| Objective: Two sensitive methods (HPLC and LC/MS/MS) had been developed to quantify JBP485, a dipeptide with antihepatitis activity, in rat biological samples. PEPT1 and PEPT2 are two important transporters in the absorption and excretion of drugs. In this study, the effects of glycylsar- cosine on uptake of JBP485 were invest- igated in vitro, to clarify whether JBP485 is a potential substrate of PEPT1/ PEPT2.Methods: In HPLC method, Samples of plasma and bile were analyzed following a single step method of protein precipitation with trichloroacetic acid (10%, v/v). JBP485 was separated on a reversed-phase Agilent TC- C18 column (5μm, 4.6×250 mm) at 20?C. The mobile phase was comprised of methanol and water (5:95, v/v). Peaks eluting from the column were detected with an ultraviolet detector at a wavelength of 203 nm. Standard curves were linear in the range 0.5–250μg/ml, and correlation coefficients were 0.999. The intra- and inter-day precision (RSD) exceeded 4% and 5%, respectively. Extraction recovery was better than 82.08% in blood. In LC/MS/MS method, Following protein-precipitation with methanol, the analyte and internal standard (JBP923) were separated from rat plasma using an isocratic mobile phase on an Elite Hypersil ODS C18 column with a mobile phase consisting of methanol-water-formic acid (3:97:0.1,V:V:V) at a flow rate of 0.5ml/min. An API 3200 tandem mass spectrometer equipped with Turbo Ion Spray ionization source was used as detector and was oper- ated in the positive ion mode. Multiple reaction monitoring transporter precursor to product ion combinations of m/z 201.1→86.1 and m/z 219. 2.1→86.1 were performed to quantify JBP485 and internal standard, respectively. After JBP485 was injected intravenously and orally to rats, blood, bile, urine and tissue were collected at different time. The plasma concentration-time curve was plotted. The main pharmacokinetic para- meters of JBP485 were obtained by 3P97 software. Uptake of JBP485 in Caco-2 cells and kidney slices were examined with or without Gly-Sar. Effects of pH and temperature on JBP485 transport was also examined in Caco-2 cells. The concentration of JBP485 was determined by LC/MS/MS. Results: Two methods were successfully applied to investigate JBP485 plasma pharmacokinetics in rats. JBP485 appeared to have a linear phar- macokinetic characterization within doses of 6.25–100 mg/kg. The half-life was 2.32±0.16 h, plasma clearance was 2.66±0.15 ml/min/kg, volume of distribution was 0.18±0.01 l/kg when comparing the plasma concentra- tion-time profile of JBP485 after i.v, oral, and portal vein injection, it was suggested that JBP485 was well absorbed from gastrointestinal lumen, and hepatic first-pass removal was minor. Renal excretion was the main excre- tory route of JBP485. Uptake of JBP485 was decreased by Gly-Sar in caco-2 cells and kidney slices as compared to JBP485 alone. pH and temperature significantly influence JBP485 uptake in Caco-2 cells, Km=0.439mM,Vmax=1.622nmol/mg protein/30min. The maximal uptake of JBP485 was found at pH= 6.0.Conclusion: Two methods were suitable for the pharmacokinetic study of JBP485 in rat with the advantages of specificity, sensitivity, accuracy and high speed. Hepatic first-pass removal was minor. Renal excretion was the main excretive route of JBP485. The results demonstrate that JBP485 is the potential substrate of the H+/oligopeptide cotransporter PEPTs.
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