In Vitro And In Vivo Evaluation Of The Effects Of Duloxetine On P-glycoprotein Function

ObjectiveTo evaluate the effect of duloxetine on P- glycoprotein function by investigating the effects of duloxetine on the function and expression of P-glycoprotein in Caco-2 cells and quantitating the effect of orally administered duloxetine on the pharmacokinetics of talinolol.Method1.The effects of duloxetine on the function and expression of P-glycoprotein in Caco-2 cells1.1 Cell culture and morphology validated of cellsCaco-2 cells were cultured with MEM(Minimal Essential Medium) which contains 10%FBS(Fetal Bovine Serum) in a humidified atmosphere of 95%air and 5%CO₂ at 37℃.The expression of p-glycoprotein in the cells was identified by flow cytometer.1.2 CytotoxicityMTT(diphenyltetrazolium bromide) assay was used to determine the non-cytotoxicity dosage of duloxetine in Caco-2 cells.The dosage at which the survival rate of cells is above 90%was considered as the highest non-cytotoxic dosage.1.3 Effects of duloxetine on the efflux of rhodamine-123 in Caco-2 cellsThe effect of duloxetine on P-glycoprotein function was analyzed using rhodamine-123 assay.Flow cytometry was used to determine the intracellular rhodamine -123 concentration and verapamil was used as the positive control.1.4 Effect of duloxetine on P-glycoprotein expressionThe expression of P-glycoprotein in Caco-2 cells was measured by flow cytometry after incubation with duloxetine for 72h.2.The effect of orally administered duloxetine on the pharmacokinetics of talinolol2.1 Dose AdministrationThe study was conducted in a self-controlled two-period experiment in a randomized,open-labeled design in 12 healthy Chinese volunteers.In the 1^st period,the volunteers received a single oral dose of 50 mg talinolol. After 1 week washout,the volunteers received 30 mg duloxetine twice daily for 6 consecutive days.On the 7^th day,they received 30 mg duloxetine and 50 mg talinlolol concomitantly.2.2 Blood samples collectionSerial blood samples were collected from an in-dwelling venous catheter(anti-coagulated with sodium heparin) at 0,0.5,1,1.5,2,3,4,5,6,7,9,12,24,36,48,60 h after talinolol administration.The plasma was separated immediately and frozen at-70℃until analysis. 2.3 Analytic MethodConcentration of talinolol in plasma was determined by HPLC-ESI-MS/MS.Results1.Effects of duloxetine on the function and expression of P-glycoprotein in Caco-2 cells.1.1 Cell Culture and Morphology ValidatedThe expression of P-glycoprotein is plentiful in Caco-2 cell and Caco-2 cell is suitable for evaluating the effect of duloxetine on P-glycoprotein function and expression.1.2 CytotoxicityThe 90%cells survived after incubation with duloxetine when the concentration of duloxetine was no more than 10μmol/L.The max concentration without cytotoxicity for duloxetine is 10μmol/L.1.3 Effects of duloxetine on the efflux of rhodamine-123 in Caco-2 cellsVarious concentrations of duloxetine decreased the efflux of rhodamine-123 significantly compared with negative control,but the differences between dose groups were not significant.1.4 The effects of duloxetine on the expression of P-glycoproteinThe P-glycoprotein expression in Caco-2 cells after incubation with various concentrations of duloxetine(0.2,5,10μmol/L) was not significant different with the negative control.So,duloxetine in these concentrations doesn't modulate the expression of P-glycoprotein in Caco-2 cells.2.The effect of duloxetine on the pharmacokinetics of talinolol2.1 Determination of talinololThe calibration curve was linear in the range of 2.0～240.0 ng/mL for talinolol.The average extraction recoveries for talinolol were above 86.6%.The methodology recoveries were between 94.3%and 105.6%. The intra-day and inter-day RSD were less than 13%and the RSD of stability test were no more than 10.5%.2.2 The effect of duloxetine on the pharmacokinetics of talinololAfter subjects treated with duloxetine,the AUC_0-60 and C_max of talinolol increased from 1348.54 ng·h/mL to 1498.30 ng·h/mL and 91.90 ng/mL to 125.21 ng/mL,respectively.The AUC_0-60 and C_max of talinolol were increased by 11%and 36%,respectively.The 90%CIs for the ratios of AUC(0.77-1.06) and C_max(0.68-1.12) were not fell within the accepted range for lack of interaction(0.80-1.25).No significant difference in any other pharmacokinetic parameters was observed between the two periods(T_max,t_1/2,CL/F,V/F).DiscussionThe study for the first time evaluated the effect of duloxetine on the P-glycoprotein function in vitro and in vivo.In vitro study showed that various concentrations of duloxetine inhibit the function of P-glycoprotein and decrease the effiux of rhodamine-123,but doesn't modulate the expression of P-glycoprotein in Caco-2 cell.This suggested that duloxetine just influence the function of P-glycoprotein.The possible reason is competitive inhibition or inhibition of ATP enzyme.In vivo study showed that a 6 -day treatment period with duloxetine increased the oral bioavailability of talinolol,but other pharmacokinetic parameters were not changed.Because talinolol is not metabolized to a relevant extent,we speculate that the increase in oral bioavailability of talinolol by co-administration of duloxetine can be attributed with high certainty to an inhibition of P-glycoprotein in the small intestine.Our results suggest that duloxetine could inhibit the function of P-gp in vitro and in vivo,and caution should be exercised when duloxetine is to be co- administered with drugs that are P-gp substrate.