Human Organic Anion Transporter Research Articles

In vitro and in silico characterization of the transport of selected perfluoroalkyl carboxylic acids and perfluoroalkyl sulfonic acids by human organic anion transporter 1 (OAT1), OAT2 and OAT3

Perfluoroalkyl carboxylic acids (PFCAs) and perfluoroalkyl sulfonic acids (PFSAs) belong to the group of poly- and perfluoroalkyl substances (PFASs), which may accumulate in humans due to their limited excretion. To provide more insight into the active renal excretion potential of PFASs in humans, this work investigated in vitro the transport of three PFCAs (PFHpA, PFOA, PFNA) and three PFSAs (PFBS, PFHxS and PFOS) using OAT1-, OAT2- or OAT3-transduced human embryonic kidney (HEK) cells. Only PFHpA and PFOA showed clear uptake in OAT1-transduced HEK cells, while no transport was observed for PFASs in OAT2-transduced HEK cells. In OAT3-transduced HEK cells only PFHpA, PFOA, PFNA, and PFHxS showed clear uptake. To study the interaction with the transporters, molecular docking and dynamics simulation were performed for PFHpA and PFHxS, for which a relatively short and long half-life in humans has been reported, respectively. Docking analyses could not always distinguish the in vitro transported from the non-transported PFASs (PFHpA vs. PFHxS), whereas molecular dynamic simulations could, as only a stable interaction of the PFAS with the inner part of transporter mouth was detected for those that were transported in vitro (PFHpA with OAT1, none with OAT2, and PFHpA and PFHxS with OAT3). Altogether, this study presents in vitro and in silico insight with respect to the selected PFASs transport by the human renal secretory transporters OAT1, OAT2, and OAT3, which provides further understanding about the differences between the capability of PFAS congeners to accumulate in humans.

Pharmacogenetic determinants of tenofovir diphosphate and lamivudine triphosphate concentrations in people with HIV/HBV coinfection.

The nucleos(t)ide analogs require phosphorylation to the pharmacologically active anabolites in cells. We investigated the hypothesis that single-nucleotide polymorphisms (SNPs) in genes that encode transporters and phosphodiesterase (PDE) enzymes involved in tenofovir (TFV), disoproxil fumarate (TDF), and lamivudine (3TC) disposition will be associated with concentrations of their phosphate anabolites and virologic response. Individuals with human immunodeficiency virus (HIV) and hepatitis B virus (HBV) coinfection receiving TDF/3TC-containing antiretroviral therapy were enrolled. Steady-state TFV diphosphate (TFV-DP) and 3TC triphosphate (3TC-TP) concentrations in peripheral blood mononuclear cells (PBMCs) and dried blood spot samples were quantified. The relationship between genetic variants and TFV-DP and 3TC-TP concentrations as well as with virologic response were examined using multivariable linear regression. Of the 136 participants (median age 43 years; 63% females), 6.6% had HBV non-suppression, and 7.4% had HIV non-suppression. The multidrug resistance protein 2 (encoded by ABCC2 rs2273697) SNP was associated with 3TC-TP concentrations in PBMCs. The human organic anion transporter-1 (encoded by SLC28A2) rs11854484 SNP was associated with HIV non-suppression, and when evaluated together with SNPs with marginal associations (ABCC2 rs717620 and PDE1C rs30561), participants with two or three variants compared to those with none of these variants had an adjusted odds ratio of 48.3 (confidence interval, 4.3-547.8) for HIV non-suppression. None of the SNPs were associated with HBV non-suppression. Our study identified ABCC2 SNP to be associated with 3TC-TP concentrations in PBMCs. Also, a combination of genetic variants of drug transporters and PDE was associated with HIV non-suppression.

SND1 Regulates Organic Anion Transporter 2 Protein Expression and Sensitivity of Hepatocellular Carcinoma Cells to 5-Fluorouracil.

Hepatocellular carcinoma (HCC) is one of the most malignant tumors in the world. Inadequate efficacy of 5-fluorouracil (5-FU) on HCC could be related to low expression of human organic anion transporter 2 (OAT2). However, the knowledge of downregulation of OAT2 in HCC remains limited. We explored the underlying mechanism focusing on protein expression regulation and attempted to design a strategy to sensitize HCC cells to 5-FU. In this study, we revealed that the 1 bp to 300 bp region of OAT2 mRNA 3' untranslated region (UTR) reduced its protein expression and uptake activity in Li-7 and PLC/PRF/5 cells. Mechanistically, it was demonstrated that staphylococcal nuclease and Tudor domain containing 1 (SND1) bound at the 1 bp to 300 bp region of OAT2 mRNA 3' UTR, leading to a decrease in OAT2 protein expression. Enrichment analysis results indicated reduction of OAT2 might be mediated by translational inhibition. Furthermore, the knockdown of SND1 upregulated OAT2 protein expression and uptake activity. Based on this, decreasing SND1 expression enhanced 5-FU-caused G1/S phase arrest in Li-7 and PLC/PRF/5 cells, resulting in suppression of cell proliferation. Additionally, the knockdown of SND1 augmented the inhibitory effect of 5-FU on PLC/PRF/5 xenograft tumor growth in vivo by increasing OAT2 protein expression and accumulation of 5-FU in the tumor. Collectively, a combination of inhibition of SND1 with 5-FU might be a potential strategy to sensitize HCC cells to 5-FU from the perspective of restoring OAT2 protein level. SIGNIFICANCE STATEMENT: We investigated the regulatory mechanism of OAT2 protein expression in HCC cells and designed a strategy to sensitize them to 5-FU (OAT2 substrate) via restoring OAT2 protein level. It found that SND1, an RNA binding protein, regulated OAT2 protein expression by interacting with OAT2 mRNA 3' UTR 1-300 bp region. Through decreasing SND1, the antitumor effect of 5-FU on HCC was enhanced in vitro and in vivo, indicating that SND1 could be a potential target for sensitizing HCC cells to 5-FU.

Interaction of the Atypical Tetracyclines Chelocardin and Amidochelocardin with Renal Drug Transporters.

Antimicrobial resistance is expected to increase mortality rates by up to several million deaths per year by 2050 without new treatment options at hand. Recently, we characterized the pharmacokinetic (PK) and pharmacodynamic properties of two atypical tetracyclines, chelocardin (CHD) and amidochelocardin (CDCHD) that exhibit no cross-resistance with clinically used antibacterials. Both compounds were preferentially renally cleared and demonstrated pronounced effects in an ascending urinary tract infection model against E. coli. Renal drug transporters are known to influence clearance into the urine. In particular, inhibition of apical transporters in renal tubular epithelial cells can lead to intracellular accumulation and potential cell toxicity, whereas inhibition of basolateral transporters can cause a higher systemic exposure. Here, selected murine and human organic cation (Oct), organic anion (Oat), and efflux transporters were studied to elucidate interactions with CHD and CDCHD underlying their PK behavior. CHD exhibited stronger inhibitory effects on mOat1 and mOat3 and their human homologues hOAT1 and hOAT3 compared to CDCHD. While CHD was a substrate of mOat3 and mOct1, CDCHD was not. By contrast, no inhibitory effect was observed on Octs. CDCHD rather appeared to foster enhanced substrate transport on mOct1. CHD and CDCHD inhibited the efflux transporter hMRP2 on the apical side. In summary, the substrate nature of CHD in conjunction with its autoinhibition toward mOat3 rationalizes the distinct urine concentration profile compared to CDCHD that was previously observed in vivo. Further studies are needed to investigate the accumulation in renal tubular cells and the nephrotoxicity risk.

Topotecan and Ginkgolic Acid Inhibit the Expression and Transport Activity of Human Organic Anion Transporter 3 by Suppressing SUMOylation of the Transporter.

Organic anion transporter 3 (OAT3), expressed at the basolateral membrane of kidney proximal tubule cells, facilitates the elimination of numerous metabolites, environmental toxins, and clinically important drugs. An earlier investigation from our laboratory revealed that OAT3 expression and transport activity can be upregulated by SUMOylation, a post-translational modification that covalently conjugates SUMO molecules to substrate proteins. Topotecan is a semi-synthetic derivative of the herbal extract camptothecin, approved by the FDA to treat several types of cancer. Ginkgolic acid (GA) is one of the major components in the extract of Ginkgo biloba leaves that has long been used in food supplements for preventing dementia, high blood pressure, and supporting stroke recovery. Both topotecan and GA have been shown to affect protein SUMOylation. In the current study, we tested our hypothesis that topotecan and GA may regulate OAT3 SUMOylation, expression, and transport function. Our data show that the treatment of OAT3-expressing cells with topotecan or GA significantly decreases the SUMOylation of OAT3 by 50% and 75%, respectively. The same treatment also led to substantial reductions in OAT3 expression and the OAT3-mediated transport of estrone sulfate, a prototypical substrate. Such reductions in cell surface expression of OAT3 correlated well with an increased rate of OAT3 degradation. Mechanistically, we discovered that topotecan enhanced the association between OAT3 and the SUMO-specific protease SENP2, a deSUMOylation enzyme, which contributed to the significant decrease in OAT3 SUMOylation. In conclusion, this study unveiled a novel role of topotecan and GA in inhibiting OAT3 expression and transport activity and accelerating OAT3 degradation by suppressing OAT3 SUMOylation. During comorbidity therapies, the use of topotecan or Ginkgo biloba extract could potentially decrease the transport activity of OAT3 in the kidneys, which will in turn affect the therapeutic efficacy and toxicity of many other drugs that are substrates for the transporter.

The regulation of human organic anion transporter 4 by insulin-like growth factor 1 and protein kinase B signaling

Human organic anion transporter 4 (hOAT4), mainly expressed in the kidney and placenta, is essential for the disposition of numerous drugs, toxins, and endogenous substances. Insulin-like growth factor 1 (IGF-1) is a hormone generated in the liver and plays important roles in systemic growth, development, and metabolism. In the current study, we explored the regulatory effects of IGF-1 and downstream signaling on the transport activity, protein expression, and SUMOylation of hOAT4. We showed that IGF-1 significantly increased the transport activity, expression, and maximal transport velocity Vmax of hOAT4 in kidney-derived cells. This stimulatory effect of IGF-1 on hOAT4 activity was also confirmed in cells derived from the human placenta. The increased activity and expression were correlated well with the reduced degradation rate of hOAT4 at the cell surface. Furthermore, IGF-1 significantly increased hOAT4 SUMOylation, and protein kinase B (PKB)-specific inhibitors blocked the IGF-1-induced regulations on hOAT4. In conclusion, our study demonstrates that the hepatic hormone IGF-1 regulates hOAT4 expressed in the kidney and placenta through the PKB signaling pathway. Our results support the remote sensing and signaling theory, where OATs play a central role in the remote communications among distal tissues.

Substrate binding and lipid-mediated allostery in the human organic anion transporter 1 at the atomic-scale

The Organic Anion Transporter 1 is a membrane transporter known for its central role in drug elimination by the kidney. hOAT1 is an antiporter translocating substrate in exchange for a-ketoglutarate. The understanding of hOAT1 structure and function remains limited due to the absence of resolved structure of hOAT1. Benefiting from conserved structural and functional patterns shared with other Major Facilitator Superfamily transporters, the present study intended to investigate fragments of hOAT1 transport function and modulation of its activity in order to make a step forward the understanding of its transport cycle. μs-long molecular dynamics simulation of hOAT1 were carried out suggesting two plausible binding sites for a typical substrate, adefovir, in line with experimental observations. The well-known B-like motif binding site was observed in line with previous studies. However, we here propose a new inner binding cavity which is expected to be involved in substrate translocation event. Binding modes of hOAT1 co-substrate α-ketoglutarate were also investigated suggesting that it may bind to highly conserved intracellular motifs. We here hypothesise that α-ketoglutarate may disrupt the pseudo-symmetrical intracellular charge-relay system which in turn may participate to the destabilisation of OF conformation. Investigations regarding allosteric communications along hOAT1 also suggest that substrate binding event might modulate the dynamics of intracellular charge relay system, assisted by surrounding lipids as active partners. We here proposed a structural rationalisation of transport impairments observed for two single nucleotide polymorphisms, p.Arg50His and p.Arg454Gln suggesting that the present model may be used to transport dysfunctions arising from hOAT1 mutations.

Perfluoroalkyl substances (PFASs) are substrates of the renal human organic anion transporter 4 (OAT4)

Poly- and perfluoroalkyl substances (PFASs) are omnipresent in the environment and have been shown to accumulate in humans. Most PFASs are not biotransformed in animals and humans, so that elimination is largely dependent on non-metabolic clearance via bile and urine. Accumulation of certain PFASs in humans may relate to their reabsorption from the pre-urine by transporter proteins in the proximal tubules of the kidney, such as URAT1 and OAT4. The present study assessed the in vitro transport of 7 PFASs (PFHpA, PFOA, PFNA, PFDA, PFBS, PFHxS and PFOS) applying URAT1- or OAT4-transfected human embryonic kidney (HEK) cells. Virtually no transport of PFASs could be measured in URAT1-transfected HEK cells. All PFASs, except PFBS, showed clear uptake in OAT4-transfected HEK cells. In addition, these in vitro results were further supported by in silico docking and molecular dynamic simulation studies assessing transporter–ligand interactions. Information on OAT4-mediated transport may provide insight into the accumulation potential of PFASs in humans, but other kinetic aspects may play a role and should also be taken into account. Quantitative information on all relevant kinetic processes should be integrated in physiologically based kinetic (PBK) models, to predict congener-specific accumulation of PFASs in humans in a more accurate manner.

Development and validation of an LC-MS/MS method to quantify kynurenic acid in human plasma.

Background: Monitoring levels of endogenous biomarkers has become an alternative approach to assess transporter-mediated drug-drug interactions in clinical trials. Among the biomarkers of interest, kynurenic acid is effective for the human organic anion transporters OAT1 and OAT3. Here, a simple and robust bioanalytical method was developed using LC-MS/MS to quantify kynurenic acid in human plasma. Results: This method achieved a LLOQ of 10nm with acceptable signal-to-noise ratio (S/N >5). In addition, an interfering agent, tryptophan, was identified and separated chromatographically. A full method validation was performed in the spirit of GLP. Conclusion: This method can serve as a tool readily available to assess potential drug-drug interactions mediated by inhibition of OAT1 and OAT3 activities.

Sensitive and valid assay for reliable evaluation of drug interactions mediated by human organic anion transporter 1 and 3 using 5-carboxyfluorescein.

Drug interactions can induce significant clinical impacts, either by increasing adverse effects or by decreasing the therapeutic effect of drugs, and thus, need to be explored thoroughly. Clinically significant drug interactions can be induced by organic anion transporter 1 (OAT1) and OAT3 when concomitant medications competitively interact with the transporters. The purposes of this study were to develop and validate a sensitive and selective analytical method for 5-carboxyfluorescein (5-CF) and optimize the experimental conditions for interaction studies. An analytical method using high-performance liquid chromatography (HPLC) equipped with a fluorescence detector was validated for accuracy, precision, matrix effect, recovery, stability, dilutional integrity, and carry-over effect. In addition, the 5-CF concentration, incubation period, and washing conditions for interaction study were optimized. Using a valid analytical method and optimized conditions, we performed an interaction study for OAT1 and OAT3 using 26 test articles. Some of the test articles showed strong inhibitory potency for the transporters, with IC50 values close to or less than 10μM. The valid analysis method and optimized systems developed in this study can be utilized to improve the predictability of drug interactions in humans and consequently aid in successful disease treatment by maintaining appropriate systemic exposures.

Functional characterization and comparison of human and mouse organic anion transporter 1 as drugs and pesticides uptake carrier

The organic anion transporter 1 (OAT1) is mainly expressed in proximal tubule cells, where it mediates the renal uptake of endogenous and exogenous compounds. Thereby, it has enormous clinical relevance particularly in drug-drug interactions. The aim of the present in vitro study was to elucidate potential species dependent disparity of human and mouse OAT1 in handling of structural diverse drugs and pesticides. A basic functional comparison of the two transporters showed a similar time-dependent uptake of the substrate para-aminohippuric acid (PAH), the affinity (Km) was 94 µM for hOAT1 and 32 µM for mOat1. Inhibition experiments for hOAT1 and mOat1 provided IC50 values for glibenclamide of 5.1 and 6.4 µM and for probenecid of 31 and 11 µM. Than the interaction of hOAT1 and mOat1 with 23 drugs and 13 pesticides was examined. Three pesticides and thirteen drugs showed high inhibitory potency of 50% or more to both transporters. Furthermore, we identified rosiglitazone as a differential active inhibitor, with stronger inhibitory properties (IC50) to mOat1 (7.7 µM) than to hOAT1 (31 µM), and olmesartan with the most pronounced difference: The IC50 of hOAT1 (0.40 µM) was 48-fold lower than of mOat1 (19 µM). In conclusion, we found a strong correlation for the inhibitory effects of most drugs and pesticides on human and mouse OAT1. But the example of olmesartan shows that species differences have to be considered when extrapolating data from mouse to human.

Insights into the structure and function of the human organic anion transporter 1 in lipid bilayer membranes

The human SLC22A6/OAT1 plays an important role in the elimination of a broad range of endogenous substances and xenobiotics thus attracting attention from the pharmacological community. Furthermore, OAT1 is also involved in key physiological events such as the remote inter-organ communication. Despite its significance, the knowledge about hOAT1 structure and the transport mechanism at the atomic level remains fragmented owing to the lack of resolved structures. By means of protein-threading modeling refined by μs-scaled Molecular Dynamics simulations, the present study provides the first robust model of hOAT1 in outward-facing conformation. Taking advantage of the AlphaFold 2 predicted structure of hOAT1 in inward-facing conformation, we here provide the essential structural and functional features comparing both states. The intracellular motifs conserved among Major Facilitator Superfamily members create a so-called “charge-relay system” that works as molecular switches modulating the conformation. The principal element of the event points at interactions of charged residues that appear crucial for the transporter dynamics and function. Moreover, hOAT1 model was embedded in different lipid bilayer membranes highlighting the crucial structural dependence on lipid-protein interactions. MD simulations supported the pivotal role of phosphatidylethanolamine components to the protein conformation stability. The present model is made available to decipher the impact of any observed polymorphism and mutation on drug transport as well as to understand substrate binding modes.

Association between proton pump inhibitors and severe hematological toxicity in patients receiving pemetrexed-based anticancer treatment: The prospective IPPEM study

ObjectivePemetrexed is associated with hematological toxicity. Drug-drug interactions (DDIs) between methotrexate and proton pump inhibitors (PPIs) induce a higher risk of hematological toxicity due to the inhibition of methotrexate excretion by PPIs. As pemetrexed and methotrexate are both excreted by human organic anion transporter 3 (hOAT3), this study investigates the hypothetical DDI between pemetrexed and PPIs in lung cancer patients. The primary objective was the occurrence of severe (grade ≥ 3) hematological toxicity. The secondary objectives were to describe the type of hematological toxicity and associated clinical consequences (NCT03537833). Materials and MethodsPPI consumption was collected for each patient receiving pemetrexed-based anticancer chemotherapy from May 2018 to October 2020 in a prospective multicentric observational and nonrandomized study. Multivariate Cox regression and propensity score (PS) adjustment, PS matching and inverse weighting on PS (IPTW) methods were used. ResultsPPI consumption (55 among 156 included patients) was associated with a significantly higher risk of severe hematological toxicity in the multivariable Cox regression model (hazard ratio HR = 2.51, 95% confidence interval [1.47–4.26]; p = 0.005). Similar results were found with PS adjustment (HR = 1.91 CI95% [1.14–3.20]; p = 0.002), PS-matching (HR = 1.93 CI95% [1.08–3.45]; p = 0.02) and IPTW method (HR = 2.06 CI95% [1.27–3.35]; p = 0.004). Severe neutropenia and anemia occurred in 32.7% and 14.1% of patients, respectively. This resulted in 48 anticancer chemotherapy postponements and 24 dose adjustments, 26 growth factor prescriptions, 24 red blood cell transfusions, and 20 hospitalizations. ConclusionsThe results strongly suggest an association between PPI consumption and pemetrexed-related severe hematological toxicity. Deprescription of PPIs when feasible should be considered to prevent this DDI.

A Human-derived Dual MRI/PET Reporter Gene System with High Translational Potential for Cell Tracking.

Reporter gene imaging has been extensively used to longitudinally report on whole-body distribution and viability of transplanted engineered cells. Multi-modal cell tracking can provide complementary information on cell fate. Typical multi-modal reporter gene systems often combine clinical and preclinical modalities. A multi-modal reporter gene system for magnetic resonance imaging (MRI) and positron emission tomography (PET), two clinical modalities, would be advantageous by combining the sensitivity of PET with the high-resolution morphology and non-ionizing nature of MRI. We developed and evaluated a dual MRI/PET reporter gene system composed of two human-derived reporter genes that utilize clinical reporter probes for engineered cell detection. As a proof-of-concept, breast cancer cells were engineered to co-express the human organic anion transporter polypeptide 1B3 (OATP1B3) that uptakes the clinical MRI contrast agent gadolinium ethoxybenzyl-diethylenetriaminepentaacetic acid (Gd-EOB-DTPA), and the human sodium iodide symporter (NIS) which uptakes the PET tracer, [18F] tetrafluoroborate ([18F] TFB). T1-weighted MRI results in mice exhibited significantly higher MRI signals in reporter-gene-engineered mammary fat pad tumors versus contralateral naïve tumors (p < 0.05). No differences in contrast enhancement were observed at 5h after Gd-EOB-DTPA administration using either intravenous or intraperitoneal injection. We also found significantly higher standard uptake values (SUV) in engineered tumors in comparison to the naïve tumors in [18F]TFB PET images (p < 0.001). Intratumoral heterogeneity in signal enhancement was more conspicuous in relatively higher resolution MR images compared to PET images. Our study demonstrates the ability to noninvasively track cells engineered with our human-derived dual MRI/PET reporter system, enabling a more comprehensive evaluation of transplanted cells. Future work is focused on applying this tool to track therapeutic cells, which may one day enable the broader application of cell tracking within the healthcare system.

Bilirubin Reduces the Uptake of Estrogen Precursors and the Followed Synthesis of Estradiol in Human Placental Syncytiotrophoblasts via Inhibition and Downregulation of Organic Anion Transporter 4.

Estrogen biosynthesis in human placental trophoblasts requires the human organic anion transporter 4 (hOAT4)-mediated uptake of fetal derived precursors such as dehydroepiandrosterone-3-sulfate (DHEAS) and 16α-hydroxy-DHEA-S (16α-OH-DHEAS). Scant information is available concerning the contribution of fetal metabolites on the impact of placental estrogen precursor transport and the followed estrogen synthesis. This study substantiated the roles of bilirubin as well as bile acids (taurochenodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid, chenodeoxycholic acid) on the inhibition of hOAT4-mediated uptake of probe substrate 6-carboxylfluorescein and DHEAS in stably transfected hOAT4-Chinese hamster ovary cells, with the IC50 of 1.53 and 0.98 μM on 6-carboxylfluorescein and DHEAS, respectively, for bilirubin, and 90.2, 129, 16.4, and 12.3 μM on 6-CF for taurochenodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, and chenodeoxycholic acid. Bilirubin (2.5-10 μM) concentration-dependently inhibited the accumulation of estradiol precursor DHEAS in human choriocarcinoma JEG-3 cells (reduced by 60% at 10 μM) and primary human trophoblast cells (reduced by 80% at 10 μM). Further study confirmed that bilirubin (0.625-2.5 μM) concentration-dependently reduced the synthesis and secretion of estradiol in primary human trophoblast cells, among which 2.5 μM of bilirubin reduced the synthesis of estradiol by 30% and secretion by 35%. In addition, immunostaining and Western blot results revealed a distinct downregulation of hOAT4 protein expression in primary human trophoblast cells pretreated with 2.5 μM of bilirubin. In conclusion, this study demonstrated that bilirubin reduced the uptake of estrogen precursors and the followed synthesis of estradiol in human placenta via inhibition and downregulation of organic anion transporter 4. SIGNIFICANCE STATEMENT: Fetal metabolites, especially bilirubin, were first identified with significant inhibitory effects on the hOAT4-mediated uptake of estrogen precursor DHEAS in hOAT4-CHO, JEG-3 and PHTCs. Bilirubin concentration-dependently suppressed the estradiol synthesis and secretion in PHTCs treated with DHEAS, which was synchronized with the decline of hOAT4 protein expression. Additionally, those identified bile acids exhibited a weaker inhibitory effect on the secretion of estradiol.

Functional characterization of human organic anion transporter 10 (OAT10/SLC22A13) as an orotate transporter

Orotate, a nutritional compound typically utilized as an intermediate in pyrimidine synthesis, has been suggested to undergo renal reabsorption. However, the detailed mechanisms involved in the process remain unclear, with only urate transporter 1 (URAT1/SLC22A12) being indicated as a transporter involved in its tubular uptake. As an attempt to identify transporters involved in that to help clarify the mechanisms, we examined a possibility that organic anion transporter 10 (OAT10/SLC22A13), which is present at the brush border membrane in renal tubular epithelial cells, could transport orotate. The operation of human OAT10 for orotate transport was demonstrated indeed and analyzed in detail in Madin-Darby canine kidney II cells introduced with this transporter by stable transfection. Orotate transport by OAT10 was found to be kinetically saturable with a biphasic characteristic and dependent on Cl−. These are unique characteristics previously unknown in its operation for the other substrates. Orotate transport by OAT10 was, on the other hand, inhibited by several anionic compounds known as OAT10 inhibitors. Finally, the rat ortholog of OAT10 was found not to be able to transport orotate, indicating animal species differences in that function. Thus, human OAT10 has been demonstrated to operate for orotate transport with unique characteristics.

Model-Based Risk Prediction of Rivaroxaban with Amiodarone for Moderate Renal Impaired Elderly Population.

Increased bleeding risk was found associated with concurrent prescription of rivaroxaban and amiodarone. We previously recommended dose adjustment for rivaroxaban utilizing a physiologically based pharmacokinetic (PBPK) modeling approach. Our subsequent in vitro studies discovered the pivotal involvement of human renal organic anion transporter 3 (hOAT3) in the renal secretion of rivaroxaban and the inhibitory potency of amiodarone. This study aimed to redefine the disease-drug-drug interactions (DDDI) between rivaroxaban and amiodarone and update the potential risks. Prospective simulations were conducted with updated PBPK models of rivaroxaban and amiodarone incorporating hOAT3-related parameters. Simulations to recapitulate previously explored DDDI in renal impairment showed a higher bleeding tendency in all simulation scenarios after integrating hOAT3-mediated clearance into PBPK models. Further sensitivity analysis revealed that both hOAT3 dysfunction and age could affect the extent of DDDI, and age was shown to have a more pivotal role on rivaroxaban in vivo exposure. When amiodarone was prescribed along with our recommended dose reduction of rivaroxaban to 10mg in moderate renal impaired elderly people, it could result in persistently higher rivaroxaban peak concentrations at a steady state. To better manage the increased bleeding risk among such a vulnerable population, a dose reduction of rivaroxaban to 2.5mg twice daily resulted in its acceptable in vivo exposure. Close monitoring of bleeding tendency is essential for elderly patients with moderate renal impairment receiving co-prescribed rivaroxaban and amiodarone. Further dose reduction is recommended for rivaroxaban to mitigate this specific DDDI risk.

Para-aminosalicylic acid significantly reduced tenofovir exposure in human subjects: Mismatched findings from in vitro to in vivo translational research.

Tenofovir and para-aminosalicylic acid (PAS) may be coprescribed to treat patients with concomitant infections of human immunodeficiency virus and Mycobacterium tuberculosis bacteria. Both drugs are known to have remarkable renal uptake transporter-mediated clearance. Owing to the lack of clinical studies on drug-drug interaction between the 2 drugs, we conducted a translational clinical study to investigate the effect of PAS on tenofovir pharmacokinetics (PK). Initially, we studied in vitro renal uptake transporter-mediated drug-drug interactions using stably transfected cells with human organic anion transporters (OAT1 and OAT3). Later, we estimated clinical drug interactions using static and physiologically based PK modelling. Finally, we investigated the effects of PAS-calcium formulation (PAS-Ca) on tenofovir disoproxil fumarate PK in healthy male Korean subjects. PAS inhibited OAT1- and OAT3-mediated tenofovir uptake in vitro. The physiologically based PK drug-drug interaction model suggested a 1.26-fold increase in tenofovir peak plasma concentration when coadministered with PAS. By contrast, an open-label, randomized, crossover clinical trial evaluating the effects of PAS-Ca on tenofovir PK showed significantly altered geometric mean ratio (90% confidence intervals) of maximum plasma concentration (Cmax ) and area under the curve (AUC0-inf ) by 0.33 (0.28-0.38) and 0.29 (0.26-0.33), respectively. Our study findings suggest that the PAS-Ca formulation significantly reduced systemic exposure to tenofovir through an unexplained mechanism, which was contrary to the initial prediction. Caution should be exercised while predicting in vivo PK profiles from in vitro data, particularly when there are potential confounders such as pharmaceutical interactions.

Upregulation of histone acetylation reverses organic anion transporter 2 repression and enhances 5-fluorouracil sensitivity in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The failure of chemotherapy in HCC patients is partly due to inadequate intracellular drug accumulation caused by abnormally expressed drug transporters. Human organic anion transporter 2 (hOAT2), a transporter mainly expressed in liver and kidney, is responsible for uptake of various antineoplastic drugs such as 5-fluorouracil (5-FU). Among 32 pairs of human HCC samples, we preliminarily found that OAT2 was suppressed in HCC tissues compared with matched tumor-adjacent tissues at both mRNA and protein levels, which resulted in 5-FU resistance in HCC. However, the epigenetic regulatory mechanisms of OAT2 downregulation have not been investigated. In this study, we first proved it was histone hypoacetylation rather than DNA hypermethylation that participated in transcriptional repression of OAT2 in two HCC cell lines (BEL-7402 and SMMC-7721). In general, there were two pathways confirmed using tissues and cells: 1) Increased histone deacetylase sirtuin 7 (SIRT7) mediated loss of histone 3 lysine 18 acetylation (H3K18ac) at the promoter of OAT2 and inhibited its transcription. 2) More histone deacetylase 7 (HDAC7) instead of lysine acetyltransferase 8 (KAT8) enrichment at the promoter of OAT2 led to low levels of histone 4 lysine 16 acetylation (H4K16ac). Further, we found that histone deacetylases inhibitor vorinostat (SAHA) could reverse histone hypoacetylation state to activate OAT2 transcription and enhance uptake of classic OAT2 substrate zidovudine. Therefore, we evaluated the effect of combining SAHA and 5-FU and the results demonstrated that SAHA could sensitize HCC cells to 5-FU. Collectively, we proposed such a combination treatment to overcome 5-FU resistance in HCC from the perspective of epigenetically restoring OAT2.

Characterization of Novel Fluorescent Bile Salt Derivatives for Studying Human Bile Salt and Organic Anion Transporters.

Bile salts, such as cholate, glycocholate, taurocholate, and glycochenodeoxycholate, are taken up from the portal blood into hepatocytes via transporters, such as the Na+-taurocholate-cotransporting polypeptide (NTCP) and organic anion-transporting polypeptides (OATPs). These bile salts are later secreted into bile across the canalicular membrane, which is facilitated by the bile salt export pump (BSEP). Apart from bile salt transport, some of these proteins (e.g., OATPs) are also key transporters for drug uptake into hepatocytes. In vivo studies of transporter function in patients by using tracer compounds have emerged as an important diagnostic tool to complement classic liver parameter measurements by determining dynamic liver function both for diagnosis and monitoring progression or improvement of liver diseases. Such approaches include use of radioactively labeled bile salts (e.g., for positron emission tomography) and fluorescent bile salt derivatives or dyes (e.g., indocyanine green). To expand the list of liver function markers, we synthesized fluorescent derivatives of cholic and chenodeoxycholic acid by conjugating small organic dyes to the bile acid side chain. These novel fluorescent probes were able to block substrate transport in a concentration-dependent manner of NTCP, OATP1B1, OATP1B3, OATP2B1, BSEP, and intestinal apical sodium-dependent bile salt transporter (ASBT). Whereas the fluorescent bile acid derivatives themselves were transported across the membrane by OATP1B1, OATP1B3, and OATP2B1, they were not transport substrates for NTCP, ASBT, BSEP, and multidrug resistance-related protein 2. Accordingly, these novel fluorescent bile acid probes can potentially be used as imaging agents to monitor the function of OATPs. SIGNIFICANCE STATEMENT: Synthetic modification of common bile acids by attachment of small organic fluorescent dyes to the bile acid side chain resulted in bright, fluorescent probes that interact with hepatic and intestinal organic anion [organic anion-transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1], bile salt uptake (Na+-taurocholate-cotransporting polypeptide, apical sodium-dependent bile salt transporter), and bile salt efflux (bile salt export pump, multidrug resistance-related protein 2) transporters. Although the fluorescent bile salt derivatives are taken up into cells via the OATPs, the efflux transporters do not transport any of them but one.