Effects Of Deoxyschizandrin And Schisandrin B On Transporting Function Of OATP1B1 And OATP1B3 And Its Mechanism

Backgroud:Schisandra chinensis is one of the major active ingredients for Shengmai formula.To date,the shengmai injection has been widely used in the clinical practice of treatment of cardiovascular disease.However,the major ingredients of shengmai injection were often co-used with the common cardiovascular agents and drug-drug interactions happens with great probability,so that resulted in a series of adverse effects.Organic Anion-Transporting Polypeptides?OATPs?belong to solute carrier organic anion transporter?SLCO?super family and ubiquitously disturb in gastrointestinal system,liver,kidney and blood-brain barrier and are important for membrane transportation of endogenous and exogenous compounds.OATP1B1 and OATP1B3 specifically express in liver basement membrane and play vital roles in drug absorbance,distribution,excretion and drug-drug interactions.So there is a clear need to investigate the pharmacokinetics of the major active components of schisandra chinensis,deoxyschizandrin and schizandrin B with OATP1B1 and OATP1B3.This study aimed to provide scientific evidences for safe and rationale use of schisandra.Objective:The purpose is to investigate the effects of the two main active components of schisandra chinensis,including deoxyschizandrin and schizandrin B,on the activities of both OATP1B1 and OATP1B3 using HEK293T cell models stably over-expressed OATP1B1 and OATP1B3.Furthermore,the interactions between these two active components and common substrates of OATP1B1 and OATP1B3were also assessed for the explanation of their effect mechanism on altered functions of OATP1B1 and OATP1B3.Methods:1.We established quantitative method of LC-MS for SchA,SchB,RSV,ATV,TMST,RV and TCNa,respectively.2.By using the established analytic LC-MS method,we explored the impairment of serial times and concentrations on the uptakes of RSV and ATV mediated by OATP1B1 and those of RSV and TMST mediated by OATP1B3 to determine the optimized conditions for the uptake of RSV and ATV in OATP1B1-HEK293T cells and RSV and TMST in OATP1B3-HEK293T cells,which could be employed in the following experiments for investigating drug-drug interaction.3.We investigated the pharmacokinetic changes in the uptakes of RSV or ATV to OATP1B1-HEK293T cells by simultaneously adding serial concentrations of SchA and SchB and calculated the EC₅₀0 and IC₅₀0 respective values by subtracting the blank control.Similarly,we observed the pharmacokinetic alternations in uptakes of RSV or TMST in OATP1B3-HEK293T cells by adding several gradient concentrations of SchA and SchB into the cells,and calculated the apparent parameters EC₅₀0 and IC₅₀by comparing with the corresponding control groups by Graphpad Prisms.4.We also studied the uptake kinetics of SchA and SchB transporting into OATP1B1-HEK293T cells and evaluated the changes in uptake behaviors of SchA and SchB when adding a series of concentrations of OATP1B1 inhibitors,ATV and Rif,and calculated the IC₅₀0 values by comparing with blank control groups.We studied the uptake kinetics of SchA and SchB into OATP1B3-HEK293T and evaluated the changes in uptake behaviors of SchA and SchB when adding a series of concentrations of OATP1B3 inhibitors,TMST and Rif,and calculate the IC₅₀0 values by comparing with blank control groups.Results:1.LC-MS analysis methods for RSV,ATV,FV,TCNa,TMST,SchA and SchB were well established.The specificity,accuracy and precision of these methods met the relevant requirements and can be well utilized for quantitatively analyzing the contents in biological samples.2.The time-dependent uptake curve shows that uptake of RSV in OATP1B1-HEK293T cells is saturated at 10 min,and values of K_m and V_max,of RSV are determined as 23.61±5.88,0.14±0.02nmol/mg protein·min^-1,respectively;Similarly,the uptake of ATV in OATP1B1-HEK293T cells is saturated at 5 min,and the parameters,K_m and V_max,of ATV are determined as 11.25±1.23?M?1.14±0.03nmol/mg protein·min-1,respectively.According to these calculated parameters,the standard uptake concentrations for RSV and ATV in the uptake experiment for OATP1B1,are set to 25?M and 10?M,respectively,and the uptake times are set to 20min and 10min,respectively,in the following sections for investigating the drug-drug interaction.The time-dependent uptake curve shows that uptake of OATP1B3-HEK293T cells is saturated at 10min,and the calculated kinetic parameters,K_m and V_max,of RSV are 13.86±2.65,0.11±0.01nmol/mg protein·min^-1,respectively,Similarly,the uptake of TMST in OATP1B3-HEK293T cells is saturated at 2 min,and the values of K_m and V_max,of TMST are determined as5.61±1.16?M?4.98±0.36nmol/mg protein·min^-1,respectively.According to these parameters,the concentrations of RSV and TMST,as substrates of OATP1B3,are set to 25?M and 10?M,respectively,and the times are set to 10 min and 2 min,respectively,in the following in the following sections for investigating the drug-drug interaction.3.SchA significantly promote the uptake of ATV,RSV and FV in OATP1B1-HEK293T cells and the promoting rates are 366.41±17.21%,102.61±15.68%,24.71±3.51%,respectively,and the EC₅₀0 values are 50.58±8.08,13.46±2.70?M,respectively,for ATV and RSV.SchA have a weak inhibitory effect on the uptake of TCNa in OATP1B1-HEK293T cells.The inhibitory rate is 19.62±0.44%.SchB significantly promote the uptake of FV and TCNa in OATP1B1-HEK293T cells and the promoting rates are 200.32±8.39%and 252.64±9.57%,respectively,and the EC₅₀0 values are 24.70±5.82 and 8.99±4.73?M.SchA and SchB have no detectable effect on the uptake of TMST and RSV.4.SchA and SchB are the potential substrates for OATP1B1.K_m values are determined as 17.61±0.43?M,18.45±1.23?M,respectively for SchA and SchB,and V_maxvaluesaredeterminedas7.21±0.28nmol/mgprotein·min^-1and1.45±0.09nmol/mg protein·min^-1,respectively for SchA and SchB.5.OATP1B1 inhibitors,ATV and Rif,have diverse inhibitory effects on the uptake of SchA and SchB in OATP1B1-HEK293T cells.The inhibitory rate of uptake of SchA by ATV is 73.59±3.40%and IC₅₀0 is 4.73±0.80?M.The inhibitory rate of SchB by ATV is 35.37±2.69%.The inhibitory rate of uptake of SchA by Rif is 61.65±8.48%and IC₅₀0 is 1.91±0.99?M.The inhibitory rate of uptake of SchB by Rif is31.94±1.98%and IC₅₀0 is 1.91±0.99?M.OATP1B3 inhibitors TMST and Rif have no detectable effect on the uptake of SchA and SchB mediated by OATP1B3.Conclusions:1.SchA can promote the OATP1B1 mediated uptakes of ATV,RSV and FV.Conversely,SchA have a weak inhibition on uptake of TCNa mediated by OATP1B1.SchB can promote the OATP1B1 mediated uptakes of ATV and RSV but not of FV.These results indicate that SchA and SchB promote the transporting activity of OATP1B1 in a substrate-selecting manner.SchA and SchB have no detectable effect on transporting activity of OATP1B3.2.OATP1B1 inhibitor ATV and Rif have inhibitory effects on the uptake of SchA and SchB mediated by OATP1B1.OATP1B1 inhibitor TMST and Rif have no detectable effect on OATP1B3 mediated uptake of SchA and SchB.3.Deoxyschizandrin and schizandrin B are the potential substrates for OATP1B1.Deoxyschizandrin and schizandrin B exhibit weaker affinity to OATP1B3 than to OATP1B1.