UGT Metabolism Of Esculetin And Methylesculetin

Background and ObjectivesBenzo-?-pyrone skeleton-containing coumarins are naturally occurring compounds,and are widely distributed throughout the plant kingdom.They have a wide variety of biological functions.Recently,it has been shown that coumarins could inhibit several types of cancer cell proliferation in-vitro and in-vivo models,and the researchs for coumarin compounds were mainly focused on its pharmacological effects,little is known about its pharmacokinetic metabolism in-invo.Therefore,this study mainly focuses on esculetin(effective components in cortex Fraxin),and methylesculetin(structure similar with esculetin),as the main research objects,deeply elucidate the mechanism UGT metabolic mechanism of these two compounds,which could help in guiding drug design,improving efficiency and safety,and developing new drug candidates of esculetin and methylesculetin.Cortex Fraxini(Chinese name Qin-pi),is the dry barks of Oleaceae plant Fraxinus rhynchophylla Hance,F.Rhynchophylla Hance,or Fraxinus paxiana,is clinically effective in the treatment of dysentery,diarrhea and inflammatory in traditional Chinese medicines.Moreover,it has been proved that coumarins,is the major bioactive constituents in Cortex Fraxini.Esculetin,as the target markers of Cortex Fraxinone.Of late,has grabbed much attention from the pharmacological and pharmaceutical area due to their broad range of therapeutical remedies.To date,it has been found to have beneficial pharmacological and biochemical activities including anti-inflammatory,antitumor,antiviral activeties.Methylesculetin,which contains methyl group at C-4 in esculetin molecules,has also exhibited variety pharmacological activities including antitumor.Thus,these two coumarins were chosen for the present study because they,could become promising anti-tumor agents,possess potent activities against cancer in vitro and in vivo.Researches found that most of coumarin derivatives,which share catechol in their skeleton,are good substrates for human UGTs,such as UGT 1A6 and UGT 1A9.Both esculetin and methylesculetin are catechol-containing compounds,which probably can be metabolited by UGTs,and this first pass effect may cause poor bioavailability and exert their pharmacological effects.In addition,both metabolizing enzyme and efflux transporters play an important role in drug disposition,which composed of the bioavailability barrier that may potential influence drug efficiency.In general,since coumarins are metabolized by intracellular metabolic enzymes inside the cells,metabolites are too polar to passively diffuse out of cells and the metabolism will be inhibited.Efflux transporters can excrete the metabolite and maintain the metabolism.Hence,the inhibition of efflux trnsporters may lead to different drug pharmacological and/or adverse effect via the reduced metabolite clearance in the intestine/liver and elevated metabolite accumulation in systemic circulation.We believe that a better understanding of these factors,can play a key role in overcoming oral bioavailability barriers.It is likely that the interplay between an efflux transporter and a phase ?enzyme will be helpful to understand and delineate the complex interplays between multiple phase II enzymes and efflux transporters while determine the drug bioavailability in-vivo.In view of this,this study aimed to investigate the metabolic mechanism of esculetin and methylesculetin.To identify the human UGT isoforms responsible for the metabolites,and to investigate the UGT1A9 metabolism and metabolite excretion on the UDP-Glucuronosyltransferase(UGT)1A9-Overexpressing Hela cell model in order to better understand the BCRP regulation on the UGT metabolism,which will provide helpful information and guidance for overcoming the oral bioavailability barriers and drug development.Methods:The metabolic mechanism of esculetin and methylesculetin was studied using UGT isoforms and chemical inhibitors in vitro by UPLC.Human liver/intestine microsomes and UGT isofoms were used to further elucidate the glucuronidation of esculetin and methylesculetin kinetics.UDP-Glucuronosyltransferase(UGT)1A9-Over expressing Hela cell model was used to investigated the UGT1A9 metabolism and metabolite excretion of esculetin and methylesculetin.All the data expressed as mean±SD.SPSS 13.0 for Windows was used as statistical software.One-way ANOVA were used to evaluate statistical difference.Results1.UGT metabolic mechanism of esculetin and methylesculetin1.1 Identification of esculetin and methylesculetin glucuronidesThe molecular weight and the structure of the metabolites were determined by quadrupole-time of flight(Q-TOF)tandem mass spectrometer with HPLC.For esculetin and methylesculetin,both of them have one mono-glucuronides,respectively.1.2 The UGT1A and UGT2B-mediated metabolism of esculetin and methylesculetin.Twelve human recombinant UGTs including UGT 1A1,UGT 1A3,UGT 1A4,UGT 1A6,UGT 1A7,UGT 1A8,UGT 1A9,UGT 1Al0,UGT 2B4,UGT 2B7,UGT 2B15 and UGT 2B17 were applied to catalyze the glucuronidation of esculetin and methylesculetin,and to identified the isozymes involved in their metabolism.The rates of metabolism by supersomes were expressed as the amount of metabolites formed(nmol/min/mg).We incubated the selected two coumarin derivatives at three concentrations(10,30,100?M)and found that UGT 1A6,UGT 1A7,UGT 1A9,UGT 2B15 were responsible for esculetin metabolism,and UGT 1A11 UGT 1A6,UGT 1A7 and UGT 1A9 were participated in methylesculetin glucuronidation.Among these recombinant UGTs,UGT1A6 and 1A9 displayed the hightest and most prominent catalytic activity.Additionally,UGT 1A1,UGT 1A6,UGT 1A7,UGT 1A9 and UGT 2B15 are highly expressed in human liver and/or intestinal meicrosomes.Therefore,we regarded liver and intestine as the main metabolic organs for esculetin and methylesculetin.1.3 Chemical inhibition studiesWe choose UGT 1A9 and UGT 1A6 as the targeted isoforms for the forward investigation of glucuronidation mechanism in human liver.To better understand the roles of UGT enzymes in hepatic glucuronidation of esculetin and methylesculetin,chemical inhibition studies with phenylbutazone and carvacrol were performed.The inhibition effects of phenylbutazone(UGT 1A6 inhibitor)and carvacrol(UGT 1A9 inhibitor)on esculetin and methylesculetin glucuronidation in pooled HLMs and recombinant UGT enzymes were evaluated.With concentration of chemical inhibition increased,there was a similar decreased tendency occurred in the formation of metabolites of these two coumarins in HLMs or UGTs,which further convinced our hypothesis that these two isoforms were target isoforms contributed to metabolism.1.4 Kinetic characterization of esculetin and methylesculetin glucuronidation in UGT 1A6 and UGT 1A9The kinetic characteristics of esculetin and methylesculetin in UGT 1A6 and UGT 1A9 were investigated.The kinetic studies showed that glucuronidation of these two coumarins by UGT 1A6 and UGT 1A9 all followed classic Michaelis-Menten kinetic.In addition,intrinsic clearance values of these two coumarins in UGT 1A6 much higher than in UGT 1A9,which means UGT 1A6 had higher affinity than that of UGT 1A9.Moreover,esculetin was metabolized faster than methylesculetin.1.5 Kinetic characterization of esculetin and methylesculetin glucuronidation in human liver and intestine microsomesThe kinetic characteristics of esculetin and methylesculetin in liver and intestine microsome were also investigated.The kinetic studies showed that glucuronidation of these two coumarins by human liver microsomesand human intestine microsomes all followed the classic Michaelis-Menten equation.The Vmax value and intrinsic clearance values of esculetin and methylesculetin in human liver microsomes was higer than that in human intestine microsomes,which means intestine might not be the main glucuronidation metabolic site but still contribute to metabolism after oral administration of these two coumarin derivatives,.In addition,esculetin was metabolized faster than methylesculetin.2.The mechanism of BCRP regulation on the UGT metabolism of esculetin and methylesculetin2.1.Enzyme kinetics study using Hela-UGT1A9 cell lysate and UGT1A9 isoformUDP-Glucuronosyltransferase(UGT)1A9-Overexpressing HeLa(Hela-UGT 1A9)was the stably transfected with UGT1A9 Hela cells.This cells line is an appropriate model to further study the interactions between exfflux transporters and metabolizing enzyme on drug disposition.Collected the cell lysate of Hela-UGT1A9,determined and compared the kinetics characteristics of esculetin and methylesculetin in the UGT 1A9 and Hela-UGT 1A9 cell lysate.Results showed that,Km values and Vmax value were similar in Hela-UGT1A9 and UGT1A9 isoform.Which means Hela-UGT1A9 cell line is suitable for the study of drug metabolism of UGT1A9 model.2.2 Investigating the metabolism and excretion characteristics of esculetin in Hela-UGT1A9 cellsIn our laboratory culture conditions,the Hela-UGT 1A9 cells were grown on 6-well plates for approximately 3 to 4 days.We tested six concentrations of esculetin including 20 ?M?40 ?M?60 ?M?80 ?M?100 ?M?120 in the Hela-UGT1A9 cells model to study the metabolism and excretion characteristics.In Hela-UGT1A9 cells,esculetin was quickly metabolized into glucuronides and then excreted.From 20 to 120 ?M,the rates of excretion of esculetin glucuronides increased with the substrate concentrations,at 120 ?M,they reached a plateau and decreased from 120?M.By contrast,the cellular clearance of the glucuronide reached a plateau at 80?M,and decreased from 80?M.With esculetin concentration increased,the amount of intracellular glucuronides increased from 20 ?M to 150 ?M,and there was a similar decreased tendency occured in Fmet and the cellular clearance of the glucuronide.2.3 Investigating the metabolism and excretion characteristics of methylesculetin in Hela-UGT1A9 cellsIn our laboratory culture conditions,the Hela-UGT1A9 cells were grown on 6-well plates for approximately 3 to 4 days.We tested six concentrations of methylesculetin including 20 ?M?40 ?M?60 pM?80 ?M?100 pM?120 in the Hela-UGT1A9 cells model to study the metabolism and excretion characteristics.In Hela-UGT1A9 cells,methylesculetin was metabolized into glucuronides and excreted quickly.The rates of excretion of methylesculetin glucuronides increased with the increasing substrate concentration.As a result,the cellular clearance of glucuronides decreased to a certain degree,and Fmet value was significantly decreased with increasing concentration.2.4 The mechanism of BCRP regulation on the UGT metabolism of esculetin in Hela-UGT1A9 cellsA specific and potent chemical inhibitor Ko143 was used to determine the role of BCRP in excretion of esculetin glucuronides in Hela-UGT1A9 cells.Three different concentrations were chosed for substrates(20,80,200 ?M)and for inhibitors(5,10 ?M),respectively.The results showed that the excretion rates of glucuronides were reduced significantly in the presence of Ko143.Compared with the control,the intracellular amount of glucuronides increased significantly,and drastically inhibited the cellular clearance of glucuronides for the meanwhile.At 10?M substrate concentration,the Fmet of esculetinglucuronides reduced by 18 and 29%in the presence of 5 and 10 ?M Ko143,respectively.At 80 or 200 ?M substrate concentration,the Fmet of esculetin glucuronides did not affect in the presence of 5 uM Ko143,had no statistical significance.2.5 The mechanism of BCRP regulation on the UGT metabolism of methylesculetin in Hela-UGT1A9 cellsA specific and potent chemical inhibitor Ko143 was used to determine the role of BCRP in excretion of methylesculetin glucuronides in Hela-UGT1A9 cells.Three different concentrations were chosed for substrates(20,80,200 ?M)and for inhibitors(5,10 ?M),respectively.The results showed that the excretion rates of glucuronides were reduced significantly in the presence of Ko143.Compared with the control,the intracellular amount of glucuronides increased significantly,and drastically inhibited the cellular clearance of glucuronides for the meanwhile.At 80 or 200 ?M substrate concentration,the Fmet of methylesculetin glucuronides reduced by 10 and 16%in the presence of 10 ?M Ko143,respectively,and 5 ?M Ko143 had limited effect on glucuronides Fmet,had no statistical significance.Conclusion:1.We characterized the in vitro metabolites of esculetin and methylesculetin,and then identified their glucuronides by UPLC-MS/MS and HRMS.2.In order to elucidate the mechanism of esculetin and methylesculetin,human liver/intestine microsomes were applied to catalyze the glucuronidation of these two coumarins,the Vmax value and intrinsic clearance values of esculetin and methylesculetin in human liver microsomes was higner than that in human intestine microsomes,which means intestine might not be the main glucuronidation metabolic site but still contribute to metabolism after oral administration of these two coumarin derivatives.In addition,the glucuronidation rate of esculetin was faster than methylesculetin,suggesting that changes in the structure,which could affect UGT metabolic characteristics;3.Esculetin and methylesculetin may be substrates for BCRP efflux tranporters with high affinity,block the function of BCRP which would result in increased the intracellular amount of glucuronides.Moreover,the interplay bewteen metabolic enzymes and efflux tranporters leads to low bioavailability for esculetin and methylesculetin.