Pharmacokinetic Study Of Aminopeptidase N Inhibitor In Rats

ObjectiveTo screen an Aminopeptidase N Inhibitor with high antitumor activities, and establish a method for determining concentrations of the Aminopeptidase N Inhibitor and its major metabolite in rats plasma and study its pharmacokinetics.Methods1. The candidate compounds were assessed by HPLC and MTT.2. Plasma samples of Bes-5FU were dealt with protein precipitation. The analytical column was a Venusil ASB C18column (4.6mm×250mm,5μm). Separation of Bes-5FU was achieved by isocratic elution using acetonitrile-0.05%formic acid (22:78, v/v). The liquid flow rate was set at1ml/min and the UV detection wavelength of Bes-5FU was262.7nm. The stability, precision, extraction recovery and calibration curve of5fluorouracil were carried out in rat plasma.2. Plasma samples of5-fluorouracil was dealt with liquid-liquid extraction. The analytical column was a Venusil ASB C18column (4.6mm×250mm,5μm). Separation of5fluorouracil was achieved by isocratic elution using methanol-water (5:95, v/v). The liquid flow rate was set at1ml/min and5FU was detected by UV absorption at266.3nm. The stability, precision, extraction recovery and calibration curve of5fluorouracil were carried out in rat plasma.3. Six male Wistar rats, weighing250g-260g, were fasted12h and with free water. Blood samples were collected in heparinized EP tubes from oculi chorioideae vein at0.033,0.083,0.167,0.333,0.5,1,2,3,4,6,8and10h after intravenous administration. The blood samples were centrifuged immediately at4℃,8609×g for5min to obtain plasmas samples which were respectively processed following the method described above. The plasma concentrations of Bes-5FU and5FU at different time points were expressed as the mean±SD and the pharmacokinetic calculations were performed by non-compartmental method using the DAS2.0statistical software (Pharmacology Institute of China).Results1. Bes-5FU was needed further research because of the high antitumor activities and good stability.2. The typical retention time of Bes-5FU was11.5min; The mean linear regression equation of calibration curve was:y=5336.1x-1702.1(r=0.9996), where y is the peak area of Bes-5FU and x is the concentration of Bes-5FU; The lower limit of quantification (LLOQ) of the method was0.3μg/ml. The extraction recoveries of Bes-5FU under the protein precipitation conditions were65.3±5.2%,66.7±4.3%and68.2±4.1%(n=6) at concentrations of0.5,5.0and10.0g/mL (QC samples), respectively; The intra-and inter-day precision of Bes-5FU determinations in plasma were less than15%(n=6).3. The typical retention time of5FU was6min; The mean linear regression equation of calibration curve was:y=37118x-9030.1(r=0.9998), where y is the peak area of5FU and x is the concentration of5FU; The lower limit of quantification (LLOQ) of the method was0.1μg/ml. The extraction recoveries of5FU under the liquid-liquid extraction conditions were70.4±7.4%,76.2±7.1%and75.7±6.8%(n=6) at concentrations of0.25,5.0and10.0μg/mL (QC samples), respectively; The intra-and inter-day precision of5FU determinations in plasma were less than10.0%(n=6).4. Bes-5FU and its major metabolite can be detected in the real rat plasma sample after intravenous administration of Bes-5FU(84mg/kg). The main pharmacokinetics parameters of5FU, for example,t1/2(h)、MRT0-t(h)、AUC0-t(μg·h/mL)、 AUC0-∞>(μg·h/mL)、AUMC0-t(μg·h2/mL)、AUMC0-∞(μg·h2/mL) were0.14±0.04,0.17±0.04,17.73±6.27,17.78±6.31,2.77±0.59,3.07±0.63, respectively.Conclusion1. Bes-5FU was needed further research because of the high antitumor activities.2. The method is accurate and simple with the high sensitivity and will be valuable for the the pharmacokinetic study of Bes-5FU and its major metabolite.3. Bes-5FU can quickly metabolize into5-fluorouracil with high antitumor activities in rats.