| 1. PurposeThis research is one part of the national natural science foundation of China projects-the research of pharmacokinetic of traditional Chinese medicine which use stable isotopes marked coupled with microdialysis techniques (NO:part of 30772791). Stable isotopes internal standard microdialysis technology is a kind new pharmacokinetic and sampling analysis technology, which can realize monitoring local and the whole drug concentration real-time continuously and accurately.The purpose of this project is to establish a stable isotopes internal standard microdialysis technology and apply it to PK and PK-PD model study of traditional Chinese medicine percutaneous preparation.The prophase basic research maked DL-nicotine as the internal standard of nicotine for microdialysis content.And we maked the nicotine addiction rat model as our test subjects. It can be more accurately to to monitor the parameters of pharmacokinetic and PK-PD through monitoring the recovery of microdialysis probe dynamically.2. Methods2.1 To establish stable isotopes internal standard microdialysis technology This part of we establish a methord to determin DL-nicotine and nicotine using LC-MS/MS, And then examining the ratio of the recovery rate of nicotine and the release of nicotine DL-nicotine in vitro:In this part we check the influence of flow velocity and concentration of perfusion fluid to the P value,and furtherly.verify the influence of the concentration of P value in vivo.2.2 Nicotine addiction, animal models building mode method research This section we established the nicotine addiction model of rats useiing healthy male SD rats.In the prophase the dose of administration increased gradually,and then it keep in a certain safety dose subcutaneous injection for about 2 weeks;In the end stop the administrating of nicotine and injectiing the ketone intraperitoneal to motivate, and then observe symptoms of addiction collaterals after 20min withdrawing,and scored it according to the scoresheets each group of independent sample values t test.2.3 The sdudy of the differences of pharmacokinetics in the blood and brain of rat with stable isotopes internal standard microdialysis technology In this section we use the nicotine addiction rat as biooxyrate animals.After administrating nicotine on abdominal in the transdermal way, perfuting the DL-nicotine solution as internal standard.Sampling the dialysate in jugular vein and striatal in the brain of rat after balance. Detecting the samples with LC-MS/MS and analyzing the data with DAS2.0 software.2.4 The study of pharmacokinetics of nicotine in local brain of living rat that application of microdialysis and stable labelled isotope Healthy SD rats is the object animal of study. Before the sampling surgery, the rat must be depilate and fixed a casing for the prote.The prote can be implanted into the striatum through the casing, perfusting the DL-Nicotine.and then sampling after administrating the nicotine on abdominal in the transdermal way analyzing the data with DAS2.0 software.Detecting the concentration of nicotine in the brain samples with LC-MS/MS and analyzing the data with DAS2.0 software.2.5 The study of PK-PD model of nicotine percutaneous preparations of addiction rat.In this part we choose three monoamine neural transmitters as the pharmacodynamic index-norepinephrine (NE) and dopamine (DA), serotonin (5-H), and also to establish a new detecting methord to detect these five componds--NE,DA,5-H,nicotine,DL-nicotine. After administrating nicotine on abdominal in the transdermal way,perfuting the DL-nicotine solution as internal standard.Sampling the dialysate in striatal of brain after balance. Detecting the dialysate samples with LC-MS/MS and analyzing the data with WINOLINE software.3. Result3.1 We have established a determination of nicotine and deuterium-nicotine with LC-MS/MS.The specific parameters are as follows:The column is Gold Hypersil C18 column (150×2.1 mm,3μm, Thermo, USA); the mobile phase is methanol-2mmol/L ammonium and acetate (70:30). the velocity is 0.21 mL/min, injection quantity is 5μl. Mass spectrometry conditions are as follows:ion source is atmospheric pressure electrospray ionization ion source (ESI), the spray voltage is 3.0 kV,the pressure of sheath gas is 25psi, the flow of auxiliary air is 5 arbitrary units, the temperature of metal ion capillary transport is 300℃, the collision energy is 25eV. MS acquisition of nicotine and DL-nicotine was performed in positive electrospray ionization SRM mode, by monitoring the reaction m/z 166/130 and 160/130 respectively. The symetry of peak of nicotine and DL-ncotine is good.the rention time of nicotine and DL-nicotine are both about 2.7min.The calibration curves were linear within the concentration range of 2.067 ng/ml～20.67μg/ml for nicotine22.47ng/ml～22.47μg/ml for DL-nicotine. In examination of P value:both value of P are not change with velocity and concentration, it keep in the value of 1. DL-nicotine can calibrate the recovery of nicotine in vivo experiments.3.2 Rats appeared symptoms after withdrawal nicotine as follows:chew/wrong tooth. twist body/pant, trembling/tremor, ptosis, yawning and comb fur and so on. This symptoms can be cured by nicotine obviously.The results of t test of scoring of each group shows that there is significant differences in each group (P< 0.01).So the results have statistical significance.3.3 The fate of nicotine in blood and brain both conforms to two compartments model:the parameters are as follows:the valure of t1/2α, t1/2βand AUC (0-∞) is 29.38min,208.51 min 152127.10 ug/L*min respectively in blood, the valure of t1/2α, t1/2βand AUC (0-∞) is 86.64min,386.00 min and 152820.90 ug/L*minrespectively in brain. The value of Tmax are both 140min. Dl-nicotine can be used as internal standard of nicotine to correcte the recovery;Stable isotopes internal standard microdialysis technology can be used for studing the whole and the local pharmacokinetic of nicotine and also provide new ideas and methods to studing the process of new drug delivery system.3.4 The fate of absorption and distribution was two compartment model and the values of t1/2αwas 147.74min,t1/2βwas 203.78 min min and the AUC(0-∞) was 170235.1 ng·L-1·min-1 respectively. At the end of sampling.It also can detect brain sample nicotine.4. Conclusion4.1 we have established a methord which determinate nicotine and DL-nicotine simultaneous,it can be used for the application of Stable isotope internal standard microdialysis technology.It is studied in the paper about in vitro and in vivo aspects of artillery reliability, and some research is made in the method of reliability analysis.4.2 All animal can appear withdrawal syndrome and little animal poison death. The method of repeated little dose subcutaneous injection decrease the mortality rate of the experimental animals acute poisoning.which is a large given by nicotine. It is in accordance with the form of the repeated intake of tobacco. This method made short time modules, has high degree of the dependence and low death rate.so it works.4.3 The pharmacokinetic parameters of two parts are similar,but the rate of disposition in the blood is fast than brain.the max concentration of nicotine in blood is more than blood.4.4 The fate of absorption in awake SD rats is confirm two compartment model but the rate of absorptionin blood is fast than in the brain-he area under cure in the blood is bigger than in the brain4.5 This experiment has established a new methord to determine the DA,NE,5-H,nicotine and DL-nicotine. This methord is high specify,with out interference, the linear relationship is nice within the limit. The quantitative, precision and stability is confirm the the biological sample analysis request.Therefore. it can be used for the PK-PD model of nicotine use the methords of stable isotopes internal standard microdialysis technology.Nicotine can inhibit the secretion of monoamine,the more incremental of concetrtion the more obvious of inhibition.4.6 It ought to consider the change of recovery when we use the internal standard methord. |
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