【作者】 陈翔；

【导师】 凌家俊；

【作者基本信息】 广州中医药大学 ， 药剂学， 2010， 硕士

【摘要】 1目的微透析技术(Microdialysis, MD)是一种在体(in vivo)、实时(realtime)和在线(online)的取样技术,该技术通过测定透析液的浓度和探针的回收率(recovery)计算组织中药物的浓度。微透析探针的回收率(Recovery)在体内试验过程中会发生变化,故单纯在试验前或试验后测定一次探针回收率(或取两次测定的平均值)无法校正它的变化。回收率测定不准确,使得由回收率计算出来的探针周围的药物真实浓度的可信性受到怀疑。因此非常有必要建立在每个取样点都能测定一次回收率的动态监测方法,从而减少回收率在试验过程中波动对结果的影响。本课题以烟碱为模型药物,探索影响微透析探针回收率波动的因素,研究内标法测定微透析探针回收率的可行性,运用上述研究成果对两种烟碱贴剂的体内药动学过程进行探索研究。2方法本课题研究主要分为以下四大部分：2.1样品HPLC分析方法的建立运用HPLC法同时检测烟碱及内标物磷酸可待因的含量,对色谱条件进行优化,并进行方法学考察。2.2回收率变化特性及影响因素研究分别考察生物碱未成盐前的分子量及pKa、灌流液前处理、灌流液流速、媒介浓度、温度等因素对烟碱体外回收率的影响,并考察微透析回收率在体内外的稳定性。考察4根探针体内试验前后体外回收率之间的变化。2.3内标法动态监测烟碱微透析回收率研究方法的建立通过考察4种生物碱的色谱峰与烟碱色谱峰的分离情况,它们的回收率与烟碱微透析回收率之间的比例P的稳定情况,筛选出合适的内标物。在体外研究中,比较三种不同情况下磷酸可待因及烟碱的回收率(或释放率)的比例P值是否一定,并研究烟碱浓度对磷酸可待因释放率的影响,最后通过体外血浆蛋白结合率来验证内标法的可行性。在体内研究中,考察在大鼠体内研究单独灌注及混合灌注这两种不同情况下磷酸可待因及烟碱的释放率的比例P值的一致性,并用t检验研究给药前后磷酸可待因的释放率是否改变。2.4两种戒烟透皮制剂的体内药动学研究采用均匀设计对丙二醇(PG)、氮酮(Azone)和油酸(OA)三种透皮促进剂进行筛选。分别把探针植入大鼠的颈静脉和背皮下,灌流液流速为1.0μl·min-1。将市售戒烟贴剂及自制戒烟巴布剂分别贴于已脱毛的大鼠背部皮肤表面(给药部位处于探针半透膜位置的上方),取样时间为16h,用磷酸可待因作为内标物动态测定烟碱的回收率并换算出组织中的烟碱浓度,绘制药时曲线,采用非房室模型计算药动学参数。3结果3.1建立了样品HPLC分析方法根据体内外试验药物浓度不同的特点分别建立体内外两种色谱条件,并对两种色谱条件进行方法学考察。结果表明两种色谱条件下药物色谱峰的分离情况良好,对称性好,线性范围及定量限、检测限符合实验要求。3.2回收率变化特性及影响因素研究五种生物碱未成盐前的分子量与其释放率呈良好的线性关系,回归方程的相关系数r=0.996。暂未发现pKa与生物碱释放率之间的关系。对灌流液超声、滤过等前处理能提高烟碱的体外回收率；灌流液的流速越大,烟碱的回收率越小；探针周围的烟碱浓度对其回收率无明显影响；烟碱的回收率随着温度的升高而增大。烟碱的体外回收率为(60.5±1.5)%,在24h内较稳定。烟碱的体内释放率为(21±7)%,波动较大。探针在经历体内实验后,体外回收率均有不同程度的下降,最高降幅达46.8%。3.3内标法动态监测烟碱微透析回收率研究方法的建立在体外测得在透析媒介为林格氏液的情况下烟碱与磷酸可待因的回收率之比P值为1.358～1.414,变化幅度为4%,P值较稳定,且不受探针周围烟碱浓度的影响。内标法及反透析法测得的大鼠体外烟碱血浆蛋白结合率分别为39.15%和39.80%,结果差别不大。在同一动物同一根探针中这两种物质的释放率的比例P相差不大,但在不同动物或不同探针间则相差较大。t检验的结果表明给药对磷酸可待因的释放率改变不大。3.4两种戒烟透皮制剂的体内药动学研究透皮促进剂的最优组合为PG:AzOne:OA=10％:4.5％:1％.药动学分析结果表明两种制剂中烟碱在血液的平均滞留时间(MRT)大于在皮下的MRT,且市售戒烟贴剂的MRT大于自制的戒烟巴布剂的MRT,说明这两种制剂中的烟碱在血液中的停留时间比皮下的要长,市售戒烟贴剂中的烟碱的停留时间比自制巴布剂的长,这说明两种制剂均较符合戒烟制剂的要求；市售戒烟贴在给药量少的情况下作用时间更长,且能长时间维持较低的血药浓度,更符合戒烟制剂的要求。经过给药量校正后,自制巴布剂与市售戒烟贴剂的AUC之比在皮下及在血液中分别为0.27和0.12。这表明自制巴布剂在生物利用方面远不如市售戒烟贴剂,自制巴布剂的工艺还需要进一步改进。4结论4.1五种生物碱未成盐前的分子量与其微透析释放率成反比,但pKa与释放率的关系不明显。4.2灌流液前处理、灌流液速度、温度等均对探针回收率有影响,探针周围浓度对探针回收率的影响不大。4.3在大鼠体内烟碱的回收率波动较大,不如体外稳定,且进行长时间的体内试验后,探针的回收率呈下降趋势。4.4确定磷酸可待因作为动态监测烟碱回收率的内标物。4.5磷酸可待因及烟碱两者微透析释放率的比值P在体外及体内试验过程中比较稳定,并不受探针周围药物浓度的影响,给药对内标物的微透析释放率无明显影响,从而确定磷酸可待因可作为测定烟碱微透析回收率的内标物。4.6市售戒烟贴剂和自制戒烟巴布剂这两种制剂中的烟碱在血液中的停留时间比皮下的长,市售戒烟贴剂中的烟碱的停留时间比自制巴布剂的长,这说明两种制剂均较符合戒烟制剂的要求；市售戒烟贴在给药量少的情况下作用时间更长,且能长时间维持较低的血药浓度,更符合戒烟制剂的要求。自制巴布剂在生物利用方面远不如市售戒烟贴剂,自制巴布剂的工艺还需要进一步改进。更多还原

【Abstract】 1 ObjectiveMicordialysis is a kind of in vivo, realtime and online sampling method from extracellular fluid (ECF). We can calculate the concentration of the drug organization using the concentration of dialysate collected in the outflow side and the recovery. Microdialysis recovery will change during the test in vivo, so the method used before testing the recovery only once before or after the experiment (or the average of the two) can not correct the changes of recovery. The concentration of the drug surrounding the probe calculated by such changing recovery is suspected. So it is necessary to establish an on line method to determine the recovery at each sampling point.The objective of this study is to explore the factors inducing the fluctuation of recovery and to evaluate the feasibility of using internal standard method for the determination of nicotine recovery in microdialysis. Pharmacokinetics of two kind of nicotine patches was study using the research results.2 MethodsThis research includes four sections:2.1 Establishment of analytical method of samplesMethod of RP-HPLC with ultraviolet detector was applied to detect the content of nicotine and codeine phosphate. The chromatographic conditions were optimized and the methodology was investigated.2.2 Study on the fluctuation of recovery and factors inducing the fluctuationThe factors including the molecular weight and pKa of Alkaloid, pre-processing of the perfusate, perfusion flow rate, media concentration around the probe and temperature on the recovery of nicotine in vitro were investigated. The stability of recovery in vitro and in vivo was also investigated. The recovery of the four probes before and after the in vivo experiment was compared in vitro.2.3 Study on evaluation of the feasibility of using internal standard method for the determination of nicotine recovery in microdialysisThe appropriate internal standard was selected in the four alkaloids by examining the separation of chromatographic peaks of internal standard and nicotine and the stability of the proportion P of the recovery (or delivery) of nicotine to that of internal standard.Nicotine and codeine phosphate were dissolved in Ringer’s solution. Nicotine, codeine phosphate and the mixture of them were perfused through the probe separately to calculate the proportion of the recovery (or delivery) of nicotine to that of codeine phosphate. And then codeine was perfused through the probe which was immersed in nicotine solution with different concentrations to calculate the proportion, too. In another condition nicotine was dissolved in rat plasma. The rat plasma protein binding rate was determined by using retrodialysis and internal standard method in vitro.Nicotine, codeine phosphate and the mixture of them were perfused through the probe separately to calculate the proportion of the recovery (or delivery) of nicotine to that of codeine phosphate in the subcutaneous tissue and plasma of rats. The delivery of codeine phosphate before and after administration of nicotine patches was compared with t test.2.4 Study on pharmacokinetic of two kinds of nicotine patches in vivoPermeation enhancer was selected by using uniform design. The optimized combination of propylene glycol (PG), Azone and oleic acid (OA) was studied.The probe was planted in the jugular vein and skin at the back of the rat. The flow rate of the perfusate was 1.0μl/min. These two kinds of nicotine patches were put on the surface of the back of the rat respectively and the dialysate sampling was collected during 16h. The concentration of nicotine in the tissue was calculated by the recovery of nicotine detected by internal standard method. The drug concentration-time curve was draw to analyse nicotine’s pharmacokinetics behavior. The pharmacokinetics parameters were calculated using non-compartment model.3 Results3.1 Establishment of analytical method of samplesTwo chromatographic conditions were established according to the different drug concentrations in vivo and in vivro. The results showed that the two chromatographic peaks of nicotine and codeine phosphate separate from each other and have good symmetry. There was good linear relation between integral quantity of peak area and sample size. The limit of quantification and the limit of detection for nicotine and codeine phosphate met the requirement. 3.2 Study on the fluctuation of recovery and factors inducing the fluctuationThe molecular weight of the five alkaloids and their delivery in vitro microdialysis showed a good linear rerationship with the correlation coefficient r= 0.996. The relationship between delivery and PKa of alkaloids was not found yet. Perfusate on the ultrasound, filtration and other pre-treatment was able to improve recovery of nicotine in vitro (n=4). Recovery of nicotine determined by gain and by loss were almost the same at each flow rate studied in vitro. Recovery decreased as the flow rate increased (n=4). Nicotine recovery was independent of concentration over the concentration range investigated (n=4). Recovery increased as the temperature increased (n=5). The result of stability test indicated that the stability period of the probe is longer than 24h. The result of recovery was (60.5±1.5)%. The recovery of nicotine detected in vivo was (21±7)%. After the experiment in vivo, the recovery in vitro decreased with the maximum decrease of 46.8%.3.3 Study on evaluation of the feasibility of using internal standard method for the determination of nicotine recovery in microdialysisThe proportion of the recovery (or delivery) of nicotine to that of codeine phosphate was 1.358～1.414 when the drugs were dissolved in Ringer’s solution, which changed in the range of 4%. The proportion was fairly stable and independent from the concentration of nicotine around the probe. Protein binding rate determined by internal standard method (39.15%) was almost the same as that determined by retrodialysis (39.80%). The P-values calculated in the same animal through the same probe were almost the same, but changed in different animals or different probes. The result of t test showed that there was no significant difference between the delivery of codeine phosphate determined before and after administration.3.4 Study on pharmacokinetic of two kinds of nicotine patches in vivoThe optimal combination was PG:Azone:OA=10%:4.5%:1%.Pharmacokinetic analysis showed that MRT of nicotine after the administration of the two kinds of nicotine patches determined in the blood was higher than that determined in the skin, and MRT of nicotine determined after the administration of the saled nicotine patches was higher than that determined after the administration of self-made nicotine cataplasm. It suggested that the retention time of nicotine in the blood is longer than that in the skin after the administration of the two kinds of nicotine patches. The retention time of nicotine determined after the administration of saled nicotine patches was longer than that determined after the administration of self-made nicotine cataplasm. It indicated that the two kinds of nicotine patches met the requirements of smoking cessation. The saled nicotine patches can long maintain a lower blood concentration in a little dose which met the requirements of smoking cessation.4 Conclusions 1. The molecular weight of the five alkaloids was in inverse proportion to their delivery in vitro microdialysis. The relationship between delivery and PKa of alkaloids was not found yet.2. Pre-treatment of perfusate, velocity of the perfusate and temperature would influence recovery. Nicotine recovery was independent of concentration over the concentration range investigated.3. Recovery of nicotine was fluctuated in rats, which was more stable in vitro. After the experiment in vivo, the recovery in vitro decreased.4. Codeine phosphate can be used as the internal standard in determining the recovery of nicotine.5. The proportion of the recovery (or delivery) of nicotine to that of codeine phosphate was fairly stable in vitro and in vivo and independent from the concentration of nicotine around the probe. The delivery of codeine phosphate was not affected by the administration. The internal standard method is an effective way in the determination of nicotine recovery.6. The retention time of nicotine in the blood is longer than that in the skin after the administration of the two kinds of nicotine patches. The retention time of nicotine determined after the administration of saled nicotine patches was longer than that determined after the administration of self-made nicotine cataplasm. It indicated that the two kinds of nicotine patches met the requirements of smoking cessation. The saled nicotine patches can long maintain a lower blood concentration in a little dose which met the requirements of smoking cessation.更多还原