研究者詳細 - 野口　幸希

論文 - 野口　幸希

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Rat-Specific Expression of Placental MATE1 Contributes to Species Difference in Fetal Transfer of Metformin and 1-Methyl-4-Phenylpyridinium

Suzuki A., Noguchi S., Tsuchitani T., Sakamoto R., Yamauchi A., Ishinabe T., Hashimoto R., Nakaguchi Y., Sakai M., Imayoshi N., Ishimoto T., Kato Y., Shirasaka Y., Katakura S., Maruyama T., Nishimura T., Tomi M.

Molecular Pharmaceutics 22 （ 12 ） 7412 - 7422 2025年12月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 15438384

Metformin readily crosses the human placenta. However, the fetal-to-maternal plasma concentration ratio (F/M ratio) of metformin has been reported to be as low as 0.057 in rats. This study investigated the underlying mechanisms of these interspecies differences by examining the expression and function of candidate transporters, organic cation transporter 3 (OCT3/SLC22A3) and multidrug and toxin extrusion protein 1 (MATE1/SLC47A1), in the placenta across species. Expression analysis at gene and protein levels using RNA-sequencing and absolute quantification by LC-MS/MS revealed minimal species differences in placental OCT3 expression (less than 4-fold). In contrast, MATE1 showed almost exclusive expression in the rat placental labyrinth, but minimal or undetectable levels were observed in samples prepared from human placental villi and mouse placental labyrinth. To evaluate the function of the placental MATE1, we compared the placental transfer of two dual substrates of MATE1 and OCT3, metformin and 1-methyl-4-phenylpyridinium (MPP+), in pregnant rats and mice in the presence and absence of pyrimethamine, a MATE1 inhibitor. The pyrimethamine was dosed to reach the plasma concentration, which specifically inhibits MATE1 based on the measured IC<inf>50</inf> of pyrimethamine to rat MATE1 and OCT3, which were 0.015 and 100 μM, respectively. The F/M ratios of metformin and MPP+ in the absence of pyrimethamine were half or less in rats compared to mice. Notably, preadministration of pyrimethamine increased the F/M ratios of metformin and MPP+ by approximately 50% in rats but not mice. These findings demonstrate that functional placental MATE1 expression is specific to rats, which, in part, contributes to the low fetal transfer of cationic compounds compared with other species. This is of significant concern, as it suggests that nonclinical developmental and reproductive toxicity studies of MATE1 substrate drugs in rats may underestimate fetal exposure and toxicity when extrapolated to humans.

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Fetal ezrin expression affects macrophages and regulatory T cells in mouse placental decidua

Nishimura T., Mizokami R., Yamanaka M., Takahashi M., Yoshida Y., Ogawa Y., Noguchi S., Tomi M.

Biochemical and Biophysical Research Communications 735 2024年11月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0006291X

Ezrin is a cross-linker protein between membrane proteins and cytosolic actin, abundantly expressed in the placenta among the ERM protein family. Ezrin gene knockout mice exhibit fetal growth restriction after gestational day (GD) 15.5. This study aimed to clarify the effect of ezrin on immune cells that influence fetal growth and immune tolerance. Ezrin heterozygous knockout (Ez+/−) mice were interbred, and the gene expressions and immune cell distributions in the placentas of wild-type (Ez+/+) and ezrin knockout (Ez−/−) fetuses were analyzed. IL-6 expression in the placenta of Ez−/− fetuses was significantly higher than in Ez+/+ fetuses at GD 15.5. The mRNA expression of IL-6 in the uterine decidua attached to Ez−/− fetuses was higher compared to that attached to Ez+/+ fetuses but not in the junctional zone and labyrinth. Classical M1 and M2 macrophages in the decidua were analyzed by flow cytometry using CD86 and CD206 as markers. M1 macrophages increased in the decidua attached to Ez−/− mice compared to Ez+/+ mice, while M2 macrophages did not increase. CD4-positive T cells showed a reduction in the decidua attached to Ez−/− fetuses. Further analysis involved the subcutaneous administration of tacrolimus in pregnant Ez+/− mice from GD 8.5 to GD 15.5, which prevented the decrease in fetal body weight and decidual CD4-positive T cells in Ez−/− mice at GD 15.5. These results suggest that impaired expression of fetoplacental-derived ezrin induces inflammatory conditions in the uterine decidua through M1 polarization of macrophages, increased IL-6, and decreased CD4-positive T cells, including Treg cells.

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

Fukazawa N., Nishimura T., Orii K., Noguchi S., Tomi M.

Pharmaceutical Research 41 （ 5 ） 849 - 861 2024年05月

研究論文（学術雑誌）, 査読有り, ISSN 07248741

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.

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

Nishimura T., Araki H., Higuchi K., Noguchi S., Saito K., Hara K., Yagishita H., Akashi R., Obata S., Tomi M.

Placenta 147 59 - 67 2024年03月

研究論文（学術雑誌）, 査読有り, ISSN 01434004

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.

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Breast Cancer Resistance Protein Limits Fetal Transfer of Tadalafil in Mice

Nishimura T., Ishii M., Tanaka H., Noguchi S., Ikeda T., Tomi M.

Journal of Pharmaceutical Sciences （Journal of Pharmaceutical Sciences） 113 （ 2 ） 486 - 492 2023年11月

研究論文（学術雑誌）, 査読有り, ISSN 00223549

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.

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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

Akashi T., Noguchi S., Takahashi Y., Nishimura T., Tomi M.

Journal of Pharmaceutical Sciences （Journal of Pharmaceutical Sciences） 112 （ 4 ） 1137 - 1144 2023年01月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 00223549

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.

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Quantitative comparison of breast cancer resistance protein (BCRP/ABCG2) expression and function between blood-brain barrier and placental barrier in mice at different gestational ages.

Fujita A, Noguchi S, Hamada R, Shimada T, Katakura S, Maruyama T, Sai Y, Nishimura T, Tomi M

Frontiers in Drug Delivery 2 2022年07月

研究論文（学術雑誌）, 共著, 査読有り

Breast cancer resistance protein (BCRP) is expressed by brain capillary endothelial cells and at the interface between two placental syncytiotrophoblast layers in rodents and serves to suppress drug distribution to the brain and the fetus. The purpose of the present study is to determine and compare the apparent impact of a single BCRP molecule on drug transfer between the maternal blood-brain barrier and placental barrier in pregnant mice at different gestation ages. BCRP protein was quantified by liquid chromatography-tandem mass spectrometry. Genistein or dantrolene was continuously administered to pregnant Bcrp−/− or wild-type (WT) mice, and the brain-to-plasma concentration ratio in the mother (K<inf>p,brain</inf>) and the fetal-to-maternal ratio of plasma concentrations (K<inf>p,fp</inf>) were determined. At gestational day 15.5 (GD15.5), the protein amount of BCRP at the murine placental barrier was estimated to be approximately three times higher than at the maternal blood-brain barrier, but the levels were approximately the same at GD17.5 due to the decline of placental BCRP expression during gestation. On the other hand, the values of Bcrp−/−/WT ratio of K<inf>p,brain</inf> for genistein and dantrolene were 6.1 and 3.8, respectively, while the K<inf>p,fp</inf> ratios were all less than 2.0. These results indicate that the apparent impact of a single placental BCRP molecule on the restriction of drug distribution is much less than that of a single brain BCRP molecule, probably because the function of placental BCRP is attenuated by bypass transfer through the connexin26 gap junctions between adjacent syncytiotrophoblast layers. The present study also found that the expression amount of BCRP protein at the human placental barrier formed by the monolayer of syncytiotrophoblasts was lower than that in mice, but this species difference appears to be functionally compensated by the murine-specific bypass route through gap junctions, at least in part.

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

Fujita A., Noguchi S., Hamada R., Inoue S., Shimada T., Katakura S., Maruyama T., Sai Y., Nishimura T., Tomi M.

Pharmaceutical Research （Pharmaceutical Research） 39 （ 7 ） 1645 - 1658 2022年07月

査読有り, ISSN 07248741

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.

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

Kurosawa K., Noguchi S., Nishimura T., Tomi M., Chiba K.

Drug metabolism and disposition: the biological fate of chemicals （Drug metabolism and disposition: the biological fate of chemicals） 50 （ 3 ） 287 - 298 2022年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 00909556

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.

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

Fokina V.M., Patrikeeva S., Wang X.m., Noguchi S., Tomi M., König J., Ahmed M.S., Nanovskaya T.

Journal of Pharmaceutical Sciences （Journal of Pharmaceutical Sciences） 111 （ 2 ） 505 - 516 2022年02月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 00223549

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.

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タダラフィルおよびシルデナフィルのマウス胎仔移行性比較と胎盤透過制御機構

石井まり, 西村友宏, 野口幸希, 田中博明, 池田智明, 登美斉俊

日本妊娠高血圧学会雑誌（(一社)日本妊娠高血圧学会） 27 58 - 58 2021年12月

ISSN 1880-3172

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

Pan X., Noguchi S., Ando M., Nishimura T., Tomi M.

Biochemical and Biophysical Research Communications （Biochemical and Biophysical Research Communications） 545 132 - 137 2021年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0006291X

© 2021 Elsevier Inc. 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.

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

Noguchi S., Takagi A., Tanaka T., Takahashi Y., Pan X., Kibayashi Y., Mizokami R., Nishimura T., Tomi M.

Biopharmaceutics and Drug Disposition （Biopharmaceutics and Drug Disposition） 42 （ 2-3 ） 85 - 93 2021年01月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1099-081X

© 2021 John Wiley & Sons Ltd. 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 [3H]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 [3H]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. [3H]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 [3H]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.

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Development of a pharmacokinetic model of transplacental transfer of metformin to predict in vivo fetal exposure

Kurosawa K., Chiba K., Noguchi S., Nishimura T., Tomi M.

Drug Metabolism and Disposition （Drug Metabolism and Disposition） 48 （ 12 ） 1293 - 1302 2020年12月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 00909556

Copyright © 2020 The Author(s). 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.

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

Noguchi S., Okochi M., Atsuta H., Kimura R., Fukumoto A., Takahashi K., Nishimura T., Tomi M.

Drug Metabolism and Pharmacokinetics （Drug Metabolism and Pharmacokinetics） 36 100363 2020年10月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 13474367

© 2020 The Japanese Society for the Study of Xenobiotics 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.

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Contribution of Prostaglandin Transporter OATP2A1/SLCO2A1 to Placenta-to-Maternal Hormone Signaling and Labor Induction

Inagaki M., Nishimura T., Nakanishi T., Shimada H., Noguchi S., Akanuma S.i., Tachikawa M., Hosoya K.i., Tamai I., Nakashima E., Tomi M.

iScience （iScience） 23 （ 5 ） 101098 - 101098 2020年05月

研究論文（学術雑誌）, 共著, 査読有り

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Quantification of ENT1 and ENT2 Proteins at the Placental Barrier and Contribution of These Transporters to Ribavirin Uptake

Nishimura T., Sano Y., Takahashi Y., Noguchi S., Uchida Y., Takagi A., Tanaka T., Katakura S., Nakashima E., Tachikawa M., Maruyama T., Terasaki T., Tomi M.

Journal of Pharmaceutical Sciences （Journal of Pharmaceutical Sciences） 108 （ 12 ） 3917 - 3922 2019年12月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 00223549

© 2019 The Authors The aims of this study are to quantify the protein levels of nucleoside transporters in placental microvillous membranes (MVMs) and to clarify the contributions of these transporters to ribavirin uptake at the placental barrier. Placental MVMs of human and rat expressed equilibrative nucleoside transporter (ENT) 1 protein, whereas the expression of ENT2 protein was obscure. Maternal-to-fetal transfer of [3H]ribavirin in rats was much higher than that of [14C]sucrose. The uptake of [3H]ribavirin by rat placental trophoblast TR-TBT 18 d-1 cells, which functionally express both ENT1 and ENT2 proteins, was saturable, and was significantly inhibited by 0.1 μM nitrobenzylthioinosine, which selectively abolishes ENT1-mediated uptake. Dipyridamole at 10 μM is capable of inhibiting ENT2 as well as ENT1, but a degree of inhibition by 10 μM dipyridamole on [3H]ribavirin uptake was not much different from that by 0.1 μM nitrobenzylthioinosine (ENT1-specific inhibitor). Therefore, ENT2 may contribute little to [3H]ribavirin uptake by these cells. Rat ENT1 cRNA-injected oocytes showed increased [3H]ribavirin uptake compared with water-injected oocytes, while rat ENT2 cRNA-injected oocytes did not. In conclusion, ENT1 protein expressed in placental MVMs appears to play a predominant role in the uptake of ribavirin.

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

Takahashi Y., Nishimura T., Higuchi K., Noguchi S., Tega Y., Kurosawa T., Deguchi Y., Tomi M.

Pharmaceutical Research （Pharmaceutical Research） 35 （ 12 ） 246 - 246 2018年10月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 07248741

© 2018, The Author(s). 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.

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LAT1が血液脳関門におけるpregabalin輸送に果たす役割

高橋優, 西村友宏, 樋口慧, 野口幸希, 手賀悠真, 黒澤俊樹, 出口芳春, 登美斉俊

日本薬剤学会年会講演要旨集（(公社)日本薬剤学会） 33年会 136 - 136 2018年05月

Cellular Uptake of Levocetirizine by Organic Anion Transporter 4

Saki Noguchi, Tomohiro Nishimura, Saya Mukaida, Leslie Z. Benet, Emi Nakashima, Masatoshi Tomi

Journal of Pharmaceutical Sciences （WILEY） 106 （ 9 ） 2895 - 2898 2017年09月

研究論文（学術雑誌）, 査読有り, ISSN 0022-3549

The pharmacokinetics of cetirizine, a nonsedating antihistamine, is profoundly affected by transporter-mediated membrane transport in the kidney. In this study, we aimed to investigate the transport mechanism of levocetirizine, the pharmacologically active enantiomer of cetirizine, via human organic anion transporter 4 (OAT4) expressed in the apical membrane of renal proximal tubules and the basal plasma membrane of placental syncytiotrophoblasts. In cells expressing human OAT4 under the control of tetracycline, levocetirizine uptake was increased by tetracycline treatment. On the other hand, OAT4 expression did not facilitate efflux of preloaded levocetirizine from the cells, either in the presence or absence of extracellular Cl-. The OAT4-mediated levocetirizine uptake was concentration-dependent with a K-m of 38 mu M. The uptake rate of levocetirizine via OAT4 was approximately twice that of racemic cetirizine, indicating stereoselective uptake of levocetirizine. On the other hand, OAT4-mediated [H-3] dehydroepiandrosterone sulfate uptake was inhibited by dextrocetirizine and levocetirizine. Overall, our findings indicate that OAT4 mediates levocetirizine uptake but is unlikely to mediate the efflux. (C) 2017 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.

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Organic Anion Transporter 4-Mediated Transport of Olmesartan at Basal Plasma Membrane of Human Placental Barrier

Noguchi Saki, Nishimura Tomohiro, Fujibayashi Ayasa, Maruyama Tetsuo, Tomi Masatoshi, Nakashima Emi

Journal of Pharmaceutical Sciences （WILEY-BLACKWELL） 104 （ 9 ） 3128 - 3135 2015年09月

研究論文（学術雑誌）, ISSN 0022-3549

Mechanisms regulating fetal transfer of olmesartan, an angiotensin-II receptor type 1 antagonist, are important as potential determinants of life-threatening adverse fetal effects. The purpose of this study was to examine the olmesartan transport mechanism through the basal plasma membrane (BM) of human syncytiotrophoblasts forming the placental barrier. Uptake of olmesartan by human placental BM vesicles was potently inhibited by dehydroepiandrosterone sulfate (DHEAS), estrone 3-sulfate, and bromosulfophthalein, which are all typical substrates of organic anion transporter (OAT) 4 localized at the BM of syncytiotrophoblasts, and was increased in the absence of chloride. In tetracycline-inducible OAT4-expressing cells, [H-3]olmesartan uptake was increased by tetracycline treatment. Olmesartan uptake via OAT4 was concentration dependent with a K-m of 20 M, and was increased in the absence of chloride. [H-3]Olmesartan efflux via OAT4 was also observed and was trans-stimulated by extracellular chloride and DHEAS. Thus, OAT4 mediates bidirectional transport of olmesartan and appears to regulate fetal transfer of olmesartan at the BM of syncytiotrophoblasts. Efflux transport of olmesartan via OAT4 from syncytiotrophoblasts to the fetal circulation might be facilitated in the presence of an inwardly directed physiological chloride gradient and extracellular DHEAS. (c) 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:3128-3135, 2015

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Role of protein kinase A in regulating steroid sulfate uptake for estrogen production in human placental choriocarcinoma cells,

Tomi Masatoshi, Miyata Yuki, Noguchi Saki, Nishimura Sachika, Nishimura Tomohiro, Nakashima Emi

Placenta （W B SAUNDERS CO LTD） 35 （ 8 ） 658 - 660 2014年08月

研究論文（学術雑誌）, ISSN 0143-4004

The purpose of this study is to assess the role of the protein kinase A (PKA) in regulating uptake of dehydroepiandrosterone sulfate (DHEAS), an estrogen precursor, by syncytiotrophoblasts. Forskolin, a PKA activator, significantly increased [H-3]DHEAS uptake and the mRNA expression levels of organic anion transporter (OAT) 4 and CYP19A1 in choriocarcinoma JEG-3 cells, while other steroid sulfate transporters present in the placenta showed no change in expression level. KT5720, a PKA inhibitor, attenuated these effects of forskolin. Accordingly, the PKA pathway appears to play an important role in estrogen synthesis by cooperatively regulating OAT4 and steroidogenic enzymes in syncytiotrophoblasts. (C) 2014 Elsevier Ltd. All rights reserved.

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