Screening In Vivo And Metabolism Research In Vivo For Dosages Form Of Tanshinone â…¡A

ObjectiveTo research bioavailability and metabolism in Vitro for a new dosage form of TanshinoneⅡA ,a monomer composition from Salvia miltiorrhiza by developing a LC-MS/MS and in Vitro method, which will provide information for clinical application.Methods①Metabolism stability of TS was studyed with rat liver microsomes. In this investigation, the samples were incubated in 96-wells microtiter plate, proces sed with tert-butylether extraction and simultaneously detected by LC-MS/MS,which supplied t1/2 and CL.②The absolute bioavailability of compound TS was determined with 4*4 crossing experiment design in beagle dog. samples detecteded by LC-MS/MS method. Pharmacokinetic parameters of TS in rats were calculated and compared by statistical moment method and software 3P97,DAS,Winnonlon . The data were analyzed by staticstic software SAS 8.2.③Metabolism phenotypes was evaluated with NADPH,FMO enzyme inhibition,P450 enzyme inhibition method.④Plasma protein binding was determined by equilibrium dialysis (18 h) at 37℃with final concentration of 2, 100 and 1000ng/mL.⑤Metabolism pathway in vitro of TS was investigated and speculated by HPLC-MSn with SD liver microsomes incubation.ResultsA LC-MS/MS method in possession of speciality, stability and extent linear range was developed to determine TS in biological matrix. a good linearity was obtained over the range 1~1000ng/mL; Bioavailability research showed that F value of suspension, mistura, capsual group was 5.4±0.61%, 7.3±0.87%,5.6±1.5%,there were no statictical difference between these groups,but F value of mistura group was higher than other groups ; metabolism stability indicated the elimination half life , clearance rate of TS were not equal to parameters in bioavailability research in beagle dog; The results of metabolism phenotypes showed that CYP3A2,CYP2E1,CYP2C11,CYP2D1 may be involved in metabolism in SD rat liver microsomes; The binding percentage of the drug remained fairly high ; Through analysed SD rat liver microsomes sample by LC/ESI-MSn method, we supposed the 10 possible metabolite of TS in vivo. it was just a kind of supposition since we have no more sufficient validation and absence of standard samples.ConclusionBy the bioavailability, metabolism stability, metabolism phenotypes, plasma protein binding and metabolism pathway in vitro research of TS, it evaluated the metabolism and pharmacokinetic features, so as to support the metabolism and pharmacokinetic study in human and offer the reference for clinical application.