Studies On The Development Of Efficent Screening Methods For P-Glycoprotein Inhibitors And The In Vitro/in Vivo Evaluation

Multidrug resistance(MDR)is a serious drug resistance phenomenon of tumor cells,which is the biggest obstacle for anticancer drug treatment.P-Glycoprotein(P-gp)is the first ABC transporter to be discovered.Overexpression of P-gp in tumor cells is one of the common mechanisms of MDR,which can lead to the failure of chemotherapy in the clinic.The combination of anticancer drugs and P-gp inhibitors can increase the accumulation of anticancer drugs in tumor cells,which can reverse MDR in cancer treatment.However,the non-specificity and non-selectivity of P-gp inhibitors is one of the most fatal shortcomings in clinical application,which can cause serious side effects.Currently,P-gp inhibitors have been developed to the third generation,but no P-gp inhibitor has passed the clinical trials due to serious side effects.Therefore,there is an urgent need for effective and low-toxic P-gp inhibitors to reverse MDR mediated by P-gp.Traditional Chinese Medicine(TCM)is the best source of P-gp inhibitor for the novel chemical structure and low toxicity.At present,the research on the interaction between small molecules and P-gp mainly stays at the cell level,which are time-consuming,laborious,and the accuracy is limited.At present,the verification of P-gp ligand is still in the in vitro,and there are relatively few in vivo studies.In response to the problems faced by P-gp inhibitors,several membrane protein stabilization strategies were used to obtain P-gp with correct conformation and good activity.Combined with surface plasmon resonance(SPR),the new method for efficient screening of P-gp inhibitors and the evaluation system for the interaction between potential inhibitors and P-gp from in vitro to in vivo have been established.The main research content of this study is divided into the following three parts:1.A rapid screening system for P-gp ligands from TCM based on lentiviral membrane protein stabilization strategy combined with SPRCombining the strategy of stabilizing membrane proteins with lentiviral particles(LVP)and SPR technology,a P-gp specific ligand screening system was constructed to screen potential P-gp inhibitors from TCM.Then its biological activity was verified in vitro.Firstly,we constructed lentiviral particles with different expression levels of P-gp and characterized the expression of P-gp.Secondly,the LVP with P-gp was immobilized on different flow cells of CM5 chip for affinity detection.The affinities of Valspodar(Val)and cyclosporin(Cs A)were detected to characterize the chip's activity,and the K_Ds of Val and Cs A were 14.09?M and 16.41?M,respectively.Forty compounds from natural products were screened using the SPR CM5 chip,three of which were screened out as potential P-gp ligands,which showing a significant response signal.The affinity assays showed the K_Ds of magnolol(Mag),honokiol(Hon),resveratrol(Res)were 15.88?M,65.44?M and 70.01?M,respectively.The results of in vitro validation experiments indicated that these three compounds could inhibit the efflux of Rh123.Besides,honokiol and resveratrol can reverse the resistance of MCF-7/ADR cells to Adr.Western blot(WB)and flow cytometry analysis showed that both magnolol and honokiol can down-regulate the expression of P-gp in MDR cancer cells.In this study,a novel P-gp inhibitor screening system based on membrane proteins stabilizing with LVP was constructed to explore the interaction of small molecules and P-gp.Compared with traditional cell experiment,the experiment time of this screening system time is greatly shortened.2.A novel method for screening potential P-gp inhibitors based on SMA membrane protein stabilization strategy combined with SPRSMA membrane protein stabilization strategy combined with SPR technology to establish a method for rapid screening of new P-gp inhibitors to reverse MDR in tumor treatment.Firstly,the breast cancer cell MCF-7 and Adriamycin(Adr)-resistant breast cancer cell MCF-7/ADR were used as models,and the membrane protein was extracted from the cell membrane by styrene-co-maleic acid(SMA)extraction technology,then,the corresponding SMA lipid particles(SMALPs)were obtained.The SMALPs were coupled on different channels of L1 chip to construct P-gp potential inhibitor screening system(P-gp-SMALP-SPR).At the same time,the pretreatment method of SMA extraction membrane protein was optimized,and the methodological investigation of the screening system was carried out,including liposome stability,chip saturation,chip specificity and interference of SMA on screening results.The results showed that the best treatment conditions for SMA pretreatment were sonication of cell membrane for 20 s,reaction time of 4 h,membrane concentration of 40 mg/m L.The affinity of positive drug Valspodar and the chip with a K_D value of 26.12?M,indicating that the chip has good specificity.The stability of liposomes on the chip was 24 hours,and the residual SMA in the sample does not interfere with the test results.This screening system was used to screen out 9compounds from 50 TCM monomers as target compounds and their biological activity in vitro was verified.It was found that tetrandrine(Tetrandrine,TET),fangchinoline,praeruptorin B,neobaicalein and icariin can significantly reduce the IC₅₀ value of MCF-7/ADR cells to Adr.Among them,TET,praeruptorin B and neobaicalein can reverse the MDR by inhibiting the function of P-gp.The established screening system can realize rapid screening for potential P-gp inhibitors with strong specificity and greatly shorten the screening time.Among them,the praeruptorin B and neobaicalein were reported to have reversing MDR activity for the first time.3.The in vivo evaluation of potential P-gp inhibitors in reversing multidrug resistance-taking tetrandrine as an exampleAt present,there are few studies on potential P-gp inhibitors in vivo,and the data of pharmacodynamics,drug interaction and toxicological characteristics are scarce.Tetrandrine(Tetrandrine,TET)is a potential P-gp inhibitor obtained by the P-gp-SMALP-SPR screening system.Cell viability experiments have confirmed that TET can reduce the IC₅₀ of Adr to MCF-7/ADR cells from 86.09±4.74?M to 13.52±0.63?M,which indicated the activity of reversing MDR was the highest in vitro.To evaluate the activity of reversing MDR of TET in vivo,MCF-7/ADR cells were used to construct the tumor bearing model.The effect of TET combined with PTX and PTX alone in the treatment of MCF-7/ADR-xenograft model was compared.According to the tumor size and weight,the tumor inhibition rate of PTX alone was 42.34%,the tumor inhibition rate of TET alone was 61.03%,and the tumor inhibition rate of PTX and TET combined was82.44%.The tumor inhibition rate of TET combined with PTX was 2 times of that of PTX alone.LC-MS/MS method was established to determine PTX level in plasma and tumor tissue.Compare the effect of TET on the distribution of PTX in plasma and tumor to comprehensively evaluate the reversal of P-gp-mediated MDR by TET.The results showed that the LC-MS/MS method for plasma and tumor tissue was with high specificity.The RSD of intra-day and inter-day precision were less than 15%and RSD of matrix effect was less than 15%,so matrix had no effect on the determination of PTX.The results shown that TET can increase the accumulation of PTX in tumor from 17.51±8.46(ng/g)to 141.75±70.15(ng/g),thus improving the inhibitory effect of PTX on MCF-7/ADR-xenograft model.At the same time,the combination of TET and PTX does not increase the concentration of anticancer drug PTX in plasma.The distribution of PTX in tumors was monitored by small animal live imaging,and the results showed that TET increased the accumulation and distribution of PTX in drug-resistant tumors.In this experiment,an in vivo evaluation system for reversing tumor MDR activity of potential P-gp inhibitors was established by the change of tumor volume and weight,the PTX plasma concentration and the cumulative amount of PTX in tumor tissues.