JBP485, A Dual Inhibitor of Organic Anion Transporters (OATs) and Renal Dehydropeptidase-I (DHP-I), Protects Against Imipenem-Induced Nephrotoxicity.

Imipenem (IMP) possesses a broad spectrum of antibacterial activity; however, nephrotoxicity limits its clinical application in patients with renal insufficiency. In our previous studies, a dipeptide, JBP485, a dipeptide with the chemical structure cyclo-trans-4-L-hydroxyprolyl-L-serine, was found to attenuate drug-induced kidney injury. The current study aimed to explore whether JBP485 could relieve IMP-induced kidney injury and clarify the potential molecular pharmacokinetic mechanism. The effects of JBP485 on IMP nephrotoxicity were evaluated in rabbits and human kidney 2 (HK-2) cells. Drug-drug interactions (DDIs) mediated by organic anion transporters (OATs) and dehydropeptidase-I (DHP-I) were explored through pharmacokinetic studies in rats, metabolism assays in the kidney, and uptake studies in OAT-over-expressing cells. The results revealed that JBP485 significantly ameliorated IMP-induced nephrotoxicity in rabbits. Further, incubation of HK-2 cells with JBP485 or cilastatin markedly improved the cell survival rate, inhibited apoptosis and attenuated mitochondrial damage by improving the stability of IMP and reducing its intracellular accumulation. This suggests that DHP-I and OATs might be involved in the protective effect of JBP485. Furthermore, coadministration with JBP485 significantly increased the IMP's plasma concentration as well as the area under the plasma concentration-time curve (AUC), while decreasing IMP renal clearance and cumulative urinary excretion. Moreover, JBP485 reduced IMP uptake in kidney slices and OAT1/3-human embryonic kidney 293 (HEK293) cells. At the same time, the metabolism of IMP by DHP-I was inhibited by JBP485 with an IC50 value of 12.15 ± 1.22 µM. Finally, the molecular docking assay revealed a direct interaction between JBP485 and OAT1/3 or DHP-I. In conclusion, JBP485 protected against IMP nephrotoxicity in rabbits and HK-2 cells by improving IMP stability and reducing its intracellular accumulation via simultaneous inhibition of renal OATs and DHP-I. JBP485 is a promising renoprotective agent and could serve as an effective supplement to reduce IMP-induced adverse renal reactions in the clinical setting.

A farewell to phlebotomy-use of placenta-derived drugs Laennec and Porcine for improving hereditary hemochromatosis without phlebotomy: a case report.

BACKGROUND: Human hepcidin, produced by hepatocytes, regulates intestinal iron absorption, iron recycling by macrophages, and iron release from hepatic storage. Recent studies indicate that hepcidin deficiency is the underlying cause of the most known form of hereditary hemochromatosis. CASE PRESENTATION: A 44-year-old Asian man who developed type 2 diabetes mellitus had elevated serum ferritin levels (10,191 ng/mL). Liver biopsy revealed remarkable iron deposition in the hepatocytes and relatively advanced fibrosis (F3). Chromosomal analysis confirmed the presence of transferrin receptor type 2 mutations (c.1100T>G, c.2008_9delAC, hereditary hemochromatosis type 3 analyzed by Kawabata). The patient received intravenous infusions of Laennec (672 mg/day, three times/week) or oral administration with Porcine (3.87 g/day) for 84 months as an alternative to repeated phlebotomy. At the end of the treatment period, serum ferritin level decreased to 428.4 ng/mL (below the baseline level of 536.8 ng/mL). Hemoglobin A1c levels also improved after treatment with the same or lower dose of insulin (8.8% before versus 6.8% after). Plural liver biopsies revealed remarkable improvements in the grade of iron deposition and fibrosis (F3 before versus F1 after) of the liver tissue. CONCLUSION: The discovery of hepcidin and its role in iron metabolism could lead to novel therapies for hereditary hemochromatosis. Laennec (parenteral) and Porcine (oral), which act as hepcidin inducers, actually improved iron overload in this hereditary hemochromatosis patient, without utilizing sequential phlebotomy. This suggests the possibility of not only improving the prognosis of hereditary hemochromatosis (types 1, 2, and 3) but also ameliorating complications, such as type 2 diabetes, liver fibrosis, and hypogonadism. Laennec and Porcine can completely replace continuous venesection in patients with venesection and may improve other iron-overloading disorders caused by hepcidin deficiency.

Placental extract ameliorates liver fibrosis in a methionine- and choline-deficient diet-induced mouse model of non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a severe form of fatty liver disease that is defined by the presence of inflammation and fibrosis, which ultimately leads to cirrhosis and hepatocellular carcinoma. We previously showed that human placental extract (hPE) was intramuscularly injected to ameliorates liver injury in a methionine- and choline-deficient (MCD) diet-induced NASH model. In the present study, we investigated the effects of hPE using dB/dB mice which exhibit obesity and insulin resistance and are thought to reproduce the pathological background of NASH. The MCD-diet induced liver atrophy accompanied by fibrosis around the liver sinusoids. hPE dose-dependently reduced the perivascular fibrosis. Moreover, αSMA-positive activated hepatic stellate cells increased in number in mice on the MCD diet, with this effect reversed by hPE treatment. hPE significantly decreased expression of Acta2, Col1a1, and Tgfb1 genes in hepatic stellate cells, and inhibited Smad phosphorylation. Moreover, hPE treatment increased the expression of the anti-oxidative genes Hmox1, Nqo1, Cat, and Sod1, and significantly enhanced nuclear factor erythroid 2-related factor 2 activity. Furthermore, hPE decreased the expression of Nox4 and attenuated the levels of intracellular reactive oxygen species. These results, along with our previous study, suggest that hPE effectively ameliorates liver fibrosis in NASH. This beneficial effect may, in part, be due to suppression of hepatic stellate cell activation.

Placental extract suppresses cardiac hypertrophy and fibrosis in an angiotensin II-induced cachexia model in mice.

Cachexia is an intractable metabolic disorder that causes extreme weight loss. It is a symptom of many chronic diseases, including cancer, liver failure, congestive heart failure and chronic kidney disease, and there is as yet no effective treatment. While the mechanisms underlying cachexia are complex, it is often accompanied by elevated angiotensin II (Ang II). Human placental extract (HPE) is a source of numerous biologically active molecules and has been used clinically to treat chronic hepatitis, liver cirrhosis and other chronic diseases. Here, we investigated the effects of HPE in an Ang II-induced cachexia model in mice. HPE treatment preserved both fat mass and lean body mass and suppressed weight loss in the cachexia model, though food intake was unaffected. Ang II infusion also caused cardiac hypertrophy and fibrosis. HPE suppressed these effects as well as Ang II-induced cardiac expression of genes related to heart failure and cardiac remodeling. HPE also reversed Ang II-induced downregulation of mitochondria-related molecules and suppressed cardiac inflammation and oxidative stress. HPE administration may thus be an effective approach to the treatment of cachexia, cardiac hypertrophy and fibrosis.

Porcine placental extract facilitates memory and learning in aged mice.

Aging induces a decline in both memory and learning ability without predisposing an individual to diseases of the central nervous system, such as dementia. This decline can have a variety of adverse effects on daily life, and it can also gradually affect the individual and the people they are surrounded by. Since recent evidence indicated that placental extract has effects on brain function such as memory, we hypothesized that placental extract could ameliorate the age-associated reduction in cognitive function in aging. Here, we investigated the effect of new modified porcine placental extract (SD-F) on memory ability in aged mice at both the behavioral and molecular levels. Our results revealed that SD-F significantly enhanced memory ability in the object recognition and object location tasks in a dose-dependent manner in aged mice relative to controls. The numbers of Nissl-positive cells in the hippocampal cornu ammonis 3 (CA3) and dentate gyrus (DG) regions were increased in SD-F-treated aged mice relative to controls. RNA-seq analysis of the hippocampus of aged mice identified 542 differentially expressed genes, of which 216 were up-regulated and 326 were down-regulated in SD-F-treated mice relative to controls. Of the 216 up-regulated genes, we identified four characteristic genes directly related to memory, including early growth response protein 1 (Egr1), growth arrest and DNA-damage-inducible, beta (Gadd45b), NGFI-A binding protein 2 (Nab2), and vascular endothelial growth factor a (Vegfa). These results suggest that the efficacy of SD-F involves upregulation of these genes.

Development of a mouse iron overload-induced liver injury model and evaluation of the beneficial effects of placenta extract on iron metabolism.

Hepatic iron deposition is seen in cases of chronic hepatitis and cirrhosis, and is a hallmark of a poorer prognosis. Iron deposition is also found in non-alcoholic steatohepatitis (NASH) patients. We have now developed a mouse model of NASH with hepatic iron deposition by combining a methione- and choline-deficient (MCD) diet with an iron-overload diet. Using this model, we evaluated the effects of human placenta extract (HPE), which has been shown to ameliorate the pathology of NASH. Four-week-old male C57BL/6 mice were fed the MCD diet with 2% iron for 12 weeks. In liver sections, iron deposition was first detected around the portal vein after 1 week. From there it spread throughout the parenchyma. Biliary iron concentrations were continuously elevated throughout the entire 12-week diet. As a compensatory response, the diet caused elevation of serum hepcidin, which accelerates excretion of iron from the body. Accumulation of F4/80-positive macrophages was detected within the sinusoids from the first week onward, and real-time PCR analysis revealed elevated hepatic expression of genes related inflammation and oxidative stress. In the model mice, HPE treatment led to a marked reduction of hepatic iron deposition with a corresponding increase in biliary iron excretion. Macrophage accumulation was much reduced by HPE treatment, as was the serum oxidation-reduction potential, an index of oxidative stress. These data indicate that by suppressing inflammation, oxidative stress and iron deposition, and enhancing iron excretion, HPE effectively ameliorates iron overload-induced liver injury. HPE administration may thus be an effective strategy for treating NASH.

Placental extract ameliorates non-alcoholic steatohepatitis (NASH) by exerting protective effects on endothelial cells.

Non-alcoholic steatohepatitis (NASH) is a severe form of fatty liver disease that is defined by the presence of inflammation and fibrosis, ultimately leading to cirrhosis and hepatocellular carcinoma. Treatment with human placental extract (HPE) reportedly ameliorates the hepatic injury. We evaluated the effect of HPE treatment in a mouse model of NASH. In the methione- and choline-deficient (MCD) diet-induced liver injury model, fibrosis started from regions adjacent to the sinusoids. We administered the MCD diet with high-salt loading (8% NaCl in the drinking water) to mice deficient in the vasoprotective molecule RAMP2 for 5 weeks, with or without HPE. In both the HPE and control groups, fibrosis was seen in regions adjacent to the sinusoids, but the fibrosis was less pronounced in the HPE-treated mice. Levels of TNF-α and MMP9 expression were also significantly reduced in HPE-treated mice, and oxidative stress was suppressed in the perivascular region. In addition, HPE dose-dependently increased survival of cultured endothelial cells exposed to 100 µM H2O2, and it upregulated expression of eNOS and the anti-apoptotic factors bcl-2 and bcl-xL. From these observations, we conclude that HPE ameliorates NASH-associated pathologies by suppressing inflammation, oxidative stress and fibrosis. These beneficially effects of HPE are in part attributable to its protective effects on liver sinusoidal endothelial cells. HPE could thus be an attractive therapeutic candidate with which to suppress progression from simple fatty liver to NASH.

JBP485 promotes corneal epithelial wound healing.

Proper wound healing is vital for maintenance of corneal integrity and transparency. Corneal epithelial damage is one of the most frequently observed ocular disorders. Because clinical options are limited, further novel treatments are needed to improve clinical outcomes for this type of disease. In the present study, it was found that placental extract-derived dipeptide (JBP485) significantly increased the proliferation and migration of corneal epithelial cells (CECs). Moreover, JBP485 accelerated corneal epithelial wound healing in vivo without inflammation and neovascularization and was found to be effective for the treatment of corneal damage. These data indicate that JBP485 efficiently activates the viability of CECs and has potential as a novel treatment for various kinds of corneal epithelial disease.

JBP485 promotes tear and mucin secretion in ocular surface epithelia.

Dry eye syndrome (DES), a multifactorial disease of the tears and ocular surface, is one of the most common ocular disorders. Tear film contains ocular mucins and is essential for maintaining the homeostasis of the wet ocular surface. Since there are a limited number of clinical options for the treatment of DES, additional novel treatments are needed to improve the clinical results. In this study, we found that placental extract-derived dipeptide (JBP485) clearly promoted the expression and secretion of gel-forming mucin 5ac (Muc5ac) in rabbit conjunctival epithelium. JBP485 also elevated the expression level of cell surface-associated mucins (Muc1/4/16) in rabbit corneal epithelium. The Schirmer tear test results indicated that JBP485 induced tear secretion in the rabbit model. Moreover, JBP485 clinically improved corneal epithelial damage in a mouse dry eye model. Thus, our data indicate that JBP485 efficiently promoted mucin and aqueous tear secretion in rabbit ocular surface epithelium and has the potential to be used as a novel treatment for DES.

Simultaneous determination of three dipeptides (JBP485, Gly-Sar and JBP923) in the cell lysates by liquid chromatography-tandem mass spectrometry: application to identify the function of the PEPT1 transfected cell.

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.

JBP485 improves gentamicin-induced acute renal failure by regulating the expression and function of Oat1 and Oat3 in rats.

We investigated the effects of JBP485 (an anti-inflammatory dipeptide and a substrate of OAT) on regulation of the expression and function of renal Oat1 and Oat3, which can accelerate the excretion of accumulated uremic toxins (e.g. indoxyl sulfate) in the kidney to improve gentamicin-induced ARF in rats. JBP485 caused a significant decrease in the accumulation of endogenous substances (creatinine, blood urea nitrogen and indoxyl sulfate) in vivo, an increase in the excretion of exogenous compounds (lisinopril and inulin) into urine, and up-regulation of the expressions of renal Oat1 and Oat3 in the kidney tissues and slices via substrate induction. To determine the effect of JBP485 on the accelerated excretion of uremic toxins mediated by Oat1 and Oat3, the mRNA and protein expression levels of renal basolateral Oats were assessed by quantitative real-time PCR, western blot, immunohistochemical analysis and an immunofluorescence method. Gentamicin down-regulated the expression of Oats mRNA and protein in rat kidney, and these effects were reversed after administration of JBP485. In addition, JBP485 caused a significant decrease in MPO and MDA levels in the kidney, and improved the pathological condition of rat kidney. These results indicated that JBP485 improved acute renal failure by increasing the expression and function of Oat1 and Oat3, and by decreasing overoxidation of the kidney in gentamicin-induced ARF rats.

OAT1 and OAT3: targets of drug-drug interaction between entecavir and JBP485.

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, t1/2ß 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, t1/2ß, 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.

Inhibitory effect of 1α,25-dihydroxyvitamin D3 on excretion of JBP485 via organic anion transporters in rats.

The aim of this study was to investigate the pharmacokinetic mechanism of interaction between JBP485 and 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. Rats were injected intraperitoneally with 0.64 nmol/kg/day 1,25(OH)(2)D(3) in 1 ml/kg corn oil for 5 days. The plasma and urine concentrations of JBP485 after intravenous administration and the uptake of JBP485 in kidney slices in vitro were determined by liquid chromatography/tandem mass spectrometry. Quantitative polymerase chain reaction, western blotting, immunohistochemical analysis and immunofluorescence were used to determine the changes in the expression of organic anion transporter (Oat)1 and Oat3 in rat kidney in response to 1,25(OH)(2)D(3) treatment. The plasma concentrations and AUCs of JBP485 were significantly increased, while the renal clearance of JBP485 and uptake of JBP485 in kidney slices were significantly decreased after 1,25(OH)(2)D(3) treatment. These results confirmed that 1,25(OH)(2)D(3) inhibited renal excretion of JBP485. Moreover, 1,25(OH)(2)D(3) decreased expression of Oat1 and Oat3 in rat kidney. Our results are novel in demonstrating an interaction between JBP485 and 1,25(OH)(2)D(3) when they are co-administered. The mechanism of interaction between JBP485 and 1,25(OH)(2)D(3) could be explained at least in part by inhibitory effect of 1,25(OH)(2)D(3) on expression of Oats in rat kidney.

Changes in expression of renal Oat1, Oat3 and Mrp2 in cisplatin-induced acute renal failure after treatment of JBP485 in rats.

The purpose of this study is to investigate whether the effect of cyclo-trans-4-l-hydroxyprolyl-l-serine (JBP485) on acute renal failure (ARF) induced by cisplatin is related to change in expression of renal Oat1, Oat3 and Mrp2 in rats. JBP485 reduced creatinine, blood urea nitrogen (BUN) and indoxyl sulfate (IS) in plasma and malondialdehyde (MDA) in kidney, and recovered the glomerular filtration rate (GFR) and the activity of superoxide dismutase (SOD) in cisplatin-treated rats. The plasma concentration of PAH (para-aminohippurate) determined by LC-MS/MS was increased markedly after intravenous administration of cisplatin, whereas cumulative urinary excretion of PAH and the uptake of PAH in kidney slices were significantly decreased. qRT-PCR and Western-blot showed a decrease in mRNA and protein of Oat1 and Oat3, an increase in mRNA and protein of Mrp2 in cisplatin-treated rats, and an increase in IS (a uremic toxin) after co-treatment with JBP485. It indicated that JBP485 promoted urinary excretion of toxins by upregulating renal Mrp2. This therefore gives in part the explanation about the mechanism by which JBP485 improves ARF induced by cisplatin in rats.

Inhibitory effect of JBP485 on renal excretion of acyclovir by the inhibition of OAT1 and OAT3.

The purpose is to investigate whether the targets of drug-drug interactions (DDIs) between JBP485 and acyclovir are OAT1 and OAT3 in kidney. Plasma concentration and accumulative urinary excretion of acyclovir in vivo, uptake of acyclovir in kidney slices and uptake of acyclovir in human (h) OAT1/ hOAT3-human embryonic kidney (HEK) 293 cells in vitro were performed to examine the effect of JBP485 on urinary excretion of acyclovir. The plasma concentration of acyclovir was increased markedly and accumulative urinary excretion and renal clearance of acyclovir were decreased significantly after intravenous administration of acyclovir in combination with JBP485. JBP485 (a substrate for OAT1 and OAT3), p-aminohippurate (PAH) (a substrate for OAT1) and benzylpenicillin (PCG) (a substrate for OAT3) could decrease the uptake of acyclovir in kidney slices and in hOAT1-/hOAT3-HEK293 cells. These results suggest that JBP485 inhibits the renal excretion of acyclovir by inhibiting renal transporters OAT1 and OAT3 in vivo and in vitro. Our results indicate the possibility of DDI between dipeptide and acyclovir.

Effect of JBP485 on obstructive jaundice is related to regulation of renal Oat1, Oat3 and Mrp2 expression in ANIT-treated rats.

The objective was to determine whether protective effects of JBP485 on biliary obstruction induced by alpha-naphthylisothiocyanate (ANIT) are mediated by the organic anion transporters Oat1, Oat3 and the multidrug resistance-associated protein Mrp2. The ANIT-induced increases in bilirubin (BIL), alanine aminotransferase (ALT) and aspartate transaminase (AST) in rat serum were inhibited significantly by oral administration of JBP485. The plasma concentration of JBP485 which is the substrate of Oat1 and Oat3 determined by LC-MS/MS was markedly increased after intravenous administration in ANIT-treated rats, whereas cumulative urinary excretion of JBP485 in vivo and the uptake of JBP485 in kidney slices were decreased remarkably. RT-PCR and Western blot showed the decreased expression of Oat1 and Oat3, increased expression of Mrp2 in ANIT-induced rats, meanwhile, the expression levels of Mrp2 and Oat1 were up-regulated after administration of JBP485. The up-regulation of Mrp2 and Oat1 was associated with a concomitant increase in urinary BIL after treatment with JBP485 in ANIT-treated rats. The mechanism for JBP485 to restore liver function might be related to improvement of the expression and function for Oat1 and Mrp2 as well as facilitation of urinary excretion for hepatoxic substance.

Construction, identification and application of HeLa cells stably transfected with human PEPT1 and PEPT2.

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.

Organic anion transporters involved in the excretion of bestatin in the kidney.

Bestatin, a dipeptide, a low molecular weight aminopeptidase inhibitor, has been demonstrated to be an immunomodulator with an antitumor activity. However, the transporter-mediated renal excretion of bestatin is not fully understood. The purpose of this study was to elucidate the transporter-mediated renal excretion mechanism for bestatin. The plasma concentration of bestatin was increased markedly and both the accumulative renal excretion and renal clearance of bestatin were decreased significantly after intravenous administration of bestatin in combination with probenecid. p-Aminohippuric acid (PAH), a substrate of organic anion transporter (OAT) 1, benzylpenicillin (PCG), a substrate of OAT3 and JBP485, a substrate of OAT1 and OAT3, reduced the uptake of bestatin in rat kidney slices and in hOAT1- or hOAT3-HEK 293 cells. The accumulation of bestatin in hOAT1-HEK and hOAT3-HEK 293 cells was significantly greater than that in vector-HEK, and the K(m) and V(max) were 0.679 ± 0.007 mM and 0.807 ± 0.006 nmol/mg protein/30s for OAT1, 0.632 ± 0.014 mM and 1.303 ± 0.015 nmol/mg protein/30s for OAT3 respectively. PAH and JBP485 inhibited significantly the uptake of bestatin in hOAT1-HEK with the K(i) values of 92 ± 9 µM and 197 ± 21 µM; and PCG, JBP485 inhibited significantly the uptake of bestatin in hOAT3-HEK 293 cells with the K(i) values of 88 ± 12 µM and 160 ± 16 µM. Our results are novel in demonstrating for the first time that OAT1 and OAT3 are involved in the renal excretion of bestatin.

Peptide cotransporter 1 in intestine and organic anion transporters in kidney are targets of interaction between JBP485 and lisinopril in rats.

The purpose of this study was to clarify the pharmacokinetic mechanism of interaction between JBP485 (cyclo-trans-4-L-hydroxyprolyl-L-serine, a dipeptide with antihepatitis activity) and lisinopril (an angiotensin-converting enzyme inhibitor) in vitro and in vivo. When JBP485 and lisinopril were administered orally simultaneously, the plasma concentrations of the two drugs were decreased significantly, but few changes were observed after simultaneous intravenous administration of the two drugs. The uptake of JBP485 and lisinopril in everted intestinal sacs and in HeLa cells transfected with human peptide cotransporter 1 (PEPT1), as well as absorption of JBP485 and lisinopril after jejunal perfusion were reduced after simultaneous drug administration, which suggested that the first target of drug interaction was PEPT1 in the intestine during the absorption process. The cumulative urinary excretions and renal clearance of the two drugs were decreased after intravenous co-administration, while uptakes of the two drugs in kidney slices and hOAT1/hOAT3-transfected HEK293 cells were decreased. These results indicated that the second target of drug-drug interaction was located in the kidney. These findings confirmed that the pharmacokinetic mechanism of interaction between JBP485 and lisinopril could be explained by their inhibition of the same transporters in the intestinal mucosa (PEPT1) and kidneys (OATs).

Inhibitory effect of zinc on the absorption of JBP485 via the gastrointestinal oligopeptide transporter (PEPT1) in rats.

The aim of this study was to investigate the pharmacokinetic mechanism of interaction between JBP485 and zinc. The plasma concentration of JBP485 after oral administration in vivo, the plasma concentration of JBP485 from the portal vein after jejunal perfusions in situ, the serosal fluid concentration of JBP485 in everted small intestine preparations and the uptake of JBP485 by HeLa-hPEPT1 cells in vitro were determined by LC-MS/MS. RT-PCR and Western blotting were used to determine the mRNA and protein levels of Pept1 in the intestinal mucosa. The AUCs of JBP485 in in vivo, in vitro and in situ studies were significantly decreased after zinc pre-administration. Kinetic analysis showed that zinc inhibits the uptake of JBP485 by decreasing the affinity of JBP485 for PEPT1 in HeLa-hPEPT1 cells. RT-PCR and Western blotting indicated that zinc had no effect on basal intestinal Pept1 expression. Our results are novel in demonstrating for the first time that zinc ions, but not zinc gluconate, can inhibit the transport activity of PEPT1. In addition, the uptake of JBP485 was not affected by changes in pH values after zinc treatment. Zinc decreases the absorption of JBP485 by inhibiting the transport activity of PEPT1; however, basal intestinal Pept1 expression does not change.