Study On Nasal PH Sensitive In Situ Gel And PK/PD Modeling Of Ligustrazine Phosphate

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.