The Molecular Pharmacokinetic Mechanisms Of Puerarin On Methotrexate With The Transporters-mediated Pharmacokinetics,Renal Toxicity In Rats And Multidrug Resistance In K562/ADR Cells

Part I Development of LC-MS/MS method for simultaneous determination of methotrexate and puerarin in biological samplesObjective:To develop a sensitive, quick, specific and high performance LC-MS/MS method for simultaneous determination of methotrexate and puerarin.Methods:After a simple protein precipitation using methanol, the analytes were separated on a Hypersil ODS-C18column. The mobile phase consisted of acetonitrile and water with0.1%(v/v) formic acid (40:60, v/v) at a flow rate of0.3mL/min. The ionization was conducted using a TurboIonspray interface in positive ion mode for bestatin and valsartan. Multiple reaction monitoring (MRM) mode was utilized to detect the compound of interest. The selected transitions were m/z455.2→308.2for methotrexate, m/z414.9→266.7for puerarin, and m/z370.4→288.1for cilostazol (internal standard).Results:The method was linear in the concentration range of1-1000ng/ml for methotrexate and puerarin. The method showed good precision and accuracy. Conclusions:A LC-MS/MS assay for simultaneous determination of methotrexate and puerarin in the biological samples was developed and validated. The method was proved suitable for the pharmacokinetic study of methotrexate and puerarin.Part Ⅱ Effect of puerarin on the pharmacokinetics of methotrexate and its molecular mechanismsObjective:The aim of this study was to investigate the effect of puerarin on the pharmacokinetics of methotrexate and its molecular mechanisms.Methods:In vivo absorption studies, in vitro everted intestinal sac preparation, kidney slices in rats and bi-directional transport assay with mock-/MDR1-MDCK cell monolayers, uptake studies in OAT1/3-HEK293cells were employed to evaluate the interaction between puerarin and methotrexate. The concentrations of puerarin and methotrexate in the biological samples were determined by LC-MS/MS.Results:After administration with puerarin in rats, the oral bioavailability of methotrexate was increased by58%, while the renal clearance was decreased significantly (from1.91±0.09ml/min to0.82±0.07ml/min) after intravenous administration. Meanwhile, in the presence of puerarin, the absorption of methotrexate by everted intestinal sac preparation was enhanced and the uptake of methotrexate by rat kidney slices was inhibited markedly. Furthermore, the efflux transport of digoxin by MDR1-MDCK cell monolayers was concentration-dependent inhibited by puerarin with IC50value of1.6μM, and the similar results was obversed in the efflux transport of methotrexate. Moreover, the uptake of puerarin by OAT1/3-HEK293cells was exhibited no significant difference compared with that by mock-HEK293cells, although the uptake of substrates (PAH and PCG, respectively) and methotrexate by OAT1/3-HEK293cells were reduced by puerarin.Conclusions:The pharmacokinetics of methotrexate could be changed greatly by puerarin, the molecular mechanism of which could be explained as the inhibitory of puerarin on the P-gp and OATs. These findings suggested that additional concern should be paid to the potential of drug interaction between puerarin and methotrexate as well as other drugs known as substrates of P-gp or OATs in the clinic.Part III Effect of puerarin on methotrexate-induced renal damage and its molecular mechanismsObjective:The purpose of present study was to investigate the effect of multiple doses of puerarin on methotrexate-induced renal damage and to explore the transporter-mediated pharmacokinetic mechanism.Methods:Methotrexate was applied to induce rat renal damage by determing serum biochemical markers and renal pathologies. Plasma and urine concentrations of methotrexate following intravenous in vivo and uptake in kidney slices were determined by LC-MS/MS. qRT-PCR analysis was used to examine the expression of transporters mRNA in rat renal.Results:Puerarin treatment significantly decreased the plasma concentration of methotrexate in vivo, and increased methotrexate uptake in rat kidney slices. RT-PCR analysis showed that puerarin induced the expressions of organic anion transporter (Oat)1/3, resulting in increased renal clearance of methotrexate. The concentrations of endogenous metabolites and toxins, such as creatinine, urea nitrogen, indoxyl sulfate in rat serum were significantly increased, and obvious morphologic damage in kidney was observed after methotrexate treatment. Combination with puerarin didn’t enhance methotrexate toxicity. Conversely, the exposure of those endogenous metabolites and toxins mentioned above and renal pathological changes were reduced by puerarin through up-regulation of Oat1/3. Meanwhile, methotrexate-induced down-regulation of BCL6and up-regulation of PEG2were partially reversed by puerarin.Conclusions:These findings suggested that puerarin could raise the expressions of Oat1/3by regulating BCL6and PEG2expressions to improve the methotrexate-induced renal toxicity.Part IV The reversal effect of puerarin on multidrug resistance of K562/ADR cells by inhibiting NF-κB pathway Objective:We aimed to investigate the reversal effect of puerarin on the multidrug resistance in K562/ADR cells and to clarify the molecular mechanisms.Methods:MTT assay was used to examine the cell survival and half-inhibitory concentrations (IC50) of K562and K562/ADR cells. The accumulations of ADR in K562and K562/ADR cells were investigated using flow cytometry. qRT-PCR and western blotting analysis were used to detect the levels of MDR1and p-κB-β.Results:K562/ADR exhibited adriamycin-resistant, and the MDRl/P-gp expression were6.58and4.62times higher in comparsion to that in K562cells. The results of qRT-PCR and western blotting experiments indicated that puerarin could significantly increase K562/ADR sensitivity to adriamycin and downregulate the expression of MDR1/P-gp in a time-dependent and concentration-dependent manner in K562/ADR cells. Morever, the phosphorylation of IκB-β was significantly suppressed by puerarin.Conclusion:Puerarin can reverse the resistance to adriamycin in K562/ADR cells by downregulating the expression of MDR1/P-gp via inhibition of NF-κB pathway.