jbp-485 and glycylsarcosine

A simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of JBP485, Gly-Sar and JBP923 in the cell lysates using methanol as a deproteinization solvent was developed and validated. Detection was performed by turbo ionspray ionization in multiple reaction monitoring mode using the transitions of m/z 147.1 → m/z 90.1 for Gly-Sar, m/z 201.1 → m/z 86.1 for JBP485, m/z 219.1 → m/z 86.1 for JBP923 and m/z 152.0 → m/z 110.0 for paracetamol (internal standard). The analytes were separated on a Hypersil ODS C18 HPLC column using isocratic elution mode with a mobile phase containing 0.1% formic acid in water-methanol (97:3, v/v) at a flow rate of 0.2 mL/min. The calibration curves were demonstrated to be linear over the concentration range of 5.00-5000 nm with coefficient of 0.9968 for Gly-Sar, 0.9975 for JBP485 and 0.9952 for JBP923. The intra- and inter-day precisions were <10.2% for each quality contro; level, and the accuracy was within ±5.6% for each analyte. The matrix effect, the extraction recovery and stabilities of LC-MS/MS analysis were also investigated. This validated method was successfully applied to the simultaneous determination of JBP485, Gly-Sar and JBP923 in the cell lysates for identification of stably transfected HeLa cells with human PEPT1.

Topics: Chromatography, High Pressure Liquid; Dipeptides; Drug Stability; HeLa Cells; Humans; Peptide Transporter 1; Peptides, Cyclic; Reproducibility of Results; Symporters; Tandem Mass Spectrometry

Entecavir and JBP485 (a dipeptide) exhibit the antihepatitis activities and it is possible for the two drugs to be coadministered in the treatment of hepatitis. We aimed to elucidate whether entecavir was a substrate of OAT1, OAT3, OCT, and PEPT1 and to investigate the targets of drug-drug interactions between entecavir and JBP485. Plasma and urine concentrations of entecavir following intravenous and oral administration in vivo, uptake of entecavir in kidney slices and transfected cells in vitro, were determined by LC-MS/MS. Following intravenous co-administration of entecavir and JBP485 in rats, entecavir AUC increased 1.93-fold, t1/2β was prolonged 2.08-fold, CLP decreased 49%, CLR decreased 73%, and accumulated urinary excretion decreased 54%. However, following oral co-administration, the entecavir Tmax and Cmax were not affected; the degree of change in other pharmacokinetic parameters (AUC, t1/2β, CLP, and accumulated urinary excretion) was similar to that of intravenous administration. The uptake of entecavir was nearly identical in hPEPT1- as in vector-HELA cells. In rat kidney slices, uptake of entecavir was markedly inhibited by p-aminohippurate, benzylpenicillin, JBP485, and tetraethyl ammonium. In hOAT1- and hOAT3-HEK293 cells, uptake of entecavir was significantly higher compared to vector-HEK293 cells and was markedly inhibited by p-aminohippurate, benzylpenicillin, and JBP485. Km and Vmax values of entecavir were 250 μM and 0.83 nmol/mg protein/30s (OAT1) and 23 μM and 1.1 nmol/mg protein/30 s (OAT3), respectively. Entecavir is the substrate of OAT1, OAT3, and OCT. Moreover, OAT1 and OAT3 are the targets of DDI between entecavir and JBP485.

Topics: Animals; Antiviral Agents; Dipeptides; Drug Interactions; Guanine; HEK293 Cells; HeLa Cells; Humans; Kidney; Male; Organic Anion Transport Protein 1; Organic Anion Transporters, Sodium-Independent; Peptide Transporter 1; Peptides, Cyclic; Rats; Rats, Wistar; Symporters

The purpose of this study was to construct stably transfected HeLa cells with human peptide transporters (hPEPT1/hPEPT2) and to identify the function of the transfected cells using the substrate JBP485 (a dipeptide) and a typical substrate for PEPTs, glycylsarcosine (Gly-Sar). An efficient and rapid method was established for the preparation and transformation of competent cells of Escherichia coli. After extraction and purification, hPEPT1/hPEPT2-pcDNA3 was transfected into HeLa cells by the liposome transfection method, respectively. HeLa-hPEPT1/hPEPT2 cells were selected by measuring the protein expression and the uptake activities of JBP485 and Gly-Sar. A simple and rapid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of JBP485 and Gly-Sar in biological samples. The Michaelis-Menten constant (K(m)) values of Gly-Sar uptake by the hPEPT1 and hPEPT2-expressing transfectants were 1.03 mM and 0.0965 mM, respectively, and the K(m) values of JBP485 uptake were 1.33 mM for PEPT1 and 0.144 mM for PEPT2. The uptake of Gly-Sar was significantly inhibited by JBP485 with a K(i) value of 8.11 mM (for PEPT1) and 1.05 mM (for PEPT2). Maximal uptake of Gly-Sar were detected at pH 5.8 (for PEPT1) and pH 6.5 (for PEPT2), suggesting that both HeLa-hPEPT1 and HeLa-hPEPT2 were H(+) dependent transporters. Stably transfected HeLa-hPEPT1/HeLa-hPEPT2 cells were constructed successfully, and the functions of hPEPT1/hPEPT2 were identified using their substrates, JBP485 and Gly-Sar. The transfected cells with transporters were used to investigate drug-drug interactions (DDIs) between JBP485 and other substrates (cephalexin or lisinopril) of PEPT1 and PEPT2.

Topics: Biological Transport; Cell Engineering; Cephalexin; Chromatography, Liquid; Dipeptides; Drug Interactions; Escherichia coli; Gene Expression; HeLa Cells; Humans; Hydrogen-Ion Concentration; Kinetics; Liposomes; Lisinopril; Peptide Transporter 1; Peptides, Cyclic; Plasmids; Symporters; Tandem Mass Spectrometry; Transfection

To investigate the effect of JBP485 (an anti-inflammatory dipeptide) on PEPT1 in indomethacin-induced intestinal injury in rats and damage in Caco-2 cells, the activity and expression of PEPT1 were examined. The effects of treatment with indomethacin and co-treatment with JBP485 were examined in terms of intestinal histological changes, MDA and MPO levels in rats; as well as LDH-release and oxidative stress in Caco-2 cells. Uptake of glycylsarcosine (Gly-Sar) by PEPT1 was determined by in vivo, in vitro and in situ studies. RT-PCR and Western blot were used to assess the expression of PEPT1 in rat intestine and Caco-2 cells. JBP485 caused a significant decrease in MDA and MPO levels, and improved the pathological condition of rat intestine, while attenuating Caco-2 cells damage induced by indomethacin. Uptake of Gly-Sar by PEPT1 was decreased by indomethacin treatment, whereas the Gly-Sar plasma concentration was markedly increased in JBP485 co-treated rats. Indomethacin down-regulated the expression of PEPT1 mRNA and protein in rat intestine and Caco-2 cells, and the effects were reversed after administration of JBP485. These results indicated that JBP485 not only improved intestinal injury and cell damage but also partially blocked the down-regulation of PEPT1 expression and function induced by indomethacin.

Topics: Animals; Blotting, Western; Caco-2 Cells; Chromatography, Liquid; Dipeptides; Humans; In Vitro Techniques; Indomethacin; Intestinal Mucosa; Intestines; Lipid Peroxidation; Male; Oxidative Stress; Peptide Transporter 1; Peptides, Cyclic; Peroxidase; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Symporters; Tandem Mass Spectrometry

To investigate the pharmacokinetics and mechanism of intestinal absorption of JBP485 in rats, the pharmacokinetics of JBP485 were investigated in vivo both intravenously and orally. The effects of glycylsarcosine (Gly-Sar) on the uptake and transepithelial transport of JBP485 were examined in everted intestinal sacs, in situ jejunal perfusion, Caco-2 cells and PEPT1 transfected Hela cells. The gastrointestinal absorption of JBP485 was rapid. T(1/2β) was 2.25 ± 0.06 h, CL(plasma) was 2.99 ± 0.002 ml/min/kg, V(d) was 0.22 ± 0.05 l/kg and bioavailability was about 30% at a dosage of 25 mg/kg. JBP485 underwent rapid distribution in the tissues. Gly-Sar significantly decreased JBP485 uptake and transport in these models. A kinetic study showed that JBP485 was transported by PEPT1 in Caco-2 cells with Km and Vmax values of 0.33 ± 0.13 mM and 0.72 ± 0.06 nmol/mg protein/10 min, respectively. JBP485 appeared to have linear pharmacokinetics at intravenous doses of 6.25-100 mg/kg with minor first-pass effect, and JBP485 was mainly distributed in the kidney; JBP485 is a substrate for PEPT1 which is involved in the absorption of JBP485 in rat intestine.

Topics: Animal Structures; Animals; Area Under Curve; Biological Availability; Caco-2 Cells; Dipeptides; HeLa Cells; Humans; Intestinal Absorption; Intestine, Small; Male; Peptide Transporter 1; Peptides, Cyclic; Perfusion; Rats; Rats, Wistar; Symporters; Tissue Distribution

It has been a desire to develop orally effective therapeutic agents that restore the liver function in chronic injury. Here we demonstrated that trans-4-L-hydroxyprolyl-L-serine (JBP923) and cyclo-trans-4-L-hydroxyprolyl-L-serine (JBP485), which was previously isolated from hydrolysate of human placenta, exhibit potent antihepatitis activity after their oral administration. The increase in bilirubin concentration and activities of liver cytosolic enzymes in serum caused by alpha-naphthylisothiocyanate intoxication in rats were significantly countered both after i.v. and oral administration of these dipeptides, whereas glycyrrhizin, which has been used in the treatment of chronic hepatitis, is active only after its i.v. administration. Antihepatitis activity of dipeptides results, at least partially, from their direct effect on hepatocytes because glutamic-oxaloacetic transaminase and lactate dehydrogenase activities in the medium of hepatotoxin-exposed primary cultured hepatocytes were reduced by these compounds. When comparing the plasma concentration-time profile of JBP923 after its i.v., oral, and portal vein injection, it is suggested that JBP923 is almost completely absorbed from gastrointestinal lumen, and hepatic first-pass removal is minor. JBP923 inhibited the proton-dependent transport of glycylsarcosine in brush-border membrane vesicles, suggesting that peptide transport system(s) may recognize JBP923. Thus, these dipeptides are potent antihepatitis reagents that are still active after oral administration and may be useful for clinical applications.

Topics: 1-Naphthylisothiocyanate; Administration, Oral; Animals; Anti-Inflammatory Agents, Non-Steroidal; Carbon Radioisotopes; Cells, Cultured; Chemical and Drug Induced Liver Injury; Cytosol; Dipeptides; Gastric Mucosa; Glycyrrhizic Acid; Intestinal Absorption; Intestinal Mucosa; Intestines; Liver; Male; Microvilli; Peptides, Cyclic; Rabbits; Rats; Rats, Wistar