Study On Joint Cavity Local PK-PD Modeling Of Sinomenine

This research is all content of the project "Study on joint cavity local PK-PD modeling of Sinomenine"(No.10451052005005581)-which was fonded by Nationnal Nature Science Fondation of Guangdong Province. Microdialysis technique is applied to samples of rabbit articular cavity, simultaneous acquisition of the rabbit antigen-induced arthritis model in vivo pharmacokinetics(PK) index and pharmacodynamic(PD) indicators, joint cavity local PK-PD modeling of Sinomenine oral and transdermal drug delivery was established, explained the mechanism of drug action,referenced to new drug development, clinical application of experimental methods and ideas.The study was consisted of three parts as following as:1Establish a study method of joint cavity local pharmacokinetic of Sinomenine1.1Joint microdialysis probe selection and implantation methodThe experiment determined the type of joint microdialysis probe:CMA/12-probe of CMA company(Active dialysis membrane length of4mm, membrane diameter of0.5mm, the molecular weight of closure value20kD);Implantation method as:rabbits with20%urethane, by dose of5mL/kg the ear marginal vein injection of anesthesia, supine position fixed on the operating table, inguinal subcutaneous injection of5000units of heparin sodium,20min later, with a needle from the highest point of the tibial tubercle and patellar lower edge of the connections among the points on both sides of the patellar ligament depression oblique45°Piercing, feel the pull out the obvious sense of breakthrough, the joint microdialysis probe catheter along the same angle of rotation from the pinhole at the piercing, move up and down the catheter, adjust the direction, without resistance, pull out the metal rod, probe carefullyinserted into the catheter can be.1.2LC-MS method for the determination of the content of sinomenine in the rabbit joints microdialysateThe experiment established LC-MS method for the determination of the the ivy alkali content of the rabbit joints microdialysate, Sinomenine concentration as the abscissa, the concentration corresponding to the peak area standard curve was drawn for the vertical axis,the regression equation was Y=6776.1X-9176.2, r=0.9994, That of sinomenine the concentration in the range of0.4375～140ng/mL and peak area showed a good linear relationship; The specificity of this method is good;The precision of Low, medium and high concentration (0.875,17.5,140ng/mL) was RSD (%)11.31%,2.36%,2.55%, indicating good precision of the instrument; The stability of Sinomenine repeated freezing and thawing three times was good, the RSD (%) of low, medium and high three concentrations were1.22%,2.79%,2.85%, the stability within7h at room temperature was good, RSD (%) was12.13%,3.08%,2.17%;The matrix effects of low, medium, high three concentrations were86.25%,89.32%,88.56%, shows that this method has no matrix effects.1.3The study of joint probe in vitro effect on the recovery factors and in vivo recovery rate stabilityThe effects of flow rate, concentration on joint probe in vitro recoveries, as well as the stability of the joint probe in vivo recovery rate within24h had been studied. The results show that when the probe around the concentration of sinomenine is certain in vitro recovery of the probe showed a downward trend, with the increase in flow rate (0.5～1.5μL/min); Under the same perfusion flow rate, probe around the concentration of sinomenine (56,140,280,700ng/mL), the recovery rate of the probe is basically the same; Joint probe in vivo recovery within24h remained stable, RSD=8.39%; Test of in vivo microdialysis sampling conditions are defined as follows:flow rate0.8μL/min,sampling interval lh,each sample volume of48μL2Establish a study method of joint cavity local pharmacodynamic of Sinomenine2.1Establish the model of rabbit antigen-induced arthritisIn this part,a method to induce an antigen-induced arthritis model in the rabbit was established:The first day of1mL sensitizer(4mg/mL ovalbumin in complete Freund adjuvant suspension) five points in the subcutaneous injection of rabbit carotid,14days later the same method to strengthen sensitization time, in the second sensitization after6days, in the rabbit knee articular injection of the0.4mL model inducer,as the modle was established,to experiment when knee swelling highest(The third day of the intra-articular injection of ovalbumin saline). The method is simple and has a high success rate.2.2The selection of the pharmacodynamic indicators and the establishment of the method for the determinationIn this study, select the arginine and citrulline as a pharmacodynamic index, indirectly reflect the level of NO changes by changes in the level of arginine and citrulline. Established a method of the OPA column derivatization high performance liquid chromatography-fluorescence detection (HPLC-FLD) for the determination of arginine and citrulline in the joint dialysate. Amino acid concentrations as the abscissa, the concentration corresponding to the peak area standard curve was drawn for the vertical axis, the arginine regression equation was Y=113.43X+1.5938, r=0.9999, showed that the concentration of arginine in0.027～6.48μg/mL range and peak area showed a good linear relationship; The citrulline regression equation was Y=106.96X+2.3846, r=0.9998, indicating that the concentration of citrulline peak area showed good linearity in the range of0.02625～6.30μg/mL; Arginine and citrulline detection limits were6.73ng/mL,7.46ng/mL,precision RSD(%) were0.86%and1.04%. The test is basically stable at room temperature within8h.3Study on joint cavity local PK-PD modeling of Sinomenine3.1Study on joint cavity local pharmacokinetic and local pharmacodynamic of SinomenineThe rabbits which has been established antigen-induced arthritis model were randomly divided into three groups:the blank model group, the oral treatment group and the percutaneous treatment group. Use the microdialysis technique to gather a group of rabbit articular cavity local microdialysis samples. Blank model group, only determination of the pharmacodynamic index, another two groups of samples divided into two, one for HPLC-FLD determination of arginine and citrulline, and another for LC-MS determination of sinomenine.The drug-time curve of the percutaneous treatment group is gentler than the the oral treatment group, Sinomenine transdermal drug delivery in the body slowly absorbed and long duration than oral administration; Sinomenine inhibited both arginine and citrulline, suggesting that sinomenine on inflammatory markers NO also inhibited the reaction of NO changes by changes in the concentration of arginine and citrulline. Arginine and citrulline are suitable for the efficacy indexes of sinomenine anti-rheumatoid arthritis.3.2Fitting of the joint cavity local PK-PD modeling of SinomenineThe pharmacokinetic and pharmacodynamic data of Oral, transdermal drug delivery group were analyzed using WinNonlin4.0.1software, according to the AIC, SBC value to determine the best pharmacokinetic model and optimal pharmacodynamic model. Results of oral and transdermal drug delivery group of intra-articular local pharmacokinetic model are the two compartment model of extravascular administration; Oral and transdermal drug delivery group pharmacodynamic models are the Inhibitory Effect Sigmoid Emax. Then the two sets of the PK-PD combination of model parameters were fitted, obtained parameters into the pharmacodynamic model, ie the quantitative equation between drug concentration and efficacy.