Effect Of Piperine On Metabolism Kinetics And Brain/Plasma Concentration Ratio Of Nortriptyline In Mice

OBJECTIVESTo establish the methods for determination nortriptyline in mice and mice; To study nortriptyline disposition kinetics and brain/plasma concentration ratio of nortriptyline after multiple dose in mice which pre-treated with physiological saline, piperine and verapamil. To analyze and compare the AUC from different groups in order to discuss whether piperine can influence of drugs across the blood-brain barrier. It lay a solid foundation on clinical therapy.METHODS1. Dose and sample collection216 male KM mice (25±3 g) were equally divided into four groups by completely random design. Each group was intragastric administration with physiological saline (B), piperine (170μg/kg), piperine (5mg/kg) and verapamil (5 mg/kg) for 7 days. In the 8th day, after 12 h fasting, Each group was intragastric administration with physiological saline (B), after 1 h every mice was injected intraperitoneally with nortriptyline (13 mg-kg-1).Then the rats were sacrificed by picking off eyeballs of rats at the time intervals of 10,20,30,45 min,1,1.5,2,3,6,8,12 h, and the cerebra were collected and weighted. At each time point, six rats were sacrificed and sampled. All samples were stored in-70℃.2 Sample analysesNortriptyline in rat plasma and brain was determined by Data processingThe dates were analysised by Excel and SPSS 13.0 software. And then the AUC brain, AUC blood, Cmax of each group was tested by one-factor analysis of variance. T1/2 of each group was tested by K Independent Samples of Nonparametric test.RESULTS1. Validation of sample analysis methodA simple, rapid, and selective method to determine the concentration of nortriptyline in human plasma and brain using High performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) was developed and validated. The detection was performed by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) at m/z 315.3-242.2 for Nortriptyline and m/z 264.2-233.2 for the internal standard, Clomipramine (IS). The analysis time was 3.5 min for plasma and 7 min for brain. Linear calibration curves ranged from 5-3000 ng mL-1 in plasma and brain. Plasma and brain samples were extracted by protein precipitation. The average extraction recoveries for Nortriptyline in plasma and brain were both above 78.34%. The intra-day and inter-day RSD were less than 9.30%, the stability test showed that the plasma and brain samples of Nortriptyline were all stable under different conditions (RSD<9.27%).2. Pharmacokinetics and brain/plasma concentration ratio after intravenous injection of Nortriptyline2.1 In blood, after intragastric administration with physiological saline (B), piperine (170μg/kg), piperine (5mg/kg) and verapamil (5 mg/kg), the AUCO-12 were 3645.77±778.60,4527.37±938.29,3989.90±307.15, 6338.88±974.8 ng-h-mL-1, respectively; AUCO-oo were 3042.05±765.31, 3825.07±1090.70,3185.37±922.61,5155.18±1205.12 ng-h-mL-1, respectively; t1/2 were 2.62±1.36,2.75±1.22,3.37±1.62,3.78±2.44 h, respectively. The AUC for V group did vary significantly different with other group (P<0.05). There was no significantly different among group for t1/2.2.2 In brain, after intragastric administration with physiological saline (B), piperine (170μg/kg), piperine (5mg/kg) and verapamil (5 mg/kg), the AUCO-12 were 19750.33±2281.08,22353.33±2751.84, 20200.07±2838.54,41162.40±7836.38 ng-h-g-1, respectively; AUCO-∞were 19219.22±2471.23,20919.67±3485.21,19660.18±2843.12, 37458.47±10339.38 ng·h·g-1, respectively;t1/2 were 1.82±0.37,2.30±1.14, 1.77±0.40,2.33±1.06 h, respectively. The AUC for V group did vary significantly different with other group (P<0.05). There was no significantly different among group for t1/2.2.3 The AUC0â†'10h of brain/plasma concentration ratio of mice were 59.61±9.93,42.35±5.48,59.03±6.99,67.47±10.10 ml·g-1, respectively. The NOR brain/plasma concentration ratios of mice from piperine (170μg/kg) was significantly lower than other groups (P<0.05).CONCLUSIONS1. MethodsIn this experiment, we describe a simple, selective and highly sensitive UPLC-MS-MS method for the determination of nortriptyline in mice plasma and brain.2. Animal experimentThe area under curve (AUCOâ†'∞) and peak concentration (Cmax) to mice treated with piperine was larger than treated with physiological saline, but there was no significantly different between them. The NOR brain/plasma concentration ratios of mice from piperine (170μg/kg) was significantly lower than other groups (P<0.05). After intragastric administration with piperine (170μg/kg) for 7 days, P-gp activity may be increased in BBB. But there is little influence treated with piperine (5 mg/kg) for 7 days.AUC and Cmax were significant higher than other groups, but the NOR brain/plasma concentration ratios was not significantly different from those of untreated mice.The NOR brain/plasma concentration ratios of mice from piperine (170μg/kg) was significantly lower than other groups and the piperine (170μg/kg) equal to a daily consumption of approximately 60-110μmol of piperine in populations that use regularly in their diets. Piperine (170μg/kg) may cause an induction of P-gp expression in BBB. So P-gp mediated drug interactions may occur in the treatment of central nervous system disease.