼠一侧大脑缺血。神经功能评分结果显示,模型大多有向瘫痪侧旋转征象,被提尾悬空时脑缺血对侧前肢呈屈曲、抬高、肩内收、肘关节伸直；大鼠脑缺血侧眼眶与对侧相比无血色,目光呆痴,反应迟钝；TTC染色结果显示模型成功大鼠大脑经TTC染色后,大脑缺血侧组织成白色。本手术操作简单易行、造模成功率高。3.2药效指标的选择与测定采用高效液相-荧光检测器系统OPA柱前衍生的方法考察了五种氨基酸的线性范围和精密度,结果表明五种氨基酸的线性关系良好。Asp的标准曲线方程：Y=83.248X+7.442,R2=0.999；Glu的标准曲线方程：Y=282.54X+26.551,R2=0.998；Gly的标准曲线方程：Y=447.9X+97.42,R2=0.997；Tau的标准曲线方程：Y=249.7X+15.57,R2=0.999；GABA的标准曲线方程：Y=297.3X+30.14,R2=0.999；各标准品的精密度良好,Asp、Glu、Gly、Tau、GABA的RSD (%)分别为0.9%、1.0%、1.9%、2.8%、1.7%；五种氨基酸的检测限分别为5.19ng/mL、2.12ng/mL、0.55ng/mL、1.89ng/mL、2.16ng/mL3.3 TMPP制剂脑局部PK/PD结合模型研究运用微透析采样技术,采集急性脑缺血模型大鼠缺血侧脑部的微透析液,试验共分为4组：TMPP溶液静脉注射给药组、TMPP溶液鼻腔给药组、TMPP凝胶剂鼻腔给药组、空白模型组。其中空白模型组的微透析样品只测定药效动力学指标,其他三个给药组的微透析样品分为两部分,一部分用于测定药物动力学指标TMPP的浓度,一部分用于测定药效动力学指标氨基酸类神经递质。测定结果显示氨基酸类神经递质中除Gly之外的其他四个指标均与模型组在变化趋势上有统计学差异。用微透析采样技术采集给药后的急性脑缺血模型大鼠脑部的微透析样品,TMPP在脑脊液中的浓度作为药物动力学指标,Asp、Glu、Tau、GABA的浓度作为药效动力学指标,将各个给药组得到的药物动力学和药效动力学数据用Winnonlin4.0.1软件进行分析,选择合适的药物动力学模型和药效动力学模型,得到的最佳药效动力学模型为Sigmoid-Emax模型,并选取适当的参数,对所得的数据进行PK/PD结合模型拟合,得到主要药效动力学参数和药物浓度与药效之间的定量关系,并得到各个给药组的各个药效指标的效应与药物浓度之间的定量方程,为临床设计药物给药方案提供参考依据,建立的Cp-E之间的关系,可用于预测(?)TMPP在脑部浓度和效应随时间变化的规律。更多还原

【Abstract】 This task executes the National Natural Science Foundation of China (No.30672669,30801551) of part of the content. This project is to work on the basis of past mentors develop in nasal in a long time, the effect of persistent tetramethylpyrazine phosphate (TMPP) new preparations-TMPP nasal pH-sensitive in situ gel. In normal rats make overall pharmacokinetics and local pharmacokinetics and acute ischemia rats for local brain PK/PD modeling.Experiment with design-Response Surface Optimization of formulation and preparation. Application of microdialysis sampling technique, the probes were implanted into the rat jugular vein and brain striatum. Visited intravenous injection, nasal administration of blood and later TMPP in animals release characteristics of the brain and the dynamic changes of concentration. Established the acute cerebral ischemia model in rats, Reserched animals in the model of intravenous injection of TMPP solution, intranasal administration of TMPP solution and nasal administration of three agents to TMPP-gel method for regional cerebral PK/PD modeling. Using the same brain microdialysis sample analysis index of its pharmacokinetics and pharmacodynamics indicators. Established the time-concentration-the quantitative relationship between efficacy and evaluation of regional cerebral PK/PD modeling. The clinical dosage regimen for the TMPP provide a more scientific basis.The study was consisted of three parts as following as: 1 preparation of TMPP nasal pH-sensitive in situ gel1.1 preparation of TMPP nasal pH-sensitive in situ gelTo the amount of Carbopol and HPMC was investigated factors. viscosity, and 5 h for the effect of the drug release index by design-Response Surface Optimization of the TMPP nasal pH-sensitive in situ gel were prepared. Using SAS 9.0 statistical software data were processed by the quadratic polynomial fitting equation has been optimized. Mapped out a response surface graph and contour map, from which you can read the study factors. According to the map gained the optimum values. In situ gel was prepared to determine their viscosity and 5 h of drug release. Deviation is smaller through measured values compared with the predicted values. indicating design-effect of surface analysis in situ gel technology has a good prediction. Determined TMPP nasal pH-sensitive in situ gel with best preparation:Take 0.5% Carbopol and 1.4% of HPMC solutions were paired with different concentrations, for 24 hours to fully swelling, dispersed and then two are mixed by a blank gel. Dissolved TMPP into distilled water and mixed with blank matrix after the ultrasound. After 5min Stir, then adjust pH value of triethanolamine to 5.5 or so that are drug-containing gel.1.2 determination of TMPP nasal pH-sensitive in situ gel of TMPP concentrationTMPP investigated using HPLC linear range and precision. The results show that the concentration of TMPP is between 8.024-206.0μg/mL that has well linear relationship. The regression equation is Y=17450X+34866 and R2=0.999. The precision RSD% was 1.2%, in addition to three batches of samples were determined according to the results.taking into account factors such as shelf life, provides the product with TMPP per lg of not less than 25mg.1.3 quality evaluation and initial stability of gelThis section of the TMPP in situ nasal gel with pH-sensitive quality was evaluated:in situ after gel opaque white liquid formulations, drugs and mixing in uniform; pH value of 5.5～6.0; a viscosity of 3.1±0.2Pa·S; stability of no anomaly; Preparation of TMPP is 31.00±1.38 mg/g of the average content.The TMPP nasal pH-sensitive in situ gel in sealed container, respectively, at room temperature and placed for three months under 40±2℃. Its stability is a preliminary exploratory study. The results showed that in situ gel content of TMPP、5h cumulative drug release and the pH value are unchanged. As temperature and time change, preparation of viscosity showed a downward trend. Under 40±2℃, the first three months after the appearance of slightly yellow color and there is little stratification, but mixing even after the restore. The stability of a downward trend indicating that the preparation is not suitable for high temperature storage.2 preparations of TMPP pharmacokinetics2.1 microdialysis probe implantation method of choice and establishmentThe results of the probe according to pre-type is that molecular interception and the length of dialysis membrane were screened to determine the final microdialysis probe for blood to CMA/20, active dialysis membrane length of 10mm, molecular retention capacity of 20000 Dalton. For the brain microdialysis probe was CMA/12, active dialysis membrane length 4mm, molecular retention capacity of 20000 Dalton. Checked the location of probe implantation in the brain. results showed that the probe is part of the brain membranes striatum, and location accurate and reliable method.2.2 Determination of Microdialysis samples of TMPP concentrationLinear range and precision of TMPP were investigated using HPLC. The results show that the concentration of TMPP in 0.0452～7.23μg/mL. It has a good linear relationship. The concentration and peak area of the regression equation is Y= 15.668X+2.991 and R2= 0.9998 with precision of 1.0% RSD. TMPP minimum quantitative limit of 17pg.2.3 In vivo microdialysis probe recovery and its stabilityA flow rate of the recovery, the impact of concentration on the recovery, recovery of stability and sampling intervals and sampling volume and other aspects were studied. study microdialysis sampling in TMPP pharmacokinetics and PK/PD feasibility. final study to the blood and brain microdialysis probe perfusion rate were 2.0μL/min with a concentration of about 1.0μg/mL determination of in vivo drug-containing perfusate recovery. The sampling time interval is 10min and sample volume is 20μL when the flow rate of perfusate is 2.0μL/min. Blood and brain microdialysis probe recovery in vivo of TMPP remained stable within 10 hours. 2.4 preparation of the overall and local pharmacokinetics of TMPP drugExperiments using microdialysis sampling technique. Synchronized with the intravenous injection of TMPP solution, intranasal administration of TMPP solution and gel after intranasal administration of TMPP in the blood and cerebrospinal fluid pharmacokinetic study of TMPP. Using Kinetica 4.4 software pharmacokinetics of drugs received power Study data were non-compartment model and compartment model analysis. The results showed that the treatment group blood and brain pharmacokinetic data are consistent with two-compartment open model and the compartment model and the non-compartment model are in the blood and brain are relatively close to the AUC respectively.From the intravenous injection of TMPP solution and nasal route of administration for two terms TMPP solution, non-compartment model of the data shows that MRT brain after nasal administration than after intravenous administration of brain MRT and the brain after nasal administration The MRT is greater than the blood of the MRT. Addition intranasal administration and intravenous administration of the brain of the latter two were more than blood AUC AUC and AUC brain after nasal administration significantly larger than the brain after intravenous administration the AUC. This result showed that after intranasal administration of TMPP in the blood faster than the elimination of the brain to eliminate slow. The brain after intranasal administration of TMPP in the elimination rate of intravenous administration than after the elimination of the more slow. Indicated that more drugs TMPP brain from the nasal cavity to reach the target site、presence of longer and more conducive to play an effective drug in the lesion sites.TMPP and TMPP gel solution were administered through the nasal cavity. From the pharmacokinetic model results, the two formulations after administration of brain TMPP concentrations were higher than the concentration of TMPP in the blood.Shows that after intranasal administration of drugs through the easy accumulation in the brain, which for ischemic cerebrovascular diseases of great help. In addition, analysis of compartment model and non-compartment model pharmacokinetic parameters obtained view that TMPP gel elimination half-life longer than TMPP solution TMPP gel group Tmax was longer than TMPP solution. The AUC is also the former than the latter. TMPP nasal gel in the brain after administration of TMPP residence time was longer than TMPP solution that TMPP gel can prolong the drug in rat brain and blood residence time.It play a slow-release effect.3 Research of Local Cerebral PK/PD modeling of TMPP3.1 Rat model of acute cerebral ischemia and EvaluationSelection for the improvement of Zea-longa suture method in rats with acute cerebral ischemia model. Put the fishing line into the common carotid artery after carotid artery and sent to the brain, causing one side of the brain ischemia in rats. The results of neurological function score are the model most had paralyzed side to the rotating sign, when vacancies were raised last contralateral limb ischemia was buckling, elevation, shoulder adduction, elbow extension. Focal cerebral ischemia and the right side of the orbit side pale in comparison, look stupid, unresponsive. TTC staining showed that the successful model of rat brain stained by TTC and ischemic brain tissue into white side. This surgical procedure is simple, the modeling success rate.3.2 The choice of indicators and determination of PharmacodynamicBy HPLC-FLD system OPA pre-column derivatization of five amino acids was investigated by the linear range and precision. The results show a good linear relationship of five amino acids. Asp of the standard curve equation:Y= 83.248X+7.442, R2=0.999. Glu standard curve equation:Y=282.54X+26.551, R2=0.998. Gly standard curve equation:Y= 447.9X+97.42, R2= 0.997. Tau standard curve equation:Y= 249.7X+15.57, R2= 0.999. GABA standard curve equation:Y= 297.3X+30.14, R2= 0.999. The standard of precision was good, Asp, Glu, Gly, Tau, GABA in the RSD (%) is 0.9%,1.0%,1.9%,2.8%,1.7%. five amino acids of the detection limits were 5.19ng/mL,2.12ng/mL,0.55ng/mL, 1.89ng/mL,2.16ng/mL.3.3 preparation of Local Cerebral PK/PD modeling of TMPPUsing microdialysis sampling, collect ischemic side of the brain microdialysate of acute cerebral ischemia rats. The testing is divided into 4 groups:TMPP solution intravenous administration group, TMPP solution nasal administration group, TMPP gel intranasal administration group, untreated group. Microdialysis samples of blank model group which only use to the determination of pharmacodynamic indices. The other three dose groups microdialysis samples were divided into two parts. One part used to measure the concentration of TMPP. The other part used to measure amino acid neurotransmitters. Measurement results show that amino acid neurotransmitters in addition to the four indicators other than Gly were changing with the model group were significantly different trend.Collected brain microdialysis samples by microdialysis sampling after administration of acute cerebral ischemia. TMPP concentrations in cerebrospinal fluid dynamics as a drug target. Asp, Glu, Tau, GABA concentrations as pharmacodynamic indicators of the various treatment group. Received the pharmacokinetics and pharmacodynamics data were analyzed using Winnonlin 4.0.1 software. select the appropriate model of the pharmacokinetics and pharmacodynamics model and get the best pharmacodynamic model the Sigmoid-Emax model. Select the appropriate parameters. The data obtained from PK/PD integration model.we obtained the quantitative relationship between the main pharmacodynamic parameters and efficacy of drug concentration. Get the quantitative equation of all treated groups in the effect of various pharmacodynamic parameters and drug concentration. The design of drug is delivered for clinical reference program. Established the relationship between Cp-E can be used to predict the concentration of TMPP in the brain and the effects of change over time rule.更多还原