Enantioselective Absorption and Metabolism of Tetrahydropalmatine
|Keywords||dl-THP l-THP HPLC Absorption in situ single pass intestinal perfusion model Bcap37 Bcap37/MDR1 Equilibrium dialysis Protein-binding Liver microsome Enzyme kinetics|
Rhizoma Corydalis（yanhusuo）is the dried tuber of Corydalis yanhusuo W.T. Wang.According to《Ben Cao Gang Mu》,《Kai Bao Ben Cao》,《Tang Ye Ben Cao》,Rhizoma Corydalis has many beneficial effects on blood flow circulation promotion,qi reinforcement and is especially effective for pain alleviation. Tetrahydropalmatine（THP）is one of the active ingredients extracted from Rhizoma Corydalis.Recent research has substantiated the fact that dl-THP has other pharmacological functions on analgesia,sedation,anti-arrhythmia and hypotension alleviation as well as thrombocyte aggregation inhibitation.There is an asymmetric carbon on the structure of dl-Tt-IP.It has been reported that the enantiomers have different pharmacological properties in vivo.The blood level of l-THP in rats is significantly higher than that of d-THP after oral administration, while the reason for this has not been clear yet.A systematic research on the influential factors of tetrahydropalmatine on pharmacokinetics can therefore,provide scientific evidence to evaluate the feasibility of new drug development as well as its direct safety and rational use on clinic.The process of pharmacokinetics includes absorption,distribution,metabolism and excretion.In the present study,we first investigated whether the dl-THP was biotransformed in rat intestinal tract.Systematic studied were performed on enteric absorption,protein-binding and liver microsome metabolism in vitro.Moreover, Bcap37,Bcap37/MDR1 cell lines were used to investigate whether the absorption difference between dl-THP and l-THP in rat intestinal tract was caused by P-gp or not.The studies are expected to provide a clear understanding of the differences between dl-THP and l-THP in enteric absorption as well as in protein-binding and liver microsome metabolism.Studies on disposition of l-THP and dl-THP in intestinefrom rats1.Studies on dl-THP metabolism in intestinal flora in vitroObjective:To investigate whether dl-THP was biotransformed in vitro intestinal flora from rat.Methods:dl-THP in rat intestinal flora was set in anaerobic culture at 37℃for 48h in vitro.After extracted by acetic ether,dl-THP was quantified by HPLC.Results:The concentration of dl-THP was not changed obviously after incubation,moreover,no metabolite was observed according to the HPLC chromatograms.Conclusion:In vitro,dl-THP was not metabolized by rat intestinal flora, therefore it was deduced that dl-THP was absorbed in the form of prototype after oral administration.2.Absorptive difference between l-THP and dl-THP in intestine from ratsObjective:To investigate the absorptive difference between I-THP and dl-THP in rat intestine as well as the mechanism of the absorption of dl-THP.Methods:In situ single pass perfusion model was used and the concentration of THP in perfusate was determined by HPLC.Results:The absorption rate constant（ka）and effective permeability values(Peff) of dl-THP had no significant difference（P＞0.05）at concentrations of 8.0,16 and 32μg·mL-1in perfusion or in four different regions of intestine of rat（duodenum, jejunum,ileum,colon）.The absorption of l-THP and dl-THP in jejunum had significant difference（P＜0.05）.The ka and Peffof dl-THP were obviously increased when verapamil was co-perfused with dl-THP,while those of 1-THP were not influenced by verapamil.Conclusion:The absorption of THP in intestine showed the passive diffusionv process and without a special absorption region.There was significant difference between the absorption of l-THP and dl-THP.Verapamil facilitated the absorption of dl-THP.The role of P-gp on the absorption of dl-THPObjective:To investigate the role of P-gp on the absorption of dl-THP.Methods:Concentrations of dl-TFIP/l-THP in Bcap37 and Bcap37/MDR1 cells when incubated with or without P-gp inhibitor verapamil/quinine were determined by reversed-phase high-performance liquid chromatography method.Results:The concentrations of dl-THP in Bcap37 and Bcap37/MDR1 cells were significantly increased when incubated with P-gp inhibitor verapamil（P＜0.05） while there was no significant difference when incubated with quinine（P＞0.05）.Conclusion:The absorptive difference betweeen l-THP and dl-THP in intestine from rats was not related with P-gp.The effect of verapamil on the absorption of dl-THP in rat intestine may result from the stereoselective effect between verapamil and d-THP.Enantioselective binding of l-THP and dl-THP to plasmaprotein Objective:To investigate the protein binding of l-THP and dl-THP to rat/ human plasma,human serum albumin（HSA）andα1-acid glycoprotein（AGP）.Methods:Equilibrium dialysis was employed and the HPLC was applied to measure the concentration of l-THP and dl-THP in rat/human plasma,human serum albumin（HSA）andα1-acid glycoprotein（AGP）.Results:The protein-binding rates of dl-THP,l-THP were relatively high when the concentration of the drug was 2.0,4.0,8.0μg.mL-1.There was no significant difference between dl-THP and l-THP binding to rat plasma（P＞0.05）.The binding rate of dl-THP and l-THP to HSA was very close to the rate to human protein while the binding rate to AGP was much lower.Conclusion:The species-dependent binding stereoselectivity was observed in the dl-THP/l-THP-protein bindingand the protein binding mainly resulted from the binding to HSA.Enzymatic kinetics disparity of I-THP and dl-THPmetabolism in rat liver microsomeObjective:To study and compare the enzyme kinetics of l-THP and dl-THP in rat liver microsome.Methods:Liver microsome of rat was prepared by calcium salts precipitation method.8.0μg·mL-1dl-THP was incubated with different protein concentrations (0.30-0.80mg·mL-1)of liver microsome at 37℃for different time（5-30min）.The concentration of dl-THP in the supernatant was determined by HPLC at proper time intervals to select the optimal concentration of liver microsome and incubation time. The different concentrations(0.50-20.0μg·mL-1)of dl-THP/l-THP were incubated with rat microsome at 37℃for the optimal incubation time,respectively,and the concentration of dl-THP/l-THP in the incubation mixture was detected.The Michaelis-Menten parameters(Michaelis constant[Km]and maximal velocity[1/V] in liver microsome were determined.The values of Km and 1/V were initially estimated by analysing Lineweave-Brurk plot.Results:With the microsomal protein of rat(0.3mg·mL-1)in incubation system spiked with 8.0μg·mL-1dl-THP,the optimal incubation time at 37℃for dl-THP in rat microsome was 15 min.The Vm and Km of dl-THP and l-THP in the rat microsomes were 0.284±0.052μg·mg-1·min-1and 4.12±0.67μg·mL-1, 0.128±0.044μg·mg-1·min-1and 3.94±0.51μg·mL-1,respectively.The Km of dl-THP and l-THP had no significant difference（P＞0.05）while the Vm and Clint had（P＜0.05）.Conclusion:d-THP、l-THP may be metabolized by one enzyme at different speed in rat liver microsome.