Sulfonation Disposition Of Acacetin:In Vitro And In Vivo

Background and ObjectivesAcacetin,one kind of flavonoid,is widely distributed in the nature.Acacetin usually has numerous pharmacological activities,such as anti-oxidation,anti-inflammatory,anti-tumor and anti atrial fibrillation,etc,and which exerts a certain therapeutic effect on breast cancer,gastric cancer and prostate cancer.However,most of flavonoids undergo extensive phase Ⅱ metabolism mediated mainly by UGT(UDP-glucuronosyltransferases)and SULT(sulfotransferases)enzymes,which leads to low bioavailability and efficacy of flavonoids.Acacetin presents favorable efficacy in pharmacological research.However,the unclear metabolic disposition mechenism of acacetin restricts severely its potential druggability.Therefore,an in-depth study of metabolic mechanism on flavonoid has great significance for its pharmacodynamical mechanism and development.Glucuronidation for acacetin has been extensively investigated in the studies with rats and recombinant human UGTs in our laboratory.However,little report is available about acacetin sulfonation.Furthermore,the increasing evidences showed that the drug metabolic enzymes and efflux transporters exert synergistic effects in the disposition of flavonoids in vivo.Some previous reports had showed that many flavonoid conjugates were the substrates of membrane-bound efflux transporters,such as P-gp,BCRP and MRPs.However,it is not well understood if sulfonation disposition of acacetin is determined by sulfotransferases or efflux transporters or both and which efflux transporters and sulfotransferases were involved in this process.Hence,elucidation of sulfonation disposition of acacetin could help to gain a comprehensive understanding for action mechanism of acacetin in vivo.Therefore,to further systematic understand the sulfonation disposition of acacetin,an in vitro and in vivo strategy was applied.The incubation in vitro,pharmacokinetics and Caco-2 cell models were adopted in the present study.A sensitive and reliable UHPLC-MS/MS method was established to determine acacetin sulfate in accurately,directly and simultaneously.By this method,the metabolic characteristics of species and isoforms were used to study the sulfonation activity of different species and ascertain the major isoforms responsible for acacetin sulfonation.The pharmacokinetics of knockout mice and wild-type mice provided insights into the effects of efflux transporters on the exposure level of acacetin sulfate.The Caco-2 cell model combined with relatively selective inhibitors was used as to imitate the metabolism and absorption process in intestine and confirm the role of efflux transporters by in vitro approach.Methods1.Identification of acacetin sulfate and development of LC-MS/MS method.By means of the Aglient UHPLC-DAD-QTOF MS,the acacetin sulfate could be identified by high resolution molecular mass and mass spectrometric ion fragmentation information.Meanwhile,the method of UV maximum absorption wavelength shift was used to comfirm the conjugated position of acacetin sulfate.In addition,a sensitive and reliable LC-MS/MS method was established to detect acacetin and its sulfate.2.The study of metabolic mechanism for acacetin sulfonation in vitro.The metabolic characteristics of acacetin sulfonation in vitro was investigated by in vitro incubation,which was mediated by applying 5 kinds of species(human,monkey,rat,mouse and dog)liver S9 fractions and 5 kinds of recombinant human SULTs(1A1,1B1,1E1,2A1 and 2B1).3.The pharmacokinetics and bioavailability studies of acacetin in mice.The oral administration or tail vein administration of wild-type FVB mice and knockout mice(Bcrp-/-,Mrp1-/-and Mrp2-/-mice)were used to study the pharmacokinetics and bioavailability of acacetin in mice.The blood sample was collected by tail vein of mouse basing on the preset time point.The concentration of acacetin and its sulfate was determined.And the pharmacokinetic parameters were calculated by applying noncompartmental model of WinNonlin(?)3.3 software to analyse.4.The enzyme kinetic studies of acacetin sulfonation mediated by mouse liver,small intestine and colon S9 fractions.The liver,small intestine and colon S9 fractions of wild-type FVB,Bcrp-/-,Mrpl-/-and Mrp2-/-mice were applied to study kinetics of acacetin sulfonation by in vitro incubation,respectively.5.The effects of efflux transporters on the disposition of acacetin sulfonation determined by Caco-2 cell model.The Caco-2 cell model combined with selective relatively transporter inbhibitor including Ko143(inhibitor of BCRP)and MK571(inhibitor of MRP1 and MPR2)was used to study the transport of MRP1,MRP2 and BCRP for acacetin sulfate.Results1.Identification of acacetin-7-sulfate and developemnt of LC-MS/MS method.By means of combining high-resolution mass spectrometry and UV spectrum strategy,the structure of acacetin sulfate can be identified and comfirmed as Aca-7-S.2.The isoforms-and species-dependent feature of acacetin sulfonation in vitro.The Aca-7-S was the main exposure form for acacetin sulfonation in vitro incubation.SULT1A1 was the major isoform responsible for acacetin sulfonation.The catalytic rate of SULTs followed the order:SULT1A1>SULT1E1>SULT1B1>SULT2A1>SULT2B1.The dog liver S9 fractions had the highest formation rate for acacetin sulfonation.The catalytic rate of species followed the order:dog>rat>human>monkey>mouse.3.The pharmacokinetic and bio availability studies of acacetin in mice.The bioavailability of acacetin in wild-type FVB mice was 7.4%,indicating that low bioavailability of acacetin in vivo.The Aca-7-S was the main exposure form in the plasma for pharmacokinetics of acacetin in mice.The AUC0-∞ value of Aca-7-S in Mrp1-/-mice were evidently smaller than wild-type FVB mice.The AUC0-∞ value of Aca-7-S in Bcrp-/-mice were significantly larger than wild-type FVB mice.The results indicated that transporter MRP1 and BCRP involve in the efflux of Aca-7-S.4.The enzyme kinetics of acacetin sulfonation mediated by mouse liver,small intestine and colon S9 fractions.The kinetic models of intestinal S9 fractions of Mrpl-/-and Bcrp-/-mice were Michaelis-Menten equation,the kinetic models of another tissues S9 fractions exhibited substrate-inhibition model.The acacetin sulfonation mediated by liver and colon tissues S9 fractions of knockout and wild-type FVB mice presented same metabolic characteristics.5.The effects of transporters MRP1 and BCRP on the transport of acacetin-7-sulfate determined by Caco-2 cell model.Compared with those of the control group,the efflux rate and clearance rate of Aca-7-S decreased significantly by MK571(inhibitor of MRP1)and Ko143(inhibitor of BCRP)in basolateral and apical side,respectively,while the intracellular concentration of Aca-7-S both increased obviously.Suggesting that transporter MRP1 and BCRP were probably responsible for efflux of Aca-7-S.This was consistent with the result of pharmacokinetics of acacetin in knockout mice,hence,the results further confirmed the efflux of MRP1 and BCRP for the Aca-7-S.ConclusionsAca-7-S was the main metabolite catalyzed mostly by SULT1A1.The Aca-7-S was transported mainly by BCRP versus MRP1.The acacetin sulfonation presents obvious species-and isoforms-dependent characteristics.The dog liver S9 had the highest metabolic rate for acacetin sulfonation.Therefore,the in vivo exposure of Aca-7,S is driven primarily by SULT1A1 and transporter BCRP and MRP1.