Microdialysis Technology Application On Pharmacokinetics And Transdermal Characteristic Study Of Sinomenine Patch

This research intended to study the Sinomenine patch pharmacokinetics from multiangle. The isotope tracer method and HPLC method was selected as analytical method. The blood drug level method and advanced pharmacokinetics sampling method, i.e. microdialysis (MD) sampling technology, was selected as pharmacokinetics study method. Normal Beagle dogs and adjuvant-induced arthritis rats were taken as experimental animal. Eventually the blood microdialysis was done by using hydrochloricum Sinomenine as model drug.In this dissertation, the related influencing factor of microdialysis sampling and in vitro transdermal absorption were investigated. The skin local pharmacokinetics of Sinomenine patch were carried out and compared the difference between liposome patch and normal patch. The in vitro diffusion and in vivo penetration correlation relation was established.The dissertation consists of eight parts: 1 Methodology Study of Microdialysis Sampling 1.1 Establishment of HPLC Analytic Method for SinomenineBy scanning the absorption wavelength in ultraviolet-visible range, 265nm was selected as the detected wavelength. Chromatography parameter was selected as follows: column was Hypersil BDS C₁₈, mobile phase methanol/water(50/50) with 0.2% quadrol as modifier, wavelength was 265nm, flow rate 1.0mL/min,and column temperature is room temperature. The retention time is approximately 10.2min. The number of theoretical plates wasn't less than1500 calculated with Sinomenine.1.2 The Influence of Microporous membrane Pore Size > Transonic Degassing Time and Perfusate Modifier on Relative RecoveryWhen the probe was perfusated with the different proportion of alcohol, the relative recovery(RR) and mass transfer coefficient k increased. The microporous membrane pore size and transonic degassing time had no significant effect on relative recovery. But the results indicated that it is better to filtrate and degas before formal experiment.1.3 The Influence of Perfusate Flow Rate and Drug Concentration on Relative Recovery and Absolute RecoveryThe Sinomenine Ringer's solution was prepared followed as concentration^.2428mg/ml ^ 0.2009mg/ml . 0.1622mg/ml respectively. The relative recovery and absolute recovery(AR) of two microdialysis probe were determined at the 4> 2> 1> 0.5> 0.2ul/min flow rate The results indicated that the relative decreased, obeyed with the exponent law. The absolute recovery increased when flow rate increased. At the same flow rate the relative recovery was approximately parallel to x-axle when the relative recovery was plotted to flow rate. It suggested that the relative recovery was independent of medium drug concentration which was the theory foundation of microdialysis sampling.1.4 The Relationship of Relative Recovery and Relative Loss and the Comparison of Probe Sampling EfficiencyThe penetration coefficient and maximum diffusion coefficient (Jmax) was calculated respectively, which was regarded as the evaluation criterion. The results showed that there lied obvious difference between probes. But the penetration coefficient was approximately equated. It indicated that recovery of every probe should be determined, the in vitro recovery doesn't take place of the in vivo recovery. The sample concentration could be calibrated by iv vivo relative loss.1.5 The Stability Investigation of Recovery1.5.1 The Stability Investigation of in vitro Relative RecoveryThe probe was immersed in Sinomenine Ringer's solution(0.201mg/mL) with the 1 ul_/min flow rate. The sampling interval was 20min and sample was collected until the 26th h. Sample was determined immediately and the recover were calculated. The mean recovery was 0.1329 and the RSD was4.14%.It showed that the in vitro recovery stability was consistent with the technology request.1.5.2 The Stability Investigation of in vitro Relative LossThe probe was immersed in Sinomenine Ringer's solution(0.195mg/mL) with the 1ul_/min flow rate. The sampling interval was 20min and sample was collected until the 26th h. The sample was determined and the recovery were calculated. The mean recovery was 0.1376 and the RSD was 8.83%. It showed that the in vitro relative loss stability was consistent with the technology request.1.5.3 The Stability Investigation of in vivo Relative LossThe linear probe was implanted in SD rat subcutaneous tissues. The high and low concentration Sinomenine Ringer's was perfusated with the flow rate of 1uL/min. The sampling interval was 20min and sample was collected until the 26th h. Sample was determined and the recover were calculated. The in vivo relative loss was 0.0913(6.76%) and 0.0889(7.20%) respectively. The results shows that in vivo loss kept stable and independent of drug concentration. It meets with the request of in vivo sampling.1.6 The Relationship between the in vitro Recovery and in vivo RecoveryThe in vitro recovery and in vivo recovery were calculated respectively. Throughout the independent sample t-test, the statistics result indicated that there lied significant difference. The in vitro recovery were higher than in vivo recovery. The in vitro recovery doesn't take place of the in vivo recovery.1.7 The Influence of Temperature on in vitro RecoveryThe recovery and mass transfer coefficient k under the 30°C. 32°C > 34°C ^ 36°C ^ 37°C constant temperature was determined. It indicated that the recovery and k increased with the increased temperature. The results accorded with Arrhenius empirical formula.2 The Correlative Influencing Factor Study of in vitro Transdermal Absorption Experiment 2.1 The Influence of Receptor Solution on Transdermal absorptionThe PBS/alcohol solution ? PBS solution(PH6.8) and 30%ethanol/saline solution were used as receptor solution. The cumulative amount was fitted as function of time with zero-orde^ first-order> Higuchi equation. The correlation coefficient and transdermal rate constant J were the evaluation criterion. Theresults shows that PBS/alcohol was the best receptor solution.2.2 The Influence of Temperature on Transdermal absorptionThe Sinomenine Transdermal rate constant under 29.5°C, 32°C > 37°C > 42°Cconstant temperature was determined.lt indicated that transdermal rate constant and the penetration coefficient increased with increased temperature. The time lag decreased. The experiment results accorded with Arrhenius empirical formula.2.3 The Influence of Skin Stratum Corneum (SC) on Transdermal absorptionThe stratum corneum-free skin was prepared by tape stripping method. When the in vitro transdermal experiment completed, patch was removed immediately and the receptor solution was replaced with fresh receptor solution. Then the skin drug-releasing sample was collected until 24h. At last the skin was homogenated by glass homogenizer and the rudimental drug of skin was assayed. The results indicated that the penetration rate and released rate of stratum corneum-free skin was higher than the untouched skin. TheSequence Of Skin depot effect Was Qback skin(stratum corneum-free )> Q abdomen skin(stratumcorneum-free >> Qback skin>Q abdomen skin-The skin stratum corneum was the main barrier of drug transdermal diffusion2.4 The Special Fitting of Transdermal Absorption Data2.4.1 Dual Exponent and Diphase FittingThe cumulative transdermal percentage was fitted as function of time with dual exponent and diphase fitting. The correlation coefficient and the AIC value were the evaluation criterion. The following is the diphase equation: 1-Q%=0.1454e-02011t+0.8479e-0008479t2.4.2 Grey Model GM(1,1) FittingThe cumulative transdermal amount of per unit area was fitted as function of time by using Grey Model. The average relative error and grey absolute association degree was the judgement criterion. The following is thegrey fitting equation: X0)(-k + ^ =i025.859429e0201916k-854.208429.3 The Feasibility Study with Blood Microdialysis Sampling onPharmacokinetics3.1 Determination of iv vitro Sinomenine-Plasma Protein Binding DegreeThree kinds of Sinomenine plasma concentration were prepared . Sixkinds of Sinomenine Ringer's solution were perfusated through probe. The difference of perfusate and dialysate was fitted as function of the initial plasma drug concentration. The slope rate of regression equation was the relative recovery(RR), the x-intercept represented the free drug concentration. The plasma-binging percentage can be calculated using the following formula: (C0-Cf)/ Co. The binding percentage kept stable at the studied concentration range. The binding percentage was approximately 16%.3.2 Determination of iv vitro Plasma Binding Degree of Hydrochloricum SinomenineThe determination procedure of Hydrochloricum Sinomenine plasma protein binding degree was as same as Sinomenine. The binding percentage kept stable in the studied concentration range. The binding percentage was approximately 26%.The results indicated that plasma protein binding percentage of Sinomenine and Hydrochloricum Sinomenine kept relative lower. It was feasible to carry out blood microdialysis sampling.4 The Transdermal Characteristic Comparison between Liposome Patch and Common PatchThe skin and blood microdialysis sampling were carried out simultaneously by implanted linear probe in subcutaneous tissue and jugular vein. The common and liposome patch in which the Sinomenine content and the applying area were same stickled to rat abdomen. The sample was collected and determined by HPLC. At the 11.5h, Thesinomenine level in common patch was lower than LOQ. The blood sample were all lower than LOQ. The cumulative sampling amount and transdermal rate constant J were calculated respectively. The results showed that the time lag of common patch was shorter and J was litter than liposome patch. It is helpful to produce drug action.5 The Correlation Study between in vitro Transdermal Diffusion and in vivo Transdermal Penetration5.1 The in vitro Transdermal Absorption Experiment by Using Franz Diffusion CellThe in vitro transdermal experiment was same as before. The blank Ringer's solution was selected as the receptor solution. The cumulative amount ofevery time point was calculated.5.2 The in vitro Transdermal Absorption Experiment by Using DiffusionCell and Sampled Using Microdialysis ProbeThe linear microdialysis probe was implanted in rat abdomen skin, which the subcutaneous fatty tissue was remained, and the skin was mounted in diffusion cell. The blank Ringer's solution was selected as the receptor solution. The cumulative amount of every time point was calculated.The correlation relationship was established between the in vitro-in vivo experiment by using cumulative amount of every time point. The results indicated that the correlation is perfect.6 Local Pharmacokinetics Study of Sinomenine Patch by Using Isotope Tracer MethodThe Sinomenine patch was prepared in which there included quantitative radioactive 3H-Sinomenine.The patch was applied on rat abdomen. The sample was continuously collected for 20h. The radioactivity intensity was assayed by using liquid flashing events-per-unit-time meter. The radioactivity intensity was calibrated ,then plotted to middle point of sampling interval.7 Pharmacokinetics Study of Sinomenine Patch by Using Blood Drug Concentration Method7.1 Pharmacokinetics Study by Using beagle Dog as Experimental AnimalThe blood sample of carotid artery was draw out at the preset sampling interval and assayed by HPLC. The pharmacokinetics parameter was obtained by using PKanalyst software package. The fitted results indicated that the physiological disposition of Sinomenine accorded with 2-compartment model with 1st order absorption.7.2 Pharmacokinetics Study by Using Adjuvant-induced Arthritis Rat as Experimental AnimalThe SD rat was divided 15 groups randomly . There were 4 rats in every group. The blood was draw out by fossa orbitalis plexus venosus at the preset sampling interval and assayed by HPLC. The fitted results indicated that the physiological disposition of Sinomenine accorded with 2-compartment model with 1st order absorption.8 Pharmacokinetics study of Hydrochloric Sinomenine Through the Different Administration route by Microdialysis Sampling Method8.1 Administration route- Caudal Vein AdministrationThe concentric probe was implanted in jugular vein toward right ventricle. The blood sample was collected at the fixed time interval and assayed by HPLC. The sample concentration was calibrated by in vivo relative recovery. The blood drug concentration was plotted to middle point time of sampling interval. The pharmacokinetics parameter was obtained by using PKanalyst software package. The fitted results indicated that the physiological disposition of Hydrochloric Sinomenine accorded with 2-compartment model with 1st order absorption.8.2 Administration route- Oral AdministrationThe experiment procedure was same as 8.1.The results indicated that Hydrochloric Sinomenine accorded with 1-compartment model with 1st order absorption.8.3 Administration route- Intramuscular Injection AdministrationThe experiment procedure was same as 8.1. The results indicated that Hydrochloric Sinomenine accorded with 1-compartment model with 1st order absorption.8.4 Administration route- Intraperitoneal Injection AdministrationThe experiment procedure was same as 8.1.The results indicated that Sinomenine accorded with 1-compartment model with 1st order absorption. ConclusionsThere were significant difference between probes, so as in vitro recovery and in vivo recovery by using in vitro simulating experiment. The probe recovery kept relative stable during whole experiment period and perfusate flow rate was the main influencing factor. It was proved that temperature and skin stratum corneum could influence the transdermal rate by using in vitro experiment. The plasma protein binding percentage of Sinomenine and hydrochloric Sinomenine was determined by using probes as sampling tool. It was feasible to deal with the blood microdialysis experiment. The liposome patch was helpful to penetrate into body by comparing the common and liposome patch. The in vivo-in vitro correlation was excellent by comparing Franz diffusion cell and microdialysis method. The local pharmacokinetics was carried out by using isotope tracer method. The results indicated that the mean residence time (MRT) was longer to generate drug action. It was helpful to treat the rheumatic disease by comparing the pharmacokinetics parameter ofnormal and pathological animal model.