Study Of Uptake Transport And Drug-Drug Interactionbetween Activated Metabolite Of Rhein And Methotrexate On HOAT3

Objective:Rhein is the in vivo active ingredient of Diacerein. Recently, coadministration of diacerein and methotrexate has been used to treat rheμMatoid arthritis (RA). However, the drug-drug interaction (DDI) of these two drugs has not been studied yet. The present study was designed to investigate the potential interaction between Rhein and methotrexate both in vitro and in vivo.Methods:1. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed for determination of Rhein and its activated metabolite Rhein acyl glucuronidation (RAG).2. Stably transfected cell lines of HEK293 were used to investigate uptake mechanisms of RAG and methotrexate.3. Stably transfected cell lines were also used to investigate the inhibition of E-3-S and methotrexate by RAG and the potential interaction between Rhein or RAG and methotrexate. The in vivoDDI studies were carried out in SD rats.Results:1. RAG was not stable in PBS and methanol. It was stable in the sample solution processed by methanol containing 3% formic acid. A good linearity of Rhein and RAG was obtained in the range of 100-25000 nM and 10-5000 nM, respectively. Intra- and inter-day variations were less than 10.7% and great accuracies of 95.8%-112% were achieved at the concentrations examined. The incubation time was optimized as 40 min.2. There was no significant difference in intracellular uptake of RAG between stably transfected hOAT3 cell lines and mock-vehicle cell lines. However, there was significant difference in the uptake of methotrexate between above two cell lines.3. In vitro study showed uptake of E-3-S and methotrexate via hOAT3 was markedly inhibited by RAG, and the IC50 value of RAG was 66.73 nM. RAG with concentration of 1μM can markedly inhibit uptake of methotrexate. DDI study of RAG and methotrexate showed that the value of AUC0-12(3402±583 ng/mL*h) and t1/2(6.35±7.78h) increased in the co-administration group (5068±637 ng/mL*h and 8.54±7.73h, respectively), which indicated RAG may influence the transport of methotrexate.Conclusion:1.The established method is stable and applicable, and can be used for the determination of Rhein and RAG in liver microsomes. Our study also provides experimental foundation for the further investigation of metabolic activation of Rhein.2. RAG was not the substrate of hOAT3 but methotrexate was the substrate of hOAT3. RAG was strong inhibitor of hOAT3 and demonstrated highly inhibition at the concentration of 1μM.3. RAG may influence the transport of methotrexate. Therefore, the co-administration of RAG andmethotrexate may result in drug-drug interaction.