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This study aimed to analyze the contributions of multiple transport mechanisms to the intestinal uptake of serotonin (5-HT) by employing a variety of in vitro experimental techniques, focusing on organic cation transporters expressed in the gastrointestinal (GI) tract, such as SERT, PMAT, THTR2, OCT3, and OCTN2. Analysis of the concentration dependence of 5-HT uptake by Caco-2 cells revealed multi-affinity kinetics with high-affinity and low-affinity components, suggesting that multiple transporters are involved in the intestinal 5-HT uptake. Comparative analysis of transporters using K m values obtained in Xenopus oocyte expression systems suggested that SERT is responsible for the high-affinity transport, while PMAT, THTR2, and OCT3 contribute to the low-affinity transport. Further analysis indicated that the relative contributions of SERT and PMAT to the intestinal 5-HT uptake (0.01 µM) are approximately 94.9% and 1.1%, respectively. Interestingly, at the concentration of 10 µM, the reported steady-state concentration of 5-HT in the human colon, the contributions of SERT, PMAT, THTR2, and OCT3 were estimated to be approximately 37.0%, 1.0%, 18.2%, and 20.5%, respectively. In conclusion, the present study indicated that the contributions of multiple transporters to 5-HT uptake in the GI tract are dependent upon the colon luminal concentration of 5-HT., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Drug-induced kidney injury (DIKI) is the major cause of acute kidney injury (AKI). Renal proximal tubular epithelial cells (RPTECs) are the primary target sites of DIKI and express transporters involved in renal drug disposition. In the present study, we focused on three-dimensionally cultured human RPTECs (3D-RPTECs) with elevated expression of drug transporters to investigate their utility in DIKI evaluation. Intracellular ATP levels in 3D-RPTECs are reduced by tenofovir and cisplatin that are substrates of an organic anion transporter 1 and an organic cation transporter 2, respectively. In addition, 3D-RPTECs were exposed to 17 and 15 drugs that are positive and negative to RPTEC toxicity, respectively, for up to 28 d. The 20 % decreasing concentration of drugs for ATP amount (EC 20 ) was obtained, and the ratio of EC 20 values and clinical maximum concentration (C max ) ≤100 were used as cut-off value to evaluate potential of DIKI. The sensitivities of 3D-RPTECs were 82.4 % and 88.2 % after 7 d and 28 d of drug exposure, respectively, and the specificities were 100 % and 93.3 %, respectively. The predictive performance of 3D-RPTECs was higher than that of two-dimensional cultured RPTECs and the kidney cell line HK-2. In conclusion, 3D-RPTECs are useful for in vitro evaluation of RPTEC injury by measuring intracellular ATP levels., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The present study was partially supported by Nikkiso Co., Ltd., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Sodium-phosphate transporter NPT4 (SLC17A3) is a membrane transporter for organic anionic compounds localized on the apical membranes of kidney proximal tubular epithelial cells and plays a role in the urinary excretion of organic anionic compounds. However, its physiological role has not been sufficiently elucidated because its substrate specificity is yet to be determined. The present study aimed to comprehensively explore the physiological substrates of NPT4 in newly developed Slc17a3 -/- mice using a metabolomic approach. Metabolomic analysis showed that the plasma concentrations of 11 biological substances, including 3-indoxyl sulfate, were more than two-fold higher in Slc17a3 -/- mice than in wild-type mice. Moreover, urinary excretion of 3-indoxyl sulfate was reduced in Slc17a3 -/- mice compared to that in wild-type mice. The uptake of 3-indoxyl sulfate by NPT4-expressing Xenopus oocytes was significantly higher than that by water-injected oocytes. The calculated K m and V max values for NPT4-mediated 3-indoxyl sulfate uptake were 4.52 ± 1.18 mM and 1.45 ± 0.14 nmol/oocyte/90 min, respectively. In conclusion, the present study revealed that 3-indoxyl sulfate is a novel substrate of NPT4 based on the metabolomic analysis of Slc17a3 -/- mice, suggesting that NPT4 regulates systemic exposure to 3-indoxyl sulfate by regulating its urinary excretion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Purpose: Plant-derived extracellular vesicles (EVs) have been reported to exert biological activity on intestinal tissues by delivering their contents into intestinal cells. We previously reported that ASBT/SLC10A2 mRNA was downregulated by apple-derived extracellular vesicles (APEVs). ASBT downregulation is effective in the treatment of cholestasis and chronic constipation, similar to the beneficial effects of apples. Therefore, this study aimed to establish the mechanism of ASBT downregulation by APEVs, focusing on microRNAs present in APEVs., Results: APEVs downregulated the expression of ASBT, but no significant effect on SLC10A2-3'UTR was observed. Proteomics revealed that APEVs decreased the expression of RARα/NR1B1. The binding of RARα to SLC10A2 promoter was also decreased by APEVs. The stability of NR1B1 mRNA was attenuated by APEVs and its 3'UTR was found to be a target for APEVs. Apple microRNAs that were predicted to interact with NR1B1-3'UTR were present in APEVs, and their mimics suppressed NR1B1 mRNA expression., Conclusions: Suppression of ASBT by APEVs was indirectly mediated by the downregulation of RARα, and its stability was lowered by microRNAs present in APEVs. This study suggested that macromolecules in food directly affect intestinal function by means of EVs that stabilize them and facilitate their cellular uptake., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2023 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

The aim of this study was to investigate the contributions of multiple transport mechanisms to the intestinal absorption of metformin, focusing on OCT3, PMAT, THTR2, SERT and OCTN2. We also assessed the impact of these transporters on the nonlinear absorption of metformin. Uptake studies with MDCKII cells expressing OCT3, PMAT, THTR2 or SERT confirmed that metformin is a substrate of these transporters. Decynium22 strongly inhibited metformin uptake mediated by all the transporters. 7-Cyclopentyl inhibited OCT3- and THTR2-mediated uptake of metformin. AG835, thiamine and paroxetine specifically inhibited PMAT-, THTR2- and SERT-mediated uptake of metformin, respectively. Using these inhibitors, the relative contributions of OCT3, PMAT, THTR2, SERT, OCTN2 and others to the intestinal permeation of metformin across Caco-2 cells were estimated to be 9.77%, 9.68%, 22.2%, 1.52%, 0% and 0.66%, respectively. Concentration-dependent analysis of metformin uptake by Caco-2 cells revealed nonlinear kinetics with the similar K m(app) value to the value for THTR2. Further in situ absorption study demonstrated that rat intestinal permeability of metformin was significantly decreased in the presence of decynium22, 7-cyclopentyl and thiamine. The present study indicated that THTR2 is the major determinant of the nonlinear absorption of metformin, although multiple transport mechanisms contribute to its intestinal absorption., Competing Interests: Conflict of Interest The authors declare no conflict of interest., (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Cluster of differentiation (CD) 38, a major enzyme for nicotinamide adenine dinucleotide (NAD + ) degradation, plays a key role in inflammation. Meanwhile, intracellular NAD + decline is also associated with inflammatory responses. However, whether CD38 activation is involved in gouty inflammation has not been elucidated. The present study aimed to clarify the role of CD38 in monosodium urate crystals (MSU)-triggered inflammatory responses. The results showed that MSU crystals increased the protein expression of CD38 in time- and concentration-dependent manner in THP-1 macrophages and mouse bone marrow-derived macrophages (BMDMs). Moreover, intracellular NAD + levels were reduced by MSU crystals along with the increased IL-1β release. However, CD38 inhibition by 78c elevated intracellular NAD + levels and suppressed IL-1β release in MSU crystals-treated THP-1 macrophages and BMDMs. Interestingly, CD38 inhibition without significant elevation of intracellular NAD + also decreased IL-1β release driven by MSU crystals in THP-1 macrophages. In conclusion, the present study revealed that MSU crystals could activate CD38 with the ensuing intracellular NAD + decline to promote inflammatory responses in THP-1 macrophages and BMDMs, while CD38 inhibition could suppress MSU crystals-triggered inflammatory responses, indicating that CD38 is a potential therapeutic target for gout., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Introduction: We clarified the renal uptake and urinary secretion mechanism of [ 99m Tc]dimercaptosuccinic acid ([ 99m Tc]DMSA) via drug transporters in renal proximal tubules., Methods: [ 99m Tc]DMSA was added to human embryonic kidney 293 cells expressing human multidrug and toxin extrusion (MATE)1 and MATE2-K, carnitine/organic cation transporter (OCTN)1 and OCTN2, and organic cation transporter (OCT)2; to Flp293 cells expressing human organic anion transporter (OAT)1 and OAT3; and to vesicles expressing P-glycoprotein (P-gp), multidrug resistance associated protein (MRP)2, MRP4, or breast cancer resistance protein with and without probenecid (OAT inhibitor for both OATs and MRPs). Time activity curves of [ 99m Tc]DMSA with and without probenecid were established using LLC-PK 1 cells. Biodistribution and single photon emission computed tomography (SPECT) imaging in mice were conducted using [ 99m Tc]DMSA with and without probenecid., Results: [ 99m Tc]DMSA uptake was significantly higher in Flp293/OAT3 than in mock cells. Uptake via OAT3 was inhibited by probenecid. [ 99m Tc]DMSA uptake into vesicles that highly expressed MRP2 was significantly higher in adenosine triphosphate (ATP) than in adenosine monophosphate (AMP), and probenecid decreased uptake to similar levels as that in AMP. In the time activity curves for [ 99m Tc]DMSA in LLC-PK 1 cells, probenecid loading inhibited accumulation from the basolateral side into LLC-PK 1 cells, whereas accumulation from the apical side into cells gradually increased. Transport of [ 99m Tc]DMSA from both sides was low. Biodistribution and SPECT imaging studies showed that [ 99m Tc]DMSA with probenecid loading resulted in significantly higher accumulation in blood, heart, liver, and bladder after [ 99m Tc]DMSA injection compared with control mice. Probenecid induced significantly lower accumulation in the kidney after [ 99m Tc]DMSA injection., Conclusions: [ 99m Tc]DMSA accumulates in renal proximal tubular epithelial cells from blood via OAT3 on the basolateral side, and then a small volume of [ 99m Tc]DMSA will be excreted in urine via MRP2. ADVANCES IN KNOWLEDGE: [ 99m Tc]DMSA accumulates via OAT3 in renal proximal tubular epithelial cells and is slightly excreted from the cells via MRP2. IMPLICATIONS FOR PATIENT CARE: [ 99m Tc]DMSA may be useful for measuring renal transport function with OAT3 in patients., Competing Interests: Declaration of competing interest There is no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

This study aimed to demonstrate usefulness of the fluorophore-labeled bile acid derivative, N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5β-cholestan-26-oyl)-2'-aminoethane sulfonate (tauro-nor-THCA-24-DBD) as a substrate of apical sodium-dependent bile acid transporter (ASBT, SLC10A2), which is expressed at distal ileum for reabsorption of bile acids and to find a novel fluorescence-based method to evaluate ASBT activity. In HPLC analysis, chromatogram of tauro-nor-THCA-24-DBD showed double peaks: R- and S-isomers of the compound. When ASBT was expressed in Xenopus laevis oocytes, their uptakes were higher than those by control oocytes, demonstrating both are transported by ASBT. Therefore, results were analyzed separately as peak 1, peak 2 and sum of them. Concentration dependent uptake of tauro-nor-THCA-24-DBD in ASBT-expressing oocytes was saturable with Km 122 μM and Vmax 1.49 pmol/oocyte/30 min for peak 1, 30.7 μM and 1.34 pmol/oocyte/30 min for peak 2, and 40.6 μM and 2.36 pmol/oocyte/30 min for sum, respectively. These uptakes were decreased in the presence of taurocholic acid and in the Na + free condition. Furthermore, in Caco-2 cells, tauro-nor-THCA-24-DBD uptake was also Na + -dependent and saturable. Additionally, these uptakes were decreased by elobixibat, a selective ASBT inhibitor. Accordingly, it was concluded that tauro-nor-THCA-24-DBD is a substrate of ASBT and useful to evaluate the intestinal ASBT transport activity., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Human OATP2B1, a member of organic anion transporting polypeptide family, is expressed in several tissues, including small intestine and liver, and contributes to cellular uptake of endogenous compounds and various drugs. Altered expression of OATP2B1 affects pharmacokinetics of substrate drugs; however, limited information is available on the regulation of OATP2B1 expression. This study aimed to explore microRNAs (miRNAs) that regulate OATP2B1 expression using HEK293 cells transfected with an expression plasmid of OATP2B1 including 3'-UTR (HEK/OATP2B1) and Caco-2 as a model of human intestine. Computational analysis predicted that three miRNAs, miR-143, miR-125b and miR-24, may bind to the 3'-UTR of OATP2B1 mRNA. A luciferase assay using a plasmid containing the 3'-UTR of OATP2B1 gene demonstrated that only miR-24 significantly reduced its expression. The overexpression of miR-24 decreased the expression of OATP2B1 mRNA and protein in HEK/OATP2B1 and Caco-2 cells and uptake of [ 3 H]estrone-3-sulfate by HEK/OATP2B1 cells. However, a statistically significant increase of endogenous OATP2B1 expression was not observed by miR-24 inhibitor in Caco-2 cells. In conclusion, it was found that miR-24 negatively regulates OATP2B1 expression, resulting in suppression of OATP2B1 activity, while its contribution to regulation of apparent expression of OATP2B1 is considered to depend on tissues and cell types., Competing Interests: Declaration of competing interest None., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Introduction: 131 I-labeled m-iodobenzylguanidine ([ 131 I]MIBG) has been used to treat neuroblastoma patients, but [ 131 I]MIBG may be immediately excreted from the cancer cells by the adenosine triphosphate binding cassette transporters, similar to anticancer drugs. The purpose of this study was to clarify the efflux mechanism of [ 131 I]MIBG in neuroblastomas and improve accumulation by inhibition of the transporter in neuroblastomas., Methods: [ 131 I]MIBG was incubated in human embryonic kidney (HEK)293 cells expressing human organic anion transporting polypeptide (OATP)1B1, OATP1B3, OATP2B1, organic anion transporter (OAT)1 and OAT2, organic cation transporter (OCT)1 and OCT2, and sodium taurocholate cotransporting polypeptide, and in vesicles expressing P-glycoprotein (MDR1), multidrug resistance associated protein (MRP)1-4, or breast cancer resistance protein with and without MK-571 and probenecid (MRP inhibitors). Time activity curves of [ 131 I]MIBG with and without MK-571 and probenecid were established using an SK-N-SH neuroblastoma cell line, and transporter expression of multiple drug resistance was measured. Biodistribution and SPECT imaging examinations were conducted using [ 123 I]MIBG with and without probenecid in SK-N-SH-bearing mice., Results: [ 131 I]MIBG uptake was significantly higher in OAT1, OAT2, OCT1, and OCT2 than in mock cells. Uptake via OCT1 and OCT2 was little inhibited by MK-571 and probenecid. [ 131 I]MIBG uptake into vesicles that highly expressed MRP1 or MRP4 was significantly higher in ATP than in AMP, and these inhibitors restored uptake to levels similar to that in AMP. Examining the time activity curves for [ 131 I]MIBG in SK-N-SH cells, higher expressions of MDR1, MRP1, MRP4, and MK-571, or probenecid loading produced significantly higher uptake than in control at most incubation times. The ratios of tumors to blood or muscle in SK-N-SH-bearing mice were significantly increased by probenecid loading in comparison with normal mice., Conclusions: [ 131 I]MIBG exports via MRP1 and MRP4 in neuroblastoma. The accumulation and tumor-to-blood or muscle ratios of [ 131 I]MIBG are improved by inhibition of MRPs with probenecid in neuroblastoma. ADVANCES IN KNOWLEDGE: [ 131 I]MIBG, widely used for treatment of neuroendocrine tumors including neuroblastoma, is excreted via MRP1 and MRP4 in neuroblastoma., Implications for Patient Care: Loading with probenecid, OAT, and MRP inhibitors improves [ 131 I]MIBG accumulation., Competing Interests: Declaration of competing interest There is no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Uric acid is biosynthesized from purine by xanthine oxidase (XO) mainly in the liver and is excreted into urine and feces. Although several transporters responsible for renal and intestinal handling of uric acid have been reported, information on hepatic transporters is limited. In the present study, we studied quantitative contribution of transporters for hepatic handling of uric acid by mathematical modeling analysis in human sandwich-cultured hepatocytes (hSCH). Stable isotope-labeled hypoxanthine, hypoxanthine- 13 C2, 15 N (HX), was incubated with hSCH and formed 13 C2, 15 N-labeled xanthine (XA) and uric acid (UA) were measured by LC-MS/MS time dependently. Rate constants for metabolism and efflux and uptake transport across sinusoidal and bile canalicular membranes of HX, XA and UA were estimated in the presence of inhibitors of XO and uric acid transporters. An XO inhibitor allopurinol significantly decreased metabolisms of HX and XA. Efflux into bile canalicular lumen was negligible and sinusoidal efflux was considered main efflux pathway of formed UA. Transporter inhibition study highlighted that GLUT9 strongly and MRP4 intermediately contribute to the sinusoidal efflux of UA with minor contribution of NPT1/4. Modeling analysis developed in the present study should be useful for quantitative prediction of uric acid disposition in liver., Competing Interests: Declaration of competing interest The authors report no conflict of interest., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Technetium-99m-labeled mercaptoacetyltriglycine ([ 99m Tc]MAG3) is widely used for evaluation of transplanted kidneys, diagnosis of tubular necrosis, and scintigraphic studies of tubular function. [ 99m Tc]MAG3 is a substrate for organic anion transporter (OAT)1 and OAT3 on the basolateral membrane side for renal secretion. We investigated the transport mechanism and affinity of [ 99m Tc]MAG3 on the apical membrane of renal proximal tubule cells for renal secretion. Adenosine triphosphate-binding cassette (ABC) transporters for renal secretion of [ 99m Tc]MAG3 were examined using ABC transporter vesicles expressing multiple drug resistance 1 (MDR1), breast cancer resistance protein (BCRP), multidrug resistance-associated protein (MRP)2, and MRP4. MK-571, a MRP inhibitor, was applied to measure the K m and V max of MRP2 and MRP4 in a vesicle transport assay. Single photon emission computed tomography (SPECT) was performed in normal rats and MRP2-deficient Eisai hyperbilirubinuria rats (EHBR) using [ 99m Tc]MAG3 with and without MK-571. [ 99m Tc]MAG3 uptake in adenosine triphosphate was significantly higher than that in adenosine monophosphate in vesicles that highly expressed MRP2 and MRP4. The affinity of [ 99m Tc]MAG3 for MRP4 was higher than that for MRP2. Renal secretion via MRP2 and MRP4 was identified by comparing normal and EHBR rats with and without MK-571 on SPECT. [ 99m Tc]MAG3 is transported via MRP2 and MRP4 on the apical membrane of renal proximal tubule cells. The affinity of MRP4 is higher than that of MRP2. SIGNIFICANCE STATEMENT: [ 99m Tc]MAG3, widely used for evaluation of transplanted kidneys, diagnosis of tubular necrosis, and scintigraphic studies of tubular function, is transported via MRP2 and MRP4 on the apical membrane of renal proximal tubule cells. The affinity of MRP4 is higher than that of MRP2., Competing Interests: Declaration of competing interest Hiroyuki Okudaira is an employee of Nihon Medi-Physics Co., Ltd., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Kidney slice has been often used as a tool reflecting basolateral transport in renal tubular epithelial cells. Recently, we reported that several important apical reabsorptive transporters such as Octn1/2, Sglt1/2, and Pept1/2 were functional in mouse kidney slices as well as transporter activities in basolateral side, which have been well accepted. Because rats are often used for preclinical pharmacodynamic and pharmacokinetic studies as well as mice, it is important to confirm applicability of rat kidney slices for evaluation of apically expressed transporters. The present study investigates usefulness of kidney slices from rats for evaluation of apical membrane transporters for efflux (multidrug resistance 1a, mdr1a) as well as influx (Octn1/2, Sglt1/2, Pept1/2). Na + -dependent uptake of ergothioneine (Octn1), carnitine (Octn2), and methyl-α-D-glucopyranoside (Sglt1/2) by rat kidney slices was observed, and the uptake was decreased by selective inhibitors. In addition, uptake of glycyl-sarcosine (Pept1/2) showed H + -dependence and was decreased by selective inhibitor. Furthermore, accumulation of mdr1a substrate azasetron was increased in the presence of zosuquidar, an mdr1a inhibitor, while strain differences existed. In conclusion, rat kidney slices should be useful for evaluation of renal drug disposition regulated by transporters in apical as well as basolateral membranes of rat renal proximal tubule cells., (Copyright © 2019. Published by Elsevier Inc.)

The inhibitor of uric acid reabsorptive transporter URAT1 in kidney is drawing attention as a drug target for hyperuricemia. However, it is difficult to evaluate efficacy of URAT1 inhibitors in vivo using laboratory animals due to species difference in uric acid metabolism. In the present study, the usefulness of exogenously administering uric acid analogues resistant to uricase was investigated for in vivo evaluation of transport activity of rUrat1 in rats. Uptake of examined four uric acid analogues by rUrat1-expressing Xenopus oocytes was significantly higher than that by water-injected oocytes. In metabolism studies, disappearance of these compounds was negligible, while uric acid was significantly decreased. When oxypurinol was administered to rats, fractional excretion (FE) was 0.4, suggesting reabsorption of oxypurinol. Moreover, FE of oxypurinol was tended to be increased, but not statistically different, by co-administration of a uricosuric agent FYU-981, while plasma concentration of oxypurinol was not affected. These results suggested that oxypurinol is a potential uric acid analogue, although it was not suitable as a probe of uric acid in in vivo study. Our findings may contribute to discovery and development of novel uricosuric agent targeting URAT1., (Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Hyperuricemia has been recognized as an independent risk factor for cardiovascular disease. Urate stimulates NADPH oxidase and induces production of reactive oxygen species (ROS); consequently, intracellular urate accumulation can induce oxidative stress leading to endothelial dysfunction. Here, we studied the mechanism involved, using human umbilical vascular endothelial cells (HUVEC) as a model. Pretreatment with 15 mg/dL unlabeled uric acid (corresponding to hyperuricemia) resulted in increased uptake of [ 14 C]uric acid at steady-state by HUVEC, whereas pretreatment with 5 mg/dL uric acid (in the normal serum concentration range) did not. However, the initial uptake rate of [ 14 C]uric acid was not affected by uric acid at either concentration. These results suggest that efflux transport of uric acid is decreased under hyperuricemic conditions. We observed a concomitant decrease of phosphorylated endothelial nitric oxide synthase. Plasma membrane expression of breast cancer resistance protein (BCRP), a uric acid efflux transporter, was decreased under hyperuricemia, though the total cellular expression of BCRP remained constant. Uric acid did not affect expression of another uric acid efflux transporter, multidrug resistance associated protein 4 (MRP4). Moreover, phosphorylation of Akt, which regulates plasma membrane localization of BCRP, was decreased. These uric acid-induced changes of BCRP and Akt were reversed in the presence of the antioxidant N-acetylcysteine. These results suggest that in hyperuricemia, uric acid-induced ROS generation inhibits Akt phosphorylation, causing a decrease in plasma membrane localization of BCRP, and the resulting decrease of BCRP-mediated efflux leads to increased uric acid accumulation and dysregulation of endothelial function., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This study aimed to investigate the interactions of 3 anticoagulants, rivaroxaban, apixaban, and dabigatran, with 5 human solute carrier transporters, hOAT1, hOAT3, hOCT2, hOATP1B1, and hOATP1B3. Apixaban inhibited hOAT3, hOATP1B1, and hOATP1B3, and rivaroxaban inhibited hOAT3 and hOATP1B3, with IC 50 values of >20 and >5 μM, respectively. The effect of dabigatran was negligible or very weak, so significant drug interactions at therapeutic doses are unlikely. Specific uptake of rivaroxaban was observed only in human and mouse OAT3-expressing cells. The K m for mouse Oat3 (mOat3) was 1.01 ± 0.70 μM. A defect in mOat3 reduced the kidney-to-plasma concentration ratio of rivaroxaban by 38% in mice. Probenecid treatment also reduced the kidney-to-plasma concentration ratio of rivaroxaban in rats by 73%. Neither mOat3 defect nor probenecid administration in rats reduced the renal clearance of rivaroxaban. The uptake of rivaroxaban by monkey kidney slices was temperature dependent and inhibited by probenecid but not by tetraethylammonium. Taken together, organic anion transporters, mainly OAT3, may mediate basolateral uptake of rivaroxaban in kidneys. hOAT3 could be an additional factor that differentiates the potential drug-drug interactions of the 3 anticoagulants in the urinary excretion process in clinical settings., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

To understand interaction of drugs with the prostaglandin transporter OATP2A1/SLCO2A1 that regulates disposition of prostaglandins, we explored the impact of 636 drugs in an FDA-approved drug library on 6-carboxyfluorescein (6-CF) uptake by OATP2A1-expressing HEK293 cells (HEK/2A1). Fifty-one and 10 drugs were found to inhibit and enhance 6-CF uptake by more than 50%, respectively. Effect of the 51 drugs on 6-CF uptake was positively correlated with that on PGE 2 uptake (r = 0.64, p < 0.001). Among those, 5 drugs not structurally related to prostaglandins, suramin, pranlukast, zafirlukast, olmesartan medoxomil, and losartan potassium, exhibited more than 90% PGE 2 uptake inhibition. Inhibitory affinity of suramin to OATP2A1 was the highest (IC 50,2A1 of 0.17 μM), and its IC 50 values to MRP4-mediated PGE 2 transport (IC 50,MRP4 ) and PGE 2 synthesis in human U-937 cells treated with phorbol 12-myristate 13-acetate (IC 50,Syn ) were 73.6 and 336.7 times higher than IC 50,2A1 , respectively. Moreover, structure-activity relationship study in 29 nonsteroidal anti-inflammatory drugs contained in the library displayed inhibitory activities of anthranilic acid derivatives, but enhancing effects of propionic acid derivatives. These results demonstrate that suramin is a potent selective inhibitor of OATP2A1, providing a comprehensive information about drugs in clinical use that interact with OATP2A1., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Crizotinib, a tyrosine kinase inhibitor, exhibits some cases of an increase in serum creatinine levels. Creatinine is excreted by not only glomerular filtration but also active secretion by organic cation transporters such as organic cation transporter 2 (OCT2). In the present study, we evaluated in vitro inhibitory effect of crizotinib on OCT2 by directly measuring creatinine uptake by OCT2. Coincubation of crizotinib reduced uptake of [ 14 C]creatinine by cultured HEK293 cells expressing OCT2 (HEK293/OCT2) in a concentration-dependent manner with IC 50 values of 1.58 ± 0.24 μM. Preincubation or both preincubation and coincubation (preincubation/coincubation) with crizotinib showed stronger inhibitory effect on [ 14 C]creatinine uptake compared with that in coincubation alone with IC 50 values of 0.499 ± 0.076 and 0.347 ± 0.040 μM, respectively. These IC 50 values of crizotinib on [ 3 H]N-methyl-4-phenylpyridinium acetate uptake by OCT2 were 10-20 times higher than those of [ 14 C]creatinine uptake. Furthermore, preincubation of crizotinib inhibited creatinine uptake by OCT2 in an apparently competitive manner. In conclusion, crizotinib at a clinically relevant concentration has the potential to inhibit creatinine transport by OCT2, suggesting an increase of serum creatinine levels in clinical use., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Skeletal muscle toxicity including rhabdomyolysis in severe case is a major side effect of low-density lipoprotein cholesterol-lowering statin drugs. We, therefore, aimed at exploring microRNA (miRNA) expression to understand molecular mechanism of statin-induced toxicity. miRNA expression profiling assay for cerivastatin (1 μM for 48 h)-treated RD cells showed more than 2-fold decrease in 26 miRNA expressions with miR-145 being downregulated prominently. When RD cells were treated with cerivastatin at 10 μM for 36 h, mitochondrial dysfunction was observed in 49.6% of the population without causing apoptosis, whereas 82% underwent apoptosis when treated at 10 μM for 48 h. In RD cells treated under the same condition (10 μM for 48 h), miR-145 expression and mRNA expressions of proapoptotic APAF1 and CASP10 genes, potential targets of miR-145, significantly decreased and increased, respectively. Moreover, enforced expression of miR-145 reduced apoptotic cell population of cerivastatin-treated RD cells (10 μM for 36 h). Because miR-145 increased in extracellular medium from cerivastatin-treated RD cells, miR-145 was suggested to be secreted in response to statin-induced toxicity. These results provide a new rationale for statin's toxicity that statin-induced apoptosis is caused by enhanced expression of proapoptotic genes mediated by decreased intracellular miR-145 due to statin-induced mitochondrial dysfunction., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The placenta is an organ that secretes prostaglandin (PG) E 2 into the fetal-placental circulation to regulate both vascular tone and remodeling of the fetal ductus arteriosus. Placental PGE 2 synthesis might be mediated by microsomal PGE synthase-1 (mPGES-1), in addition to cyclooxygenase (COX) isoforms. Thus, the purpose of this study is to clarify the temporal and spatial expression patterns of mPGES-1, together with COX-1 and COX-2, in murine placenta. We found that mPGES-1 and COX-1 protein levels continuously increased in the placental labyrinth from gestational day (GD) 13.5 to GD19.5, becoming higher than in the decidua or the junctional zone by GD17.5. The PGE 2 level at GD17.5 was also highest in the labyrinth. Immunofluorescence stainings for mPGES-1 and COX-1 in the labyrinth at GD17.5 overlapped and were located on the fetal side of the signals for connexin 26, which forms gap junctions between maternal-facing (SynT-I) and fetal-facing (SynT-II) syncytiotrophoblast layers, and on the maternal side of the signals for glucose transporter 1 on the basal plasma membrane of SynT-II. On the other hand, the signals for COX-2 did not overlap with those for mPGES-1. These results indicate that COX-1 and mPGES-1 are co-localized in murine placental SynT-II, facing the fetal-placental circulation. Therefore, SynT-II could contribute to placental synthesis of PGE 2 for release into the fetal-placental circulation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The goal of this study was to improve the intestinal mucosal cell membrane permeability of the poorly absorbed guanidino analogue of a neuraminidase inhibitor, oseltamivir carboxylate (GOC) using a carrier-mediated strategy. Valyl amino acid prodrug of GOC with isopropyl-methylene-dioxy linker (GOC-ISP-Val) was evaluated as the potential substrate for intestinal oligopeptide transporter, hPEPT1 in Xenopus laevis oocytes heterologously expressing hPEPT1, and an intestinal mouse perfusion system. The diastereomers of GOC-ISP-Val were assessed for chemical and metabolic stability. Permeability of GOC-ISP-Val was determined in Caco-2 cells and mice. Diastereomer 2 was about 2 times more stable than diastereomer 1 in simulated intestinal fluid and rapidly hydrolyzed to the parent drug in cell homogenates. The prodrug had a 9 times-enhanced apparent permeability (P(app)) in Caco-2 cells compared with the parent drug. Both diastereomer exhibited high effective permeability (P(eff)) in mice, 6.32 ± 3.12 and 5.20 ± 2.81 × 10(-5) cm/s for diastereomer 1 and 2, respectively. GOC-ISP-Val was found to be a substrate of hPEPT1. Overall, this study indicates that the prodrug, GOC-ISP-Val, seems to be a promising oral anti-influenza agent that has sufficient stability at physiologically relevant pHs before absorption, significantly improved permeability via hPEPT1 and potentially rapid activation in the intestinal cells., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The stereoselective pharmacokinetics of fexofenadine are associated with OATP2B1-mediated transport, and grapefruit juice (GFJ) is an inhibitor of OATP2B1. Therefore, in this study, we aimed to investigate whether and to what extent GFJ ingestion affected the pharmacokinetics of fexofenadine enantiomers in healthy subjects. In a randomized, two-phase, open-label, crossover study, 14 subjects received 60 mg of racemic fexofenadine simultaneously with water or GFJ. Ingestion of GFJ significantly decreased the areas under the plasma concentration-time curve (AUC0-24) for (R)- and (S)-fexofenadine by 39% and 52%, respectively. Subsequently, GFJ increased the mean R/S ratio of the AUC0-24 from 1.58 to 1.96 (P < 0.05). Although GFJ greatly reduced the amounts of (R)- and (S)-fexofenadine excreted into the urine (Ae0-24) by 52% and 61%, respectively, the mean R/S ratios of Ae0-24 and the renal clearances of both enantiomers were unchanged between the control and GFJ phases. GFJ, an OATP2B1 inhibitor, significantly reduced the plasma concentrations of fexofenadine enantiomers, exhibiting clinically moderate effects. The present results suggested that changes in OATP2B1 activity by GFJ may alter the stereoselective pharmacokinetics of fexofenadine and that reduced intestinal OATP2B1 activity may affect the stereoselectivity of fexofenadine., (Copyright © 2015 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

TAS-102, which is effective for refractory metastatic colorectal cancer, is a combination drug of anticancer trifluridine (FTD; which is derived from pyrimidine nucleoside) and FTD-metabolizing enzyme inhibitor tipiracil hydrochloride (TPI) at a molecular ratio of 1:0.5. To evaluate the intestinal absorption mechanism of FTD, the uptake and transcellular transport of FTD by human small intestinal epithelial cell (HIEC) monolayer as a model of human intestinal epithelial cells was investigated. The uptake and membrane permeability of FTD by HIEC monolayers were saturable, Na(+) -dependent, and inhibited by nucleosides. These transport characteristics are mostly comparable with those of concentrative nucleoside transporters (CNTs). Moreover, the uptake of FTD by CNT1-expressing Xenopus oocytes was the highest among human CNT transporters. The obtained Km and Vmax values of FTD by CNT1 were 69.0 μM and 516 pmol/oocyte/30 min, respectively. The transcellular transport of FTD by Caco-2 cells, where CNT1 is heterologously expressed, from apical to basolateral side was greater than that by Mock cells. In conclusion, these results demonstrated that FTD exhibits high oral absorption by the contribution of human CNT1., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Five-aminolevulinic acid (ALA) is a prodrug to generate phototoxic protoporphyrin IX (PPIX) for photodynamic cancer therapy. It remains unclear how PPIX accumulates in cancer cells; therefore, we aimed to clarify determinant factors by assessing ALA uptake, PPIX biosynthesis, conversion of PPIX to heme (ferrochelatase activity), and PPIX efflux, independently, in 10 human cancer cell lines. ALA-induced PPIX accumulation was not correlated with ALA uptake clearance. ALA uptake rates were far greater than maximum conversion rates of ALA to PPIX in the five cell lines, where ALA uptake activity was detected. A negative correlation of PPIX accumulation with ferrochelatase activity was found, but not statistically significant among all cell lines. As PPIX accumulation was restored in MCF-7 and DU145 cells by adding an inhibitor of PPIX efflux transporter BCRP, a compartment model incorporating PPIX synthesis, ferrochelatase activity, and PPIX efflux, was established, and hybrid parameters (π index) calculated using the model were significantly correlated with ALA-induced PPIX accumulation (r = 0.90, p = 0.005). Accordingly, kinetic analyses indicate that ALA-induced PPIX levels are determined by the three processes of PPIX biosynthesis, conversion of PPIX to heme, and PPIX efflux, suggesting that π index is a useful to predict ALA-induced PPIX accumulation., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Hepatic arterial infusion (HAI) chemotherapy with gemcitabine (GEM) is expected to be more effective and safer method to treat hepatic metastasis of pancreatic cancer compared with intravenous administration, because it affords higher tumor exposure with lower systemic exposure. Thus, a key issue for dose selection is the saturability of hepatic uptake of GEM. Therefore, we investigated GEM uptake in rat and human isolated hepatocytes. Hepatic GEM uptake involved high- and low-affinity saturable components with Km values of micromolar and millimolar order, respectively. The uptake was inhibited concentration dependently by S-(4-nitrobenzyl)-6-thioinosine (NBMPR) and was sodium-ion-independent, suggesting a contribution of equilibrative nucleoside transporters (ENTs). The concentration dependence of uptake in the presence of 0.1 μM NBMPR showed a single low-affinity binding site. Therefore, the high- and low-affinity sites correspond to ENT1 and ENT2, respectively. Our results indicate hepatic extraction of GEM is predominantly mediated by the low-affinity site (hENT2), and at clinically relevant hepatic concentrations of GEM, hENT2-mediated uptake would not be completely saturated. This is critical for HAI, because saturation of hepatic uptake would result in a marked increase of GEM concentration in the peripheral circulation, abrogating the advantage of HAI over intravenous administration in terms of severe adverse events., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)

A lower exposure of mycophenolic acid (MPA) in patients receiving MPA-mofetil in combination with cyclosporin A (CsA) is thought to be due to the inhibition of enterohepatic circulation of phenyl-glucuronide of MPA (MPAG). This study aimed to evaluate the interaction of CsA with hepatic disposition of MPA and MPAG in sandwich-cultured human hepatocytes (SCHH) by a mathematical modeling approach. In addition, the inhibition of CsA for glucuronidation of MPA to MPAG was examined in human liver microsomes. Inhibitory parameters of CsA for hepatic disposition of MPAG were estimated using a non-linear mixed effect model program, NONMEM. As a result, CsA did not influence the conversion of MPA to MPAG in either SCHH or human liver microsomes. In contrast, CsA inhibited the basolateral uptake of MPAG with an estimated maximum inhibitory effect (Imax) of 32.4%. CsA also inhibited basolateral efflux and biliary excretion of MPAG formed in SCHH, and the concentration producing 50% of Imax (IC50) for biliary excretion was lower than that for basolateral efflux. Our modeling approach suggests that CsA inhibits both basolateral uptake and biliary excretion of MPAG and leads to changes in systemic exposure of MPA and MPAG in humans., (Copyright © 2014 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Estrone-3-sulfate (E1S) is thought to be a major estrogen precursor in estrogen receptor (ER)-positive breast cancer. Since E1S is a hydrophilic compound, the uptake of E1S into cancer cells is probably mediated by transporters, such as organic anion-transporting polypeptide (OATP, SLCO) family. In this study, we investigated the relationship between expression of OATP2B1 and cell proliferation in ER-positive breast cancer. Cell-based assays were carried out in MCF-7 cells both with and without overexpression of OATP2B1. Normal breast and tumor tissues were collected and used in this study. Cell proliferation, ER-mediated transcriptional activities and estradiol secretion were stimulated by addition of E1S to the culture medium of MCF-7 cells. These stimulatory effects were significantly greater in MCF-7 cells overexpressing OATP2B1 than in control cells. The expression level of SLCO2B1 mRNA was significantly correlated with histological grade, Ki-67 labelling index and mRNA expression of steroid sulfatase. The expression level of SLCO2B1 mRNA in luminal B-like cancers was higher than that in luminal A-like cancers. Uptake of E1S resulted in down-regulation of ERα protein and induction of Ki-67 in MCF-7 cells. The present study suggests that OATP2B1 is involved in cell proliferation by increasing the amount of estrogen in ER-positive breast cancer cells., (Copyright © 2014 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Since drug-drug interaction (DDI) can affect organic anion-transporting polypeptide (OATP) and cause clinical events, prediction of such DDI is important in early clinical development. Although statins are useful probes for OATP-mediated DDI, endogenous probes would be more practical for predicting such DDI. In this study, we investigate the possible use of dehydroepiandrosterone sulfate (DHEAS), an endogenous OATP substrate, in predicting OATP-mediated DDI in cynomolgus monkeys as a first step toward in human assessment. In in vitro experiments, both human and cynomolgus monkey hepatocytes showed a time- and temperature-dependent DHEAS uptake. Rifampicin (RIF), a typical OATP inhibitor, inhibited this uptake, indicating the involvement of OATP in DHEAS uptake. In in vivo experiments, the area under the plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) of DHEAS were significantly increased following administration of RIF 10 mg/kg, although the extent of this increase was lower than that observed with the test-statins used in this study. However, based on the results of in vitro hepatic DHEAS uptake, changes in DHEAS concentration are expected to be more prominent in human than in monkey. This shows for the first time that DHEAS may be used as endogenous probe for predicting OATP-mediated DDI., (Copyright © 2015 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Introduction: In clinical hepatobiliary scintigraphy, (99m)Tc-N-pyridoxyl-5-methyltryptophan ((99m)Tc-PMT) is an effective radiotracer among the (99m)Tc-pyridoxylaminates. However, the mechanisms of human hepatic uptake and bile excretion transport of (99m)Tc-PMT have not been determined. We thus investigated the transport mechanisms of human hepatic uptake and bile excretion in hepatobiliary scintigraphy with (99m)Tc-PMT., Methods: Four solute carrier (SLC) transporters involved in hepatic uptake were evaluated using human embryonic kidney (HEK) and HeLa cells with high expression of SLC transporters (organic anion transporting polypeptide (OATP)1B1, OATP1B3, OATP2B1, organic anion transporters (OAT)2 and organic cation transporters (OCT)1) after 5 min of (99m)Tc-PMT incubation. Metabolic analysis of (99m)Tc-PMT was performed using pooled human liver S9. Adenosine triphosphate (ATP)-binding cassette (ABC) transporters for bile excretion were examined using hepatic ABC transporter vesicles human expressing multiple drug resistance 1 (MDR1), multidrug resistance-associated protein 2 (MRP2), breast cancer resistance protein or bile salt export pump. (99m)Tc-PMT was incubated for 1, 3 and 5 min with ATP or adenosine monophosphate and these vesicles. SPECT scans were performed in normal and Eisai hyperbilirubinemic (EHBR) model rats, deficient in Mrp2 transporters, without and with verapamil (rat Mdr1 and human MDR1 inhibitor) after intravenous injection of (99m)Tc-PMT., Results: Uptake of (99m)Tc-PMT in HEK293/OATP1B1 and HeLa/OATP1B3 was significantly higher than that in HEK293- and HeLa-mock cells. (99m)Tc-PMT was not metabolized in the human liver S9. In vesicles with high expression of ABC transporters, uptake of MDR1 or MRP2 was significantly higher at all incubation times. Bile excretion of (99m)Tc-PMT was also identified by comparison between normal and EHBR rats with and without verapamil on in-vivo imaging., Conclusions: Human hepatic uptake of (99m)Tc-PMT was transferred by OATP1B1 and OATP1B3, and excretion into bile canaliculi via MDR1 and MRP2. (99m)Tc-PMT hepatobiliary scintigraphy may be a useful ligand as a noninvasive method of visualizing and quantifying hepatobiliary transporter functionality, which could predict drug pharmacokinetics., (© 2014.)

Bile salt export pump (BSEP) plays a major role in biliary secretion of bile salts; therefore, drug-induced cholestasis could occur because of BSEP inhibition by drugs. Drug interaction on hepatic bile canalicular transporters such as BSEP with prodrugs that are rapidly metabolized has not been evaluated well. In the present study, candesartan cilexetil (CIL) was used as a model compound and its inhibitory potential against BSEP was determined in sandwich-cultured human hepatocytes (hSCH) as well as in BSEP-expressing membrane vesicles. CIL exhibited potent BSEP inhibition with an IC50 value of 6.2 µM in the transport assay using membrane vesicles. In contrast, BSEP inhibition by CIL was not observed in hSCH after 120 min exposure. This discordance is possibly explained by metabolic elimination of CIL in hSCH because BSEP inhibition became reversely pronounced under the conditions where CIL metabolism was suppressed by diisopropyl fluorophosphates. The results observed in hSCH are consistent with the fact that liver dysfunction or jaundice occurs with low frequency in clinical use of CIL, which may not be obtained by membrane vesicle study on the effect of CIL on BSEP.

We aimed to explore the major active components in grapefruit juice (GFJ), orange juice (OJ), and apple juice (AJ) that are responsible for OATP2B1-mediated drug interactions, by means of in vitro studies using Xenopus oocytes expressing OATP2B1 with a typical OATP2B1 substrate, estrone-3-sulfate. All three juices inhibited OATP2B1-mediated estrone-3-sulfate uptake with half-maximum inhibition (IC50 ) values of 0.222% (GFJ), 0.807% (OJ), and 2.27% (AJ). Eight major flavonoids (naringin, naringenin, hesperidin, hesperetin, phloridzin, phloretin, quercetin, and kaempferol) contained in the juices inhibited OATP2B1-mediated estrone-3-sulfate uptake with IC50 values of 4.63, 49.2, 1.92, 67.6, 23.2, 1.31, 9.47, and 21.3 μM, respectively. When the concentration-IC50 ratios ([C]/IC50 ) of these flavonoids in GFJ, OJ, and AJ were calculated, values of [C]/IC50 ≥ 100 were obtained for naringin in GFJ and hesperidin in OJ. No flavonoid in AJ showed a ratio higher than unity. However, significant inhibition of OATP2B1 was observed with a mixture of phloridzin, phloretin, hesperidin, and quercetin at the concentrations present in AJ. In conclusion, our results indicate that naringin and hesperidin are the major OATP2B1 inhibitors in GFJ and OJ, respectively, whereas a combination of multiple components appears to be responsible for OATP2B1 inhibition by AJ., (Copyright © 2013 Wiley Periodicals, Inc.)

Ipratropium bromide (IPR) is an anticholinergic used to treat chronic obstructive pulmonary disease (COPD), and is a substrate of organic cation transporters. The present study aimed to assess the contribution of organic cation transporters to tracheobronchial absorption of IPR in vivo by directly injecting [(3) H]IPR into the tracheal lumen of mice and measuring its accumulation in tracheal tissue. RT-PCR and immunohistochemical analysis showed that Octn1, Octn2, and Oct2 were localized at epithelial cells in the respiratory tract. Electron-microscopic immunohistochemistry indicated that Octn1 and Octn2 were localized at the apical portions of ciliated epithelial cells of trachea. In vitro uptake studies in HEK293 cells expressing these transporters demonstrated that IPR is a preferred substrate of Octn2. Inhibition of mouse tracheal accumulation of [(3) H]IPR by carnitine was concentration-dependent, reaching a maximum of 42% at 1 mM, whereas inhibition by 0.1 mM MPP(+) amounted to 62%. Tracheal accumulation of [(3) H]IPR was unchanged when mice were simultaneously injected with Octn1 substrate ergothioneine and organic anion transporter substrate estrone sulfate. These results suggest that Octn2 is involved in membrane permeation of IPR in the respiratory tract in vivo. Targeting organic cation transporters may be an effective strategy for delivery of cationic anti-COPD drugs to patients., (Copyright © 2013 Wiley Periodicals, Inc.)

Introduction: Trans-1-amino-3-[(18)F]fluorocyclobutanecarboxylic acid (anti-[(18)F]FACBC) is a promising amino acid positron emission tomography (PET) radiotracer for visualizing prostate cancer. We previously showed that anti-FACBC is transported by amino acid transporters, especially by alanine-serine-cysteine transporter 2 (ASCT2), which is associated with tumor growth. We studied this affinity to assess the mechanism of anti-FACBC transport in prostate cancer cells., Methods: Kinetic assays for trans-1-amino-3-fluoro-[1-(14)C]cyclobutanecarboxylic acid ([(14)C]FACBC) were performed in Xenopus laevis oocytes over-expressing either ASCT2 or sodium-coupled neutral amino acid transporter 2 (SNAT2), both of which are highly expressed in prostate cancer cells. We also examined the kinetics of [(14)C]FACBC uptake using mammalian cell lines over-expressing system L amino acid transporter 1 or 2 (LAT1 or LAT2). Results: ASCT2 and SNAT2 transported [14C]FACBC with Michaelis–Menten kinetics Km values of 96.7 ± 45.2 μM and 196.5 ± 19.7 μM, respectively. [correted]. LAT1 and LAT2 transported [(14)C]FACBC with Michaelis-Menten Km values of 230.4 ± 184.5 μM and 738.5 ± 87.6 μM, respectively., Conclusions: Both ASCT2 and SNAT2 recognize anti-FACBC as a substrate. Anti-FACBC has higher affinity for ASCT2 than for SNAT2, LAT1, or LAT2. The ASCT2-preferential transport of anti-[(18)F]FACBC in cancer cells could be used for more effective prostate cancer imaging., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We aimed to explore the major active components in grapefruit juice (GFJ), orange juice (OJ), and apple juice (AJ) that are responsible for OATP2B1-mediated drug interactions, by means of in vitro studies using Xenopus oocytes expressing OATP2B1 with a typical OATP2B1 substrate, estrone-3-sulfate. All three juices inhibited OATP2B1-mediated estrone-3-sulfate uptake with half-maximum inhibition (IC(50) ) values of 0.222% (GFJ), 0.807% (OJ), and 2.27% (AJ). Eight major flavonoids (naringin, naringenin, hesperidin, hesperetin, phloridzin, phloretin, quercetin, and kaempferol) contained in the juices inhibited OATP2B1-mediated estrone-3-sulfate uptake with IC(50) values of 4.63, 49.2, 1.92, 67.6, 23.2, 1.31, 9.47, and 21.3 µM, respectively. When the concentration-IC(50) ratios ([C]/IC(50) ) of these flavonoids in GFJ, OJ, and AJ were calculated, values of [C]/IC(50) ≥ 100 were obtained for naringin in GFJ and hesperidin in OJ. No flavonoid in AJ showed a ratio higher than unity. However, significant inhibition of OATP2B1 was observed with a mixture of phloridzin, phloretin, hesperidin, and quercetin at the concentrations present in AJ. In conclusion, our results indicate that naringin and hesperidin are the major OATP2B1 inhibitors in GFJ and OJ, respectively, whereas a combination of multiple components appears to be responsible for OATP2B1 inhibition by AJ., (Copyright © 2012 Wiley Periodicals, Inc.)

Urate is mainly excreted into urine in humans. Serum urate level is regulated by a urate transport system located on the renal proximal tubule. Urate transporter 1 (URAT1) is located on the apical side of the renal proximal tubule and is responsible for the reabsorption of urate from the luminal side into tubular cells. At the same site, it has been hypothesized that sodium-coupled monocarboxylate transporters (SMCTs) are responsible for the transportation of monocarboxylates such as lactate and nicotinate, which are exchanged for urate transport via URAT1. Accordingly, SMCTs could enhance URAT1-mediated urate reabsorption by providing monocarboxylates for the exchange. The present study was carried out to clarify the hypothesized functional cooperative relationship between URAT1 and SMCTs in the reabsorptive transport of urate. By preloading nicotinate in SMCT1/URAT1-coexpressing Xenopus oocytes, URAT1-mediated urate transport was stimulated. Nicotinate was taken up by SMCT1 but not by URAT1. When removing sodium ions from the uptake medium, the stimulation effect was decreased. When adding SMCT1 inhibitors, the stimulation effect was also reduced. The results from this study indicate the cooperative relationship of URAT1 and SMCT1, and that SMCT1 is a potential target for the alteration of renal handling of urate indirectly.

There is convincing evidence that many organic anion transporting polypeptide (OATP) transporters influence the pharmacokinetics and pharmacological efficacy of their substrate drugs. Each OATP family member has a unique combination of tissue distribution, substrate specificity and mechanisms of gene expression. Among them, OATP1B1, OATP1B3 and OATP2B1 have been considered as critical molecular determinants of the pharmacokinetics of a variety of clinically important drugs. Liver-specific expression of OATP1B1 and OATP1B3 contributes to the hepatic uptake of drugs from the portal vein, and OATP2B1 may alter their intestinal absorption as well as hepatic extraction. Accordingly, changes in function and expression of these three OATPs owing to genetic polymorphisms may lead to altered pharmacological effects, including decreased drug efficacy and increased risk of adverse effects. Association of genetic polymorphisms in OATP genes with alterations in the pharmacokinetic properties of their substrate drugs has been reported; however, there still exists a degree of discordance between the reported outcomes in different clinical settings. For better understanding of the clinical relevance of genetic polymorphisms of OATP1B1, OATP1B3 and OATP2B1, the present review focuses on the association of the genotypes of these OATPs with in vitro activity changes and in vivo clinical outcomes of substrate drugs.

The purpose of this study was to examine whether the presence of multiple binding sites can explain the pleiotropy of substrate recognition by OATP2B1, using Xenopus oocytes expressing OATP2B1. OATP2B1-mediated uptake of estrone-3-sulfate apparently exhibited biphasic saturation kinetics, with Km values of 0.10 ± 0.05 and 29.9 ± 12.1 µM and Vmax values of 14.1 ± 6.4 and 995 ± 273 fmol/min/oocyte for high- and low-affinity sites, respectively. Contribution analysis revealed that transport of estrone-3-sulfate mediated by high- and low-affinity sites on OATP2B1 could be evaluated at the concentrations of 0.005 and 50 µM, respectively. pH-dependence study of OATP2B1-mediated estrone-3-sulfate uptake suggested that high- and low-affinity sites show different pH sensitivity. When the inhibitory effect of 12 compounds on estrone-3-sulfate uptake by high- and low-affinity sites on OATP2B1 was examined, 4 compounds appeared to be inhibitors of the high-affinity site on OATP2B1. Two other compounds appeared to be inhibitors for the low-affinity site and four others were inhibitory at both sites. These results indicated the presence of multiple binding sites on OATP2B1 with different affinity for drugs. Accordingly, it is likely that drug-drug and drug-beverage interactions occur only when two drugs share the same binding site on OATP2B1.

We previously showed that oseltamivir, a prodrug of the influenza virus neuraminidase inhibitor Ro 64-0802, is a substrate of proton-coupled oligopeptide transporter (PEPT1), and its intestinal absorption in rats is markedly inhibited by administration with milk. To investigate the importance of PEPT1 for oseltamivir absorption in humans, and the characteristics of the drug-milk interaction, a crossover clinical study was conducted in healthy volunteers, who received 75 mg of oseltamivir with 400 mL of water or milk. Milk significantly reduced the maximum plasma concentration (C(max) ) and the area under the plasma concentration-time curve from 0 to 2 h (AUC(0-2) ) of both oseltamivir and Ro 64-0802 (oseltamivir, 68.9% and 34.5%; Ro 64-0802, 69.5% and 14.2%, respectively, vs. water), but had no significant effect on the apparent terminal half-life (t(1/2) ) or AUC(0-∞) . Urinary recovery of oseltamivir and Ro 64-0802 was significantly reduced to 77.5% of the control by milk. The early reduction of oseltamivir absorption might be through the PEPT1 inhibition by milk peptides. However, the extent of interaction in humans was limited as compared with that in rats, possibly because of species difference in the PEPT1 expression and its contribution. This might be the first report suggesting the clinical drug-food interaction via PEPT1., (Copyright © 2011 Wiley-Liss, Inc.)

The purpose of this study is to examine the contributions of organic anion transporting polypeptide (Oatp) and/or P-glycoprotein (P-gp) to grapefruit juice (GFJ) interaction with two statins, pravastatin and pitavastatin, which undergo negligible metabolism in rats. The two statins were found to be substrates of both Oatp1a5 and Oatp2b1, whereas pitavastatin, but not pravastatin, was a substrate of P-gp. The plasma concentration of pravastatin after oral administration was significantly decreased by GFJ and naringin, whereas that of pitavastatin was significantly increased. Naringin inhibited Oatp1a5- and Oatp2b1-mediated uptake of pravastatin and Oatp1a5-mediated, but not Oatp2b1-mediated, uptake of pitavastatin. Naringin also inhibited P-gp-mediated transport of pitavastatin. These results suggested that the decrease of pravastatin absorption in the presence of GFJ is due to the inhibitory effect of naringin on Oatp, whereas the increase of pitavastatin is due to the inhibition of P-gp. These observations are consistent with the results of in situ absorption studies. In conclusion, Oatp and/or P-gp contribute to the intestinal absorption of statins, and the differential effect of GFJ on pravastatin and pitavastatin absorption is at least partly accounted for by the different inhibitory effects of naringin on these transporters., (Copyright © 2011 Wiley-Liss, Inc.)

Many solute carrier transporters that interact with anticancer agents and contribute to their pharmacokinetics have been shown to be differentially upregulated in cancer cells as a result of adaptive response to altered nutritional requirements. This review focuses on pathophysiological function of membrane transporters responsible for the influx of physiological substances including oligopeptides, amino acids, and organic cations and anions, and summarizes the recent knowledge regarding mechanisms in their gene expressions. Broad substrate specificity of enhanced oligopeptide H(+) /peptide cotransporter 1 activity in cancer cells is useful for tumor tissue-specific delivery of chemotherapeutic agents and positron emission tomography diagnostic probes. Amino acid transporters such as LAT1 and ASCT2 are upregulated in human cancer cells and are thought to stimulate tumor growth by regulating mammalian target of rapamycin through nutrient pathway. Especially, LAT1 could be a molecular target to deprive cancer cells of amino acids in combination with aminopeptidase inhibitors. As organic anion transporting polypeptides carry estrone-3-sulfate that is intracellularly hydrolyzed to estrone, their overexpression may provide a pharmacological merit to treat hormone-responsive breast tumors. Therefore, it is important to understand the pathophysiological significance and gene expression in cancer to develop new rationales for drug delivery and pharmacological interventions for chemotherapy., (Copyright © 2011 Wiley-Liss, Inc.)

The purpose of this study was to investigate the involvement of organic anion transporting polypeptide (OATP/Oatp) and P-glycoprotein (P-gp)/multidrug resistance 1 (MDR1/Mdr1) in intestinal absorption of pitavastatin. Pitavastatin was found to be a substrate for human OATP1A2, OATP2B1, and MDR1 and rat Oatp1a5, Oatp2b1, and Mdr1a in experiments using transporter-expressing Xenopus oocytes and LLC-PK1 cell systems. Naringin inhibited Oatp1a5- and Mdr1a-mediated transport of pitavastatin with IC(50) values of 18.5 and 541 µM, respectively. The difference in the IC(50) values of naringin for Oatp1a5 and Mdr1a-mediated pitavastatin transport may account for the complex concentration-dependent effect of naringin on the intestinal absorption of pitavastatin. Rat intestinal permeability of pitavastatin measured by the in situ closed-loop perfusion method was indeed significantly reduced by 200 µM naringin, but was significantly increased by 1000 µM naringin. Furthermore, the permeability was significantly increased by elacridar, a potent inhibitor of Mdr1, while the permeability was significantly decreased in the presence of both elacridar and naringin, suggesting that Oatp1a5 and Mdr1a are both involved in intestinal absorption of pitavastatin. Our present results indicate that OATP/Oatp and MDR1/Mdr1 play roles in the intestinal absorption of pitavastatin as influx and efflux transporters, respectively.

To study transport of the carnitine precursor gamma-butyrobetaine (GBB) by rat liver-specific GABA transporter 2 (rGat2), we measured the uptake of deuterated GBB (d(3)-GBB) by Xenopus oocytes expressing rGat2. rGat2-mediated d(3)-GBB uptake was Na(+) and Cl(-) dependent, and was saturable with a K(m) value of 70.6 ± 34.8 µM. In carnitine transporter Octn2-deficient Jvs mice, mRNA expression of mGat3, the mouse ortholog of GAT2, decreased in liver, but increased in brain, while mRNA expression of mBbox1, which hydroxylates GBB to carnitine, increased in kidney and brain. These results indicate that GAT2 plays a role in transmembrane transport of GBB.

The purpose of the present study was to examine whether the intestinal absorption of low-permeability drugs could be improved by utilization of the intestinal influx transporter PEPT1. We investigated whether peptide derivatives of poorly absorbable nonamino acid-like drugs might be substrates of PEPT1, using rebamipide (Reb) as a model drug. We synthesized several peptide derivatives of rebamipide and examined their inhibitory effect on the uptake of [(3)H]Gly-Sar by PEPT1-expressing HeLa cells. Some of the peptide derivatives inhibited PEPT1-mediated uptake of [(3)H]Gly-Sar. Next, uptake of the inhibitory peptide derivatives was evaluated in PEPT1-expressing Xenopus oocytes and HeLa cells. Ser(Reb)-Gly exhibited significantly increased uptake by PEPT1-expressing cells in comparison with that by mock cells. The permeability of Ser(Reb)-Gly across a Caco-2 cell monolayer was significantly higher than that of rebamipide itself, and the transport was decreased in the presence of PEPT1 substrates. Further, a rat intestinal perfusion study revealed increased absorption of Ser(Reb)-Gly compared with rebamipide. These results demonstrate that the addition of a dipeptide moiety to a poorly absorbable nonpeptide/nonamino acid-like drug can result in absorption via the intestinal transporter PEPT1, though there is some selectivity as regards the structure of the added peptide moiety.

Intestinal membrane permeability is an important factor affecting the bioavailability of drugs. As a strategy to improve membrane permeability, membrane transporters are useful targets since essential nutrients are absorbed efficiently via specific transporters. For example, there are reports that intestinal hexose transporters could be used as a tool to improve permeability; however, there has been no direct evidence that the transporter protein, sodium/glucose cotransporter 1 (SGLT1), is involved in the transport of hexose analogs. Accordingly, we examined directly whether the intestinal membrane permeability of hexose analogs can be improved by utilizing SGLT1. Three hexose-quinoline derivatives were synthesized and their interactions with SGLT1 were evaluated. Among the three derivatives, the glucose-quinoline molecule exhibited an inhibitory effect on D-glucose uptake by both rat intestinal brush-border membrane vesicles (BBMVs) and Xenopus oocytes expressing SGLT1. In addition, significant uptake of the glucose-quinoline derivative by Xenopus oocytes expressing SGLT1 was observed by both an electrophysiological assay and direct measurement of the uptake of the compound, while the galactose-quinoline derivative did not show significant uptake via SGLT1. Thus, it was directly demonstrated that SGLT1 could be used as a tool for the improvement of intestinal membrane permeability of drugs by modification to the glucose analogs.

Since rat organic cation transporter 1 (Oct1, Slc22a1) is expressed mainly in the liver and mediates drug transport, its activity may determine the hepatic handling of cationic drugs. Here, we studied the regulation mechanism of the expression of rat Oct1, focusing on the nuclear receptors. Various nuclear receptors are considered to regulate expressions of many genes for membrane transporters and enzymes that are involved in the drug absorption and disposition. Previously, we demonstrated that some ligands of nuclear receptors affected the transcriptional regulation of rat Oct1 when examined in the primary cultured rat hepatocytes. In the present study, dexamethasone, a ligand of glucocorticoid receptor, down-regulated the expression of rat Oct1. In addition, the transport activity of rat Oct1, evaluated by the uptake of substrates of rat Oct1, was decreased by treatment of dexamethasone in comparison with untreated rat hepatocytes, showing a good agreement with the change in mRNA level. In conclusion, these observations suggested that the expression of rat Oct1 gene and the apparent organic cation uptake activity of rat hepatocytes are down-regulated by dexamethasone presumably via a glucocorticoid receptor.

Elevated serum uric acid level has been associated with increased cardiovascular risk in hypertensive patients. Several angiotensin II receptor blockers exhibit differential effects on regulation of serum uric acid level in humans. We have demonstrated that some angiotensin II receptor blockers trans-stimulate the uptake of uric acid by human URAT1 and others inhibit the transport of uric acid mediated by human URAT1, OAT1, OAT3 and MRP4 in vitro. This study investigated the effects of candesartan, pratosartan and telmisartan on renal handling of uric acid in rats in vivo and in vitro. Candesartan (0.1 mg/kg) significantly decreased the urinary excretion of uric acid and increased the plasma uric acid concentration. The kidney candesartan level after low-dose treatment is close to that required to trans-stimulate uric acid uptake in vitro. Pratosartan exhibited dose-dependent hypouricemic and uricosuric effects, while telmisartan showed no effects on plasma uric acid level. Furthermore, we confirmed the effects of the tested drugs on uric acid transport by rat renal brush border membrane transporter(s) and basolateral Oat1 and Oat3. Effects of angiotensin II receptor blockers in rats may be mainly determined by their intrinsic effects (cis-inhibition and trans-stimulation) on uric acid reabsorption transporter(s) and their pharmacokinetic properties in rats.

We recently showed that arginine transport via cationic amino acid transporter 1 (CAT1), which transports arginine, lysine, ornithine, and histidine, is essential for erythropoiesis. In the present study, to confirm the importance of both arginine and CAT1 in erythropoiesis, we investigated the relationship of arginine uptake activity and differentiation and proliferation of blood cells by knockdown of the CAT1 gene using shRNA. Five short hairpin RNA (shRNA)-transfected K562 cell clones, in which the CAT1 mRNA expression level is decreased, and a vector-transfected clone were obtained. The differentiation to erythrocytes and proliferation of K562 cells were decreased by knockdown of CAT1. In addition, the initial uptake rate of [(3)H]arginine was decreased in the shRNA-transfected cell clones. The ratio of differentiation of CAT1-knockdown K562 cells was well correlated with the uptake activity for arginine by the cells (R(2)=0.59). These findings indicate that CAT1 is directly involved in erythropoiesis through supplying arginine to the blood cells.