Regulation And Molecular Mechanism Of Flavonoids On Drug Transporters BCRP And OATP1B1

I Regulation and Molecular Mechanisms of Flavonoids on Drug Transporter BCRPFlavonoids are a class of natural products widely distributed in vegetables,fruits and herbal products,with extensive pharmacological activities,such as anti-tumor,immune regulation,anti-inflammatory,anti-oxidation and so on.Several studies have shown that flavonoids are responsible for the beneficial health effects,and consumption of flavonoids plays vital roles in the prevention of cancer and neurodegenerative diseases.However,when they are co-administered with prescription drugs in clinical,the risk of drug-drug interactions(DDI)could increase accordingly.These interactions could result from the modulation of drug transporters,especially breast cancer resistance protein(BCRP).BCRP,an important subtype of ABC transporter superfamily that widely distributed in human intestine,liver,blood-brain barrier,placenta barrier and so on,plays an important function in drugs and xenotoxins disposition.The pharmacokinetic characteristics of its substrates,such as plasma concentration,bioavailability and clearance rate,will be affected by regulating BCRP mediated efflux,leading to DDI.In addition,overexpression of BCRP in tumor cells is also one of the main reasons for multi-drug resistance(MDR)in chemotherapy.So it is possible to reverse MDR of tumor cells and restore chemosensitivity by inhibiting BCRP-mediated efflux.In our present study,the BCRP-MDCK Ⅱ cell model stably transfected with the human BCRP gene was employed to investigate the effect of 99 flavonoids,which are main components in frequently used traditional Chinese medicine(TCM),vegetables and fruits,on BCRP,and their biological effects were further explored in cell models.The whole animal model was adopted to evaluate the effect of falvonoids on the pharmacokinetic of mitoxantrone.The molecular mechanism of inhibitory effect of flavonoids on BCRP was assessed by molecular docking.Finally,the structure-activity relationship between flavonoids and BCRP was established by pharmacophore calculation.Taken together,our research can be helpful to optimize the structure of flavonoids and to predict the potential risk of food/herb-drug interaction.The results were shown as follows:1.Effect of 99 flavonoids on BCRP activity1.1 Uptake studies in BCRP-MDCK Ⅱ cells showed that 11 flavonoids exhibited significant inhibitory effect on BCRP(>50%),including amentoflavone,apigenin,biochanin A,chrysin,diosmin,genkwanin,hypericin,kaempferol,kaempferide,licochalcone A and naringenin.Another 49 flavonoids showed weaker inhibitory effects(20-50%),and the rest 39 flavonoids exhibited little or no inhibitory effects on BCRP(<20%)in BCRP-MDCK Ⅱ cells model at 50 μM or at nontoxic concentration in the study.1.2 In BCRP-MDCK Ⅱ cells,the IC50 value of flavonoids with significant inhibitory effect on BCRP(>50%)was determined,results showed that amentoflavone(IC50=4.33 μM)exhibited strongest inhibitory effect on BCRP among the tested flavonoids,followed by kaempferide(IC50=4.73 μM),hypericin(IC50=6.64 μM),kaempferol(IC50=15 μM),diosimin(IC50=16.65 μM),naringenin(IC50=19.41 μM),chrysin(IC50=20.03 μM),apigenin(IC50=23.51 μM),biochanin A(IC50=24.13 μM),licochalcone A(IC50=33.48 μM)and genkwanin(IC50=36.64 μM).2.Evaluation of biological effects of flavonoids on BCRP2.1 In BCRP-MDCK Ⅱ cells,the 11 flavonoids mentioned-above and KO143,the positive inhibitor of BCRP,could increase the cytotoxicity of doxorubicin,with the cell survival rate reduced.Among them,four flavonoids,chrysin,hypericin,licochalcone A and apigenin caused a sharp downward shift of the cell viability curves even at 0.025 μM,which indicated that these four flavonoids have stronger inhibitory activity against BCRP when concurrent use with doxorubicin in BCRP-MDCK Ⅱ cells.These results suggest that flavonoid inhibitors may reduce the efflux of cytotoxic drugs by inhibiting BCRP,thus enhancing the cytotoxicity of doxorubicin.2.2 In the glioma cells U251,temozolomide(TMZ)-resistant U251T and TMZ-resistant T98G,the 11 flavonoids mentioned-above and KO143 can significantly increase the cytotoxicity of TMZ in U251T and T98G cells,with downward shifts of cell viability curves,and the influence are stronger than on U251 cells.Among them,licochalcone A and genkwanin inhibit BCRP most significantly(>95%),with sharp downward shifts of cell viability curves and obviously decreased IC50 values of TMZ much more than KO143,from 939.9μM to 1.22μM and 1.31μM in U251T cells and from 1385μM to 20.7 μM and 44.92μM in T98G cells,respectively.These results suggest that flavonoid inhibitors could reverse TMZ-resistance in U251T and T98G by inhibiting BCRP.2.3 The biological evaluation of the inhibitory effect of flavonoid inhibitors on BCRP in SD rats showed that,when pretreated intragastrically with a single dose of tested flavonoids,the AUC0-t of mitoxantrone increased in different extents compared with the blank control group in SD rats.Among them,apigenin,naringenin,licochalcone A,kaempferol and chrysin increased the AUC0-t from 30.10%to 81.97%,which were much more than the positive BCRP inhibitor KO143(25.62%).These results suggest that the regulation of flavonoid inhibitors on BCRP could affect the exposure of mitoxantrone in SD rats.3.The molecular mechanism and structure-activity relationship of flavonoids inhibiting BCRP3.1 Molecular docking could clarify the interaction mechanism between the ligands and receptor proteins at molecular level.A computational docking model(CDOCKER)was employed,the results showed that the Pi-Pi stacked interactions with Phe439 and/or Pi-Alkyl interactions with Va1546 may be critical for the stronger inhibition of flavonoids to BCRP.The number of these bonds may be related to the inhibition activity,while the conventional hydrogen bond was supposed to be not related to the inhibition activity.3.2 The structure-activity relationship between flavonoids and BCRP was elucidated by pharmacophore calculation.The results indicated that the critical pharmacophores of BCRP inhibitors are aromatic ring,hydrophobic groups and hydrogen bond acceptors.The aromatic ring B may play a vital role in the potency of inhibition on BCRP,while the methoxy at 4’position,hydroxyl and/or other hydrophobic groups at 5-position and 7-position are important for the inhibition,and when these positions binded with other groups with larger steric hindrance such as glucose,the inhibitory effect of flavonoids on BCRP would decrease or even disappear,such as apigenin and apioside,naringenin and naringin.These results would contribute to elucidate the structure-activity relationship between flavonoids and BCRP and provide valuable information for further structure optimization of flavonoids.To sum up,in this study,99 flavonoids were screened and evaluated using stably transfected BCRP-MDCK Ⅱ cells.Molecular docking was employed to elucidate the molecular mechanism of stronger inhibition of flavonoids on BCRP.The pharmacophore calculation preliminarily clarified the structure-activity relationship between flavonoids and BCRP,which provid a reference for guiding the structural optimization of flavonoids to develope selective BCRP inhibitors with high-efficiency and low toxicity to reverse MDR of tumors.Ⅱ Regulation and Molecular Mechanisms of Flavonoids on Drug Transporter OATP1B1Organic anion transporting polypeptides 1B1(OATP1B1),a liver specific uptake transporter exclusively expressed at the basolateral membrane of human hepatocytes,plays a critical role in mediating the uptake of various endogenous substances and drugs into the liver.Its functional uptake processes can result in intracellular accumulation of substrate drugs and bile components,leading to cholestasis and drug-induced liver injury(DILI).It is of great significance to develop OATP1B1 inhibitor with high efficacy and low toxicity to alleviate the DILI mediated by OATP1B1.Flavonoids,a kind of natural products with various pharmacological activities,are widely distributed in food and herbs,and have an exceptional safety record.However,it has been reported that some flavonoids can cause DDI by regulating OATP1B1.In the present study,OATP 1B1-HEK293 cells stably transfected with the human OATP 1B1 gene was employed to study the effects of 99 flavonoids that are rich in foods and herbs on OATP1B1,and their biological effects were further explored in cell models.The whole animal model was employed to evaluate the effect of falvonoids on the pharmacokinetic of MTX.The bosentan-induced liver injury rat model was used to assess the hepatoprotective effect of flavonoids.Finally,the structure-activity relationship between flavonoids and OATP1B1 was further studied by pharmacophore calculation.Taken together,our research can be helpful to optimize the structure of flavonoids and to predict the potential risk of food/herb-drug interaction.The results were shown as follows:1.Effect of 99 flavonoids on OATP1B1 activity1.1 Uptake studies in OATP1B1-HEK293 cells showed that 8 flavonoids exhibited significant inhibitory effect on OATP1B1(>50%),including biochanin A,hispidulin,isoliquiritigenin,isosinensetin,kaempferol,licochalcone A,luteolin and sinensetin.Another 59 flavonoids showed weaker inhibitory effects(20-50%),and the rest 32 flavonoids exhibited little or no inhibitory effects on OATP1B1(<20%)in OATP1B1-HEK293 cells model at 50 μM or at nontoxic concentration in the study.1.2 In OATP1B1-HEK293 cells,the IC50 value of flavonoids with significant inhibitory effect on OATP1B1(>50%)was determined,results showed that licochalcone A(IC50=7.96 μM)exhibited strongest inhibitory effect on OATP1B1 among the tested flavonoids,followed by luteolin(IC50=22.03 μM),biochanin A(IC50=22.28 μM),hispidulin(IC50=32.7 μM),kaempferol(IC50=33.05 μM),isoliquiritigenin(IC50=33.14 μM),sinensetin(IC50=40.15 μM)and isosinensetin(IC50=47.48 μM).2.Evaluation of biological effects of flavonoids on OATP1B12.1 In OATP1B1-HEK293 cells,the 8 flavonoids mentioned-above and rifampin,the positive inhibitor of OATP1B1,could reduce the cytotoxicity of MTX in OATP1B1-HEK293 cells significantly,with the cell survival rate increased.Among them,luteolin has the most obvious effect,which caused upward shift of the cell viability curves at 50-100μM.These results suggest that flavonoid inhibitors may reduce the influx of cytotoxic drugs by inhibiting OATP1B1,thus reducing the cytotoxicity of MTX.2.2 The biological evaluation of the inhibitory effect of flavonoid inhibitors on OATP1B1 in SD rats showed that,when pretreated intragastrically with a single dose of tested flavonoids,the AUC0-1 of MTX increased in different extents,from 30.10%to 81.97%,which were all weaker than rifampin(208.21%).These results suggest that the regulation of flavonoid inhibitors on OATP1B1 could affect the exposure of MTX in SD rats.2.3 In bosentan-induced liver injury rat model,the 8 flavonoids mentioned-above could significantly reduce the level of total bile acid(TBA)in the serum of rats,and the tested flavonoids decreased the concentration of bosentan in liver in different extents,from 7.12%to 54.17%.These results suggest that flavonoid inhibitors could alleviate bosentan induced liver injury by inhibiting OATP1B1-mediated bosentan uptake.3.The structure-activity relationship of flavonoids with OATP1B13.1 The structure-activity relationship between flavonoids and OATP1B1 was elucidated by pharmacophore calculation,and the results indicated that the hydrogen bond receptors at 4’ and 5-position and hydrogen bond donors at 7-position of flavonoids are the critical pharmacophores for flavonoids to inhibit the activity of OATP1B1.When these positions binded with other groups with larger steric hindrance such as glucose,the inhibitory effect of flavonoids on OATP1B1 would significantly decrease or even disappear,such as isoliquiritigenin and isoliquiritin,kaempferol and kaempferitrin.These results would contribute to elucidate the structure-activity relationship between flavonoids and OATP1B1 and provide valuable information for further structure optimization of flavonoids.Taken together,99 flavonoids were screened and evaluated using stably transfected OATP1B1-HEK293 cells in the present study.The bosentan-induced rat model was employed to assess the hepatoprotective effect of flavonoids.The pharmacophore calculation preliminarily clarified the structure-activity relationship between flavonoids and OATP1B1,which provide a valuable experimental basis for guiding the structural optimization of flavonoids to develop specific OATP1B1 inhibitors with high-efficiency and low toxicity to alleviate DILI.Ⅲ Effects of Ginkgo tablets on the pharmacokinetics of substrates of BCRP and/or OATP1BGinkgo tablets are made of Ginkgo biloba leaf extract by modern pharmaceutical technology,with the effect of promoting blood circulation,removing blood stasis and dredging collaterals.It has been widely used in the treatment of chest pain,stroke and other diseases.One of its main components are ginkgo flavonoids,including quercetin,kaempferol,isorhamnetin,and so on.Our previous studies showed that kaempferol has a strong inhibitory effect on BCRP and OATP1B1,hence,it is necessary to evaluate the effect of kaempferol-rich Ginkgo tablets on BCRP and OATP1B1,thus to better guide the rational use of Ginkgo tablets in clinic.In this study,commercial Ginkgo tablets,the BCRP substrate mitoxantrone,the OATP1B1 substrate bosentan,and BCRP and OATP1B1 common substrate fluvastatin sodium,were employed to study the effect of Ginkgo tablets on the pharmacokinetic of BCRP or OATP1B1 substrates in rats,in order to provide reference for clinical rational use of Ginkgo tablets.The results were shown as follows:1.The effects of Ginkgo tablets on the pharmacokinetic of mitoxantrone in SD rats When pre-treated intragastrically with KO143,kaempferol or Ginkgo tablets,the AUC0-t of mitoxantrone increased by 34.80%,22.78%,and 26.63%respectively,and there was significant difference compared with the mitoxantrone single treated group.These results suggest that the regulation of Ginkgo tablets on BCRP could affect the exposure of mitoxantrone in SD rats.2.The effects of Ginkgo tablets on the pharmacokinetic of bosentan in SD rats When pre-treated intragastrically with rifampin or kaempferol,the AUC0-t of bosentan increased by 219.43%and 9.56%,respectively.While the AUC0-t of bosentan was decreased by 56.72%after co-administration of bosentan with ginkgo tablets,this might be the combined effect of multiple active ingredients in Ginkgo tablets.These results suggest that the combined effect of multiple active ingredients in Ginkgo tablets may decrease the exposure of bosentan in SD rats,the mechanism remains to be further clarified.3.The effects of Ginkgo tablets on the pharmacokinetic of fluvastatin sodium in SD ratsWhen pre-treated intragastrically with rifampin,KO143 or kaempferol,the AUC0-t of fluvastatin sodium increased by 51.99%,23.02%and 5.18%,respectively.While the AUC0-t of fluvastatin sodium decreased by 30.75%after co-administration of fluvastatin sodium with Ginkgo tablets,which might be the combined effect of multiple active ingredients in Ginkgo tablets.These results suggest that the combined effect of multiple active ingredients in Ginkgo tablets may decrease the exposure of fluvastatin sodium in SD rats,the mechanism remains to be further clarified.In conclusion,the present study investigated the effects of Ginkgo tablets on the pharmacokinetics of mitoxantrone,bosentan,and fluvastatin,the substrates of BCRP and/or OATP1B1 in rats,which provide reference for the combined use of Ginkgo tablets with mitoxantrone,bosentan and fluvastatin sodium in clinic.