Intestinal Disposition Affected The Pharmacokinetic Characteristics Of Esculetin And 4-methylesculetin

ObjectiveEsculetin(6,7-dihydroxy-coumarin,ET)and 4-methylesculetin(6,7-dihydroxy-4-methylcoumarin,4-ME)are coumarin derivatives that have been demonstrated to have beneficial pharmacological and biochemical functions,such as anti-inflammatory,analgesic,antioxidant,anti-inflammatory,antitumor,vascular protection.Coumarins have very low bioavailability(2-6%).Nevertheless,there was few report in regard to the bioavailability,absorption and metabolism of them in vivo,especially about 4-ME.It has reported that catechol-containing compounds could be likely metabolited by UDP-glucuronosyltransferases(UGT),which may affect their pharmacological effects.Meanwhile,efflux transporters play an important role in drug disposition as well as metabolizing enzymes.Therefore,it's first step that studying the pharmacokinetic characteristics of ET and 4-ME,then,the study focusd on the investigation of the characteristics and mechanism of the disposition of ET and 4-ME,as well as their glucuronides(ET-G and 4-ME-G,respectively)in intestinal tract which affected their pharmacokinetic characteristic.It will provide valuable imformation and guidance for drug development,preclinical study and safe clinical medication,etc.Methods1.The blood samples of rats after oral or intravenous administration were collected from the orbital venous sinus at certain time.The plasma concentrations of esculetin,4-methylesculetin and their glucuronides were analyzed to study their pharmacokinetic parameters as well as bioavailability.2.An in situ rat intestinal perfused model was used for the study of absorption and metabolism of esculetin and 4-methylesculetin in the duodenum,jejunum,ileum and colon.The liquid chromatography-mass spectrometry(LC-MS/MS)was used for analyzing amounts in the perfusate,bile and plasma to assess the difference of absorption and metabolism among intestinal segments,chemical structures.The effect of ?-glucuronidase(?-gus)inhibitor on the disposition of esculetin or 4-methylesculetin was investigated to study whether enteric recycling involved in.In addition,We assessed the effect of efflux transporters-breast cancer resistance protein(BCRP),multidrug resistance-associated protein 2(MRP2),P-glycoprotein(P-gp)on disposition of glucuronides of esculetin or 4-methylesculetin by using a certain chemical inhibitor Ko143(10 ?M),MK571(20 ?M),verapamil(50 ?M),respectively.3.Enzyme kinetics of esculetin and 4-methylesculetin in Caco-2 cell lysates were conducted.The Caco-2 cell monolayer model was used to study the bidirectional transport of esculetin and 4-methylesculetin,and determine efflux tranporters involved in excretion of glucuronides of esculetin and 4-methylesculetin by using a certain chemical inhibitor Ko143(10 ?M),MK571(20 ?M)? verapamil(50 ?M),respectively.Results1.After oral administration,the AUC0-t values of esculetin glucuronide were approximately 44 folds than values of esculetin,the AUC0-t values of 4-methylesculetin glucuronide were approximately 9 folds than values of 4-methylesculetin,and the AUC0-t values of 4-methylesculetin were approximately 5 folds than values of esculetin,the half-time values of 4-methylesculetin were approximately 4 folds than values of esculetin.After intravenous administration,the AUC0-t values of esculetin glucuronide were approximately 3 folds than values of esculetin,the AUC0-t values of 4-methylesculetin were approximately 2 folds than values of 4-methylesculetin glucuronide,the half-time values of 4-methylesculetin were approximately 6 folds than values of esculetin.The absolute bioavailability of esculetin was calculated as 10.48%,and the absolute bioavailability of 4-methylesculetin was calculated as 7.59%.2.The apparent permeabilities of esculetin and 4-methylesculetin in intestinal segments approximately approach to 2,the maximum amounts of their glucuronides excreted were in jejunum,and the excretion rates in colon were significantly lower than these in other intestine segments.The amounts of their glucuronides in bile and plasma were abundant in a time-dependent manner which aglycones couldn't be detected,the concentrations of esculetin glucuronide in plasma were higher than 4-methylesculetin(P<0.05).Both of esculetin and 4-methylesculetin were hydrolyzed to aglycone by ?-gus,and P-gus could lower the excretion of 4-methylesculetin glucuronide in colon while esculetin glucuronide couldn't(P<0.05).Inhibitors of efflux transporter could decline the excretion rates of glucuronides,while couldn't affect their absorption rates.3.For Caco-2 cell lysates,the Km values of esculetin and 4-methylesculetin were 141.67 ?M and 103.48 ?M,respectively.In the transport study of Caco-2 cell,The Papp values of esculetin and 4-methylesculetin were higher than 1.50×10-5 cm/s.In inhibition study,the efflux rates and cellular clearances of glucuronides were decreased,nevertheless,the intracellular concentrations were dramatically increased in the presence of Ko143.In addition,verapamil mostly significantly reduced the efflux rate of 4-methylesculetin glucuronide while significantly enhanced the intracellular amount of it(P<0.05).Conclusions1.Esculetin and 4-methylesculetin have poorly bioavailability that the absolute bioavailability were 10.48%and 7.59%,respectively.The AUC0-t values of glucuronides were significantly more than values of aglycones after oral administration,which indicates esculetin and 4-methylesculetin extensively metabolized by UGTs after absorbed through the body.2.Esculetin and 4-methylesculetin could well absorbed in intestine via passive diffusion,after enteral absorption,they are mainly in the form of their glucuronides exposed to the plasma and bile,simultaneously.In addition,some significant differences between esculetin and 4-methylesculetin existed.4-Methylesculetin has lower affinity with UGT metabolic enzymes than esculetin,and the clearance of esculetin was greater than that of 4-methylesculetin in vivo.3.Both of mono-glucuronides of esculetin and 4-methylesculetin are substrates for BCRP,MRP2 may be involved in the transport of their glucuronides similarly,and 4-methylesculetin glucuronide may be substrate for P-gp.Therefore,we speculate the coupling of UGTs and efflux transporters(such as BCRP,MRP2,P-gp)regulates the intestine disposion of esculetin and 4-methylesculetin so that forms a serious first-pass effect and hinders their development.