Conversion of Olmesartan to Olmesartan Medoxomil, A Prodrug that Improves Intestinal Absorption, Confers Substrate Recognition by OATP2B1.

PURPOSE: Olmesartan medoxomil (olmesartan-MX), an ester-type prodrug of the angiotensin II receptor blocker (ARB) olmesartan, is predominantly anionic at intestinal pH. Human organic anion transporting polypeptide 2B1 (OATP2B1) is expressed in the small intestine and is involved in the absorption of various acidic drugs. This study was designed to test the hypothesis that OATP2B1-mediated uptake contributes to the enhanced intestinal absorption of olmesartan-MX, even though olmesartan itself is not a substrate of OATP2B1. METHODS: Tetracycline-inducible human OATP2B1- and rat Oatp2b1-overexpressing HEK 293 cell lines (hOATP2B1/T-REx-293 and rOatp2b1/T-REx-293, respectively) were established to characterize OATP2B1-mediated uptake. Rat jejunal permeability was measured using Ussing chambers. ARBs were quantified by liquid chromatography-tandem mass spectrometry. RESULTS: Significant olmesartan-MX uptake was observed in hOATP2B1/T-REx-293 and rOatp2b1/T-REx-293 cells, whereas olmesartan uptake was undetectable or much lower than olmesartan-MX uptake, respectively. Furthermore, olmesartan-MX exhibited several-fold higher uptake in Caco-2 cells and greater permeability in rat jejunum compared to olmesartan. Olmesartan-MX uptake in hOATP2B1/T-REx-293 cells and in Caco-2 cells was significantly decreased by OATP2B1 substrates/inhibitors such as 1 mM estrone-3-sulfate, 100 µM rifamycin SV, and 100 µM fluvastatin. Rat Oatp2b1-mediated uptake and rat jejunal permeability of olmesartan-MX were significantly decreased by 50 µM naringin, an OATP2B1 inhibitor. Oral administration of olmesartan-MX with 50 µM naringin to rats significantly reduced the area under the plasma concentration-time curve of olmesartan to 76.9%. CONCLUSION: Olmesartan-MX is a substrate for OATP2B1, and the naringin-sensitive transport system contributes to the improved intestinal absorption of olmesartan-MX compared with its parent drug, olmesartan.

Transplantation of bioengineered liver capable of extended function in a preclinical liver failure model.

Unlimited organ availability would represent a paradigm shift in transplantation. Long-term in vivo engraftment and function of scaled-up bioengineered liver grafts have not been previously reported. In this study, we describe a human-scale transplantable liver graft engineered on a porcine liver-derived scaffold. We repopulated the scaffold parenchyma with primary hepatocytes and the vascular system with endothelial cells. For in vivo functional testing, we performed auxiliary transplantation of the repopulated scaffold in pigs with induced liver failure. It was observed that the auxiliary bioengineered liver graft improved liver function for 28 days and exhibited upregulation of liver-specific genes. This study is the first of its kind to present 28 days of posttransplant evaluation of a bioengineered liver graft using a preclinical large animal model. Furthermore, it provides definitive evidence for the feasibility of engineering human-scale transplantable liver grafts for clinical applications.

Transplacental Pharmacokinetic Model of Digoxin Based on Ex Vivo Human Placental Perfusion Study.

Digoxin is used as first-line therapy to treat fetal supraventricular tachycardia; however, because of the narrow therapeutic window, it is essential to estimate digoxin exposure in the fetus. The data from ex vivo human placental perfusion study are used to predict in vivo fetal exposure noninvasively, but the ex vivo fetal-to-maternal concentration (F:M) ratios observed in digoxin perfusion studies were much lower than those in vivo. In the present study, we developed a human transplacental pharmacokinetic model of digoxin using previously reported ex vivo human placental perfusion data. The model consists of maternal intervillous, fetal capillary, non-perfused tissue, and syncytiotrophoblast compartments, with multidrug resistance protein (MDR) 1 and influx transporter at the microvillous membrane (MVM) and influx and efflux transporters at the basal plasma membrane (BM). The model-predicted F:M ratio was 0.66, which is consistent with the mean in vivo value of 0.77 (95% confidence interval: 0.64-0.91). The time to achieve the steady state from the ex vivo perfusion study was estimated as 1,500 minutes, which is considerably longer than the reported ex vivo experimental durations, and this difference is considered to account for the inconsistency between ex vivo and in vivo F:M ratios. Reported digoxin concentrations in a drug-drug interaction study with MDR1 inhibitors quinidine and verapamil were consistent with the profiles simulated by our model incorporating inhibition of efflux transporter at the BM in addition to MVM. Our modeling and simulation approach should be a powerful tool to predict fetal exposure and DDIs in human placenta. SIGNIFICANCE STATEMENT: We developed a human transplacental pharmacokinetic model of digoxin based on ex vivo human placental perfusion studies in order to resolve inconsistencies between reported ex vivo and in vivo fetal-to-maternal concentration ratios. The model successfully predicted the in vivo fetal exposure to digoxin and the drug-drug interactions of digoxin and P-glycoprotein/multidrug resistance protein 1 inhibitors in human placenta.

Limited Impact of Murine Placental MDR1 on Fetal Exposure of Certain Drugs Explained by Bypass Transfer Between Adjacent Syncytiotrophoblast Layers.

PURPOSE: Multidrug resistance protein 1 (MDR1) is located at the interface between two syncytiotrophoblast layers in rodent placenta, and may influence fetal drug distribution. Here, we quantitatively compare the functional impact per single MDR1 molecule of MDR1 at the placental barrier and blood-brain barrier in mice. METHODS: MDR1A and MDR1B proteins were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Paclitaxel or digoxin was continuously administered to pregnant Mdr1a-/-/Mdr1b-/- or wild-type mice, and the drug concentrations in the maternal and fetal plasma and maternal brain were quantified by LC-MS/MS. RESULTS: MDR1A and MDR1B proteins are expressed in the membrane of mouse placental labyrinth, and total MDR1 at the placental barrier amounts to about 30% of that at the blood-brain barrier. The fetal-to-maternal plasma concentration ratio of digoxin was only marginally affected in Mdr1a-/-/Mdr1b-/- mice, while that of paclitaxel showed a several-fold increase. No such difference between the two drugs was found in the maternal brain distribution. The impact per single MDR1 molecule on the fetal distribution of digoxin was calculated to be much lower than that on the brain distribution, but this was not the case for paclitaxel. Our pharmacokinetic model indicates that the impact of placental MDR1 is inversely correlated to the ratio of permeability through gap junctions connecting the two syncytiotrophoblast layers to passive diffusion permeability. CONCLUSION: Our findings indicate that murine placental MDR1 has a minimal influence on the fetal concentration of certain substrates, such as digoxin, due to bypass transfer, probably via connexin26 gap junctions.

MicroRNA-126 suppresses the invasion of trophoblast-model JEG-3 cells by targeting LIN28A.

Inadequate trophoblast invasion and impaired trophoblast-induced vascular remodeling are features of preeclampsia. In this context, an angiogenesis-related microRNA, miR-126, is abnormally expressed in preeclampsia placentas, but its role in trophoblast development remains unclear. The purpose of this study was to investigate the roles of miR-126 in the proliferation, migration, and invasion processes of trophoblast cells using the human choriocarcinoma-derived JEG-3 cell line as a model. The mRNA expression profiling of JEG-3 cells with and without miR-126 overexpression, in combination with bioinformatics analysis, identified LIN28A as a putative target of miR-126. The results of real-time RT-PCR and luciferase assay were consistent with this idea. Overexpression of miR-126 in JEG-3 cells decreased the invasive ability of the cells without affecting proliferation or migration. The invasiveness of JEG-3 cells was significantly reduced to a similar extent by knockdown of LIN28A with siRNA and by miR-126-overexpression-induced downregulation of LIN28A, although the level of LIN28A protein was much lower in the siLIN28A-transfected cells. These results indicate that miR-126 suppresses JEG-3 cell invasion by targeting LIN28A, and suggest that miR-126-mediated downregulation of LIN28A might contribute to the onset/deterioration of preeclampsia.

Fluorouracil uptake in triple-negative breast cancer cells: Negligible contribution of equilibrative nucleoside transporters 1 and 2.

Equilibrative nucleoside transporters (ENTs) 1 and 2 reportedly accept fluorouracil as a substrate. Here, we evaluated ENT1/2 expression at the messenger RNA (mRNA), protein, and functional levels in a panel of four triple-negative breast cancer (TNBC) cell lines, BT-549, Hs578T, MDA-MB-231, and MDA-MB-435, and we examined the relationship of the observed profiles to fluorouracil sensitivity. Nitrobenzylthioinosine (NBMPR) at 0.1 µM inhibits only ENT1, while dipyridamole at 10 µM or NBMPR at 100 µM inhibits both ENT1 and ENT2. We found that the uptake of [3 H]uridine, a typical substrate of ENT1 and ENT2, was decreased to approximately 40% by 0.1 µM NBMPR. At 100 µM, NBMPR almost completely blocked the saturable uptake of [3 H]uridine, but this does not imply a functional role of ENT2, because 10 µM dipyridamole showed similar inhibition to 0.1 µM NBMPR. Expression of ENT1 mRNA was almost 1 order of magnitude higher than that of ENT2 in all TNBC cell lines. Liquid chromatography-tandem mass spectrometry(LC-MS/MS) LC-MS/MS-based targeted protein quantification showed that ENT1 protein levels were in the range of 9.3-30 fmol/µg protein in plasma membrane fraction of TNBC cell lines, whereas ENT2 protein was below the detection limit. [3 H]Fluorouracil uptake was insensitive to 0.1 µM NBMPR and 10 µM dipyridamole, suggesting a negligible contribution of ENT1 and ENT2 to fluorouracil uptake. The levels of ENT1 mRNA, ENT1 protein, ENT2 mRNA, and ENT1-mediated [3 H]uridine uptake in the four TNBC cell lines showed no correlation with fluorouracil sensitivity. These results indicate that neither ENT1 nor ENT2 contributes significantly to the fluorouracil sensitivity of TNBC cell lines.

Development of a Pharmacokinetic Model of Transplacental Transfer of Metformin to Predict In Vivo Fetal Exposure.

Two types of systems are used in ex vivo human placental perfusion studies to predict fetal drug exposures, that is, closed systems with recirculation of the maternal and fetal buffer and open systems using a single-pass mode without recirculation. The in vivo fetal/maternal (F:M) ratio of metformin, a cationic drug that crosses the placenta, is consistent with that reported in an open system ex vivo but not with that in a closed system. In the present study, we aimed to develop a pharmacokinetic (PK) model of transplacental transfer of metformin to predict in vivo fetal exposure to metformin and to resolve the apparent inconsistency between open and closed ex vivo systems. The developed model shows that the difference between open and closed systems is due to the difference in the time required to achieve the steady state. The model-predicted F:M ratio (approx. 0.88) is consistent with reported in vivo values [mean (95% confidence interval): 1.10 (0.69-1.51)]. The model incorporates bidirectional transport via organic cation transporter 3 (OCT3) at the basal plasma membrane, and simulations indicate that the use of trimethoprim (an OCT3 inhibitor) to prevent microbial growth in the placenta ex vivo has a negligible effect on the overall maternal-to-fetal and fetal-to-maternal clearances. The model could successfully predict in vivo fetal exposure using ex vivo human placental perfusion data from both closed and open systems. This transplacental PK modeling approach is expected to be useful for evaluating human fetal exposures to other poorly permeable compounds, besides metformin. SIGNIFICANCE STATEMENT: We developed a pharmacokinetic model of transplacental transfer of metformin, used to treat gestational diabetes mellitus, in order to predict in vivo fetal exposure and resolve the discrepancy between reported findings in open and closed ex vivo perfusion systems. The discrepancy is due to a difference in the time required to reach the steady state. The model can predict in vivo fetal exposure using data from both closed and open systems.

Transport of Pregabalin Via L-Type Amino Acid Transporter 1 (SLC7A5) in Human Brain Capillary Endothelial Cell Line.

PURPOSE: The anti-epileptic drug pregabalin crosses the blood-brain barrier (BBB) in spite of its low lipophilicity. This study was performed to determine whether L-type amino acid transporters (LAT1/SLC7A5 and LAT2/SLC7A8) contribute to the uptake of pregabalin. METHODS: Pregabalin uptake by LATs-transfected HEK293 cells or hCMEC/D3 cells, an in vitro human BBB model, was measured by LC-MS/MS analysis. Expression of LAT1 mRNA in hCMEC/D3 cells was determined by quantitative RT-PCR analysis. RESULTS: Overexpression of LAT1, but not LAT2, in HEK293 cells significantly increased the cellular uptake of pregabalin, and the LAT1-mediated uptake was saturable with a Km of 0.288 mM. LAT1-mediated amino acid uptake was inhibited specifically and almost completely in the presence of 1 mM pregabalin. The uptake of pregabalin by hCMEC/D3 cells was sodium-independent, saturable (Km = 0.854 mM), and strongly inhibited by large amino acids at 1 mM, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, a specific system L inhibitor, at 1 mM and by JPH203, a LAT1-selective inhibitor, at 10 µM. Pregabalin uptake in hCMEC/D3 cells was also decreased by 75% by the silencing of LAT1 gene using LAT1 siRNA. CONCLUSIONS: Our results indicate that LAT1, but not LAT2, recognizes pregabalin as a substrate. It is suggested that LAT1 mediates pregabalin transport at the BBB.

Hypotaurine Is a Substrate of GABA Transporter Family Members GAT2/Slc6a13 and TAUT/Slc6a6.

Hypotaurine is a precursor of taurine and a physiological antioxidant that circulates in adult and fetal plasma. The purpose of the present study was to clarify whether hypotaurine is a substrate of Slc6a/gamma-aminobutyric acid (GABA) transporter family members. Radiolabeled hypotaurine was synthesized from radiolabeled cysteamine and 2-aminoethanethiol dioxygenase. The uptakes of [3H]GABA, [3H]taurine, and [14C]hypotaurine by HEK293 cells expressing mouse GAT1/Slc6a1, TAUT/Slc6a6, GAT3/Slc6a11, BGT1/Slc6a12, and GAT2/Slc6a13 were measured. TAUT and GAT2 showed strong [14C]hypotaurine uptake activity, while BGT1 showed moderate activity, and GAT1 and GAT3 showed slight but significant activity. Mouse TAUT and GAT2 both showed Michaelis constants of 11 µM for hypotaurine uptake. GAT2-expressing cells pretreated with hypotaurine showed resistance to H2O2-induced oxidative stress. These results suggest that under physiological conditions, TAUT and GAT2 would be major contributors to hypotaurine transfer across the plasma membrane, and that uptake of hypotaurine via GAT2 contributes to the cellular resistance to oxidative stress.

Contributions of system A subtypes to α-methylaminoisobutyric acid uptake by placental microvillous membranes of human and rat.

System A consists of three subtypes, sodium-coupled neutral amino acid transporter 1 (SNAT1), SNAT2, and SNAT4, which are all expressed in the placenta. The aim of this study was to evaluate the contributions of each of the three subtypes to total system A-mediated uptake in placental MVM of human and rat, using betaine and L-arginine as subtype-selective inhibitors of SNAT2 and SNAT4, respectively. Appropriate concentrations of betaine and L-arginine for subtype-selective inhibition in SNAT-overexpressing cells were identified. It was found that 10 mM betaine specifically and almost completely inhibited human and rat SNAT2-mediated [14C]α-methylaminoisobutyric acid ([14C]MeAIB) uptake, while 5 mM L-arginine specifically and completely inhibited [3H]glycine uptake via human SNAT4, as well as [14C]MeAIB uptake via rat SNAT4. In both human and rat placental MVM vesicles, sodium-dependent uptake of [14C]MeAIB was almost completely inhibited by 20 mM unlabeled MeAIB. L-Arginine (5 mM) partly inhibited the uptake in humans, but hardly affected that in rats. Betaine (10 mM) partly inhibited the uptake in rats, but hardly affected it in humans. These results suggest that SNAT1 is most likely the major contributor to system A-mediated MeAIB uptake by human and rat MVM vesicles and that the remaining uptake is mainly mediated by SNAT4 in humans and SNAT2 in rats. Thus, inhibition studies using betaine and L-arginine are useful to characterize the molecular mechanisms of system A-mediated transport.

System A amino acid transporter SNAT2 shows subtype-specific affinity for betaine and hyperosmotic inducibility in placental trophoblasts.

Betaine uptake is induced by hypertonic stress in a placental trophoblast cell line, and involvement of amino acid transport system A was proposed. Here, we aimed to identify the subtype(s) of system A that mediates hypertonicity-induced betaine uptake. Measurement of [(14)C]betaine uptake by HEK293 cells transiently transfected with human or rat sodium-coupled neutral amino acid transporters (SNATs), SNAT1, SNAT2 and SNAT4 revealed that only human and rat SNAT2 have betaine uptake activity. The Michaelis constants (Km) of betaine uptake by human and rat SNAT2 were estimated to be 5.3 mM and 4.6 mM, respectively. Betaine exclusively inhibited the uptake activity of SNAT2 among the rat system A subtypes. We found that rat SNAT1, SNAT2 and SNAT4 were expressed at the mRNA level under isotonic conditions, while expression of SNAT2 and SNAT4 was induced by hypertonicity in TR-TBT 18d-1 cells. Western blot analyses revealed that SNAT2 expression on plasma membrane of TR-TBT 18d-1 cells was more potently induced by hypertonicity than that in total cell lysate. Immunocytochemistry confirmed the induction of SNAT2 expression in TR-TBT 18d-1 cells exposed to hypertonic conditions and indicated that SNAT2 was localized on the plasma membrane in these cells. Our results indicate that SNAT2 transports betaine, and that tonicity-sensitive SNAT2 expression may be involved in regulation of betaine concentration in placental trophoblasts.

Breast Cancer Resistance Protein Limits Fetal Transfer of Tadalafil in Mice.

Tadalafil, a phosphodiesterase 5 (PDE5) inhibitor, is a candidate therapeutic agent for fetal growth restriction and hypertensive disorders of pregnancy. In this study, we elucidated the fetal transfer of tadalafil in comparison with that of sildenafil, the first PDE5 inhibitor to be approved. We also examined the contributions of multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP) to fetal transfer. Tadalafil or sildenafil was administered to wild-type, Mdr1a/b-double-knockout or Bcrp-knockout pregnant mice by continuous infusion from gestational day (GD) 14.5 to 17.5, and the fetal-to-maternal plasma concentration ratio of unbound drug (unbound F/M ratio) was evaluated at GD 17.5. The values of unbound F/M ratio of tadalafil and sildenafil in wild-type mice were 0.80 and 1.6, respectively. The unbound F/M ratio of tadalafil was increased to 1.1 and 1.7 in Mdr1a/b-knockout and Bcrp-knockout mice, respectively, while the corresponding values for sildenafil were equal to or less than that in wild-type mice, respectively. A transcellular transport study revealed that basal-to-apical transport of both tadalafil and sildenafil was significantly higher than transport in the opposite direction in MDCKII-BCRP cells. Our research reveals that tadalafil is a newly identified substrate of human and mouse BCRP, and it appears that the fetal transfer of tadalafil is, at least in part, attributed to the involvement of BCRP within the placental processes in mice. The transfer of sildenafil to the fetus was not significantly constrained by BCRP, even though sildenafil was indeed a substantial substrate for BCRP.

Involvement of GAT2/Slc6a13 in hypotaurine uptake at fetal-facing plasma membrane of syncytiotrophoblasts at mid-to-late gestation in rats and mice.

INTRODUCTION: Hypotaurine, a precursor to taurine, is known for its antioxidant properties and is prominently present in fetal plasma and the placenta. Our previous research revealed that ezrin-knockout mice experience fetal growth retardation, coinciding with reduced hypotaurine levels in fetal plasma. This study aims to elucidate the expression and role of hypotaurine transporters within the placenta. METHODS: We employed quantitative RT-PCR to measure mRNA expression of GAT transporter family members in the placenta during mid-to-late gestation. LC/MS/MS was used to analyze the distribution of hypotaurine in different placental subregions. Immunohistochemistry was utilized to examine the localization of GAT2 in mice. Placental hypotaurine uptake from fetal circulation was studied via umbilical perfusion in rats. RESULTS: Among hypotaurine transporters, GAT2 exhibited increased mRNA and protein expression in murine placenta during mid-to-late gestation. Notably, GAT2/Slc6a13 mRNA and hypotaurine were most concentrated in the labyrinth of murine placenta. In contrast, enzymes responsible for hypotaurine synthesis, such as cysteine dioxygenase, cysteine sulfinic acid decarboxylase, and 2-aminoethanethiol dioxygenase, showed minimal expression in the labyrinth. These findings suggest that GAT2 is a key determinant of hypotaurine levels in the placental labyrinth. Immunohistochemical examination unveiled that GAT2 was predominantly localized on the fetal-facing plasma membrane within syncytiotrophoblasts, which co-localized with ezrin. In rat umbilical perfusion experiments, the GAT2/3 and TauT inhibitor, SNAP-5114, significantly reduced hypotaurine extraction from fetal circulation to the placenta. DISCUSSION: The results suggest that GAT2 plays a pivotal role in the concentrative uptake of hypotaurine from fetal plasma within syncytiotrophoblasts of the placenta.

Hypoxic adaptation engages the CBP/CREST-induced coactivator complex of Creb-HIF-1α in transactivating murine neuroblastic glucose transporter.

We have shown in vitro a hypoxia-induced time-dependent increase in facilitative glucose transporter isoform 3 (GLUT3) expression in N2A murine neuroblasts. This increase in GLUT3 expression is partially reliant on a transcriptional increase noted in actinomycin D and cycloheximide pretreatment experiments. Transient transfection assays in N2A neuroblasts using murine glut3-luciferase reporter constructs mapped the hypoxia-induced enhancer activities to -857- to -573-bp and -203- to -177-bp regions. Hypoxia-exposed N2A nuclear extracts demonstrated an increase in HIF-1α and p-Creb binding to HRE (-828 to -824 bp) and AP-1 (-187 to -180 bp) cis-elements, respectively, in electromobility shift and supershift assays, which was confirmed by chromatin immunoprecipitation assays. In addition, the interaction of CBP with Creb and HIF-1α and CREST with CBP in hypoxia was detected by coimmunoprecipitation. Furthermore, small interference (si)RNA targeting Creb in these cells decreased endogenous Creb concentrations that reduced by twofold hypoxia-induced glut3 gene transcription. Thus, in N2A neuroblasts, phosphorylated HIF-1α and Creb mediated the hypoxia-induced increase in glut3 transcription. Coactivation by the Ca⁺⁺-dependent CREST and CBP proteins may enhance cross-talk between p-Creb-AP-1 and HIF-1α/HRE of the glut3 gene. Collectively, these processes can facilitate an adaptive response to hypoxic energy depletion targeted at enhancing glucose transport and minimizing injury while fueling the proliferative potential of neuroblasts.

L-type Amino Acid Transporter 1 (SLC7A5)-Mediated Transport of Pregabalin at the Rat Blood-Spinal Cord Barrier and its Sensitivity to Plasma Branched-Chain Amino Acids.

Pregabalin is an anti-neuropathic pain drug inhibiting the α2δ subunit of the voltage-dependent calcium channel in the spinal cord. The aim of this study is to characterize the transport mechanism of pregabalin at the blood-spinal cord barrier (BSCB) by means of in vivo experiments in rats and in vitro studies using primary-cultured rat spinal cord endothelial cells. We isolated endothelial cells by culturing rat spinal cord tissue in the presence of puromycin, and confirmed the expression of BSCB markers such as Cd31, Mdr1a, and Claudin-5. The uptake of pregabalin by primary-cultured rat spinal cord endothelial cells was sodium-independent and was significantly inhibited by L-leucine, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, and JPH203. These results suggest the involvement of L-type amino acid transporter (LAT) 1. LAT1 mRNA and protein was expressed in primary-cultured rat spinal cord endothelial cells, which is consistent with LAT1 expression at the BSCB. In the in vivo study, the transfer of pregabalin to rat spinal cord and brain was significantly decreased by the pre-administration of branched chain amino acids (BCAAs), which are endogenous substrates of LAT1. Our results indicate that pregabalin transport across the BSCB is mediated at least in part by LAT1 and is inhibited by plasma BCAAs.

Role of Uptake Transporters OAT4, OATP2A1, and OATP1A2 in Human Placental Bio-disposition of Pravastatin.

Pravastatin is currently under evaluation for prevention of preeclampsia. Factors contributing to placental disposition of pravastatin are important in assessment of potential undesirable fetal effects. The purpose of this study was to identify the uptake transporters that contribute to the placental disposition of pravastatin. Our data revealed the expression of organic anion transporting polypeptide 1A2 (OATP1A2) and OATP2A1 in the apical, and OATP2B1 and OATP5A1 in the basolateral membranes of the placenta, while organic anion transporter 4 (OAT4) exhibited higher expression in basolateral membrane but was detected in both membranes. Preloading placental membrane vesicles with glutarate increased the uptake of pravastatin suggesting involvement of glutarate-dependent transporters such as OAT4. In the HEK293 cells overexpressing individual uptake transporters, OATP2A1, OATP1A2 and OAT4 were determined to accept pravastatin as a substrate at physiological pH, while the uptake of pravastatin by OATP2B1 (known to interact with pravastatin at acidic pH) and OATP5A1 was not detected at pH 7.4. These findings led us to propose that OATP1A2 and OATP2A1 are responsible for the placental uptake of pravastatin from the maternal circulation, while OAT4 mediates the passage of the drug across placental basolateral membrane in the fetal-to-maternal direction.

Decellularized Organ-Derived Scaffold Is a Promising Carrier for Human Induced Pluripotent Stem Cells-Derived Hepatocytes.

Human induced pluripotent stem cells (hiPSCs) are a promising cell source for elucidating disease pathology and therapy. The mass supply of hiPSC-derived cells is technically feasible. Carriers that can contain a large number of hiPSC-derived cells and evaluate their functions in vivo-like environments will become increasingly important for understanding disease pathogenesis or treating end-stage organ failure. hiPSC-derived hepatocyte-like cells (hiPSC-HLCs; 5 × 108) were seeded into decellularized organ-derived scaffolds under circumfusion culture. The scaffolds were implanted into immunodeficient microminiature pigs to examine their applicability in vivo. The seeded hiPSC-HLCs demonstrated increased albumin secretion and up-regulated cytochrome P450 activities compared with those in standard two-dimensional culture conditions. Moreover, they showed long-term survival accompanied by neovascularization in vivo. The decellularized organ-derived scaffold is a promising carrier for hiPSC-derived cells for ex vivo and in vivo use and is an essential platform for regenerative medicine and research.

Substrate recognition of renally eliminated angiotensin II receptor blockers by organic anion transporter 4.

Organic anion transporter (OAT) 4, which is localized at the apical membrane of human renal proximal tubules, transports olmesartan, an angiotensin II receptor blocker (ARB). Many ARBs, including olmesartan, undergo partial tubular secretion as active forms, and inhibit OAT4-mediated uptake activity. Here, we examined the substrate recognition of various ARBs by OAT4 in order to assess whether OAT4 might be involved in the renal handling of ARBs. Concentration-dependent OAT4-mediated uptake of azilsartan, candesartan, carboxylosartan, losartan, and valsartan was observed with Km values of 6.6, 31, 7.2, 13, and 1.7 µM, respectively, in the absence of extracellular Cl-. In the presence of extracellular Cl-, OAT4-mediated uptake of dianionic ARBs (azilsartan, candesartan, carboxylosartan, and valsartan) was lower and reached a steady state faster than in the absence of extracellular Cl-. Thus, OAT4 is proposed to use extracellular Cl- as a counterpart for anion efflux. Our results suggest that OAT4 may play a role in the excretion of azilsartan, candesartan, carboxylosartan, and valsartan, as well as olmesartan. In contrast, OAT4-mediated uptake of losartan, a monoanionic ARB, was little affected by extracellular Cl-, suggesting that only OAT4-mediated dianion transport is Cl--sensitive.

Proton-coupled erythromycin antiport at rat blood-placenta barrier.

The aim of the present study was to characterize the mechanism of erythromycin transport at the blood-placenta barrier, using TR-TBT 18d-1 cells as a model of rat syncytiotrophoblasts. [(14)C]Erythromycin was taken up by TR-TBT 18d-1 cells with a Michaelis constant of 466 microM. Although the uptake was not dependent on extracellular Na(+) or Cl(-), it was increased at weakly alkaline pH. Significant overshoot of [(14)C]erythromycin uptake by placental brush-border membrane vesicles was observed in the presence of an outwardly directed proton gradient. These results indicate that erythromycin is transferred by the H(+)-coupled transport system in syncytiotrophoblasts. To address the physiological transport of erythromycin in rat placenta, fetal-to-maternal transport clearance was estimated by means of the single placental perfusion technique. Clearance of [(14)C]erythromycin was higher than that of [(14)C]inulin, a paracellular pathway marker, and was decreased by the addition of 5 mM erythromycin, indicating that saturable efflux system from fetus to mother is involved. The effect of various transporter inhibitors on [(14)C]erythromycin efflux from TR-TBT 18d-1 cells was evaluated. cyclosporin A, fumitremorgin C, and probenecid had no effect, whereas ethylisopropylamiloride, a specific inhibitor of Na(+)/H(+) exchangers (NHEs), was significantly inhibitory. These results suggest that erythromycin efflux transport at the rat blood-placenta barrier is mediated by an erythromycin/H(+) antiport system, driven by H(+) supplied by NHEs.

Involvement of system A in the retina-to-blood transport of l-proline across the inner blood-retinal barrier.

The purpose of the present study was to elucidate the mechanisms of retina-to-blood transport of l-proline across the blood-retinal barrier (BRB) in vivo and in vitro, and to identify the responsible transporter(s). The vitreous humor/retina-to-blood transport of [(3)H]l-proline across the BRB was evaluated by microdialysis. Transport mechanisms of [(3)H]l-proline were investigated by cellular uptake using an in vitro model of the inner BRB (TR-iBRB2 cells). The mRNA level of system A was determined by quantitative real-time PCR analysis with specific primers. [(3)H]l-Proline and [(14)C]d-mannitol, which is a bulk flow marker, were bi-exponentially eliminated from the vitreous humor after vitreous bolus injection. The elimination rate constant of [(3)H]l-proline during the terminal phase was 1.6-fold greater than that of [(14)C]d-mannitol. The terminal elimination rate constant difference between [(3)H]l-proline and [(14)C]d-mannitol was reduced in the retinal presence of 3 mM l-proline and 5 mM alpha-methylaminoisobutyric acid, suggesting that l-proline is transported via a carrier-mediated retina-to-blood transport process across the BRB. [(3)H]l-Proline uptake by TR-iBRB2 cells appeared to be mediated through a saturable and Na(+)-dependent process. The corresponding Michaelis-Menten constant was 392 muM. This process was reduced by substrates for system A, suggesting that system A is involved in l-proline uptake. Of the isoforms of system A, ATA1, ATA2, and ATA3, ATA2 mRNA is predominantly expressed in TR-iBRB2 cells and isolated rat retinal endothelial cells. In conclusion, system A, most likely ATA2, is responsible for the retina-to-blood transport of l-proline across the inner BRB and may play a role in maintaining the concentration of small neutral amino acids in the retina.