Interactions Between Apigenin And Acyclovir Or Adefovir Mediated By OAT1

BackgroundAs is known to all,OAT1 is a member of the organic anion transporting family identified in the past decade and is mainly expressed in the kidney,whose responsibilities are to mediate the uptake of substrates from the blood into the renal tubular cells and then contribute to the secretion form the renal tubules.Besides,OAT1 has been proved to be associated with the nephrotoxicity caused by a variety of anionic xenobiotics.As one of the main anion transporters in the kidney,OAT1 has been playing important roles in the pharmacokinetics of drugs,drug-drug interactions,and toxicology of drugs due to the facts that its substrates are so rich and that OAT1 itself is the primary anion transporter located in the kidney.However,currently,because of the safety problems caused by the clinical use of OAT1 inhibitors,screening of potent OAT1 inhibitors with low toxicity will be of great value in reducing thenephrotoxicity mediated by OAT 1 or avoiding OAT 1-mediated drug-drug interactions.Flavonoids have long been the focus of researchers because of their high safety and rich activities.Flavonoids have been used in combination with some medicinal drugs as adjuvant drugs to achieve the aim of increasing the efficacy of the drugs in clinic,but the drug-drug interactions(DDI)caused by this combination are often overlooked.Factually,DDI are mainly caused by those drug-metabolizing enzymes and drug transporters.The DDI mediated by drug-metabolizing enzymes have been well studied over the last few decades,however,the DDI caused by the drug transporters have not been widely-reported up to now.The studies on the interactions between the drugs and transporters are quite urgent and can provide guidance for the clinical uses of the drugs because of the increasing DDI events occurred in the clinic,which were related to the drug transporters.Apigenin,as a potent flavonoid being widely studied,has been reported to have interactions with several drug transporters,such as organion-transporting polypeptide 1B1(OATP1B1)and glucose Transportase-1(GLUT-1).The aim of this study was to furtherly investigate the interactions of apigenin and the substrates of OAT1.OBJECTIVE:To establish a cell model stably expressing human organic anion transporter 1(hOAT1)by using lentiviral infection and screen potent inhibitors of hOATl cells and conduct the studies on the drug-drug interactions based on hOAT1 in order to provide experimental basis to improve the clinical safety and rational use of drugs.METHODS:Lentiviral recombinant plasmids containing hOAT1 gene were constructed in vitro and were afterwards transfected into MDCK cells.The mRNA and protein expression of hOATl in MDCK cells stably expressing hOAT1 were examined by RT-qPCR and Western-blot,respectively.Besides,the uptake ability of 6-CFL taken place in both cells was examined.UPLC-MS/MS methods were applied to evaluate the effects of potent hOAT1 inhibitors on the uptake of acyclovir mediated by OAT1 in vitro and in vivo by UPLC-MS/MS.Additionally,MTT assays were conducted to test the protective effects of potent hOAT1 inhibitor on the cytotoxicity caused by adefovir,which has been reported to be a substrate of OAT 1.Results:The constructed recombinant plasmids were proved to be correct after being digested and sequenced.The results of RT-qPCR and Western-blot showed that the mRNA of hOATl was 7204.02 times of that in the control cells,and the protein level was significantly higher than that of the control cells.And the uptake abilitiy of OAT 1-overexpressing cells was 18.12 fold of that in the mock cells.Results from the inhibitors screening assays indicated that apigenin was an effective inhibitor of hOAT1(IC50=0.737μM).The pre-incubation of apigenin enhanced its inhibitory activity against hOAT1 with the IC50 value decreasing from 0.737μM to 0.295μM.Apigenin(1μM)has been proved to reduce the uptake of acyclovir in MDCK-hOAT1 cells by 55%(p<0.001).Studies in vivo showed that apigenin(70mg/kg)in combinaton with acyclovir(35mg/kg)could increase the concentration of acyclovir in SD rats.Besides,after oral administration of apigenin,the AUC(o-t)of acyclovir has increased from 10230.36±212.38mg/L*min to 21109.581±2867.39 mg/L*min(p<0.0001),MRT has increased from 65.384±10.73min to 109.06±14.64min(p<0.001),ti/2 has lengthened from 98.626±29.00min to 125.86±45.05min。Futhermore,apigenin could effectively reduce the cytotoxicity of adefovir mediated by hOAT1 in a dose-dependent manner.In the presence of 50μM of apigenin,the viability of cells treated with 200μM adefovir considerably increased from 50.6%to 112.62%.Conclusion:A cell model stably-expressing hOAT1 has been successfully established.The cell model can be used in the study of drug-drug interactions mediated by hOAT1.Among those tested compounds,apigenin was found to be the most potent inhibitor of OAT1.Apignein,as the results have shown can interact with acyclovir and adefoivr.Based on the results mentioned above,as an potent inhibitor of OAT1,apigenin might affect the pharmacokinetics of the substrates of OAT1 such as acyclovir.Additionally,apigenin could be used as a nephroprotective agent of some antiviral drugs,such as adefovir and cidofovir,which might lead to nephrotoxicity owing to their interactions with OAT1.