Mechanism Of Interaction Between Quetiapine, Aripiprazole And P-Glycoprotein

OBJECTIVETo study the mechanism of interaction between aripiprazole,quetiapine and P-glycoprotein by assessing the effects of aripiprazole,quetiapine on the function and expression of P-glycoprotein and theeffects of P-glycoprotein on transportation of quetiapine and aripiprazoleacross Caco-2 monolayer model.METHODS1 Methods in the Studies of the Effects of Aripiprazole, Quetiapine onthe Function and Expression of P-glycoprotein(1) Cell Culture and Establishment of Caco-2 Cell Monolayer ModelCaco-2 cell and ECV304 cell were cultured with DMEM (Dulbecco'sModified Eagle's Medium) which contains 10% FBS(Fetal Bovine Serum)in a humidified atmosphere of 95% air and 5% CO₂ at 37℃. Theexpression of P-glycoprotein in the cells was identified byimmunofluorescence assy. The Caco-2 cell was cultured in transwellplates to establish monolayer model. The EVOM epithelial voltohmeter,fluorescein and propranolol were used to test the function of themonolayer model.(2) Cytotoxicity In VitroMTT (Methyl thiazolyl tetrazolium) assay and living cells counting method were used. The concentrations for aripiprazole and quetiapinewere from 0.01～100μmol/L in MTT assay, while 1～50μmol/L in livingcells counting method.(3) Effects of Quetiapine and Aripiprazole on the Function ofP-glycoproteinECV304 cell was used as negative cell control. Verapamil was used aspositive control of the inhibitory effect on the function of P-glycoprotein.Fluorescence spectrophotometric method and flowcytometry were used tostudy the effects of quetiapine and aripiprazole on the efflux ofrhodamine123 which is the substrate of P-gp in Caco-2 cell.(4) Effects of Quetiapine and Aripiprazole on the Expression ofMDR1 Gene mRNA and P-glycoproteinCyclosporin A was used as positive control of up regulation effect onthe expression in mRNA level of MDR1 gene. Caco-2 cell without drugdealing was used as negative control. RT-PCR (ReverseTranscription-Polymerase Chain Reaction) was used to measure theexpression of MDR1 gene mRNA in Caco-2 cell. Constitutive GAPDH(glyceraldehyde-3-phosphate dehydrogenase) gene was determined as acontrol. Flowcytometry was used to measure the expression ofP-glycoprotein in Caco-2 cell.2 Methods in the Studies of the Effects of P-glycoprotein on theTransportation of Quetiapine and Aripiprazole(1) In vitro P-glyeoprotein Affinity for Quetiapine andAripiprazoleThe affinity of quetiapine, aripiprazole and a proven substrate, verapamil, in vitro for P-glycoprotein was assessed by examining theirP-gp-dependent ATPase activity. The P-gp-dependent ATPase activitywas quantified by determining the change of concentration of inorganicphosphate (Pi) that released from ATP hydrolysis process during theenergy-dependent P-gp drug transportation process. TheMichaelis-Menten equation was used to analyse the data.(2) Effects of P-glycoprotein on the transportation of Quetiapine andAripiprazoleTo estiblish the ultra-performance^TM liquid chromatography-electrospray tandem mass spectrometry (uplc-ESI-MS/MS) method forthe determination of quetiapine and aripiprazole in in vitro samples, uplcseparation was achieved using an Acquity UPLCTM BEH C₁₈ column(100mm×2.1mm, i.d., 1.71μm particle size), maintained at 40℃, with amobile phase flow rate of 0.3 ml.min^-1. The mobile phase contained 63%acetonitrile and 37% ammonium acetate at a final concentration of30mmol/L. The sample volume injected was 4μl. The determination wasperformed using a Waters Micromass Quattro Premier XE tandemquadrupole mass spectrometer with an electrospray source in positivemode and in multiple reaction monitoring (MRM) mode. The sample wasredissolved in 1.5ml 1-chlorobutane-triethylarnine (5: 0.5).To study the effects of P-glycoprotein on transportation of quetiapineand aripiprazole across Caco-2 monolayer model. The cell monolayermodel on transwell plate was used. The effects of duration, drugconcentration, P-gp inhibitor (verapamil) on the transportation ofquetiapine and aripiprazole was detected by studying the bi-directiontransportation of the two drugs through Transwell plate. The amount oftransportation of quetiapine and aripiprazole was detected withuplc-MS/MS and the apparent permerbility coefficient (P_app) wasobtained. RESULTS1 Results in the Studies of the Effects of Aripiprazole, Quetiapine onthe Function and Expression of P-glycoprotein(1) Cell Culture and Establishment of Caco-2 Cell MonolayerModelThe expression of p-glycoprotein is plentiful in Caco-2 cell but sparsein ECV304 cell. The Caco-2 cell monolayer mode is tight and intact.Caco-2 cell monolayer mode is established successfully. Caco-2 cell andmonolayer model are fit for the study of interaction between newerantypical antipsychotic drugs and P-glycoprotein, and ECV304 cell is fitto be negative control.(2) Cytotoxicity In VitroThe cells died when the concentrations of quetiapine and aripiprazolewere 50μmol/L. The cells survived when the concentrations were nomore than 30μmol/L. The max concentrations without cytotoxicity forquetiapine and aripiprazole are 30μmol/L, which is fit for the study of theeffects of quetiapine and aripiprazole on the function and expression ofP-glycoprotein.(3) Effects of Quetiapine and Aripiprazole on the Function ofP-glycoproteinWhen the concentration is 0.01μmol/L, there was no differencebetween quetiapine, Verapamil and negative control group in theintracellular fluorescence (P＞0.05), but the intracellular fluorescence ofaripiprazole group was significantly higher than that of negative controlgroup (P＜0.05). when the concentration≥0.1μmol/L, the concentrationwas higher, the intracellular fluorescence of aripiprazole, quetiapine and verapamil group was higher. The intracellular fluorescence of the threedrugs was all significantly higher than that of negative control group(P＜0.05). At low concentration (0.01μmol/L), quetiapine and verapamilhave no effects on the function of P-glycoprotein, but aripiprazole inhibitsthe function of P-glycoprotein; At high concentration (≥0.1μmol/L),quetiapine, aripiprazole and verapamil inhibit the function ofP-glycoprotein.(4) The Effects of Quetiapine and Aripiprazole on the Expression ofMDR1 Gene mRNA and P-glycoproteinThe expression of MDR1 gene mRNA in quetiapine group was higherthan that in negative control group(P＜0.05); the expression of MDR1gene mRNA in aripiprazole group was lower than that in negative controlgroup. Quetiapine up regulates the expression of MDR1 gene mRNA.Aripiprazole down regulates the expression of MDR1 gene mRNA.When the concentration of drugs was between 0.01～1μmol/L, therewas no difference between aripiprazole group and negative control in thefluorescence (P＞0.05); When the concentration of drugs was between10～30μmol/L, the fluorescence of aripiprazole group was significantlylower than that of negative control group (P＜0.05); When theconcentration of drugs was between0.01～30μmol/L, the fluorescence ofquetiapine group was significantly higher than that of negative controlgroup (P＜0.05). In low concentration (0.01～1μmol/L), aripiprazole hasno effects on the expression of P-glycoprotein. In high concentration(≥10μmol/L), aripiprazole down-regulates the expression ofP-glycoprotein. Between 0.01～30μmol/L, quetiapine up-regulates theexpression of P-glycoprotein. 2 Results in the Studies of the Effects of P-glyeoprotein on thetransportation of Quetiapine and Aripiprazole(1) In vitro P-glycoprotein Affinity for Quetiapine andAripiprazoleThe rank order of the V_max/K_m ratio was: verapamil (2.16)＞aripiprazole (1.29)＞quetiapine (1.15).(2) Effects of P-glyeoprotein on the transportation of Quetiapine andAripiprazoleThe uplc-ESI-MS/MS method is accurate, sensitive, and simple forthe analysis of quetiapine and aripiprazole in in vitro samples. Peaks werevery sharp and the peak widths at half height (w_h) were around 2.5 s. Arun time is less than 3 min. The instrumental LODs lower than 0.005μg/L.The calibration curves were linear in the ranges of 0.05～5μg/L forquetiapine and aripiprazole. Quetiapine: C=0.113R-0.140 (r=0.997);Aripiprazole: C=1.213R-0.002 (r=0.999).The P_app increased with the elongation of duration and finally reached asaturation point. The P_app decreased with the increased concentration ofquetiapine and aripiprazole. P_app.ab Value increased significantly (P＜0.05)when the verapamil was added, while P_app.ba value decreased significantly(P＜0.05). The P-gp inhibitor (verapamil) can influence the transportationof quetiapine and aripiprazole. The transportation of quetiapine andaripiprazole through Caco-2 cell model probably is mediated byP-glycoprotcin. CONCLUSIONAripiprazole (ARI) and quetiapine (QTP) both are substrates of P-gpand have affinity of P-gp. P-gp participates the transportation of ARI andQTP through promoting the effiux of the two drugs.ARI inhibits the function and expression of P-gp. The effiux of ARI byP-gp decreases when the drugs is used for a long time, this is one of themain reasons why there is no ARI resistance phenomenon in clinic. Ourresult suggests that drug interaction will occur when ARI and othersubstrate of P-gp are used together. ARI probably increases intracellularconcentration of other substrate of P-gp, and the therapeutic and toxiceffect of ARI will enhance. So the dosage of ARI should be reduced whenARI and other substrate of P-gp are used together in clinic.QTP can inhibit the function of P-gp, but QTP can induce theexpression of P-gp. The synthetic effect of QTP on P-gp is that the effiuxof QTP increases when QTP is used for a long time. This is one of themain reason why there is QTP resistance phenomenon in clinic. Ourresult indicates that drug interaction will occur when QTP and othersubstrate of P-gp are used together. QTP probably reduces intracellularconcentration of other substrate of P-gp through induce the expression ofP-gp, so the therapeutic and toxic effect of QTP will decrease. Our resultsuggests that the dosage of QTP should increase appropriately after a longtime use or when id used with other substrate of P-gp.