The Study Of P-glycoprotein Mediated Amitriptyline And Nortriptyline Interactions In Central Nervous System In Vitro And In Vivo

ObjectiveTo study the effect of dexamethasone (Dex) and verapamil(Ver) on the function and expression of P-glycoprotein, and their effect on the efflux ratio and the distritribution into brain of amitriptyline (Ami) and nortriptyline(Nor). To investigate the correlation between in vitro and in vivo results of drug interactions. And to assess the feasibility of predicting outcomes of interactions in central nervous system based on the results in vitro and improving the efficacy and decreasing adverse reactions by utilizing P-glycoprotein mediated interactions.Methods1. Blood samples were prepared with protein precipitation, and Halo C18column was performed with the mobile phase consisting water and acetonitrile, and samples were detected on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM).2. Caco-2cells were cultured with MEM which contains10%FBS(Fetal Bovine Serum) in a humidified atmosphere of95%air and5%CO2at37℃. The expression of P-glycoprotein in the cells was identified by immunofluorescence assay. Activity of Caco-2cells was assessed by MTT (Methyl thiazolyl tetrazolium) assay. Fluorescence spectrophotometric method and flowcytometry were used to study the effects of Ver and Dex on the efflux of Rhodamine123. The expression of P-glycoprotein in Caco-2cells was measured by immunofluorescence method.3. The efflux ratios (RE) of Ami and Nor were investigated with Caco-2cells monolayer model in transwell plates.4.396Kunming male mice were divided into three groups, and the concentration of Ami and Nor in plasma and brain tissues were detected by UPLC-MS/MS after simultaneous intraperitoneal injection Ami or Nor and a single dose or multiple doses of Dex or Ver.Results1. UPLC-ESI-MS/MS in the MRM model methodology provides a highly sensitive, selective, reliable and rapid method for determination of Ami and Nor. The linear calibration curves of Ami and Nor were obtained in the concentration10ng mL-1～2500ng mL-1. The extraction recoveries were74.3%-78.6%, intra-and inter-day precision of the assay at three concentrations were2.8%-14.1%with accuracy of95%-105%.2. Caco-2cells grew very well and the expression of p-glycoprotein was plentiful in Caco-2cells. Dex and Ver did not affect the activity of Caco-2cells when the concentrations were less than100μmol-L-1. Dex can decrease the fluorescence intensity obout20-50%, and there was a significant negative correlation between fluorescence intensity and the dosage when the concentration of Dex was less than25μmol·L-1. Ver can increase the fluorescence intensity in a dose-dependent manner.The study of expression of P-gp showed that there was no difference between Dex group(within0.01～0.1μmol·L-1) and control group in the fluorescence (P>0.05). When the concentration of Dex was within1～50μmol·L-1, the fluorescence of Dex group was significantly higher than that of control group (P<0.05); When the concentration of Ver was within0.1～50μmol·L-1, the fluorescence of Ver group was significantly higher than that of control group (P<0.05).3. The study of monolayer model showed that Ami and Nor are the poor substrates of P-glycoprotein and the efflux ratio of Ami and Nor were increased about10to60percents in presence of Dex and decreased about10to20percents in presence of Ver.4. Dex can decrease the value of AUC of brain tissue in mice about40to50percent and Ver increased the AUC about10to50percents. There were no differences in Cmax between Dex group, Ver group and control group.ConclusionDex can induce the function and the expression of P-gp, and Ver can inhibit the function and induce the expression of P-glycoprotein. Ami and Nor both are the poor substrates of P-gp. The efflux ratio of Ami and Nor can be increased by Dex and decreased by Ver. Results in vivo showed that distribution of Ami and Nor into the brain was increased by Ver and decreased by Dex.The studies showed that the results in vitro can predict the outcomes of drug-drug interactions in central nervous system. Inducer and inhibitor can modulate the function and expression of P-glycoprotein, which indicates that rational use inducer and inhibitor can improve the curative effect and decrease adverse reactions.