Uptake, Transport And Regulation Of JBP485 By PEPT1 In Vitro And In Vivo

Objective: Cyclo-trans-4-L-hydroxyprolyl-L-serine ( JBP485 ) is a dipeptide with anti-hepatitis activity that has been chemically synthesized. Previous experiments in rats showed that JBP485 was well absorbed by the intestine after oral administration. The human peptide transporter (PEPT1) is expressed in the intestine and recognizes compounds such as dipeptides and tripeptides. The purposes of this study were to determine if JBP485 acted as a substrate for intestinal PEPT1, and to investigate the characteristics of JBP485 uptake ,transport and regulation by PEPT1.Methods : A rapid and selective liquid chromatographic/tandem mass spectrometric method has previously been developed to determine JBP485 in biological samples. (1): The time and pH dependent studies of 0.5 mM JBP485 were taken in Caco-2 cells. The concentration dependence of the initial uptake of JBP485 was studied over the range from 0.05– 5 mM. The Michaelis-Menten constant ( Km) and the maximal velocity( Vmax ) were calculated by Eadie-Hofstee plot. Then the uptake of 0.5 mM JBP485 was measured in the absence and presence of various concentrations of glycylsarcosine (Gly-Sar, a typical substrate for PEPT1 transporters), JBP923 (a derivative of JBP485), and cephalexin (CEX, aβ-lactam antibiotic and a known substrate of PEPT1) to assess the interactions of these compounds with PEPT1-mediated JBP485 uptake in Caco-2 cells. (2): The transport of 0.5 mM JBP485 across cell monolayers cultured on permeable filters was measured at pH6.0 at the apical and pH7.4 at the basolateral side. The potential role of PEPT1 in JBP485(0.5 mM)transport was assayed using Gly-Sa(r6 mM), JBP923 (5 mM), and CEX(5 mM)as specific inhibitors of the transport system. (3): The regulations of Ca2+ channel blockers, insulin, zinc and alcohol on the uptake of 0.25 mM JBP485 were investigated in Caco-2 cells.(4): The regulation of verapamil on JBP485 absorption was determined in rats when coadministered with 20 mg/kg verapamil and 25 mg/kg JBP485. Then the relative effect of acetaldehyde ( AcH ) on JBP485 absorption was also determined. The rats were administered a bolus oral dose of JBP485(25 mg/kg)on the sixth day of ethanol treatment. (5): Cells were incubated with 5 mM JBP485 for 24 h and total RNA was extracted to determin the change of PEPT1 expression.Results: JBP485 uptake at pH6.0 was higher than that at pH 7.4. JBP485 uptake was also significantly inhibited by Gly-Sar, JBP923, and CEX, with Ki values of 16.94 mM, 0.77 mM, and 0.26 mM,respectively. The uptake was saturable at 37°C, and Km and Vmax were 1.7(6mM) and 1.7(1nmol/mg protein per 10 min). The rate of apical-to-basolateral transepithelial transport of JBP485 was 1.84 times higher than that for basolateral-to-apical transport. JBP485 transport was obviously inhibited by Gly-Sar, JBP923 and CEX. The uptake of JBP485 was increased by verapamil and inhibited by the presence of Zinc or AcH. Also the absorption of JBP485 was increased by verapamil and decreased by ethanol in vivo. PEPT1 mRNA levels were strongly enhanced after exposure of the cells to JBP485 for 24 h, compared to control.Conclusion: (1): These findings strongly suggest that JBP485 is a substrate of PEPT1, and that its absorption is actively mediated by PEPT1. (2): The uptake of JBP485 was increased by verapamil and inhibited by the presence of Zn2+ or the toxic metabolite of ethanol, acetaldehyde (AcH), and this phenomenon could have important meaning to rational guide the use of drugs in clinic. (3): JBP485 could increase the expression of PEPT1 at mRNA level.