In Vitro And In Vivo Treatment Of Glucosinolates And Drug Interactions Based On CYP Enzymes

Traditional Chinese medicines(TCMs)have been an essential part of the health care system in China and been increasingly incorporated into Western health care.With the increasing use in disease treatment or as dietary supplements,TCMs are often administered concomitantly with conventional medications,leading to drug-related toxicity or untoward effect caused by herb-drug interactions(HDI).Evaluation of HDI liability is always challenging due to uncertainty and complexity of the active constituents in TCM,often scant knowledge of active constituent pharmacokinetics and lack of interaction mechanism between active constituent and cytochrome P450(CYP)enzymes or transporters.Evaluation of HDI liability is an important part for the development of TCM and is crucial to ensure the safety and efficacy in clinical.Hence,it is necessary to systematically investigate the metabolism and disposition in vivo and in vitro of active constituents in TCM,and the interaction mechanism of active constituents on CYPs and transporters.The study focused on Radix Angelica dahuricae(RAD)(Chinese name:Baizhi)to investigate the HDI liability of active constituents in TCM.RAD is a well-known TCM and traditionally used for the treatment of headaches,toothaches,nose congestion,abscesses,furunculoses,and acne.It is also used as the principal material in a number of Chinese herbal formulas.Furanocoumarins are a group of bioactive chemicals naturally existing in RAD and exert multiple pharmacological effects.In vivo and in vitro studies indicated that most of these furanocoumarins underwent extensive metabolism.The metabolism was mainly mediated by CYP enzymes.RAD is also one of frequently reported TCMs showing the HDI potential.The inhibition or induction of furanocoumarins on CYP enzymes was likely the main reason for the HDI of RAD.However,in vitro and in vivo disposition of RAD franocoumarins,as well as the HDI liability,was not thoroughly investigated.The previous studies mainly focused on a few major components,such as imperatorin.The studies with active components can not present the characteristics of RAD that is a typical TCM with "multiple constituents and targets".In the present study,the metabolism and disposition of RAD franocoumarins were fully investigated in vivo and in vitro for better understanding the characteristics of pharmacokinetics,absoption,distribution and excretion,and for identifying the marked furanocoumarins in circulation and target organs.The present study also evaluated the HDI libility of furanocoumarins in RAD by determining the pharmacokinetic changes of drugs co-administrated with RAD,investigating the inhibition and induction of furanocoumarins on CYP enzymes,and further understanding the relationship between HDI and the property of metabolism and disposition in vivo and in vitro.The study would provide valuable data for the prediction of HDI in human,and also offered the useful information for safe and effective use of RAD in clinic.The experiments and results of the study are summarized as follows:1.A sensitive LC-MS/MS method for simultaneous quantification of nine furanocoumarins,including imperatorin(IM),isoimperatorin(?M),bergapten(BER),oxypeucedanin hydrate(OXYH),oxypeucedanin(OXY),xanthotoxol(XANT),xanthotoxin(XAN),isopimpinellin(IPIM)and psoralen(PSO),was developed.The results of the method validation indicated that it was sensitive,rapid and reliable,and met the requirement of the present study.2.The pharmacokinetics and excretion of nine furanocoumarins were investigated after oral administratin of RAD extract in rats.Most of the RAD furanocoumarins were absorbed and eliminated rapidly.IM,IIM,BER and OXYH were found to be the major bioactive furanocoumarins in rat plasma,representing more than 90%exposure of all the tested furanocoumarins.The result suggested that the four furanocoumains were the marked constituents contributed to the pharmacological effects and HDI of RAD.The excretion of ?M??M?BER?XAN?IPIM and PSO from urine and feces were lower than 3%,indicating that they underwent extensive metabolism and mainly excreted as the metabolites.3.After oral administration of RAD extract,furanocoumarins could widely distribute into tissues.IM,IIM,BER and OXYH were found to be the major bioactive furanocoumarins in rat plasma and tissues,representing more than 90%exposure for all the tested furanocoumarins.Liver was the most concentrative organ with total tissue-to-plasma exposure ratio(K_p,AUC)of 5.1,6.7 and 4.7 for ?M,?M and BER,and 2.2 for OXYH,respectively.The protein binding study showed that IM,IIM,BER and OXYH had high protein binding in tissues and plasma,and the protein binding in tissues was higher than in plama.The unbound concentration in tissues and plasma were calculated by unbound fraction(fu).The unbound tissue-to-plasma exposure ratios(K_p,fu)were siginificantly reduced,which indicated that higher protein binding of the furanocoumarins in tissues partially contributed to their higher tissue exposure.?M,?M and BER had higher unbound exposure concentration in liver than in plasma and other tisssues with K_p,fu>2.6,indicating the liver-specific distribution property of these components.4.The hepatic uptake of ?M,?M,BER and OXYH was evaluated in suspended rat primary hepatocytes by the oil-spin method.The hepatic uptake of the four furanocoumarins was temperature-dependent,with considerably higher uptake at 37? than at 4?.Uptake kinetics indicated that the hepatic uptake of IM,IIM,BER and OXYH involved both active transport and passive diffusion processes.For IM,IIM and BER,the contribution of the active transport was greater than the passive process,with the CLactive/CLuptake>72%.The hepatic uptake of IM,IIM,BER and OXYH was further pretreated with known inhibitors of organic anion transporting polypeptide(Oatp),organic anion transporters(Oat)and organic cation transporter(OCT).The results showed that the hepatic uptake of ?M and BER was significantly inhibited by Oatp inhibitors and hepatic uptake of ?M was remarkably inhibited by Oat inhibitor.Oatp and Oat mediate active hepatic uptake of ?M,?M and BER played the primal role in the pecific distribution of ?M,?M and BER in liver.5.The exposure of phenacetin in rat plasma was significantly increased by coadministration of a single dose of RAD extract.The plasma AUC of phenacetin were increased to 9-fold and 12-fold of control group in low-dose group(RAD 0.46 g/kg)and high-dose group(RAD 2.3 g/kg),respectively.The T1/2 and MRT0-t were longer,and metabolic clearance rate significantly reduced.Meanwhile,the production of metabolite acetaminophen by phenacetin-O-deethylation was significantly inhibited,with the Cmax of acetaminophen reduced to 14-22%of control group,and T1/2 and MRT0-t significantly prolonged.These results indicated that a single oral dose of RAD extract can reduce the metabolic clearance of phenacetin by CYP1 A2inhibition.6.The effect of furanocoumarins in RAD extract on main CYP enzymes activity in HLM and RLM was investigated by "Cocktail" probe substrates.The results showed that RAD furanocoumarins can inhibit activities of a number of CYP enzymes in HLM and RLM.IM,IIM,BER,OXY,XAN,IPIM and PSO were the potent inhibitors on CYP1A2 in HLM with IC50 between 0.023 and 0.25 ?mol/L.While OXYH and XANT were moderate inhibitors.A compatison of the inhibitory effects on human CYP1A2 between the RAD extract and the mixture of the tested furanocoumarins were further conducted.It was found that the inhibitory activity of the mixture was comparable to that of the RAD extract.The integrated IC50 values were calculated based on the IC50 values of single furanocoumarin and their concent percentage in mixture.The results showed that the integrated IC50 values of single furanocoumarin were comparative to the real IC50 values of the mixture,indicating that the method of integrated IC50 values can be applied to the evaluation of inhibition of multiple constituents.The study further predicted the HDI potential by IC50 values and drug concentration in liver.The[?]/K1 values of ?M,?M and BER were 19.6,4.02 and 2.0 respectively,indicating the high HDI potential in rat.The result had been verified in rat.The inhibition of IM,IIM,BER and OXYH on CYP1A2 in human was stronger than in rat,indicating the higher CYP1A2 based HDI potential in human than in rat.7.IM,IIM,OXYH and BER are the main active constituents in blood circulation and liver,so the study of inhibition mechanism was focused on these four furanocoumarins.The results of IC50 shift experiment showed that the inhibition of IIM,OXYH and BER on CYPIA2 in HLM was time-dependent.More than 4-fold shift was observed in NAD PH preincubation group.The inhibition of IM on CYP1A2 was not changed with or without NADPH preinhibition,indicating that its inhibition was reversible.The mechanism-based inhibition(MBI)of CYPA2 by IIM,OXYH and BER was further confirmed with the results of time-dependent and concentration-dependent inhibition on CYP1A2 in HLM.The kinetic parameter KI obtained was 2.08,1.96 and 6.71 ?mol/L,respectively,and kinact was 0.07,0.04 and 0.07 min-1,respectively.8.The exposure of phenacetin in rat plasma was significantly reduced after multiple dose of RAD extract.Compared with control group,the plasma AUC was reduced by 45%and 62%in low-dose group and high-dose group respectively,and the metabolic clearance was increased to 1.76-and 2.65-folds of control group respectively.Meanwhile,the formation of acetaminophen was significantly increased.These results indicated that multiple oral administration of RAD extract can enhance the metabolic clearance of phenacetin by CYP1A2 induction.9.The induction of activity and mRNA expression of CYP1A,CYP2B and CYP3A were observed after multiple oral administration of RAD extract.The CYP1A2 activity increased to 1.9-and 2.2-folds of control group in low-dose and high-dose group,respectively.The mRNA expression of CYP1A1 enhanced to 8.4-and 43.5-folds of control group,while CYP1A2 enhanced to 2.7-and 16.8-folds of control group respectively,indicating the induction effect of RAD on CYP1A1 was higher than CYP1A2.10.Using SimCypTM PBPK modeling software and parameters obtained from the study,the rat PBPK models of ?M,?M,OXYH and BER were established.The rat models were simulated and validated by the experimental data.On the basis of the validated rat model,the human PBPK models were developed with the human papameters obtained form our in vitro experiments.The human pharmacokinetics and drug-drug interactions(DDI)of IM,IIM,OXYH and BER were then predicted.The results showed that the notable DDI potential of RAD furanocoumarins when they coadministrated with CYP1A2 substrates.The predicted AUCi/AUC of caffeine,phenacetin and theophylline when coadministrated with or without IM,IIM and BER were 2.0,1.7 and 1.3 respectively in health population.The ratios were higher in heavy smoke population.These results indicated the DDI risk of RAD furanocoumarins in both health population and heavy smoker population.The novel points or new findings of this study are illustrated as follows:1.IM,IIM,BER and OXYH were the major components detected in both plasma and tissues,and presented the majority of the RAD furanocoumarins.They could be used as the marked princinples for further pharmacokinetic and pharmacodynamic study of RAD.This finding provided essential scientific basis for understanding the material of pharmacology and drug interaction of RAD.2.?M,?M,BER and OXYH could specifically concentrated in liver.Oatp and Oat mediated active hepatic uptake played an important role in the hepatic specific distribution.Protein binding was lalso one of cause for the high hepatic exposure of the four furanocoumarins..3.The study revealed that interaction of RAD with CYP1A2 was bidirectional.Single dose of RAD showed the inhibitory effect on CYP1A2,while multiple doses not only reversed the inhibition and also showed the induction effect on the activity and mRNA expression of CYP1A2.This finding offered the basis for understanding the HDI mechanism of RAD and provided useful information guide for the safe and effective use of RAD in clinic.4.The method of integrated IC50 values was developed and validated in the study,which can be applied to the integral evaluation on the CYP inhibition of multiple constituents in TCM.It may be used an evaluation method in vitro for the HDI of multiple constituents of TCM.5.IM is a potent CYP1A2 inhibitor with the reversible inhibitory mechanism.?M,BER and OXYH are CYP1A2 inactivator with MBI mechanism.The human DDI prediction using PBPK model indicated that RAD furanocoumarins possess notable CYP1A2 based HDI risk in human.In the clinic,it is necessary to monitor the possible HDI caused pharmacokinetic and pharmacodynamic alteration when RAD is coadministrated with drugs of CYP1A2 substrates.