The Modulation Effects Of Flavonoids On CYP3A4 And P-glycoprotein And Molecular Mechanism Study

I Activation effects of flavonoids on cytochrome P450 3A4 and molecular mechanism studyFlavonoids,with the basic structure of diphenyl chromone,are produced by photosynthesis of plants and ubiquitously distributed among vegetables,fruits and herbs.Flavonoids are known for their wide variety of pharmacological activities,including antioxidant,anti-lipidemic,cardioprotective and free radical elimination activity et al.In recent years,with the development of modern medicine,flavonoids have been extensively investigated due to their wide variety of pharmacological activities.Now it has become more and more common that flavonoids combination with drugs,which greatly increases the possibility of food drug and drug drug interactions(DDIs).In the mechanism of metabolic DDIs,drug metabolizing enzyme and drug transporter mediated DDIs are the two most important mechanisms,that is,an exogenous substance could cause the change of blood drug concentration of the coadiministrated drug through regulating the activity of drug metabolizing enzyme or drug transporter,leading to toxic reaction or drug efficacy reduction.Since drug metabolizing enzymes and drug transporters,with substrates cross,play a leading role in the metabolism of xenobiotic substances,the interaction between xenobiotic substances and metabolizing enzymes or transporters has become one of the focuses of modern medical research.Cytochrome P450(CYP450)is the important phase I enzymes in human and plays a critical role in the metabolism of endogenous components and xenobiotics,such as hormones,bile acids,drugs and dietary compounds.CYP450 is mainly distributed in liver,kidney and small intestine.CYP3A4 is one of the most important isozymes of CYP450 superfamily,which can metabolize nearly 50%of clinical drugs.The change of CYP3A4 activity is likely to cause DDIs.CYP3A4 enzyme activity changes are including inhibition,induction and activation,also induction and inhibition have been reported in many literatures.Compared to inhibition and induction,heterotropic activation could result in transient and intense effects on enzymes activity.However,there are few studies on the activation of enzyme activity,mainly due to the poor correlation between the activation in vitro and in vivo,and the molecular mechanism is still unclear.Therefore,our present study was focused on the activation on CYP3A4 of flavonoids and its molecular mechanism.The present study was designed to systematically investigate the heterotropic activation effects on CYP3A4 of 75 flavonoids in human liver microsomes(HLM)and CYP3A4-HepG2 cells,which are likely to be the major components of daily diet or frequently used traditional Chinese medicines.The activation effects were assessed in cells and the whole animal model.The underlying activation mechanisms were assessed using the molecular docking analysis and circular dichroism(CD)measurements.Finally,the 3D-and 2D-QSAR model was established to elucidate the structure-activity relationships of flavonoids with CYP3A4,thus would be helpful to predict the potential interactions between untested flavonoids and drugs which metabolized by CYP3A4.The results were shown as follows:1.The primary screening and activation biological effects of 75 flavonoids on CYP3A41.1.In the primary screening in HLM,8 out of 75 flavonoids exhibited significant activation(activation rates>50%)on CYP3A4,including artemisetin,α-naphthoflavone,tangeretin,6-methylflavone,6-hydroxyflavone,β-naphthoflavone,5-hydroxyflavone and flavone.Another 8 flavonoids showed weaker activation effects(activation rates 20-50%),including 3-hydroxyflavone,7-hydroxyflavone,nobiletin,sinensetin,chrysin,galangin,isosinensetin and eupatilin.And the rest 59 flavonoids exhibited little or no active effects on CYP3A4(activation rates<20%)in HLM.1.2.The concentration-dependent activation on CYP3A4 showed that artemisetin was the strongest flavonoids activator of CYP3A4(EC50=2.17 μM),followed byα-naphthoflavone(EC50=3.12μM),tangeretin(EC50=3.22 μM),6-methylflavone(EC50=4.10 μM),6-hydroxyflavone(EC50=6.10 μM),β-naphthoflavone(EC50=6.42 μM),5-hydroxyflavone(EC50=7.99 μM)and flavone(EC50=9.22μM).1.3.In order to assess the activation biological effects in cells,three CYP3A4 substrate clinical drugs were chosen as target drugs,including carbamazepine,diosbulbin B and dronedarone(DND),finally DND was selected as the target drug,and its suitable incubation concentration and time were 10 μM and 6 h,respectively.1.4.The results of activation biological effects in CYP3A4-HepG2 cells showed tangeretin,sinensetin,flavone and 6-hydroxyflavone could decrease the DND-induced cytotoxicity and increase the cell viability by increasing the metabolism of DND.1.5.Besides substrate dependent,activation effects are species dependent.The similar results were only observed in Syrian golden hamster liver microsomes compared with HLM,but not in liver microsomes of SD rats and ICR mouse.Finally hamsters were selected as the animal model for activation.1.6.The results of activation biological effects in hamsters in vivo suggested that tangeretin,sinensetin,flavone and 6-hydroxyflavone could activate the DND metabolism,the AUC0-t and Cmax increased in different extents,from 2.36 to 2.87 folds and 1.57 to 2.71 folds,respectively.1.7.The Chinese patent medicine containing Chenpi had no influence to the metabolism of DND in hamsters,indicating there are no or less significant metabolic drug-drug interactions in clinical practice,but needs further study.2.The activation mechanisms and structure-activity relationships of flavonoids with CYP3A42.1.The molecular docking results suggested that the primary force between CYP3A4 and flavonoids were Van der Waals force and Pi interactions,and the amino acid residues Cys 442 is the common binding residues for flavone,tangeretin,sinensetin and 6-hydroxyflavone.2.2.The CD spectrum interaction results of flavonoids with CYP3A4 showed that flavone,tangeretin,sinensetin and 6-hydroxyflavone could change the secondary structure of CYP3A4,and the content ratio of α-helix/β-turn were increased,which would induce more stable conformation and eventually lead to the increase of the enzyme activity.2.3.The 3D-QSAR of flavonoids with CYP3A4 results suggested that the common feature pharmacophores of CYP3A4 activator are B aromatic ring,hydrophobic groups at 7-position and hydrogen bond acceptors at 4-position.2.4.The 2D-QSAR model established using ADMET Predictor showed that the Q2 of training set and test set were all above 0.7,and the model predictability was well,showing the deviation between the predicted value and the measured value is within 0.33 to 3 flods.The 2D-QSAR model would be helpful for predicting the efficacy of untested flavonoids.In conclusion,the present study investigated the activation effects of 75 flavonoids on CYP3A4,established the in vivo hamster activation model,explore the molecular mechanism using molecular docking and CD analysis.Finally,the established structure-activity relationships pharmacophore model would provide useful information for predicting the potential risks of flavonoid-containing food/herb-drug interactions in humans.II Inhibitory effects of flavonoids on P-glycoprotein and structure-activity relationshipsP-glycoprotein(P-gp)is one of the most important drug transporters,can efflux foreign substances such as poisons and drugs from intracellular and play the barrier role of body.P-gp is highly expressed in liver,kidney,small intestine and in capillary endothelial cells of some important physiological barriers such as blood-brain barrier and placental barrier.Therefore,for the substrate drugs of P-gp,the change of P-gp activity may cause the change of blood drug concentration.For the drugs with narrow therapeutic window,the small fluctuation of blood drug concentration may lead to the occurrence of toxic reaction or the change of clinical efficacy.In addition to normal tissues,P-gp is also highly expressed in tumor cells,which is one of the main reasons for multidrug resistance of tumor drugs.Therefore,inhibition of P-gp activity may improve multidrug resistance.Our present study was designed to investigate the inhibitory effects on P-gp of 75 flavonoids,which are probably the main constituents in daily diet or frequently used in traditional Chinese medicine prescriptions,further explore the inhibitory biological effects of flavonoids in MDR1-MDCKII cells and Taxol-resistant MX-1/T cells,as well the whole animal model.The underlying inhibitory mechanism of flavonoids was also assessed using the molecular docking analysis.Eventually,the pharmacophore model was established to elucidate the structure-activity relationships of flavonoids with P-gp,thus would be helpful to predict the DDIs in clinical treatment.1.Inhibitory effects of flavonoids on P-glycoprotein1.1.The transport assays results in MDR1-MDCKII cells showed that 6 out of 75 flavonoids exhibited significant inhibition on P-gp(inhibition rates>50%),including nobiletin,isosinensetin,tangeretin,sinensetin,sciadopitysin and oroxylin A.Another 23 flavonoids showed weaker inhibitory effects(inhibition rates 20-50%),and the rest 46 flavonoids exhibited little or no inhibitory effects on P-gp(inhibition rates<20%).1.2.The concentration-dependent inhibition on P-gp showed that nobiletin was the strongest inhibitor of P-gp(IC50=2.21μM),followed by isosinensetin(IC5O=4.2μM),tangeretin(IC50=12.66 μM),sinensetin(IC50=18.9 μM),sciadopitysin(IC50=53.42 μM)and oroxylin A(IC50=78.33 μM).1.3.In MDR1-MDCKII cells,isosinensetin,tangeretin,sinensetin,sciadopitysin and oroxylin A could increase the cytotoxicity of paraquat,especially when paraquat at 750 μM,the cytotoxicity of paraquat increased by 54.51%and 59.65%in oroxylin A and sciadopitysin groups,respectively.1.4.In MX-1 and MX-1/T(taxol resistant cells)cells,the above 6 flavonoids could increase the cytotoxicity of taxol.Compared with the wild MX-1 cells,tangeretin and sciadopitysin could significantly increase taxol cytotoxicity in MX-1/T cells.So tangeretin and sciadopitysin are expected to overcome,at least in part,drug resistance in the clinical treatment.1.5.In flavonoids pre-treated SD rats groups,including nobiletin,isosinensetin,tangeretin,sinensetin,sciadopitysin and oroxylin A,the AUC0-t and Cmax of digoxin were increased in different extents.Especially in nobiletin group,the AUC0-t and Cmax of digoxin increased 2.03 folds and 4 folds,respectively.1.6.The Chinese patent medicine containing Chenpi and Ginkgo had no influence to the plasma concentration of digoxin in SD rats,indicating there are no or less significant metabolic drug-drug interactions in clinical practice,but needs further study.2.The inhibition mechanisms and structure-activity relationships of flavonoids with P-gp2.1.The results of molecular docking of flavonoids to P-gp suggested that different amino acid residues and interaction forces were responsible for the binding of flavonoids and P-gp.And the results of analogs docking with P-gp suggested that the inhibitory effect of flavonoids might be related to Pi interactions,but not hydrogen bonds.2.2.The results of 3D-QSAR pharmacophores model suggested that the critical pharmacophores of P-gp inhibitors are B aromatic ring,hydrogen bond acceptors at 4-position,and hydrophobic groups at 6-position,7-position and 4’-position.In conclusion,six of tested 75 flavonoids exhibited significant inhibition of P-gp in vitro and/or in vivo,which can increase the cell toxicity in MDR1-MDCKII cells and taxol-resistant MX-1/T cells.Moreover,molecular docking assays elucidated the inhibitory effect of flavonoids might be related to Pi interactions,but not hydrogen bonds.The established structure-activity relationships model would provide useful information for predicting the potential risks of food/herb-drug interactions in humans,and help to optimize flavonoid structure and obtain promising compounds to overcome,at least in part,drug resistance in the clinical treatment.