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Background and purpose The metabolic activity of cytochrome P450 (CYP) 2D6 is highly variable and CYP2D6 genotypes insufficiently explain the extensive and intermediate metabolic phenotypes, limiting the prediction of drug response plus adverse drug reactions. Since CYP2D6 prototypic substrates are positively charged, the aim of this study was to evaluate the organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs) as potential contributors to the variability of CYP2D6 hydroxylation of debrisoquine, dextromethorphan, diphenhydramine, perhexiline and sparteine. Experimental approach OCT1/SLC22A1-, OCT2/SLC22A2-, OCT3/SLC22A3-, MATE1/SLC47A1-, and MATE2K/SLC47A2-overexpressing cell lines were used to investigate the transport of the selected drugs. Individuals from a study cohort, well defined with respect to CYP2D6 genotype and sparteine pharmacokinetics, were genotyped for the common OCT1 variants rs12208357 (OCT1-R61C), rs34130495 (OCT1-G401S), rs202220802 (OCT1-Met420del), rs34059508 (OCT1-G465R), OCT2 variant rs316019 (OCT2-A270S) and MATE1 variant rs2289669. Sparteine pharmacokinetics was stratified according to CYP2D6 and OCT1, OCT2 or MATE1 genotype. Key results OCTs and MATE1 transport sparteine and debrisoquine with high affinity in vitro, but OCT- and MATE1-dependent transport of dextromethorphan, diphenhydramine and perhexiline was not detected. Sparteine and debrisoquine transport depends on OCT1 genotype; however, sparteine pharmacokinetics is independent from OCT1 genotype. Conclusions and implications Some drugs that are substrates of CYP2D6 are also substrates of OCTs and MATE1, suggesting overlapping specificities. Variability in sparteine hydroxylation in extensive and intermediate metabolizers cannot be explained by OCT1 genetic variants indicating presence of other factors. Dose-dependent toxicities of dextromethorphan, diphenhydramine and perhexiline appear to be independent from OCTs and MATEs.

Introduction: Alzheimer’s disease (AD) is a severe neurodegenerative disorder of the brain characterized by degeneration of cholinergic neurons which is directly linked to cognitive decline. Nerve growth factor (NGF) is the most potent protective factor for cholinergic neurons, additionally the NMDA antagonist memantine blocks glutamate-mediated excitotoxic activity. Quinidine is an inhibitor of organic cation transporter 2 (OCT2). OCT2 is located on cholinergic neurons and plays a role in presynaptic reuptake and recycling of acetylcholine in the brain. We hypothesize that quinidine can modulate the protective effects of NGF and memantine on cholinergic neurons in organotypic brain slices of the nucleus basalis of Meynert (nBM). Methods: Organotypic brain slices of nBM were incubated with 100 ng/mL NGF, 10 µM memantine, 10 µM quinidine, and combinations of these treatments for 2 weeks. Cholinergic neurons were immunohistochemically stained for choline acetyltransferase (ChAT). Results: Our data show that NGF as well as memantine counteracted the cell death of cholinergic nBM neurons. Quinidine alone had no toxic effect on cholinergic neurons but inhibited the protective effect of NGF and memantine when applied simultaneously. Discussion/Conclusion: Our data provide evidence that quinidine modulates the survival of cholinergic nBM neurons via OCT2.

The transport efficiency (TE) describes the performance of a transport protein for a specific substrate. To compare the TE of different transporters, the number of active transporters in the plasma membrane must be monitored, as it may vary for each transporter and experiment. Available methods, like LC–MS quantification of tryptic peptides, fail to discriminate inactive intracellular transporters or, like cell-surface biotinylation followed by affinity chromatography and Western blotting, are imprecise and very laborious. We wanted to normalize active transporters by the activity of a second transporter. A transporter tandem, generated by joining two transporter cDNAs into a single open reading frame, should guarantee a 1 : 1 stoichiometry. Here we created a series of tandems with different linkers between the human ergothioneine (ET) transporter ETT (gene symbol SLC22A4) and organic cation transporter OCT2 (SLC22A2). The linker sequence strongly affected the expression strength. The stoichiometry was validated by absolute peptide quantification and untargeted peptide analysis. Compared with wild-type ETT, the normalized ET clearance of the natural variant L503F was higher (f = 1.34); G462E was completely inactive. The general usefulness of the tandem strategy was demonstrated by linking several transporters with ETT; every construct was active in both parts. Transporter tandems can be used - without membrane isolation or protein quantification — as precise tools for transporter number normalization, to identify, for example, relevant transporters for a drug. It is necessary, however, to find suitable linkers, to check the order of transporters, and to verify the absence of functional interference by saturation kinetics.

Tucatinib is a potent tyrosine kinase inhibitor selective for human epidermal growth factor receptor 2 (HER2) approved by the US Food and Drug Administration for the treatment of HER2‐positive metastatic breast cancer and in development for other HER2‐positive solid tumors. Modest, reversible serum creatinine (SCr) elevations have been observed in tucatinib clinical trials. SCr is conveyed by the renal drug transporters organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1) and 2‐K (MATE2‐K) and can increase in the presence of inhibitors of these transporters. In vitro, tucatinib inhibited OCT2‐, MATE1‐, and MATE2‐K‐mediated transport of metformin, with IC50 values of 14.7, 0.340, and 0.135 µM, respectively. Tucatinib also inhibited OCT2‐ and MATE1‐mediated transport of creatinine, with IC50 values of 0.107 and 0.0855 µM, respectively. A phase 1 study with metformin administered orally in the absence and presence of tucatinib was conducted in 18 healthy subjects. Renal function was assessed by measuring glomerular filtration rate (GFR; based on iohexol plasma clearance) and endogenous markers (SCr, cystatin C‐based estimated glomerular filtration rate [eGFR]) with and without tucatinib. Metformin exposure increased (1.4‐fold) and renal clearance decreased (29.99‐17.64 L/h) with tucatinib, with no effect on metformin maximum concentration. Creatinine clearance transiently decreased 23% with tucatinib. GFR and eGFR, which are unaffected by OCT2 and/or MATE1/2‐K transport, were unchanged with tucatinib. These data demonstrate that tucatinib inhibits OCT2‐ and MATE1/2‐K‐mediated tubular secretion of creatinine, which may manifest as mild SCr elevations that are not indicative of renal impairment.

Introduction: Cisplatin induced nephrotoxicity may limit the clinical use of this drug. The aim of this study was to investigate the mechanism of the possible renoprotective effect of curcumin in cisplatin nephrotoxicity. Methods: Thirty male Wistar rats were randomly divided into five groups: 1- control (0.5ml normal saline ip, daily for 10 constitutive days); 2- cisplatin (10mg/kg ip, single dose at the first day); 3- cisplatin + curcumin (10mg/kg ip, dissolved in 5% DMSO, daily for 10 constitutive days); 4- cisplatin + vehicle (5% DMSO, 0.3ml ip) and 5- curcumin (10mg/kg ip). At the end of the study, urine and blood samples were obtained for biochemical (BUN, creatinine, sodium and potassium) analysis. The right kidneys were kept in 10% formalin for H&E and TUNEL staining, and the left kidneys were used for type 2 organic cation transporter (OCT2) and type 3 glutathione peroxidase (GPx3) gene expression and malondialdehyde measurements. Results: Cisplatin significantly increased serum creatinine, BUN, potassium and kidney lipid peroxidation. However, the effect of cisplatin on Gpx3 and OCT2 gene expression was not statistically significant. Curcumin treatment decreased serum creatinine, BUN, and kidney lipid peroxidation, but increased GPx3 and OCT2 gene expression. Moreover, curcumin significantly reversed the cisplatin-induced kidney tissue injury and decreased apoptosis. Conclusion: It is concluded that the ameliorative effect of curcumin in cisplatin nephrotoxicity was assumed to be due to antioxidant effect of this agent. The role of curcumin in mediating uptake of cisplatin is still unclear.

There is a growing interest in using endogenous compounds as drug transporter biomarkers to facilitate drug-drug interaction (DDI) risk assessment in early phase I clinical trials. Compared to other drug transporters, however, no valid biomarker for hepatic organic cation transporter (OCT) 1 has been described to date. The present work represents the first report of an endogenous compound, isobutyryl-l-carnitine (IBC), as a potential clinical OCT1 biomarker for DDI assessment. A hydrophilic interaction chromatography (HILIC)-mass spectrometry/high resolution mass spectrometry (MS/HRMS) assay with a simple sample preparation method was developed. The assay is capable of simultaneously quantifying multiple endogenous compounds, including IBC, thiamine, N1-methylnicotinamide (1-NMN), creatinine, carnitine, and metformin, which is a probe for OCT1 and OCT2 and MATE1 and MATE2K (multidrug and toxin extrusion proteins) in clinical studies. The HRMS assay was fit-for-purpose validated in human plasma and demonstrated good linearity, accuracy, and precision for all analytes. It was further applied to two phase I clinical trials to evaluate potential biomarkers for OCT1 and additional cation transporters (renal OCT2, MATE1, and MATE2K). The clinical data demonstrated that plasma IBC changes correlated well with in vitro data and supported its use as a liver OCT1 biomarker. The described HILIC-MS/HRMS assay can be used as a "biomarker cocktail" to simultaneously assess clinical DDI risk for the inhibition of OCT1/2 and MATEs in clinical studies with new drug candidates.

Made available in DSpace on 2020-12-12T01:18:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-08-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Deutsche Forschungsgemeinschaft Gabapentin (GBP) is an organic cation mainly eliminated unchanged in urine, and active drug secretion has been suggested to contribute to its renal excretion. Our objective was to evaluate the potential drug-drug interaction between GBP and cetirizine (CTZ), an inhibitor of transporters for organic cations. An open-label, 2-period, crossover, nonrandomized clinical trial was conducted in patients with neuropathic pain to evaluate the effect of CTZ on GBP pharmacokinetics. Twelve participants were treated with a single dose of 300 mg GBP (treatment A) or with 20 mg/d of CTZ for 5 days and 300 mg GBP on the last day of CTZ treatment (treatment B). Blood sampling and pain intensity evaluation were performed up to 36 hours after GBP administration. The interaction of GBP and CTZ with transporters for organic cations was studied in human embryonic kidney (HEK) cells expressing the organic cation transporters (OCTs), multidrug and toxin extrusion proteins (MATEs), and OCTN1. CTZ treatment resulted in reduced area under the concentration-time curve and peak concentration compared with treatment A. In treatment B, the lower plasma concentrations of GBP resulted in reduced pain attenuation. GBP renal clearance was similar between treatments. GBP has low apparent affinity for OCT2 (concentration of an inhibitor where the response [or binding] is reduced by half [IC50] 237 µmol/L) and a high apparent affinity for hMATE1 (IC50 1.1 nmol/L), hMATE2-K (IC50 39 nmol/L), and hOCTN1 (IC50 2.1 nmol/L) in HEK cells. At therapeutic concentrations, CTZ interacts with hMATE1 and OCTN1. In summary, CTZ reduced the systemic exposure to GBP and its effect on neuropathic pain attenuation. However, CTZ × GBP interaction is not mediated by the renal transporters. School of Pharmaceutical Sciences of Ribeirão Preto USP–São Paulo University School of Pharmaceutical Sciences UNESP–São Paulo State University School of Medicine of Ribeirão Preto USP–São Paulo University Experimental Nephrology Medicine Clinic D University Hospital Münster School of Pharmaceutical Sciences UNESP–São Paulo State University CNPq: 142247/2014-6 CNPq: 290076/2017-0 Deutsche Forschungsgemeinschaft: CI 107/11-1

The intestine epithelium is considered to be the most critical obstacle for nanoparticles for oral delivery of water-insoluble and poorly absorbed drugs. Based on the specific transporters located on the apical membrane of the intestinal epithelium, the carnitine-conjugated polymeric micelles targeting to the carnitine/organic cation transporter 2 (OCTN2) were developed by combining carnitine-conjugated poly(2-ethyl-2-oxazoline)-poly(d,l-lactide) with monomethoxy poly(ethylene-glycol)-poly(d,l-lactide). The carnitine-conjugated micelles with favorable stability in gastrointestinal fluid were validated to remarkably increase the cellular internalization and transcellular transport, while these were not the cases in the presence of free carnitine. These were further confirmed by more distribution of the micelles within epithelial cells, on the apical and basolateral side of the epithelium in mice. Additionally, identification of the carnitine-conjugated micelles by OCTN2 was detected to facilitate cellular uptake of the micelles via fluorescence immunoassay. Both clathrin and caveolae/lipid rafts pathways mediated endocytosis and transcellular transport of the carnitine-conjugated micelles, implying the enrichment of endocytic and transcellular transport pathway compared with that of carnitine-unconjugated micelles. Further, the intracellular trafficking process of the carnitine-conjugated micelles was tracked under confocal laser scanning microscopy, which involved in intracellular compartments such as late endosomes, lysosomes, endoplasmic reticulum, and Golgi apparatus as well. In conclusion, the current study provided an efficient strategy to facilitate the oral absorption of water-insoluble and poorly absorbed agents using intestinal transporter-mediated polymeric micelles.

Various drug transporters are widely expressed throughout the intestine and play important roles in absorbing nutrients and drugs, thus providing high quality targets for the design of prodrugs or nanoparticles to facilitate oral drug delivery. In particular, intestinal carnitine/organic cation transporter 2 (OCTN2) and mono-carboxylate transporter protein 1 (MCT1) possess high transport capacities and complementary distributions. Therefore, we outline recent developments in transporter-targeted oral drug delivery with regard to the OCTN2 and MCT1 proteins in this review. First, basic information of the two transporters is reviewed, including their topological structures, characteristics and functions, expression and key features of their substrates. Furthermore, progress in transporter-targeting prodrugs and nanoparticles to increase oral drug delivery is discussed, including improvements in the oral absorption of anti-inflammatory drugs, antiepileptic drugs and anticancer drugs. Finally, the potential of a dual transporter-targeting strategy is discussed., Graphical abstract In this review, we outline the recent developments in transporter-targeting oral drug delivery, especially on intestinal carnitine/organic cation transporter 2 (OCTN2) and monocarboxylate transporter protein 1 (MCT1).Image, graphical abstract

Organic cation transporters (OCTs) are poly-specific carriers for endogenous and exogenous cationic compounds. These are widely distributed in the nervous system and mediate neuronal activities. As antineoplastic cationic drugs accumulate in the dorsal root ganglion (DRG), OCT function has been studied mainly in cultured DRG neurons. However, the histological distribution of OCTs in the DRG is unclear. This study investigated the localization of OCT2 (a member of OCTs) in mouse DRG neurons and determined their histochemical properties. OCT2 expression was found in about 20% of DRG neurons, which were small to medium size. OCT2-expressing neurons were labeled with markers for peptidergic nociceptive (substance P or calcitonin gene-related peptide) and tactile/proprioceptive (neurofilament 200 or tropomyosin receptor kinase B or C) neurons. OCT2 was also expressed in cholinergic DRG neurons identified by choline acetyltransferase promoter-derived Cre expression. In the spinal dorsal horn, OCT2 was distributed in superficial to deep laminae. OCT2 immunoreactivity was punctate in appearance and localized in the nerve terminals of sensory afferents with labeling of neurochemical markers. Our findings suggest that OCT2 as a low-affinity, high-capacity carrier may take up substrates including cationic neurotransmitters and drugs from the extracellular space around cell bodies in DRG neurons.

The human organic cation transporter 2 (hOCT2) mediates renal and neuronal cellular cisplatin and oxaliplatin uptake, and therefore plays a significant role in the development of side effects associated with these chemotherapeutic drugs. Autophagy is induced by cisplatin and oxaliplatin treatment and is believed to promote cell survival under stressful conditions. We examined in vitro the role of hOCT2 on autophagy induced by cisplatin and oxaliplatin. We also explored the effect of autophagy on toxicities of these platinum derivatives. Our results indicate that autophagy, measured as LC3 II accumulation and reduction in p62 expression level, is induced in response to cisplatin and oxaliplatin in HEK293-hOCT2 but not in wild-type HEK293 cells. Furthermore, inhibition of autophagy is associated with higher toxicity of platinum derivatives, and starvation was found to offer protection against cisplatin-associated toxicity. In conclusion, activation of autophagy could be a potential strategy to protect against unwanted toxicities induced by treatment with platinum derivatives.

MATE1 (multidrug and toxin extruder 1) and OCT2 (organic cation transporter 2) play critical roles in organic cation excretion by the human kidney. The transporter turnover rate (TOR) is relevant to understanding both their transport mechanisms and interpreting the in vitro–in vivo extrapolation (IVIVE) required for physiologically-based pharmacokinetic (PBPK) modeling. Here, we use a quantitative western blot method to determine TORs for MATE1 and OCT2 proteins expressed in CHO cells. MATE1 and OCT2, each with a C-terminal V-5 epitope tag, were cell surface biotinylated and the amount of cell surface MATE1 and OCT2 protein was quantified by western analysis, using standard curves for the V5 epitope. Cell surface MATE1 and OCT2 protein represented 25% and 24%, respectively, of the total expression of these proteins in CHO cells. The number of cell surface transporters was ~55 fmol cm−2 for MATE1 and ~510 fmol cm−2 for OCT2. Dividing these values into the different Jmax values for transport of MPP, metformin, and atenolol mediated by MATE1 and OCT2 resulted in calculated TOR values (±SE, n = 4) of 84.0 ± 22.0 s−1 and 2.9 ± 0.6 s−1; metformin, 461.0 ± 121.0 s−1 and 12.6 ± 2.4 s−1; atenolol, 118.0 ± 31.0 s−1, respectively. These values are consistent with the TOR values determined for a variety of exchangers (NHEs), cotransporters (SGLTs, Lac permease), and uniporters (GLUTs, ENTs).

The balance of cisplatin uptake and efflux, mediated mainly by organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 (MATE1), respectively, determines the renal accumulation and nephrotoxicity of cisplatin. Using transporter-mediated cellular uptake assay, we identified wedelolactone (WEL), a medicinal plant-derived natural compound, is a competitive inhibitor of OCT2 and a noncompetitive inhibitor of MATE1. Wedelolactone showed a selectivity to inhibit OCT2 rather than MATE1. Cytotoxicity studies revealed that wedelolactone alleviated cisplatin-induced cytotoxicity in OCT2-overexpressing HEK293 cells, whereas it did not alter the cytotoxicity of cisplatin in various cancer cell lines. Additionally, wedelolactone altered cisplatin pharmacokinetics, reduced kidney accumulation of cisplatin, and ameliorated cisplatin-induced acute kidney injury in the Institute of Cancer Research mice. In conclusion, these findings suggest a translational potential of WEL as a natural therapy for preventing cisplatin-induced nephrotoxicity and highlight the need for drug–drug interaction investigations of WEL with other treatments which are substrates of OCT2 and/or MATE1.

The renal secretory clearance for organic cations (neurotransmitters, metabolism products and drugs) is mediated by transporters specifically expressed in the basolateral and apical plasma membrane domains of proximal tubule cells. Here, human organic cation transporter 2 (hOCT2) is the main transporter for organic cations in the basolateral membrane domain. In this study, we stably expressed hOCT2 in Madin-Darby Canine Kidney (MDCK) cells and cultivated these cells in the presence of an extracellular matrix to obtain three-dimensional (3D) structures (cysts). The transport properties of hOCT2 expressed in MDCK cysts were compared with those measured using human embryonic kidney cells (HEK293) stably transfected with hOCT2 (hOCT2-HEK cells). In the MDCK cysts, hOCT2 was expressed in the basolateral membrane domain and showed a significant uptake of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) with an affinity (Km) of 3.6 ± 1.2 µM, similar to what was measured in the hOCT2-HEK cells (Km = 3.1 ± 0.2 µM). ASP+ uptake was inhibited by tetraethylammonium (TEA+), tetrapentylammonium (TPA+), metformin and baricitinib both in the hOCT2-HEK cells and the hOCT2- MDCK cysts, even though the apparent affinities of TEA+ and baricitinib were dependent on the expression system. Then, hOCT2 was subjected to the same rapid regulation by inhibition of p56lck tyrosine kinase or calmodulin in the hOCT2-HEK cells and hOCT2- MDCK cysts. However, inhibition of casein kinase II regulated only activity of hOCT2 expressed in MDCK cysts and not in HEK cells. Taken together, these results suggest that the 3D cell culture model is a suitable tool for the functional analysis of hOCT2 transport properties, depending on cell polarization.

The organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1) mediate the renal secretion of drugs. Recent studies suggest that ondansetron, a 5-HT3 antagonist drug used to prevent nausea and vomiting, can inhibit OCT2- and MATE1-mediated transport. The purpose of this study was to test the ability of five 5-HT3 antagonist drugs to inhibit the OCT2 and MATE1 transporters. The transport of the OCT2/MATE1 probe substrate ASP+ was assessed using two models: (1) HEK293 kidney cells overexpressing human OCT2 or MATE1, and (2) MDCK cells transfected with human OCT2 and MATE1. In HEK293 cells, the inhibition of ASP+ uptake by OCT2 listed in order of potency was palonosetron (IC50: 2.6 μM) &gt, ondansetron &gt, granisetron &gt, tropisetron &gt, dolasetron (IC50: 85.4 μM) and the inhibition of ASP+ uptake by MATE1 in order of potency was ondansetron (IC50: 0.1 μM) &gt, palonosetron = tropisetron &gt, dolasetron (IC50: 27.4 μM). Ondansetron (0.5–20 μM) inhibited the basolateral-to-apical transcellular transport of ASP+ up to 64%. Higher concentrations (10 and 20 μM) of palonosetron, tropisetron, and dolasetron similarly reduced the transcellular transport of ASP+. In double-transfected OCT2-MATE1 MDCK cells, ondansetron at concentrations of 0.5 and 2.5 μM caused significant intracellular accumulation of ASP+. Taken together, these data suggest that 5-HT3 antagonist drugs may inhibit the renal secretion of cationic drugs by interfering with OCT2 and/or MATE1 function.

Summary: The transcription factors (TFs) that regulate inducible genes in activated neutrophils are not yet completely characterized. Herein, we show that the genomic distribution of the histone modification H3K27Ac, as well as PU.1 and C/EBPβ, two myeloid-lineage-determining TFs (LDTFs), significantly changes in human neutrophils treated with R848, a ligand of Toll-like receptor 8 (TLR8). Interestingly, differentially acetylated and LDTF-marked regions reveal an over-representation of OCT-binding motifs that are selectively bound by OCT2/POU2F2. Analysis of OCT2 genomic distribution in primary neutrophils and of OCT2-depletion in HL-60-differentiated neutrophils proves the requirement for OCT2 in contributing to promote, along with nuclear factor κB (NF-κB) and activator protein 1 (AP-1), the TLR8-induced gene expression program in neutrophils. Altogether, our data demonstrate that neutrophils, upon activation via TLR8, profoundly reprogram their chromatin status, ultimately displaying cell-specific, prolonged transcriptome changes. Data also show an unexpected role for OCT2 in amplifying the transcriptional response to TLR8-mediated activation.

Cisplatin (CP) is one of the most effective chemotherapeutics in the treatment of human cancers. However, the beneficial effects of CP are limited by the toxic effects, especially nephrotoxicity. Fluorofenidone (AKFPD) is a promising multifunctional antifibrosis pyridinone drug discovered by our group. But there is no evidence of its protective effects against acute kidney injury (AKI). Therefore, we investigated the protective effects of AKFPD on CP-induced AKI

Cisplatin (CDDP) is one of the most important chemotherapeutic drugs in modern oncology. However, its use is limited by severe toxicities, which impair life quality after cancer. Here, we investigated the role of organic cation transporters (OCT) in mediating toxicities associated with chronic (twice the week for 4weeks) low-dose (4mg/kg body weight) CDDP treatment (resembling therapeutic protocols in patients) of wild-type (WT) mice and mice with OCT genetic deletion (OCT1/2−/−). Functional and molecular analysis showed that OCT1/2−/− mice are partially protected from CDDP-induced nephrotoxicity and peripheral neurotoxicity, whereas ototoxicity was not detectable. Surprisingly, proteomic analysis of the kidneys demonstrated that genetic deletion of OCT1/2 itself was associated with significant changes in expression of proinflammatory and profibrotic proteins which are part of an OCT-associated protein network. This signature directly regulated by OCT consisted of three classes of proteins, viz., profibrotic proteins, proinflammatory proteins, and nutrient sensing molecules. Consistent with functional protection, CDDP-induced proteome changes were more severe in WT mice than in OCT1/2−/− mice. Laser ablation-inductively coupled plasma-mass spectrometry analysis demonstrated that the presence of OCT was not associated with higher renal platinum concentrations. Taken together, these results redefine the role of OCT from passive membrane transporters to active modulators of cell signaling in the kidney.

Following oral administration, dabigatran etexilate (DABE) is rapidly hydrolyzed to its active form, dabigatran. DABE, but not dabigatran, presents as a P-glycoprotein (P-gp) substrate and has increasingly been used as a probe drug. Therefore, although dosed as DABE, a P-gp drug-drug interaction (DDI) is reported as a dabigatran plasma concentration ratio (perpetrator versus placebo). Because the majority of a DABE dose (80 to 85%) is recovered in urine as unchanged dabigatran (renal active secretion is ∼25% of total clearance), dabigatran was evaluated in vitro as a substrate of various human renal transporters. Active (pyrimethamine-sensitive) dabigatran uptake was observed with human embryonic kidney (HEK) 293 cells expressing multidrug and toxin extrusion protein 1 (MATE1) and 2K (MATE2K), with Michaelis-Menten constant (Km) values of 4.0 and 8.0 μM, respectively. By comparison, no uptake of 2 μM dabigatran (versus mock-transfected HEK293 cells) was evident with HEK293 cells transfected with organic cation transporters (OCT1 and OCT2) and organic anion transporters (OAT1, 2, 3, and 4). The efflux ratios of dabigatran across P-gp- and BCRP (breast cancer resistance protein)-MDCK (Madin-Darby canine kidney) cell monolayers were 1.5 and 2.0 (versus mock-MDCK cell monolayers), suggesting dabigatran is a relatively poor P-gp and BCRP substrate. Three of five drugs (verapamil, ketoconazole, and quinidine) known to interact clinically with dabigatran, as P-gp inhibitors, presented as MATE inhibitors in vitro (IC50 = 1.0 to 25.2 μM). Taken together, although no basolateral transporter was identified for dabigatran, the results suggest that apical MATE1 and MATE2K could play an important role in its renal clearance. MATE-mediated renal secretion of dabigatran needs to be considered when interpreting the results of P-gp DDI studies following DABE administration.

Flecainide, an anti-arrhythmic drug, undergoes renal excretion through active renal tubular secretion in addition to passive glomerular filtration. The contribution of renal uptake and efflux transporters in active renal tubular secretion of flecainide remains unclear except that flecainide is a substrate of human multidrug resistance protein 1 (MDR1). To elucidate renal efflux and uptake transporters involved with active renal tubular secretion of flecainide, we conducted in vitro interaction studies of flecainide using organic cation transporter 2 (OCT2), multidrug and toxin extrusion (MATE) 1, and MATE2-K. Uptake transporter inhibition assays using hOCT2-Chinese hamster ovary (CHO), hMATE1-CHO, and hMATE2-K-Madin Darby canine kidney strain II (MDCKII) cells revealed that flecainide (2.5 µM) inhibited hMATE1-mediated transport by 40% with an IC50 value of 6.7 µM; however, it showed no or weak inhibitory effects on hOCT2- and hMATE2-K-mediated transport. For investigating flecainide as a substrate of hMATE1, the accumulation of flecainide in hMATE1-CHO was compared with that in control cells. Uptake transporter substrate assay revealed that flecainide (1 µM) showed 1.11-fold accumulation though the hMATE1-related active transport was significantly decreased in the presence of quinidine (42.0 ± 23.9 vs. 11.8 ± 4.1 pmol/mg in transfected cells; p < 0.05). These results suggest that flecainide is a weak substrate of hMATE1, which is involved in the renal tubular secretion of cationic drugs, and hMATE1 may be less important in the pharmacokinetic drug-drug interaction for renal excretion of flecainide. However, in vivo drug-drug interaction studies of flecainide with substrates of hMATE1 may be needed because flecainide has the potential to inhibit hMATE1.

The anticholinergic drug trospium is secreted into urine and, to a smaller extent, into bile. Chemically, it is an organic cation, and it is a substrate of the uptake transporters OCT1 and OCT2 as well as for the export proteins MATE1 and MATE2-K as determined in uptake studies using HEK293 cells. So far, neither MATE-mediated export nor the interplay of OCT-mediated uptake and MATE-mediated export have been investigated. Therefore, we used polarized monolayers of single- and double-transfected MDCKII cells (MDCK-OCT1, MDCK-OCT2, MDCK-MATE1, MDCK-OCT1-MATE1, and MDCK-OCT2-MATE1) and the respective control cells (MDCK-Co) for transcellular transport assays. We demonstrate that the transcellular, basal-to-apical transport of trospium is significantly higher in all cell lines compared to control cells over nearly the complete concentration range tested. The transcellular transport mediated by double-transfected MDCK-OCT1-MATE1 and MDCK-OCT2-MATE1 exceeded that in the single-transfected cells (MDCK-OCT1-MATE1 vs MDCK-OCT1: 2.2-fold; MDCK-OCT1-MATE1 vs MDCK-MATE1: 1.7-fold; MDCK-OCT2-MATE1 vs MDCK-OCT2: 6.1-fold; MDCK-OCT2-MATE1 vs MDCK-MATE1: 1.8-fold at a trospium concentration of 1.0 μM; p0.001 each). Thus, we show that MATE1 does not only mediate the uptake of trospium into HEK293 cells but also the efflux of trospium out of polarized MDCKII-cells. Furthermore, our results indicate that OCT1 or OCT2 as uptake transporters and MATE1 as an export protein contribute to the transcellular transport of trospium at concentrations normally reached during trospium therapy. These data suggest that both, OCT-mediated uptake as well as MATE1-mediated efflux may contribute to trospium renal and biliary elimination.

1. Jatrorrhizine is an active ingredient found in various traditional Chinese medicinal plants. Based on our previous finding that jatrorrhizine was a potent inhibitor of OCT2 and OCT3, the aim of the present study was to explore whether jatrorrhizine has an antidepressant-like action action via inhibition of uptake-2 transporters. 2. In vitro uptake tests showed that jatrorrhizine strongly inhibited PMAT-mediated MPP+ uptake with an IC50 value of 1.05 μM and reduced 5-HT and NE uptake mediated by hOCT2, hOCT3 and hPMAT with IC50 values of 0.1–1 μM (for OCT2 and OCT3) and 1–10 μM (for PMAT). 3. In mouse synaptosomes, jatrorrhizine suppressed 5-HT and NE uptake in a concentration dependently manner, where the role of uptake-2 inhibition is significant. 4. The antidepressant-like action of jatrorrhizine was evaluated by mouse tail suspension test (TST). The TST showed that one week of jatrorrhizine (5, 10 and 20 mg/kg, i.p.) or venlafaxine (20 mg/kg, i.g.) can significantly reduce the duration of immobility when compared with vehicle control group. 5. The concentration of jatrorrhizine shows a dose-dependent increase in brain tissues. 6. Our study suggested that jatrorrhizine might be used as an antidepressant agent via inhibition of uptake-2 transporters.

Metformin and berberine are often combined for treating diabetes. In the present study, we evaluated the drug-drug pharmacokinetic interaction between metformin and berberine after oral co-administration in vivo and the underlying mechanism. As revealed by comparison with the metformin-only group, berberine significantly decreased the maximum plasma concentration (Cmax), area under the curve from 0 to 4 h (AUC0–4h), and urinary and bile excretion, and increased the kidney tissue concentration of metformin in rats. The non-everted intestinal sac study showed that berberine inhibited the absorption of metformin, and in transfected Madin-Darby canine kidney (MDCK)-rat organic cation transporter 1 (MDCK-rOCT1), MDCK-rat organic cation transporter 2 (MDCK-rOCT2), and MDCK-rat multidrug and toxin extrusion 1 (MDCK-rMATE1) cells, berberine significantly inhibited metformin transport mediated by OCT1, OCT2, and MATE1 in a concentration-dependent manner with half-maximal inhibitory concentration (IC50) values of 18.8, 1.02, and 10.7 μM, respectively. In contrast, co-administration of metformin increased the Cmax and AUC0-4h of berberine with no significant difference in pharmacokinetics parameters between co-administration and berberine-only groups. Furthermore, metformin increased kidney and liver concentrations and reduced the urinary and biliary excretion of berberine. Metformin (≥1 or ≥0.3 mM) decreased berberine transport in MDCK-rOCT1, MDCK-rOCT2, and MDCK-rMATE1 cells. However, metformin did not affect berberine concentration in MDCK-multidrug resistance protein 1 cells. These results suggest that the combination of metformin and berberine induced a pharmacokinetic interaction by cooperatively inhibiting OCT and MATE1-mediated transport.

Growing evidence supports involvement of low-affinity/high-capacity organic cation transporters (OCTs) and plasma membrane monoamine transporter (PMAT) in regulating clearance of monoamines. Currently decynium-22 (D22) is the best pharmacological tool to study these transporters, however it does not readily discriminate among them, underscoring a need to develop compounds with greater selectivity for each of these transporters. We developed seven D22 analogs, and previously reported that some have lower affinity for α1-adrenoceptors than D22 and showed antidepressant-like activity in mice. Here, we extend these findings to determine the affinity of these analogs for OCT2, OCT3 and PMAT, as well as serotonin, norepinephrine and dopamine transporters (SERT, NET and DAT) using a combination of uptake competition with [(3)H]methyl-4-phenylpyridinium acetate in overexpressed HEK cells and [(3)H]citalopram, [(3)H]nisoxetine and [(3)H]WIN 35428 displacement binding in mouse hippocampal and striatal preparations. Like D22, all analogs showed greater binding affinities for OCT3 than OCT2 and PMAT. However, unlike D22, some analogs also showed modest affinity for SERT and DAT. Dual OCT3/SERT and/or OCT3/DAT actions of certain analogs may help explain their ability to produce antidepressant-like effects in mice and help account for our previous findings that D22 lacks antidepressant-like effects unless SERT function is either genetically or pharmacologically compromised. Though these analogs are not superior than D22 in discriminating among OCTs/PMAT, our findings point to development of compounds with combined ability to inhibit both low-affinity/high-capacity transporters, such as OCT3, and high-affinity/low-capacity transporters, such as SERT, as therapeutics with potentially improved efficacy for treatment of psychiatric disorders.

Abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6, is indicated for metastatic breast cancer treatment. Reversible increases in serum creatinine levels of ~15-40% over baseline have been observed following abemaciclib dosing. This study assessed the in vitro and clinical inhibition of renal transporters by abemaciclib and its metabolites using metformin (a clinically relevant transporter substrate), in a clinical study that quantified glomerular filtration and iohexol clearance. In vitro, abemaciclib inhibited metformin uptake by organic cation transporter 2, multidrug and toxin extrusion (MATE)1, and MATE2-K transporters with a half-maximal inhibitory concentration of 0.4-3.8 μM. Clinically, abemaciclib significantly increased metformin exposure but did not significantly affect measured glomerular filtration rate, serum neutrophil gelatinase-associated lipocalin (NGAL), serum cystatin-C, or the urinary markers of kidney tubular injury, NGAL and kidney injury molecule-1.

The successful prospective incorporation of in vitro transporter kinetics in physiologically based pharmacokinetic (PBPK) models to describe drug disposition remains challenging. Although determination of scaling factors to extrapolate in vitro to in vivo transporter kinetics has been facilitated by quantitative proteomics, no robust assessment comparing membrane recoveries between different cells/tissues has been made. HEK293 cells overexpressing OCT2, MATE1, and MATE2K or human kidney cortex were homogenized and centrifuged to obtain the total membrane fractions, which were subsequently subjected to liquid-liquid extraction followed by centrifugation and precipitation to isolate plasma membrane fractions. Plasma membrane recoveries determined by quantitation of the marker Na+/K+-ATPase in lysate and plasma membrane fractions were ≤20% but within 3-fold across different cells and tissues. A separate study demonstrated that recoveries are comparable between basolateral and apical membranes of renal proximal tubules, as measured by Na+/K+-ATPase and γ-glutamyl transpeptidase 1, respectively. The plasma membrane expression of OCT2, MATE1, and MATE2K was quantified and relative expression factors (REFs) were determined as the ratio between the tissue and cell concentrations. Corrections using plasma membrane recovery had minimal impact on REF values (

The present study aimed at incorporating active renal excretion via the organic cation transporter 2 (OCT2) into a generic rat physiologically based kinetic (PBK) model using an in vitro human renal proximal tubular epithelial cell line (SA7K) and mepiquat chloride (MQ) as the model compound. The Vmax (10.5 pmol/min/mg protein) and Km (20.6 μM) of OCT2 transport of MQ were determined by concentration-dependent uptake in SA7K cells using doxepin as inhibitor. PBK model predictions incorporating these values in the PBK model were 6.7-8.4-fold different from the reported in vivo data on the blood concentration of MQ in rat. Applying an overall scaling factor that also corrects for potential differences in OCT2 activity in the SA7K cells and in vivo kidney cortex and species differences resulted in adequate predictions for in vivo kinetics of MQ in rat (2.3-3.2-fold). The results indicate that using SA7K cells to define PBK parameters for active renal OCT2 mediated excretion with adequate scaling enables incorporation of renal excretion via the OCT2 transporter in PBK modelling to predict in vivo kinetics of mepiquat in rat. This study demonstrates a proof-of-principle on how to include active renal excretion into generic PBK models.

Imeglimin is a novel oral antidiabetic drug to treat type 2 diabetes, targeting the mitochondrial bioenergetics. In vitro, imeglimin was shown to be a substrate of human multidrug and toxic extrusion transporters MATE1 and MATE2-K and organic cation transporters OCT1 and OCT2. The objective of the study was to assess the potential drug–drug interaction between imeglimin and cimetidine, a reference inhibitor of these transporters. A phase 1 study was carried out in 16 subjects who received a single dose of 1500 mg imeglimin alone on day 1 followed by a 6-day treatment (day 5 to day 10) with cimetidine 400 mg twice daily. On day 8, a single dose of imeglimin was co-administered with cimetidine. Blood and urine samples were collected up to 72 h after each imeglimin administration. Pharmacokinetic parameters were determined using non-compartmental methods. Imeglimin maximum plasma concentration (Cmax) and area under the plasma concentration–time curve (AUC) were 1.3-fold [90% CI (1.12–1.62) and (1.10–1.46) for Cmax and AUC0–last, respectively] higher when imeglimin was co-administered with cimetidine but this increase was not considered clinically relevant. This increase could be mainly explained by a reduction in renal elimination, mediated through the cimetidine inhibition of renal MATE1 transporter. Imeglimin taken alone or with cimetidine was safe and well tolerated in all subjects. No clinically significant drug–drug interaction exists between imeglimin and cimetidine, a reference inhibitor of MATE1, MATE2-K, OCT1 and OCT2 transporters. EudraCT 2018-001103-36.

Drug elimination alteration has been well reported in acute lymphoblastic leukemia (ALL). Considering that transporters and glomerular filtration influence, to different extents, the drug disposition, and possible side effects, we evaluated the effects of ALL on major renal transporters and glomerular filtration mediated pharmacokinetic changes, as well as expression of renal drug transporters. ALL xenograft models were established and intravenously injected with substrates of renal transporters and glomerular filtration separately in NOD/SCID mice. The plasma concentrations of substrates, after single doses, were determined using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). With the development of ALL, protein expression of MDR1, OAT3 and OCT2 were increased by 2.62-fold, 1.70-fold, and 1.45-fold, respectively, whereas expression of MRP2 and MRP4 were significantly decreased by 30.98% and 45.28% in the kidney of ALL groups compared with control groups. Clearance of MDR1-mediated digoxin, OAT3-mediated furosemide, and OCT2-mediated metformin increased by 3.04-fold, 1.47-fold, and 1.26-fold, respectively. However, clearance of MRPs-mediated methotrexate was reduced by 39.5%. These results are consistent with mRNA expression. Clearance of vancomycin and amikacin, as markers of glomerular filtration rate, had a 2.14 and 1.64-fold increase in ALL mice, respectively. The specific alteration of renal transporters and glomerular filtration in kidneys provide a rational explanation for changes in pharmacokinetics for ALL.

Farnesoid X receptor (FXR) is a ligand-activated transcription factor highly expressed in the liver and kidneys. Activation of FXR decreases organic cation transporter (OCT) 1-mediated clearance of organic cation compounds in hepatocytes. The present study investigated FXR regulation of renal clearance of organic cations by OCT2 modulation and multidrug and toxin extrusion proteins (MATEs). The role of FXR in OCT2 and MATEs functions was investigated by monitoring the flux of 3H&ndash, MPP+, a substrate of OCT2 and MATEs. FXR agonists chenodeoxycholic acid (CDCA) and GW4064 stimulated OCT2-mediated 3H&ndash, MPP+ uptake in human renal proximal tubular cells (RPTEC/TERT1 cells) and OCT2-CHO-K1 cells. The stimulatory effect of CDCA (20 &micro, M) was abolished by an FXR antagonist, Z-guggulsterone, indicating an FXR-dependent mechanism. CDCA increased OCT2 transport activity via an increased maximal transport rate of MPP+. Additionally, 24 h CDCA treatment increased MATEs-mediated 3H-MPP+ uptake. Moreover, CDCA treatment increased the expression of OCT2, MATE1, and MATE2-K mRNA compared with that of the control. OCT2 protein expression was also increased following CDCA treatment. FXR activation stimulates renal OCT2- and MATE1/2-K-mediated cation transports in proximal tubules, demonstrating that FXR plays a role in the regulation of OCT2 and MATEs in renal proximal tubular cells.

To date, relatively little is known about the interactions of pharmaceutical excipients with hepatic and renal drug uptake transporters. The present study was designed to systematically evaluate the effects of 16 commonly consumed excipients on human organic cation transporter 1 and 2 (hOCT1 and hOCT2), human organic anion transporter 1 and 3 (hOAT1 and hOAT3) and human organic anion transporting polypeptide 1B1 and 1B3 (hOATP1B1 and hOATP1B3).The inhibitory effects and mechanisms of excipients on transporters were investigated using in vitro uptake studies, cell viability assays, concentration-dependent studies, and the Lineweaver–Burk plot method.Triton X-100 is a non-competitive inhibitor for all six transporters. Tween 20 inhibits hOCT2, hOAT1, hOAT3, and hOATP1B3 in a mixed way, whereas it competitively inhibits hOATP1B1. The inhibition of Tween 80 is competitive for hOCT2, non-competitive for hOATP1B1 and hOATP1B3, and mixed for hOAT1 and hOAT3. Concentration-dependent studies identify Triton X-100 as a strong inhibitor of hOCT1 and hOCT2 with IC50 values of 20.1 and 4.54 μg/mL, respectively. Additionally, Triton X-100, Tween 20, and Tween 80 strongly inhibit hOAT3 with IC50 values ≤31.0 μg/mL.The present study is significant in understanding the excipient–drug interactions and provides valuable information for excipient selection in drug development. To date, relatively little is known about the interactions of pharmaceutical excipients with hepatic and renal drug uptake transporters. The present study was designed to systematically evaluate the effects of 16 commonly consumed excipients on human organic cation transporter 1 and 2 (hOCT1 and hOCT2), human organic anion transporter 1 and 3 (hOAT1 and hOAT3) and human organic anion transporting polypeptide 1B1 and 1B3 (hOATP1B1 and hOATP1B3). The inhibitory effects and mechanisms of excipients on transporters were investigated using in vitro uptake studies, cell viability assays, concentration-dependent studies, and the Lineweaver–Burk plot method. Triton X-100 is a non-competitive inhibitor for all six transporters. Tween 20 inhibits hOCT2, hOAT1, hOAT3, and hOATP1B3 in a mixed way, whereas it competitively inhibits hOATP1B1. The inhibition of Tween 80 is competitive for hOCT2, non-competitive for hOATP1B1 and hOATP1B3, and mixed for hOAT1 and hOAT3. Concentration-dependent studies identify Triton X-100 as a strong inhibitor of hOCT1 and hOCT2 with IC50 values of 20.1 and 4.54 μg/mL, respectively. Additionally, Triton X-100, Tween 20, and Tween 80 strongly inhibit hOAT3 with IC50 values ≤31.0 μg/mL. The present study is significant in understanding the excipient–drug interactions and provides valuable information for excipient selection in drug development.

Creatinine is widely used as a biomarker of glomerular filtration, and, hence, renal function. However, transporter-mediated secretion also contributes to its renal clearance, albeit to a lesser degree. Inhibition of these transporters causes transient serum creatinine elevation, which can be mistaken as impaired renal function. The current study developed mechanistic models of creatinine kinetics within physiologically based framework accounting for multiple transporters involved in creatinine renal elimination, assuming either unidirectional or bidirectional-OCT2 transport (driven by electrochemical gradient). Robustness of creatinine models was assessed by predicting creatinine-drug interactions with 10 perpetrators; performance evaluation accounted for 5% intra-individual variability in serum creatinine. Models showed comparable predictive performances of the maximum steady-state effect regardless of OCT2 directionality assumptions. However, only the bidirectional- OCT2 model successfully predicted the minimal effect of ranitidine. The dynamic nature of models provides clear advantage to static approaches and most advanced framework for evaluating interplay between multiple processes in creatinine renal disposition.

Drug-induced kidney injury is a major clinical problem and causes drug attrition in the pharmaceutical industry. To better predict drug-induced kidney injury, kidney in vitro cultures with enhanced physiologic relevance are developed. To mimic the proximal tubule, the main site of adverse drug reactions in the kidney, human-derived renal proximal tubule epithelial cells (HRPTECs) were injected in one of the channels of dual-channel Nortis chips and perfused for 7 days. Tubes of HRPTECs demonstrated expression of tight junction protein 1 (zona occludens-1), lotus lectin, and primary cilia with localization at the apical membrane, indicating an intact proximal tubule brush border. Gene expression of cisplatin efflux transporters multidrug and toxin extrusion transporter (MATE) 1 (SLC47A1) and MATE2-k (SLC47A2) and megalin endocytosis receptor increased 19.9 ± 5.0-, 23.2 ± 8.4-, and 106 ± 33-fold, respectively, in chip cultures compared with 2-dimensional cultures. Moreover, organic cation transporter 2 (OCT2) (SLC22A2) was localized exclusively on the basolateral membrane. When infused from the basolateral compartment, cisplatin (25 µM, 72 hours) induced toxicity, which was evident as reduced cell number and reduced barrier integrity compared with vehicle-treated chip cultures. Coexposure with the OCT2 inhibitor cimetidine (1 mM) abolished cisplatin toxicity. In contrast, infusion of cisplatin from the apical compartment did not induce toxicity, which was in line with polarized localization of cisplatin uptake transport proteins, including OCT2. In conclusion, we developed a dual channel human kidney proximal tubule-on-a-chip with a polarized epithelium, restricting cisplatin sensitivity to the basolateral membrane and suggesting improved physiologic relevance over single-compartment models. Its implementation in drug discovery holds promise to improve future in vitro drug-induced kidney injury studies. SIGNIFICANCE STATEMENT: Human-derived kidney proximal tubule cells retained characteristics of epithelial polarization in vitro when cultured in the kidney-on-a-chip, and the dual-channel construction allowed for drug exposure using the physiologically relevant compartment. Therefore, cell polarization-dependent cisplatin toxicity could be replicated for the first time in a kidney proximal tubule-on-a-chip. The use of this physiologically relevant model in drug discovery has potential to aid identification of safe novel drugs and contribute to reducing attrition rates due to drug-induced kidney injury.

Drug induced kidney injury (DIKI) is a common reason for compound attrition in drug development pipelines with proximal tubule epithelial cells (PTECs) most commonly associated with DIKI. Here, we investigated freshly isolated human (hPTECs) as an in vitro model for assessing renal tubular toxicity. The freshly isolated hPTECs were first characterized to confirm gene expression of important renal transporters involved in drug handling which was further corroborated by confirming the functional activity of organic cation transporter 2 and organic anion transporter 1 by using transporter specific inhibitors. Additionally, functionality of megalin/cubilin endocytic receptors was also confirmed. A training set of 36 compounds was used to test the ability of the model to classify them using six different endpoints which included three biomarkers (Kidney Injury Molecule-1, Neutrophil gelatinase-associated lipocalin, and Clusterin) and three non-specific injury endpoints (ATP depletion, LDH leakage, and barrier permeability via transepithelial electrical resistance) in a dose-dependent manner across two independent kidney donors. In general, biomarkers showed higher predictivity than non-specific endpoints, with Clusterin showing the highest predictivity (Sensitivity/Specificity - 65.0/93.8 %). By using the thresholds generated from the training set, nine candidate internal Takeda compounds were screened where PTEC toxicity was identified as one of the findings in preclinical animal studies. The model correctly classified four of six true positives and two of three true negatives, showing validation of the in vitro model for detection of tubular toxicants. This work thus shows the potential application of freshly isolated primary hPTECs using translational biomarkers in assessment of tubular toxicity within the drug discovery pipeline.

Cisplatin is used widely for the treatment of multiple solid tumors. Cisplatin-induced nephrotoxicity is caused by renal accumulation of cisplatin via human organic cation transporter 2 (hOCT2). As lansoprazole, a proton pump inhibitor, is known to inhibit hOCT2 activity, lansoprazole might ameliorate cisplatin-induced nephrotoxicity. A previous study showed that concomitant lansoprazole administration ameliorated nephrotoxicity in patients receiving cisplatin. However, the detailed mechanism remains to be clarified. In the present study, the drug-drug interaction between lansoprazole and cisplatin was examined using hOCT2-expressing cultured cells and rat renal slices. Moreover, the effect of lansoprazole on cisplatin-induced nephrotoxicity and the pharmacokinetics of cisplatin in rats was investigated. In the uptake study, lansoprazole potently inhibited the uptake of cisplatin in hOCT2-expressing cultured cells and rat renal slices. The in vivo rat study showed that concomitant lansoprazole significantly ameliorated cisplatin-induced nephrotoxicity and reduced the renal accumulation of platinum up to approximately 60% of cisplatin alone at 72 h after cisplatin intraperitoneal administration. Furthermore, the renal uptake of platinum at 3 min after intravenous cisplatin administration in rats with cisplatin and lansoprazole decreased to 78% of rats with cisplatin alone. In addition, there was no significant difference in the plasma platinum concentration between rats treated with and without lansoprazole at 3 min after cisplatin intravenous administration. These findings suggested that concomitant lansoprazole ameliorated cisplatin-induced nephrotoxicity by inhibiting rOCT2-mediated cisplatin uptake in rats, thus decreasing cisplatin accumulation in the kidney. The present findings provided important information for the establishment of novel protective approaches to minimize cisplatin-induced nephrotoxicity.

Organic cation transporters (OCTs) are membrane proteins with relevant physiological (because they accept neurotransmitters as substrate) and pharmacological (because of their interaction with drugs) roles. The human OCTs hOCT1 (SLC22A1/hOCT1) and hOCT2 (SLC22A2/hOCT2) are highly expressed in hepatic (hOCT1) and in renal and neuronal tissue (hOCT2), suggesting a possible role in modulating neurotransmitter activity in the liver, kidney, and brain, and their clearance from the blood. Even though there are several data demonstrating that OCTs are regulated under various patho-physiological conditions, it remains largely unknown which proteins directly interact with OCTs and thereby influence their cellular processing, localization, and function. In this work, using a mating-based split-ubiquitin yeast two-hybrid system, we characterized the potential interactome of hOCT1 and 2. It became evident that these OCTs share some potential interaction partners, such as the tetraspanins CD63 and CD9. Moreover, we confirmed interaction of hOCT2 with CD9 by fluorescence-activated cell sorting coupled with Forster resonance energy transfer analysis. Together with other proteins, tetraspanins build "tetraspanins webs" in the plasma membrane, which are able to regulate cellular trafficking and compartmentalization of interacting partners. While CD63 was demonstrated to mediate the localization of the hOCT2 to the endosomal system, we show here that co-expression of hOCT2 and CD9 led to strong cell surface localization of the transporter. These data suggest that tetraspanins regulate the cellular localization and function of OCTs. Co-localization of CD9 and hOCT was confirmed in tissues endogenously expressing proteins, highlighting the potential biological relevance of this interaction.

Renal accumulation and exposure of cadmium originating from pollution in agricultural land and the prevalence of cigarette smoking remains an unneglectable human health concern. Whereas cadmium exposure has been correlated with increased incidence of a variety of kidney diseases, little is known pertaining to its effect on renal drug disposition and response in patients. Here, we report that cadmium exposure significantly increased the activity of organic cation transporter 2 (OCT2), a critical renal drug transporter recommended in United States Federal Drug Administration guidance for assessment during drug development. Cadmium enhanced OCT2 trafficking to the cell membrane both in vitro and in vivo. Mechanistically cadmium-mediated OCT2 translocation was found to involve protein-protein interaction between serine/threonine-protein kinase AKT2, calcium/calmodulin and the AKT substrate AS160 in in vitro cellular studies. The formed protein complex could selectively facilitate phosphorylation of AKT2 at T309, which induced translocation of OCT2 to the plasma membrane. Moreover, cadmium exposure markedly exacerbated nephrotoxicity induced by cisplatin, an OCT2 substrate, by increasing its accumulation in the mouse kidney. Consistently, there was a significant correlation between plasma cadmium level and alteration of renal function in cervical cancer patients who underwent chemotherapy with cisplatin. Thus, our studies suggest that membrane transporter distribution induced by cadmium exposure is a previously unrecognized factor for the broad variation in renal drug disposition and response.

Organic cation transporters play a critical role in mediating the distribution of cationic pharmaceuticals. Indeed, organic cation transporter (OCT)2 is the initial step in the renal secretion of organic cations and consequently plays a defining role in establishing the pharmacokinetics of many cationic drugs. Although a hallmark of OCTs is their broad selectivity, this characteristic also makes them targets for unwanted, adverse drug-drug interactions (DDIs), making them a focus for efforts to develop models of ligand interaction that could predict and preempt these adverse interactions. This review discusses the molecular characteristics of these transporters as well as the evidence that established the OCTs as key players in the distribution of organic cations. However, the primary focus is the present understanding of the complexity of ligand interaction with OCTs, particularly OCT2, including evidence for the presence of multiple ligand-binding sites and the influence of substrate structure on the affinity of the transporter for inhibitory ligands. This leads to a discussion of the complexities associated with the development of protocols for assessing the inhibitory potential of new molecular entities to perpetrate unwanted DDIs, the criteria that should be considered in the interpretation of the results of such protocols, and the challenges associated with development of models capable of predicting unwanted DDIs.

Cimetidine decreases the renal clearance of metformin by inhibition of renal tubular cation transport, and the underlying molecular mechanisms are still not fully understood. We investigated polarized metformin transport without and with the addition of cimetidine as well as polarized cimetidine transport in double-transfected MDCK-OCT2-MATE1 cells that mimic organic cation transport processes in proximal renal tubule cells and in MDCK vector control and single-transfected MDCK-OCT2 and MDCK-MATE1 cells. At all tested concentrations (1, 10, 100 μM), the intracellular accumulation of cimetidine after administration to the basal compartment was considerably higher in MDCK-OCT2 cells compared to that in all other cells ( p < 0.001). Whereas cimetidine transcellular, basal-to-apical transport was only slightly higher in MDCK-OCT2 cells, the presence of MATE1 in the apical membrane caused a pronounced translocation of cimetidine in both single- and double-transfected cells ( p < 0.001). Transcellular, basal-to-apical metformin net transport was reduced by 89.1, 74.5, and 91.0% in MDCK-OCT2-MATE1 cells after the addition of cimetidine (100 μM) to the basal, the apical, or both compartments ( p < 0.001). In MDCK-MATE1 and MDCK-OCT2-MATE1 cells, transcellular net transport of metformin was inhibited by cimetidine with IC50 values of 8.0 and 6.6 μM, respectively. Our data confirm the relevance of MATE1 and suggest the relevance of OCT2 for the cimetidine-metformin interaction, primarily because OCT2 mediates uptake of the perpetrator cimetidine into renal proximal tubular cells and thereby to the site of the metformin exporter MATE1. This work supports the notion that a thorough understanding of transporter-mediated drug-drug interactions may require investigations on the impact of transporters on cellular uptake and transcellular transport of victim as well as perpetrator drugs.

The role of plasma membrane transporters in the nephrotoxicity of two antiretroviral drugs, cidofovir and tenofovir, was studied in primary cultures of human proximal tubular (hPT) cells. Cells were grown on Transwell filter inserts to maintain epithelial polarity and access to either the apical or basolateral plasma membrane. The function of relevant membrane transporters, organic anion transporter 1 and 3 (OAT1/3), P-glycoprotein (multidrug resistance protein-1; P-gp or MDR1), and organic cation transporter 2 (OCT2), was validated by measurements of apical-to-basolateral and basolateral-to-apical fluxes of furosemide, digoxin, and metformin, respectively. Acute cytotoxicity of cidofovir (0, 10, 50, 150, or 300 μM) in the absence or presence of 500 μM probenecid, tenofovir disoproxil fumarate (0, 20, 90, 180, or 360 μM) in the absence or presence of 500 μM probenecid, or cisplatin (0, 20, 90, 180, or 360 μM) as a positive control in the absence or presence of 500 μM cimetidine, was assessed after 4-h incubations by determinations of release of lactate dehydrogenase (LDH), γ-glutamyltransferase (GGT), N-acetyl-β-d-glucosaminidase (NAG), or Kidney Injury Molecule-1 (KIM-1). Cell death generally agreed with each of the four biomarkers, was generally greater when cidofovir or tenofovir was added to the upper compartment, and was markedly diminished in the presence of the appropriate transport inhibitor. Additionally, the extent of cytotoxicity caused by the two antiviral drugs was similar to that caused by cisplatin. The results demonstrate the importance of plasma membrane transport of antiviral drugs to elicit cytotoxicity in the hPT cell.

The human organic cation transporter 2 (OCT2) is a multispecific transporter with cholesterol-dependent allosteric features. The present work elucidates the role of evolutionarily conserved cholesterol recognition/interaction amino acid consensus sequences (CRAC and CARC) in the allosteric binding to 1-methyl-4-phenylpyridinium (MPP+) in human embryonic kidney 293 cells stably or transiently expressing OCT2. Molecular blind simulations docked two mirroring cholesterol molecules in the 5th putative transmembrane domain, where a CARC and a CRAC sequence lie. The impact of the conserved amino acids that may constitute the CARC/CRAC mirror code was studied by alanine-scanning mutagenesis. At a saturating extracellular concentration of substrate, at which the impact of cholesterol depletion is maximal, five mutants transported MPP+ at a significantly lower rate than the wild-type OCT2 (WT), resembling the behavior of the WT upon cholesterol depletion. MPP+ influx rate as a function of the extracellular concentration of substrate was measured for the mutants R234A, R235A, L252A and R263A. R234A kinetic behavior was similar to that of the WT, whereas R235A, L252A and R263A activity shifted from allosteric to one-binding site kinetics, very much like the WT upon cholesterol depletion. The impact of cholesterol on protein thermal stability was assessed for WT, R234A and R263A. While the thermal stability of WT and R234A was improved by the supplementation with cholesterol, R263A was not sensitive to the presence of cholesterol. To conclude, the disruption of the CARC/CRAC mirror code in the 5th putative transmembrane domain is sufficient to abolish the allosteric interaction between OCT2 and MPP+.

Purpose Pretreatment with magnesium (Mg) has been reported to attenuate cisplatin (CDDP)-induced nephrotoxicity (CIN). This attenuation involves modulation of the expression of renal transporters, resulting in reduced renal platinum accumulation after a single round of CDDP treatment. In this study, we investigated whether Mg co-administration ameliorates CIN after multiple doses of CDDP as effectively as after a single dose. Methods Rats were divided into control, Mg alone, CDDP alone, and CDDP with Mg groups. Rats received CDDP (2.5 mg/kg), MgSO4 (40 mg/kg), or saline once per week for three weeks. Seven days after the third round of treatment, the kidneys were excised, and the expression of renal transporters and renal platinum accumulation were analyzed. Results CDDP significantly elevated serum creatinine levels, which were significantly reduced by Mg co-administration. Renal platinum accumulation was significantly lower in the CDDP-Mg group than in the CDDP group. Expression of renal organic cation transporter 2 (rOct2) and multidrug and toxin extrusion protein 1 (rMate1), which are involved in CDDP transport, did not differ between the groups. However, the expression of copper transporter 1 (rCtr1) was significantly downregulated after Mg co-administration. Conclusion Mg co-administration significantly attenuated CIN by reducing renal platinum accumulation even after multiple rounds of treatment with CDDP as effectively as in a model of a single CDDP administration. However, the specific underlying mechanism was different between single and multiple administrations, further studies will be needed to identify what contributes to this difference and to elucidate how Mg regulates the expression of renal transporters.

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 [14C]creatinine by cultured HEK293 cells expressing OCT2 (HEK293/OCT2) in a concentration-dependent manner with IC50 values of 1.58 ± 0.24 μM. Preincubation or both preincubation and coincubation (preincubation/coincubation) with crizotinib showed stronger inhibitory effect on [14C]creatinine uptake compared with that in coincubation alone with IC50 values of 0.499 ± 0.076 and 0.347 ± 0.040 μM, respectively. These IC50 values of crizotinib on [3H]N-methyl-4-phenylpyridinium acetate uptake by OCT2 were 10-20 times higher than those of [14C]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.

Chronic kidney disease (CKD) is characterized by the accumulation of uremic solutes; however, little is known about how these solutes affect drug absorption and disposition. The goal of this study is to evaluate the effect of uremic solutes on the organic cation transporter, OCT2, which plays a key role in the renal secretion of many basic drugs. As a second goal, we reviewed the literature to determine whether there was evidence for the effect of CKD on the renal secretion of basic drugs. We first screened 72 uremic solutes as inhibitors of [14C]-labeled metformin uptake by OCT2. Seven were identified as inhibitors and 3 of them were determined to be clinically relevant. Of the 7 solutes, dimethylamine, malondialdehyde, trimethylamine, homocysteine, indoxyl-β-d-glucuronide, and glutathione disulfide were novel OCT2 inhibitors. For 6 drugs that are known OCT2 substrates, both secretory clearance and glomerular filtration rate declined in parallel with progression of CKD from stage 2 to 4, suggesting that selective effects of uremic solutes on net tubular secretion of organic cations do not occur. Further clinical studies are warranted with a broader range of OCT2 substrates to determine whether CKD may differentially affect tubular secretion of drugs especially in patients with advanced CKD.

Creatinine is a widely accepted biomarker for renal toxicity, but its renal clearance via transporter-mediated active secretion is significant. For a given new chemical entity, therefore, elevations in serum creatinine (SCr) can be caused by the inhibition of renal transporter(s) without renal toxicity. In the present study, an effort was made to assess the correlation between the inhibition of renal transporters in vitro and elevations in SCr. A total of 15 compounds were chosen based on their known effect on SCr and minimal impact on glomerular filtration rate. Their inhibition potencies against the major creatinine renal transporters, including organic cation transporter 2, organic anion transporter 2, and 2 forms of multidrug and toxin extrusion (MATE1 and MATE2K), were assessed in transporter-transfected cell lines using creatinine as a probe substrate. Collectively, the data suggest that the observed elevations in SCr can be attributed to the inhibition of renal transporter(s), but inhibition of renal transporters does not necessarily lead to elevated SCr. Thus, renal transporter inhibition data can be used to rationalize SCr changes. Additionally, differing renal transporter inhibition potencies using creatinine and metformin as probe substrates suggest that substrate-dependent inhibition exists for some compounds.

ObjectivesTo study the functional consequences of the human and rat forms of OCT2 in the presence of phenothiazines.MethodsMDCK cells expressing human or rat OCT2 were established, and MPP+ transport was determined by uptake assays. Concentration dependency was studied for the stimulatory/inhibitory effects of phenothiazines on MPP+ transport.Key findingsAmong the 11 phenothiazines examined, the majority were found to have comparable effects on transporter function between the orthologous forms, while three phenothiazines, particularly mesoridazine, had complex impacts on transporter function. For rOCT2, mesoridazine stimulated transport at 0.1 and 1 μmMPP+ with the mesoridazine concentration–uptake curve becoming bell-shaped. This conditional effect became less pronounced at 30 μmMPP+, resulting in an inhibition curve with a typical profile. For hOCT2, mesoridazine behaved as a typical inhibitor of transporter function at all MPP+ concentrations, although the kinetics of inhibition were still affected by the substrate concentration.ConclusionsThe conditional stimulation by mesoridazine in rOCT2, and the lack thereof in hOCT2, may be a manifestation of the interaction of phenothiazine with substrate binding at the high-affinity site of the OCT2. As OCT2 was previously indicated in some drug–drug interactions, the conditional stimulation of OCT2 and its potential species-differences may be of practical relevance.

Membrane transporters play a significant role in the transport of many endogenous and exogenous compounds. The knowledge of transporter substrate requirements has allowed further development of drugs that utilize them to ensure tissue permeation. In this study, we demonstrate that inclusion of a biguanide functionality can potentiate uptake by the organic cation transporters 1 and 2 (OCT1 and OCT2). We synthesized 18 pairs of structurally diverse compounds, each pair consisting of a parent amino compound and its biguanide analog; and then assessed their cellular uptake in HEK293 cells overexpressing human OCT1 or OCT2. Our results show that addition of the biguanide significantly improved OCT1- and OCT2- mediated transport for the majority of compounds. The biguanides also inhibited the uptake of prototypical substrates of both transporters, 1-methyl-4-phenylpyridinium (MPP+) and metformin. We found that molecular weight, molecular volume, Log D (pH 7.4), and accessible surface area were important determinants of OCT2 substrates, but none of these parameters was a significant factor for OCT1. More so, the inhibition of MPP+ uptake correlated linearly with that of metformin uptake for the tested biguanides in both cell lines. Taken together, we conclude that the inclusion of the biguanide scaffold in nonsubstrates of OCT1 and OCT2 increase their propensity to become substrates and inhibitors for these transporters.

Previously, we reported that specific lower dose of sodium 2,3-dimercapto-1-propanesulfonic acid (DMPS) which is an antidote to heavy metal intoxication, inversely enhanced cisplatin (CDDP)-induced antitumor activity to S-180 cell-bearing mouse. This activity was only weak with meso-2,3-dimercaptosuccinic acid (DMSA), however. This study investigated the effects of lower doses of DMPS or DMSA on the nephrotoxicity and kinetics of CDDP. Kidney and blood isolated from female mice which received CDDP with or without DMPS or DMSA once daily for 4 days were provided for measuring levels of blood urea nitrogen (BUN) and transporter proteins (OCT2: organic cation transporter; MATE1: multidrug and toxin extrusion) mRNA, and CDDP-originated platinum, and TUNEL staining of renal tubular cells. DMPS or DMSA reduced effectively CDDP-induced BUN, and caused a moderate reduction of platinum in kidney. Additionally, both dimercapto-compounds restored the CDDP-reduced mRNA levels of transporter proteins (OCT2 and MATE1), and apparently suppressed the CDDP-induced apoptosis. These results suggest that DMPS, as well as DMSA, at approximate 17-fold dose (μmol/kg) of CDDP, has an enough potential to reverse the CDDP nephrotoxicity, and concomitant use of DMPS considering both dose and timing for administration is potentially useful for preventing nephrotoxicity and enhancing antitumor activity during CDDP chemotherapy.

Author(s): Taghavi, Taraneh; St Helen, Gideon; Benowitz, Neal L; Tyndale, Rachel F | Abstract: ObjectivesNicotine metabolism rates differ considerably among individuals, even after controlling for variation in the major nicotine-metabolizing enzyme, CYP2A6. In this study, the impact of genetic variation in alternative metabolic enzymes and transporters on nicotine and cotinine (COT) pharmacokinetics and smoking was investigated.MethodsWe examined the impact of UGT2B10, UGT2B17, FMO3, NAT1, and OCT2 variation on pharmacokinetics and smoking (total nicotine equivalents and topography) before and after stratifying by CYP2A6 genotype in 60 African American (AA) smokers who received a simultaneous intravenous infusion of deuterium-labeled nicotine and COT.ResultsVariants in UGT2B10 and UGT2B17 were associated with urinary glucuronidation ratios (glucuronide/free substrate). UGT2B10 rs116294140 was associated with significant alterations in COT and modest alterations in nicotine pharmacokinetics. These alterations, however, were not sufficient to change nicotine intake or topography. Neither UGT2B10 rs61750900, UGT2B17*2, FMO3 rs2266782, nor NAT1 rs13253389 altered nicotine or COT pharmacokinetics among all individuals (n=60) or among individuals with reduced CYP2A6 activity (n=23). The organic cation transporter OCT2 rs316019 significantly increased nicotine and COT Cmax (P=0.005, 0.02, respectively) and decreased nicotine clearance (P=0.05). UGT2B10 rs116294140 had no significant impact on the plasma or urinary trans-3'-hydroxycotinine/COT ratio, commonly used as a biomarker of CYP2A6 activity.ConclusionWe found that polymorphisms in genes other than CYP2A6 represent minor sources of variation in nicotine pharmacokinetics, insufficient to alter smoking in AAs. The change in COT pharmacokinetics with UGT2B10 rs116294140 highlights the UGT2B10 gene as a source of variability in COT as a biomarker of tobacco exposure among AA smokers.