Transporter-Based Effect Of Resveratrol On Absorption Of Bestatin And Drug-Drug Interaction Between Resveratrol And Methotrexate As Well As Its Molecular Pharmacokinetics Mechanisms

Part Ⅰ Development of LC-MS/MS methods for simultaneous determination of bestatin and methotrexate in biological samplesObjective:To develop a sensitive,quick,specific and high performance LC-MS/MS method for simultaneous determination of bestatin and methotrexate(MTX).Methods:After a simple protein precipitation using acetonitrile,the analytes were separated on a Hypersil ODS-C18 column.The mobile phase consisted of 60%(v/v)acetonitrile,40%(v/v)water with 0.1%formic acid for bestatin,20%(v/v)acetonitrile,10%(v/v)methanol,70%(v/v)water with 0.1%formic acid for MTX at a flow rate of 0.4 mL/min.The ionization was conducted using a Turbolonspray interface in positive ion mode for bestatin and MTX.The selected transitions of m/z were m/z 309.2→120.0 for bestatin,m/z 455.2→307.8 for MTX,and m/z 370.4→288.1 for cilostazol(internal standard).Results:The method was linear in the concentration range of 2.00 to 2000.00 ng/mL for bestatin and MTX.The method showed good precision and accuracy.Conclusions:A LC-MS/MS assay for simultaneous determination of bestatin and MTX in the biological samples was developed and validated.The method was proved suitable for the pharmacokinetic study of bestatin and MTX.Part Ⅱ Transporter-Based effect of resveratrol on absorption of bestatin and Its Molecular Pharmacokinetic MechanismsObjective:The purpose of present study was to assess the enhancing effect of resveratrol(Res)on the absorption of bestatin and clarify the related molecular mechanism.Methods:In vivo absorption studies,in situ jejunal perfusion technique and uptake studies in Caco-2 cells were used to evaluate the intestinal absorption of bestatin.The LC-MS/MS assay for bestatin biologic samples was developed.Results:Res facilitated bestatin absorption by down-regulating both protein and gene levels of multidrug resistance 1(Mdrl)and Multidrug resistance-associated protein 2(Mrp2),and up-regulating oligopeptide transporter 1(Pept1)protein and mRNA expression in rat intestine.In the same manner,Res increased penetration of bestatin via significantly activating mRNA and protein expression of PEPT1 in Caco-2 cells.Conversely,mRNA and protein expression levels of MDR1,MRP2 and phosphorylation level of Insulin-like growth factor 1 receptor(IGF-1R)were inhibited by Res in Caco-2 cells.Moreover,Res also altered the phosphorylation of extracellular signal-regulated kinase(ERK)and protein kinase B(AKT).Res enhanced the intracellular concentration of bestatin by down-regulating MDR1 and MRP2 expression through a mechanism that involves IGF-1R/AKT/ERK signaling pathway inhibition in Caco-2 cells.Conclusion:Res enhances bestatin absorption by regulating PEPT1,MDR1 and MRP2 both in vivo and in vitro.Part Ⅲ Transporter-Based Drug-Drug Interaction between resveratrol and methotrexate and Its Molecular Pharmacokinetic MechanismsObjective:The purpose of present study was to investigate the ability of Res to alter MTX pharmacokinetics and clarify the related molecular mechanism.Methods:In vivo absorption studies,in vitro everted intestinal sac preparation,kidney slices in rats and bi-directional transport assay with MDR1-/MRP2-MDCK cell monolayers,uptake studies in hOAT1/3-HEK293 cells were employed to evaluate the interaction between Res and MTX.Results:Res significantly increased rat intestinal absorption of MTX in vivo and in vitro.Res inhibited MTX efflux transport in MDR1-MDCK and MRP2-MDCK cell monolayers.Furthermore,there was a significant decrease in renal clearance of MTX after simultaneous intravenous administration.Similarly,MTX uptake was markedly inhibited by Res in rat kidney slices and hOAT1/3-HEK293 cell.In addition,concomitant administration of Res decreased cytotoxic effects of MTX in hOAT1/3-HEK293 cells,renal morphologic injury and creatinine and blood urea nitrogen(BUN)levels.However,Res can not decrease the increased levels of MDA and MPO caused by MTX and intestinal damage caused by MTX was not exacerbated after Res treatment.Conclusion:Res enhances MTX absorption and decrease MTX elimination by inhibiting P-gp,MRP2,OAT1 and OAT3 in vivo and in vitro.Additional attention should be paid to the alternative exposure of MTX when MTX was co-administered with Res in clinic.