Role Of P-glycoprotein And Breast Cancer Resistance Protein In The Transport Of Paroxetine Across Blood Brain Barrier

In recent years, depression has become the world’s fourth largest disease, which is a serious threat to human health. paroxetine, an antidepressant of the selective serotonin reuptake inhibitor, is a major drug in the treatment of depression. The oral bioavailability appears to be less than 50% due to extensive first-pass metabolism. Drug resistance appeared during the process of treatment and ultimately developed into treatment resistant depression(TRD). However, the mechanism of TRD remains unclear. Since ABC efflux transporters at BBB attenuate the efficacy of most CNS drugs, we speculate that depression resistance is likely to be related to ABC transporters mediated efflux. Therefore, this study aimed to study the role of the main BBB efflux transporters, P-gp and BCRP, in the brain penetration of paroxetine, and to clarify the potential transporters based mechanism of depression resistance. The transfected cells and mouse brain microvascular endothelial cell(BCEC) as in vitro BBB models, along with in situ brain perfusion and whole animal experiments as in vivo models, were used to evaluate the role of P-gp and/or BCRP in the cellular uptake and transport of paroxetine across the BBB. In addition, the antidepressant-like effect was investigated to illuminate the pharmacodynamics impact of P-gp/BCRP on efficacy of paroxetine using tail suspension test(TST) in mice.Firstly, a rapid and sensitive high performance liquid chromatography-fluorescence(HPLC-FLU) quantitative analytical method was established for determination of paroxetine in different biological matrices. Using propranolol as internal standard, plasma and brain homogenate were extracted with ethyl acetate. The analysis was conducted by HPLC-FLU detector on a Eurospher 100-5 C18 column. The mobile phase consisted of acetonitrile-0.5% triethylamine(v/v, 35:65, pH 3.5 adjusted by phosphoric acid). The excitation and emission wavelength were 295 nm and 350 nm, respectively. The run time was 8 min, which ensures the high-throughput analysis of samples. The assay was fully validated with dynamic ranges of 5-500 ng/m L for plasma, brain homogenate and dialysate. The intra- and inter-day precision all met the acceptance criteria. The stability of paroxetine was verified under different storage conditions. In summary, this rapid, simple, sensitive and accurate method can be used to determine paroxetine in biological samples.Uptake studies were performed in MDCK-MDR1 and MDCK-BCRP transfected cells to inspect the role of P-gp/BCRP in the penetration of paroxetine. Time- and concentration-dependence studies showed the uptake of paroxetine could be saturated, and the uptake rate of paroxetine in MDCK-MDR1 cells was less than MDCK-WT cells. Furthermore, P-gp inhibitors increased the intracellular levels of paroxetine, indicating that P-gp was involved in the efflux of paroxetine. However, BCRP played a minor role in the efflux of paroxetine. Since P-gp and BCRP are highly expressed in BCEC, mouse BCEC were estabolished as another in vitro BBB model to verify the above results. Time- and concentration-dependence studies showed processes of non-linear kinetics, indicating the involvement of transporters. Combination of paroxetine and efflux transporter inhibitors proved that P-gp mainly contributed to the efflux of paroxetine, which was in good agreement with the results obtained from transfected cells.To further investigate the transport mechanism of paroxetine across BBB, both in situ brain perfusion and in vivo pharmacokinetics-brain distribution experiments were performed to research the effect of P-gp/BCRP on the brain distribution of paroxetine. In situ mouse brain perfusion study indicated that both brain volume of distribution and transport coefficient of paroxetine showed a good linear relationship with time and perfusion concentration. P-gp inhibitors, verapamil and CsA, increased the brain volume of distribution and transport coefficient of paroxetine, while BCRP inhibitor pantoprazole did not show such effect, which revealed that P-gp expressed at BBB significantly restricted the brain penetration of paroxetine. In vivo pharmacokineticsbrain distribution experiments were carried out to study the effect of verapamil, CsA and pantoprazole on the plasma and brain exposure of paroxetine. The results showed that verapamil and CsA significantly increased the accumulation of paroxetine in the brain, but the plasma concentration remained unchanged or increased slightly. Pantoprazole did not alter plasma or brain concentration levels of paroxetine. The brain/plasma concentration(B/P) ratios were used to highlight the role of efflux transporters at BBB. Expectedly, verapamil and CsA obviously increased B/P ratios, while pantoprazole did not alter the B/P ratios. Since unbound drug showes efficacy in the target tissue, there is a need to determine the protein binding to research the unbound drug exposure of paroxetine in mouse plasma and brain tissue. The equilibrium dialysis results showed paroxetine had very high protein binding. The plasma bound fraction was around 95%, while that for brain was as high as 99%. The free plasma and brain drug concentrations were calculated using the total drug concentrations and unbound fraction. Verapamil and CsA increased free drug levels in brain without altering the plasma drug concentration and pantoprazole didn’t alter the free drug levels in brain or plasma.Finally, TST was used to investigate the impact of P-gp/BCRP on pharmacodynamics of paroxetine in mice. The immobility time within 6 min under the desperate state was observed as an efficacy indicator. The potential influence of paroxetine and corresponding inhibitors on spontaneous activity was measured in mice employing an open-filed test(OFT). Data showed that neither paroxetine nor inhibitors changed the spontaneous activity. However, paroxetine significantly reduced the immobility time, while co-administration with P-gp inhibitors verapamil and CsA strengthened the response to paroxetine in mice. The pantoprazole pre-treatment group showed similar result with control group. The concentration of paroxetine in specific parts of the brain tissue and plasma were subsequently determined. In accordance with pharmacodynamics results, verapamil and CsA enhanced the concentration of paroxetine in all brain parts of interest, while plasma concentration remained unchanged, suggesting that increased paroxetine delivery to brain caused by P-gp inhibition resulted in enhanced antidepressant-like activity.In summary, we studied the mechanism of P-gp and BCRP mediated transport of paroxetine across BBB. The results showed that P-gp rather than BCRP was the major transporter contributing to the efflux of paroxetine. P-gp inhibitors, such as verapamil and CsA, could increase the brain distribution of paroxetine and thus improve the efficacy. We suggest that combined administration with P-gp inhibitors may represent a beneficial approach to augment paroxetine therapy in TRD.