Study On Pharmacokinetics And Pharmacodynamics Of Xiongbing Microemusion By Microdialysis Technique

Xingbing is empirical formula that optimized from NaoXingⅡthat from the first affiliated hospital of GuangZhou University of TCM. It's widely used in clinical for Acute Ischemic Stroke(AIS), is made up of Rrhizome Ligustici Chuanxiong and borneol.To improve the curative and brain targeting effect, increasing absorption, we had optimized the Xiongbing microemusion drug delivery system.This research is the sub-item of the project The Construction and Evaluation of local PK/PD Model's on chinese medicine by Microdialysis Technique (No.30873443)-which was funded by National Nature Science Foundation of China.In this study, microdialysis probe was plant in the vein and brain of rat to determinate the concentration of xiongbing microemusion in the blood and local brain, and the differences of parameters of xiongbing microemusion and tetramethylpyrazine moicroemucion was compared. In this study, we also establised the cerebral ischemia rat model by thread occlusion and study Pharmacokinetics and pharmacodynamics of xiongbing microemusion by microdialysis technique.The study was consisted of four parts as following as:1 Investigation of recovery and stability in microdialysis of Ferulic acid and Tetramethylpyrazine1.1 simultaneous determination the of Ferulic acid and Tetramethylpyrazine by HPLCEstablished an HPLC method for simultaneous determination the contents of Ferulic acid and Tetramethylpyrazine. The mixed titrating solution of the Ferulic acid and Tetramethylpyrazine has good correlation with the peak area within the range of 0.0368-1.84μg·mL-1and 0.0614-3. 07μg·mL-1, respectively. The method is simple, accurate and selective1.2 Recovery and stability in microdialysis of Ferulic acid and TetramethylpyrazineThe effect of concentration and flow rate on recovery has studied, and the stability of relative loss in vivo and difference of recovery in use of 5 times was invested. The result indicated that with the increase of flow rate, the recovery is decreasing.But the concentration has no obvious effect on relative recovery, there is no significant difference between relative recovery and relative loss in the same flow rate. The relative loss had good stability in 10 hours in vivo. The recovery are different in different conditions. Miecodialysis sampling can be used for the pharmacokinetic study of ferulic acid/tetramethylpyrazine and retrodialysis can be used for the determination of the drugs'recovery, but the relative recovery of every experiment should be determined.2 The establishment of microdialysis sampling methodThe new probe should be perfused in blank perfusate for 2 hours at the 2μL·min-1flow rate to soak the membrance and cleaning the micromolecules. Warning to prevent the probe damaged by blood clotting,it should be immersed in heparin sodium 20min before of use of it.This study choose the jugular vein as the place of blood probe to planted in. Separated jugular vein and ligate the distal end, and open a incision in the side of near the cordis. The blood probe insert in vein and along it to the cordis for 2.4cm, suture the wound. The plant mothed of brain probe was in accordance with cerebrum atlas.Make the puncture of anterior fontanel as the cardinal point, the values of the coordinate are AP:+0.2mm; ML:+3.2mm; DV:-7.0mm, which the site was the corpus striatum of the left brain.Under the flow rate of 1.5μL·min-1 we can gain a satisfactory recovery of the drugs,the sampling interval was 10-20min, the volume and drug concentration of sample can meet the need of detect. 3 Study on pharmacokinetic of xiongbing microemusion in rat3.1 simultaneous determination the of Ferulic acid and Tetramethylpyrazine by HPLC in the sampleThe analysis was performed on platisil ODS column (150 mm×4.6 mm,5μm).The mobile phase was consisted of acetonitrile-0.5% acetic acid (volume ratio was 36:64) with a flow rate of 1.0mL·min-1.307nm was selected as the detected wavelength. The liner range was test to be good as under the amount range of 0.011-3.52ug·mL-1μg·mL-1 and 0.03031-4.33μg·mL-1of Ferulic acid and Tetramethylpyrazine respectively. Result of precision test showed that RSD were 1.23%and 1.37%, detectability were 12 and 23pg of two drugs respectively.3.2 Study on pharmacokinetic of xiongbing microemusion of blood and brain in rat by microdialysis sampling techniqueThe blood pharmacokinetic and brain pharmacokinetic of xiongbing microemusion were viewed by microdialysis when the drug was administrated orally or intravenous injection, and the Ligustrazine microemusion was taked as an control group. DAS2.1 was used to calculate the parameters with compartment and statistical moment.The result showed that Tetramethylpyrazine was two compartment model with first order absorption after intravenous injection while it was one compartment with first order absorption after the drug was administrated orally. Compare with Ligustrazine microemusion, the bioavailability of Tetramethylpyrazine in xiongbing microemusion increased 33%.After oral administration, the MRT and the ratio of AUCbrain/AUCblood of xiongbing increased 9% and 81.6% respectively. These result indicated that the composing prescriptions of xiongbing was reasonable in view of its effect on the Tetramethylpyrazine's pharmaeokinetic and brain targeting efficiency.4 Study on oral administration's pharmacokinetic/pharmacodynamic link model of xiongbing microemusion4.1 Establishment of in vitro model of cerebral ischemiaestablishment of cerebral ischemia rat models made by improved mothed of thread occlusion.The achievement ratio of the model was determined by the functional defects in rat central nervous system and 2,3,5-triphenyhetrazolkml chloride(TTC)-staining. The result indicated that the improved rat model was a good method, but a successful rat model depends on the suitable weighted rats, the satisfactory manufature of thread and skilled operation.4.2 Determination the of Tetramethylpyrazine by HPLC-UV in the sampleThe mobile phase was consisted of acetonitrile-0.5% acetic acid (volume ratio was 36:64) with a flow rate of 1.OmL/min.295nm was selected as the detected wavelength. The liner range was test to be good as under the amount range of 0.0155-0.465μg·mL-1 and the sample has no interference.4.3 the determination of animo acid concentration of in sampleThe levels of EAA (Excitatory Amino Acids, Glu, Asp) and IAA (Inhibitory Amino Acids, Gly, GABA, Tau) were dynamically measured by phthalie diearboxaldehyde (OPA) pre-column derivatization and high performance liquid chromatography-fluorescence detector (HPLC-FLD)Under the optimized separation condition, the correlation coefficients between the peak area of amino acid and the concentration of amino acid were all above 0.99, resolution and detectability are contended to the concentration determination of our sample. The sample were stable within a week kept in-20℃.4.4 Pharmacokinetic/Pharmacodynamic modeling of xiongbing microemusion in cerebral ischemia ratsSD rats were randomly divided into xiongbing-treatment and cerebral ischemia-control groups. The former group received drug and the later group had Sodium Chloride only.After the probes planted, several samples were acquired for the determination of baseline pharmacodynamic index after 2h perfusion for balance. when it is performed, starting to model cerebral ischemia and then dosage drug and sampling instantaneous. The success of modeling were verificated by TTC-staining, and only the data from succeed model were applied. Every sample was divided into two parts, and one for pharmacokinetic and another for pharmacodynamic. But the cerebral ischemia-control group only used to determinate pharmacodynamic index but not for pharmacokinetic.The result indicated that the concentration of EAA and IAA were increased all, but xiongbing microemusion can decrease the contents EAA and raise the contents of IAA, and it may be the mechanism of the drug on the neuroprotective effect.WinNonlin 4.0.1 was used to calculate the parameter of PK and was used for PK/PD modeling. The result showed that tetramethylpyrazine was one compartment with first order absorption after the drug was administrated orally in brain of cerebral ischemia rats. The value of tmax and AUC were 32. 0min and 86590. 1μg/L*min respectively.The brain concentration of tetramethylpyrazine were pharmacokinetic index and the contents of animo acid were observed as effect index. The Emax model was use to analyze the relationship between the effect and brain concentration, and the time. The PK/PD model of xiongbing microemusion and the relationship among the effect, brain concentration and time were established.