Regulation Of Acacetin By Glucuronidation Metabolism And Efflux Transporters

Purpose:It has been reported that acacetin could be easily metabolized by uridine diphosphoglucuronosyl transferases(UGTs)in vitro.Studies on in vivo pharmacokinetics suggest that acacetin glucuronides are more than acacetin exposed to blood,indicating that the bioavailability of acacetin is probably low.In addition,acacetin glucuronides are possibly the substrates for breast cancer resistance protein(BCRP/Bcrp)and multidrug resistance proteins(MRPs/Mrps).However,systematic researches on in vivo disposition of acacetin are still lacking.Regulation of acacetin glucuronides by BCRP and MRPs remains unclear.In this study,pharmacokinetic model,intestinal perfusion model and hepatic distribution were used to study the in vivo disposition of acacetin.The potential effect of modulation of Bcrp and Mrp2 on the pharmacokinetics and intestinal transport was also elucidated in vivo.Methods:1.Evaluation on pharmacokinetics of acacetin in FVB mice and knockout mice.The blood samples were collected at different time points after FVB mice and Bcrpl knockout(Bcrp 1(-/-))mice and Mrp2 knockout(Mrp2(-/-))mice were orally administered with a dose of 5 mg/kg acacetin.The pharmacokinetic parameters were calculated by WinNonLin 3.3 using non-compartment model Statistical difference of these parameters between wild-type FVB mice and knockout mice was determined.2.Evaluation on intestinal disposition of acacetin in FVB mice and knockout miceAfter the mouse was anaesthetized,the bile duct was ligated,the small intestine and the colon were perfused with 5?M of the acacetin solution.The perfusate was collected per 20 min.When the perfusion was ended,the bile in the gallbladder was collected in a tube.The tail was cut off and the blood was taken.The concentrations of acacetin and the metabolites in perfusate,bile and plasma were measured after these samples were treated.Statistical difference of the concentration of the metabolite in these samples was determined.3.Study on hepatic tissue distribution of acacetin in miceFVB mice,Bcrpl(-/-)and Mrp2(-/-)mice were used for determining the hepatic distribution levels of acacetin.5 minute after oral administration of acacetin,the mice were anesthetized and the blood was taken by extracting the eyeball.The livers were then removed from the animals.The concentrations of acacetin and its metabolites in the plasma and blood were determined after sample treatment.4.Effects of knockout on activities of hepatic and intestinal UGT enzymesHepatic and intestinal S9 fractions were prepared from FVB mice,Bcrpl(-/-)mice and Mrp2(-/-)mice.These S9 fractions at the concentration of 0.25 mg/kg were incubated with acacetin at 2.5?M,10?M and 40?M at 37?C.The reaction was terminated by organic solvent and the metabolites were measured.5.Statistical analysisData are expressed as mean ± standard deviation.Student's test or One-way ANOVE was used for statistical evalution.Significance standard ?=0.05.p<0.05 was regarded as statistically significant.Results:1.Pharmacokinetics of acacetin in FVB mice and knockout miceAfter oral administration of acacetin,the time to reach maximum concentration was quite fast.However,the concentration in the plasma was rather low.Acacetin could be quickly metabolized into acacetin-7-glucuronide(Aca-7-Glu)and acacetin-5-glucuronide(Aca-5-Glu),and these metabolites could be mostly found in the plasma.The concentration of Aca-7-Glu was 5-fold to 8-fold higher than Aca-5-Glu.Compared with wild-type FVB mice,the area under the curve(AUC)of Aca-7-Glu was increased by 5-fold(p<0.05)and 1-fold in Mrp2(-/-)mice and Bcrpl(-/-)mice,respectively,whereas the clearance(CL)was significantly decreased(p<0.05).The knockout of Mrp2 resulted in significantly increased half-life(T1/2).The AUC of Aca-5-Glu in Bcrp1(-/-)mice was significantly enhanced(p<0.05).2.Intestinal disposition of acacetin in FVB mice and knockout miceWe selected two segments of intestines,namely,small intestine and colon to pefuse with acacetin.After perfusion,effective permeability coefficient of the two intestines was greater than 1 in FVB mice and knockout mice,indicating that acacetin was well absorbed.Aca-7-Glu could be measured in perfusate,bile and plasma.The concentration of Aca-5-Glu was below the lower limit of quantitation.Significant amounts of Aca-7-Glu in the small intestine were discovered,while much less in colon.In Bcrp1(-/-)or Mrp2(-/-)mice,amounts of Aca-7-Glu in the small intestine were significantly lower than those of wild-type FVB mice(p<0.05).In Bcrpl(-/-)mice,Aca-7-Glu could not be detected in colon.3.Hepatic tissue distribution characteristic of acacetin in FVB mice and knockout miceThe liver possessed high levels of acacetin and Aca-7-Glu.The concentration of acacetin in the liver of Mrp2(-/-)was 2-fold lower than wild-type FVB mice(p<0.05),and there was no difference in the plasma.The concentration of Aca-7-Glu was 4-fold higher in the liver(p<0.05)and 8-fold higher in the plasma(p<0.05)than wild-type FVB mice.4.Study on UGT enzyme activities in Bcrpl(-/-)and Mrp2(-/-)miceThe hepatic enzyme activity in Bcrpl(-/-)and Mrp2(-/-)mice was remarkably decreased at all tested substrate concentrations(p<0.05).The intestinal enzyme activity was increased at low substrate concentration(p<0.05),and no significant difference was found at middle and high concentrations.Conclusions:1.Acacetin was mosty exposed as glucuronides in the plasma.The knockout of Bcrp and Mrp2 significantly enhanced the AUC of Aca-7-Glu,but remarkably decreased CL.2.Acacetin could be sufficiently metabolized into Aca-7-Glu in small intestine.Aca-7-Glu excreted to lumen was significantly reduced when Bcrp and Mrp2 were knocked out.3.The liver possessed high levels of acacetin and Aca-7-Glu after oral administration.The knockout of Mrp2 significantly increased the amounts of Aca-7-Glu in the liver and plasma.4.The hepatic enzyme activities were found to be decreased when Bcrp and Mrp2 were knocked out.