Study On Transmembrane Transport And Drug Resistance Of Parthenolide Derivative ACT001 Based On Transporter

ObjectiveBy studying the effect of parthenolide derivative ACT001 on the activity of drug transporters,identify the transporters that may have an effect on ACT001 efficacy.Then further study the affinity of ACT001 with the transporter and the regulatory effect on the transporter to explore the transmembrane transport and drug resistance mechanism of ACT001.Methods1.The inhibition of Parthenolide derivative ACT001 on the transport activity of transporters were preliminary screened at a series of concentration.The half maximal inhibitory concentration(IC₅₀)of ACT001 to transport activity of transporters was also determined.2.The Caco-2 cell model was used to study the intestinal absorption mechanism of ACT001.Use the stably transfected efflux transporter MDCK cell model to study the efflux mechanism of ACT001.The data will help to predict the effect of efflux transporters on ACT001intestinal transmembrane transport,further predict the drug resistance that may occur in the clinical use of ACT001.3.Using drug transporters induction methods in vitro,RT-PCR,Western blot and other technologies to study the regulation and ability of ACT001 on efflux transporters,infer the drug transporter-mediated ACT001 drug resistance and drug interactions,and further predict the method to reverse the resistance.4.U87 human glioma cells were used to study the antitumor effect of the first-in-class anti-glioma agent ACT001,and the effective methods to enhance efficacy and even reverse the resistance of ACT001 were deduced and investigated.Results1.Parthenolide derivative ACT001 can inhibit the transport activity of efflux transporter BCRP with IC₅₀ of 48.6?mol/L;ACT001 has no effect on the transport activities of OAT1,OAT3,OCT1,OCT2,OATP1B1,OATP1B3,P-gp and BSEP with IC₅₀are all greater than100?mol/L.2.The efflux rate of ACT001 on the monolayer of Caco-2 cells was 2.95.Add in BCRP inhibitor Ko143 decreased the efflux rate to 0.807.The P-gp inhibitor verapamil hydrochloride has no effect on the efflux rate.It is suggested that ACT001 is a substrate of the efflux transporter BCRP as well as a competitive inhibitor.3.Parthenolide derivative ACT001 can induce the expression of BCRP and P-gp.In human colon adenocarcinoma cell LS-180,ACT001 10?mol/L increased the m RNA expression levels of P-gp and BCRP by 8.89-fold and 8.21-fold after induction for 72 h,respectively.Under the same conditions,the protein amount of the two transporters was increased by3.76-fold and 2.92-fold,respectively.It is suggested that ACT001 may produce drug resistance in clinical application.4.Cell proliferation inhibition experiments showed that the presence of BCRP inhibitor,Ko143,at 5?M,reduced the IC₅₀ value from 33?M to 26?M?P<0.05?refers to the anti-glioma effect of ACT001.Conclusions1.Parthenolide derivative ACT001 is a substrate of the efflux transporter BCRP.BCRP is highly expressed in the blood-brain barrier and tumor cells.When ACT001 reaches the blood-brain barrier and tumor tissue,it will be recognized by BCRP for efflux,then the ability to cross the blood-brain barrier is reduced.As a result,the effective blood drug concentration cannot be achieved at the target point and the efficacy is reduced.2.Parthenolide derivative ACT001 is an inducer of efflux transporters P-gp and BCRP,which can increase the m RNA and protein expression levels of the two transporters and enhance their efflux activity in LS-180 cell;BCRP and P-gp are highly expressed in the blood-brain barrier and tumor tissue,and the induction of the two proteins will further lead to a decrease in drug efficacy and drug resistance,resulting in treatment failure.3.BCRP and P-gp are widely distributed.When ACT001 is used in combination with inhibitors or substrates of two proteins,it will affect the transport of other drugs and drug-drug interactions will occur due to competition and induction.4.ACT001 is a substrate of BCRP,which will be discharged into the intestinal cavity in the intestine,reduce the blood concentration,limit absorption,and reduce the oral bioavailability.5.BCRP inhibitors can inhibit BCRP pump ACT001,increase blood concentration and brain exposure,then enhance its antitumor effect.6.This study provides a reasonable and effective reference for ACT001 clinical application.