Sulfotransferases And Efflux Transporters Determine The Intestinal Disposition Of Calycosin

Backgroud and ObjectivesDrug metabolizing enzymes(DMEs)and efflux transporter(ETs)play an important role on the absorption and disposition of drug in vivo.UDP-glucuronosyltransferases(UGTs)and sulfotransferases(SULTs)are the main phase ? enzymes in liver and small intestine,which extensively participate in the metabolism of various drugs.The efflux transporters,such as P-glycoprotein(P-gp),breast cancer resistance protein(BCRP),multidrug resistance protein 2(MRP2),and multidrug resistance protein 1(MRP1),are major determinants of the excretion of many drugs.Thus,it is meaningful to study the function of DMEs and ETs on the pharmacokinetics characters and efficacy of drugs in vivo.Calycosin(C)is an active isoflavone compound in the extract of Astragali radix.It has attracted much attention for its various biological effects including anti-oxidation,cardiovascular protective effect,hepatoprotective effect,renal protective effects,and anti-cancer effects.Our previously studies have investigated the pharmacokinetics studies and the enterohepatic disposition of the water extracts of Astragali radix and calycosin-7-O-?-glucoside(CG).The results showed that CG is mainly hydrolyzed into calycosin,which to be the main part of the 'fate' of CG in vivo.However,there were little studies performed on the pharmacokinetic characteristics and the intestinal disposition of calycosin.Therefore,the FVB mice and efflux transporter knock-out mice(KO mice)were chosen as the animal models to investigate the importance of SULTs and ETs on the pharmacokinetic characteristics and intestinal disposition of calycosin.It will provide useful informations on the clinical guidances for the use of Astragali radix and further researches of the new drugs.Methods1.The sulfated conjugate was identified by multiple analytical methods,including high resolution mass spectrum(HMRS)and nuclear magnetic resonance hydrogen spectrum(1H-NMR),analyzing the accurate molecular weight,mass spectrum information and comparing the 1H-NMR information with calycoisn.And we developed sensitive and reliable UHPLC-MS/MS methods to determine calycosin and its phase ? metabolites(C-3'-G and C-3'-S)in various experiments.2.FVB mice,Bcrpl(-/-)micee Mrp2(-/-)mice and Mrp1(-/-)mice,were used as animal models in the present studies.The pharmacokinetics and in situ intestinal perfusion studies have been performed on these animal models to clearly clarify the importance of efflux transporters on the pharmacokinetic characteristics and intestinal disposition of calycosin.In addition,we used the recombinant SULT isoforms and mice hepatic/intestinal S9 fractions to investigate the sulfation characteristics of calycosin in vitro.3.The bi-directional transport studies on Caco-2,MDCK ?/WT,and MDCK ?/BCRP cell lines were performed to determine the absorption and disposition of calycosin in both directions.Further,the over-expressed BCRP and the inhibitor of BCRJP(Ko143)were used to further clarify the dominant importance of efflux transporter of BCRP on the disposition of calycosin and its sulfated metabolites.Results1.Identification of calycosin sulfated conjugateThe structure of the sulfated metabolite of calycosin was identified by HRMS and 1H-NMR information.The result showed that the sulfted metabolite of calycosin,which obtained via mice intestinal S9 and mice intestinal perfusion stuides,was identified as calycosin-3'-sulfate(C-3'-S).2.The pharmacokinetics studies of calycosin in FVB mice and knockout miceAfter oral administration of 20 mg/kg calycosin in FVB mice,Bcrpl(-/-)mice and Mrpl(-/-)mice,calycosin was sufficiently and rapidly absorbed and intensively metabolized into phase ? metabolites.Calycosin climbed to the plasma concentration peak within 5 min,and it was eliminated very fast(T1/2?10.3±1.88 min,FVB mice).There were detected C,calycosin-3'-glucuronide(C-3'-G),and C-3'-S in mice plasma.The plasma concentration of calycosin was considerablely low.However,the plasma concentration of phase ? metabolites(C-3'-G and C-3'-S)was much higher than the plasma concentration of calycosin.And C-3'-G showed the highest plasma concentration in vivo.Compared with FVB mice,the Cmax,AUC0-24h,and T1/2 of C-3'-G in Bcrpl(-/-)mice were increased by 1-,2.9-,and 1.4-fold,respectively(p<0.01);the Cmax,AUCO-24h,and T-1 of C-3'-S in Bcrpl(-/-)mice were increased by 15.8-,41.3-,and 1.8-fold,respectively(p<0.01);the Cnax and AUC0-24h of calycosin in Bcrpl(-/-)mice were increased by 6-,and 3-fold,respectively(p<0.05).The deletion of Mrpl has no effects on the pharmacokinetics parameters of calycosin.3.The intestinal absorption and disposition of calycosinThe absorption amounts of calycosin in small intestine were significantly higher than those in colon.Calycosin was extensively happened glucuronidation and sulfation metabolism.There were deetected the phase ? metabolites of C-3'-G,and C-3'-S in perfusate,plasma,and bile samples.The excreted amounts of C-3'-S and C-3'-G in small intestine were markablely higher than those in colon(p<0.01).And the intestinal excreted amounts of C-3'-S were higher than C-3'-G.With the comparision of FVB mice,the intestinal excreted amounts of C-3'-S in Bcrpl(-/-)mice were markedly decreased by 82.3%-91.3%in small intestine and 97.6-98.2%in colon(p<0.01).But there were no significant differences in Mrpl(-/-)and Mrp2(-/-)mice.The intestinal excreted amounts of C-3'-G in Mrpl(-/-)were higher than those in FVB mice.And the deletion of Bcrpl has no influences on the excretion of C-3'-G.Compared with FVB mice,Mrp2(-/-)and Bcrpl(-/-)mice showed significantly decrease in the biliary excretion of C-3'-G(p<0.01).We used five commercially available recombinant SULTs to determine the main SULT isoforms responsible for metabolizing calycoisn.At three calycosin concentration tested(50,150,and 500 nM),results showed that SULT1A1,SULTIBI,and SULT1E1 are the main isoform for the metabolism of calycosin into C-3'-S.The formation rate of C-3'-S by liver S9 showed highest,followed by colon and liver S9.The liver and colon S9 fractions in mice,which mediated C-3'-S formation,exhibited substrate inhibition kinetic characteristics.Aind the small intestine S9 was exhibited classic Michealis-Menten kinetic profile,as evidenced by Eadie-Hofstee plot.4.Role of BCRP on the disposition of calycosin sulfated conjugateCalycosin was rapidly absorbed and then metabolized into C-3;-S in Caco-2,MDCK?/WT,and MDCK ?/BCRP bi-directional studies.Kol43 led to a significant decrease in the apical excreted amount(44.7-65.7%),efflux rate(54.2-66.4%),and clearance(33.9-78.2%)of C-3'-S(p<0.01).The Fmet value did not altered in the presence of Kol43,indicating Ko 143 did not change the enzyme activities in the Caco-2 cells.A marked increase of the intracellular concentration(48.5-81.0%)of C-3'-S were observed with the the presence of Kol43(p<0.01).Western bloting results showed that the protein expression level of SULT1A1 and SULT1B1 were founded in both MDCK ?/WT and MDCK ?/BCRP cells.And the expression level of BCRP was much higher than that in MDCK ?/WT cells.In contrast with MDCK IIAVT cells,the apical excreted amount,and efflux rate of C-3'-S were significantly increased by 10.5-13 folds and 11.3-15.6 folds in MDCK ?/BCRP cells,respectively(p<0.01).Kol43 led to a substantial decrease of the apical excreted amounts(39.7-88.1%),efflux rate(51.0-87.9%),and clearance(89.1-97.2%)of C-3'-S in MDCK?/BCRP cells(p<0.01).Though the Fmet values were altered by Kol43,the intracellular concentration of C-3'-S was still increased by 3.2-6.4 folds.Conclusions1.BCRP plays a dominant role on the disposition of calycosin in vivo and in vitro,the excretion of C-3'-S was mainly regulated by BCRP.And MRP2 and BCRP play important role on the biliary excretion of C-3'-G.2.Calycosin is rapidly and sufficiently absorbed by passive diffusion.Extensive glucurnidation and sulfation were happened in small intestine,and the main metabolites were C-3'-G and C-3'-S.SULT1A1 and SULT1B1 are the main isoforms to catalyze the formation of C-3'-S.