Regulation of solute carriers oct2 and OAT1/3 in the kidney: a phylogenetic, ontogenetic, and cell dynamic perspective.

Over the course of more than 500 million years, the kidneys have undergone a remarkable evolution from primitive nephric tubes to intricate filtration-reabsorption systems that maintain homeostasis and remove metabolic end products from the body. The evolutionarily conserved solute carriers organic cation transporter 2 (OCT2) and organic anion transporters 1 and 3 (OAT1/3) coordinate the active secretion of a broad range of endogenous and exogenous substances, many of which accumulate in the blood of patients with kidney failure despite dialysis. Harnessing OCT2 and OAT1/3 through functional preservation or regeneration could alleviate the progression of kidney disease. Additionally, it would improve current in vitro test models that lose their expression in culture. With this review, we explore OCT2 and OAT1/3 regulation from different perspectives: phylogenetic, ontogenetic, and cell dynamic. Our aim is to identify possible molecular targets both to help prevent or compensate for the loss of transport activity in patients with kidney disease and to enable endogenous OCT2 and OAT1/3 induction in vitro in order to develop better models for drug development.

A novel pathogenic variant in the carnitine transporter gene, SLC22A5, in association with metabolic carnitine deficiency and cardiomyopathy features.

BACKGROUND: Primary carnitine deficiency (PCD) denotes low carnitine levels with an autosomal recessive pattern of inheritance. Cardiomyopathy is the most common cardiac symptom in patients with PCD, and early diagnosis can prevent complications. Next-generation sequencing can identify genetic variants attributable to PCD efficiently. OBJECTIVE: We aimed to detect the genetic cause of the early manifestations of hypertrophic cardiomyopathy and metabolic abnormalities in an Iranian family. METHODS: We herein describe an 8-year-old boy with symptoms of weakness and lethargy diagnosed with PCD through clinical evaluations, lab tests, echocardiography, and cardiac magnetic resonance imaging. The candidate variant was confirmed through whole-exome sequencing, polymerase chain reaction, and direct Sanger sequencing. The binding efficacy of normal and mutant protein-ligand complexes were evaluated via structural modeling and docking studies. RESULTS: Clinical evaluations, echocardiography, and cardiac magnetic resonance imaging findings revealed hypertrophic cardiomyopathy as a clinical presentation of PCD. Whole-exome sequencing identified a new homozygous variant, SLC22A5 (NM_003060.4), c.821G > A: p.Trp274Ter, associated with carnitine transport. Docking analysis highlighted the impact of the variant on carnitine transport, further indicating its potential role in PCD development. CONCLUSIONS: The c.821G > A: p.Trp274Ter variant in SLC22A5 potentially acted as a pathogenic factor by reducing the binding affinity of organic carnitine transporter type 2 proteins for carnitine. So, the c.821G > A variant may be associated with carnitine deficiency, metabolic abnormalities, and cardiomyopathic characteristics.

Modulating the Activity of the Human Organic Cation Transporter 2 Emerges as a Potential Strategy to Mitigate Unwanted Toxicities Associated with Cisplatin Chemotherapy.

Cisplatin (CDDP) stands out as an effective chemotherapeutic agent; however, its application is linked to the development of significant adverse effects, notably nephro- and ototoxicity. The human organic cation transporter 2 (hOCT2), found in abundance in the basolateral membrane domain of renal proximal tubules and the Corti organ, plays a crucial role in the initiation of nephro- and ototoxicity associated with CDDP by facilitating its uptake in kidney and ear cells. Given its limited presence in cancer cells, hOCT2 emerges as a potential druggable target for mitigating unwanted toxicities associated with CDDP. Potential strategies for mitigating CDDP toxicities include competing with the uptake of CDDP by hOCT2 or inhibiting hOCT2 activity through rapid regulation mediated by specific signaling pathways. This study investigated the interaction between the already approved cationic drugs disopyramide, imipramine, and orphenadrine with hOCT2 that is stably expressed in human embryonic kidney cells. Regarding disopyramide, its influence on CDDP cellular transport by hOCT2 was further characterized through inductively coupled plasma isotope dilution mass spectrometry. Additionally, its potential protective effects against cellular toxicity induced by CDDP were assessed using a cytotoxicity test. Given that hOCT2 is typically expressed in the basolateral membrane of polarized cells, with specific regulatory mechanisms, this work studied the regulation of hOCT2 that is stably expressed in Madin-Darby Canine Kidney (MDCK) cells. These cells were cultured in a matrix to induce the formation of cysts, exposing hOCT2 in the basolateral plasma membrane domain, which was freely accessible to experimental solutions. The study specifically tested the regulation of ASP+ uptake by hOCT2 in MDCK cysts through the inhibition of casein kinase II (CKII), calmodulin, or p56lck tyrosine kinase. Furthermore, the impact of this manipulation on the cellular toxicity induced by CDDP was examined using a cytotoxicity test. All three drugs-disopyramide, imipramine, and orphenadrine-demonstrated inhibition of ASP+ uptake, with IC50 values in the micromolar (µM) range. Notably, disopyramide produced a significant reduction in the CDDP cellular toxicity and platinum cellular accumulation when co-incubated with CDDP. The activity of hOCT2 in MDCK cysts experienced a significant down-regulation under inhibition of CKII, calmodulin, or p56lck tyrosine kinase. Interestingly, only the inhibition of p56lck tyrosine kinase demonstrated the capability to protect the cells against CDDP toxicity. In conclusion, certain interventions targeting hOCT2 have demonstrated the ability to reduce CDDP cytotoxicity, at least in vitro. Further investigations in in vivo systems are warranted to ascertain their potential applicability as co-treatments for mitigating undesired toxicities associated with CDDP in patients.

Amino acids in transmembrane helix 1 confer major functional differences between human and mouse orthologs of the polyspecific membrane transporter OCT1.

Organic cation transporter 1 (OCT1) is a membrane transporter that affects hepatic uptake of cationic and weakly basic drugs. OCT1 transports structurally highly diverse substrates. The mechanisms conferring this polyspecificity are unknown. Here, we analyzed differences in transport kinetics between human and mouse OCT1 orthologs to identify amino acids that contribute to the polyspecificity of OCT1. Following stable transfection of HEK293 cells, we observed more than twofold differences in the transport kinetics of 22 out of 28 tested substrates. We found that the ß2-adrenergic drug fenoterol was transported with eightfold higher affinity but at ninefold lower capacity by human OCT1. In contrast, the anticholinergic drug trospium was transported with 11-fold higher affinity but at ninefold lower capacity by mouse Oct1. Using human-mouse chimeric constructs and site-directed mutagenesis, we identified nonconserved amino acids Cys36 and Phe32 as responsible for the species-specific differences in fenoterol and trospium uptake. Substitution of Cys36 (human) to Tyr36 (mouse) caused a reversal of the affinity and capacity of fenoterol but not trospium uptake. Substitution of Phe32 to Leu32 caused reversal of trospium but not fenoterol uptake kinetics. Comparison of the uptake of structurally similar ß2-adrenergics and molecular docking analyses indicated the second phenol ring, 3.3 to 4.8 Å from the protonated amino group, as essential for the affinity for fenoterol conferred by Cys36. This is the first study to report single amino acids as determinants of OCT1 polyspecificity. Our findings suggest that structure-function data of OCT1 is not directly transferrable between substrates or species.

Contribution and Expression of Organic Cation Transporters and Aquaporin Water Channels in Renal Cancer.

The body homeostasis is maintained mainly by the function of the kidneys, which regulate salt and water balance and excretion of metabolism waste products and xenobiotics. This important renal function is determined by the action of many transport systems, which are specifically expressed in the different parts of the nephron, the functional unit of the kidneys. These transport systems are involved, for example, in the reabsorption of sodium, glucose, and other important solutes and peptides from the primary urine. They are also important in the reabsorption of water and thereby production of a concentrated urine. However, several studies have shown the importance of transport systems for different tumor entities. Transport systems, for example, contributed to the proliferation and migration of cancer cells and thereby on tumor progression. They could also serve as drug transporters that could enable drug resistance by outward transport of, for example, chemotherapeutic agents and other drugs. Although many renal transporters have been characterized in detail with respect to the significance for proper kidney function, their role in renal cancer progression is less known. Here, we describe the types of renal cancer and review the studies that analyzed the role of organic cation transporters of the SLC22-family and of the aquaporin water channel family in kidney tumors.

Inhibitory interaction of flavonoids with organic cation transporter 2 and their structure-activity relationships for predicting nephroprotective effects.

Organic cation transporter 2 (OCT2) is mainly responsible for the renal secretion of various cationic drugs, closely associated with drug-induced acute kidney injury (AKI). Screening and identifying potent OCT2 inhibitors with little toxicity in natural products in reducing OCT2-mediated AKI is of great value. Flavonoids are enriched in various vegetables, fruits, and herbal products, and some were reported to produce transporter-mediated drug-drug interactions. This study aimed to screen potential inhibitors of OCT2 from 96 flavonoids, assess the nephroprotective effects on cisplatin-induced kidney injury, and clarify the structure-activity relationships of flavonoids with OCT2. Ten flavonoids exhibited significant inhibition (>50%) on OCT2 in OCT2-HEK293 cells. Among them, the six most potent flavonoid inhibitors, including pectolinarigenin, biochanin A, luteolin, chrysin, 6-hydroxyflavone, and 6-methylflavone markedly decreased cisplatin-induced cytotoxicity. Moreover, in cisplatin-induced renal injury models, they also reduced serum blood urea nitrogen (BUN) and creatinine levels to different degrees, the best of which was 6-methylflavone. The pharmacophore model clarified that the aromatic ring, hydrogen bond acceptors, and hydrogen bond donors might play a vital role in the inhibitory effect of flavonoids on OCT2. Thus, our findings would pave the way to predicting the potential risks of flavonoid-containing food/herb-drug interactions in humans and optimizing flavonoid structure to alleviate OCT2-related AKI.

The Role of Organic Cation Transporters in the Pharmacokinetics, Pharmacodynamics and Drug-Drug Interactions of Tyrosine Kinase Inhibitors.

Tyrosine kinase inhibitors (TKIs) decisively contributed in revolutionizing the therapeutic approach to cancer, offering non-invasive, tolerable therapies for a better quality of life. Nonetheless, degree and duration of the response to TKI therapy vary depending on cancer molecular features, the ability of developing resistance to the drug, on pharmacokinetic alterations caused by germline variants and unwanted drug-drug interactions at the level of membrane transporters and metabolizing enzymes. A great deal of approved TKIs are inhibitors of the organic cation transporters (OCTs). A handful are also substrates of them. These transporters are polyspecific and highly expressed in normal epithelia, particularly the intestine, liver and kidney, and are, hence, arguably relevant sites of TKI interactions with other OCT substrates. Moreover, OCTs are often repressed in cancer cells and might contribute to the resistance of cancer cells to TKIs. This article reviews the OCT interactions with approved and in-development TKIs reported in vitro and in vivo and critically discusses the potential clinical ramifications thereof.

Monoamine oxidase A and organic cation transporter 3 coordinate intracellular ß1AR signaling to calibrate cardiac contractile function.

We have recently identified a pool of intracellular ß1 adrenergic receptors (ß1ARs) at the sarcoplasmic reticulum (SR) crucial for cardiac function. Here, we aim to characterize the integrative control of intracellular catecholamine for subcellular ß1AR signaling and cardiac function. Using anchored Förster resonance energy transfer (FRET) biosensors and transgenic mice, we determined the regulation of compartmentalized ß1AR-PKA signaling at the SR and plasma membrane (PM) microdomains by organic cation transporter 3 (OCT3) and monoamine oxidase A (MAO-A), two critical modulators of catecholamine uptake and homeostasis. Additionally, we examined local PKA substrate phosphorylation and excitation-contraction coupling in cardiomyocyte. Cardiac-specific deletion of MAO-A (MAO-A-CKO) elevates catecholamines and cAMP levels in the myocardium, baseline cardiac function, and adrenergic responses. Both MAO-A deletion and inhibitor (MAOi) selectively enhance the local ß1AR-PKA activity at the SR but not PM, and augment phosphorylation of phospholamban, Ca2+ cycling, and myocyte contractile response. Overexpression of MAO-A suppresses the SR-ß1AR-PKA activity and PKA phosphorylation. However, deletion or inhibition of OCT3 by corticosterone prevents the effects induced by MAOi and MAO-A deletion in cardiomyocytes. Deletion or inhibition of OCT3 also negates the effects of MAOi and MAO-A deficiency in cardiac function and adrenergic responses in vivo. Our data show that MAO-A and OCT3 act in concert to fine-tune the intracellular SR-ß1AR-PKA signaling and cardiac fight-or-flight response. We reveal a drug contraindication between anti-inflammatory corticosterone and anti-depressant MAOi in modulating adrenergic regulation in the heart, providing novel perspectives of these drugs with cardiac implications.

Clinical characteristics of primary carnitine deficiency: A structured review using a case-by-case approach.

A broad spectrum of signs and symptoms has been attributed to primary carnitine deficiency (PCD) since its first description in 1973. Advances in diagnostic procedures have improved diagnostic accuracy and the introduction of PCD in newborn screening (NBS) programs has led to the identification of an increasing number of PCD patients, including mothers of screened newborns, who may show a different phenotype compared to clinically diagnosed patients. To elucidate the spectrum of signs and symptoms in PCD patients, we performed a structured literature review. Using a case-by-case approach, clinical characteristics, diagnostic data, and mode of patient identification were recorded. Signs and symptoms were categorized by organ involvement. In total, 166 articles were included, reporting data on 757 individual patients. In almost 20% (N = 136) of the cases, the diagnosis was based solely on low carnitine concentration which we considered an uncertain diagnosis of PCD. The remaining 621 cases had a diagnosis based on genetic and/or functional (ie, carnitine transporter activity) test results. In these 621 cases, cardiac symptoms (predominantly cardiomyopathy) were the most prevalent (23.8%). Neurological (7.1%), hepatic (8.4%), and metabolic (9.2%) symptoms occurred mainly in early childhood. Adult onset of symptoms occurred in 16 of 194 adult patients, of whom 6 (3.1%) patients suffered a severe event without any preceding symptom (five cardiac events and one coma). In conclusion, symptoms in PCD predominantly develop in early childhood. Most newborns and mothers of newborns detected through NBS remain asymptomatic. However, though rarely, severe complications do occur in both groups.

Role of human organic cation transporter-1 (OCT-1/SLC22A1) in modulating the response to metformin in patients with type 2 diabetes.

BACKGROUND: Organic cation transporter 1 primarily governs the action of metformin in the liver. There are considerable inter-individual variations in metformin response. In light of this, it is crucial to obtain a greater understanding of the influence of OCT1 expression or polymorphism in the context of variable responses elicited by metformin treatment. RESULTS: We observed that the variable response to metformin in the responders and non-responders is independent of isoform variation and mRNA expression of OCT-1. We also observed an insignificant difference in the serum metformin levels of the patient groups. Further, molecular docking provided us with an insight into the hotspot regions of OCT-1 for metformin binding. Genotyping of these regions revealed SNPs 156T>C and 1222A>G in both the groups, while as 181C>T and 1201G>A were found only in non-responders. The 181T>C and 1222A>G changes were further found to alter OCT-1 structure in silico and affect metformin transport in vitro which was illustrated by their effect on the activation of AMPK, the marker for metformin activity. CONCLUSION: Taken together, our results corroborate the role of OCT-1 in the transport of metformin and also point at OCT1 genetic variations possibly affecting the transport of metformin into the cells and hence its subsequent action in responders and non-responders.

Clinical Aspects of Drug-Drug Interaction and Drug Nephrotoxicity at Renal Organic Cation Transporters 2 (OCT2) and Multidrug and Toxin Exclusion 1, and 2-K (MATE1/MATE2-K).

The organic cation transporter 2 (OCT2) belongs to the SLC22 family, while the multidrug and toxin extrusion 1 and 2-K (MATE1/MATE2-K) belong to the SLC47 family, are localized to the basolateral and apical membrane of human renal proximal tubular epithelial cells, respectively. They are polyspecific transporters that enable the transit of structurally diversified drugs with overlapping selectivity across plasma membranes. OCT2 and MATE1/2-K are critically involved in renal secretion, pharmacokinetics (PK), and toxicity of cationic drugs. Drug-drug interactions (DDIs) at OCT2 and/or MATE1/2-K have been shown to result in clinical impacts on PK, therapeutic efficacy and are probably involved in the renal accumulation of drugs. Sites of OCT2 and MATE1/2-K expression and function play an essential role in the pharmacokinetics and toxicity of drugs, such as cisplatin. Thus, knowing the sites (basolateral vs. apical) of the interaction of two drugs at transporters is essential to understanding whether this interaction helps prevent or enhance drug-induced nephrotoxicity. In this work, an overview of OCT2 and MATE1/2-K is presented. Primary structure, membrane location, functional properties, and clinical impact of OCT2 and MATE1/2-K are presented. In addition, clinical aspects of DDIs in OCT2 and MATE1/2-K and their involvement in drug nephrotoxicity are compiled.

Mirogabalin, a novel α2δ ligand, is not a substrate of LAT1, but of PEPT1, PEPT2, OAT1, OAT3, OCT2, MATE1 and MATE2-K.

Mirogabalin is a α2δ ligand as well as pregabalin. The aim of this study was to clarify whether mirogabalin is a substrate of human LAT1, which involved in absorption and disposition of pregabalin, and to investigate transporters involved in renal secretion and absorption of mirogabalin using transporter-expressing cells and fresh human kidney slices.We employed uptake assay of [3H]mirogabalin by HEK293T or HEK293 cells transiently overexpress human OAT1, OAT3, OCT2, LAT1/4F2hc, LAT2/4F2hc, PEPT1, and PEPT2 proteins. Transport assay of MDCKII cells transiently overexpress OCT2/MATE1, and OCT2/MATE2-K proteins was conducted. Contribution of transporters to renal secretion was investigated by uptake assay using human kidney slices.Uptake clearances of [3H]mirogabalin by OAT1-, OAT3-, OCT2-, PEPT1-, and PEPT2-expressing cells were higher than that by vector cells, but by LAT1/4F2hc and LAT2/4F2hc-expressing cells were not. In transport assay using OCT2/MATE1 and OCT2/MATE2-K cells, [3H]mirogabalin showed directional transport from basolateral to apical side. Contribution of OAT1, OAT3, and OCT2 was observed by uptake of [3H]mirogabalin into the kidney slices.These results indicate that mirogabalin is not a substrate of LAT1, but of PEPT1 and PEPT2 involved in absorption and of OAT1, OAT3, OCT2, MATE1 and/or MATE2-K involved in its urinary secretion.

Expression status of p53 and organic cation transporter 1 is correlated with poor response to preoperative chemotherapy in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a highly malignant neoplasm. DNA-damaging drugs, such as cisplatin (CDDP) and 5-fluorouracil (5-FU), are most frequently used in preoperative chemotherapy for ESCC. However, the response to preoperative chemotherapy varies among patients. p53, encoded by TP53, participates in apoptotic pathways following chemotherapy with DNA-damaging drugs, and mutation of TP53 contributes to chemoresistance. Organic cation transporter 1 (OCT1) participates in the uptake of CDDP, and its reduced expression is associated with CDDP resistance. The aim of this study was to evaluate the predictive impact of the expression status of p53 and OCT1 in response to preoperative chemotherapy in ESCC. METHODS: We retrospectively assessed 66 ESCC patients who received preoperative chemotherapy with CDDP/5-FU (CF) or docetaxel/CDDP/5-FU (DCF). p53 and OCT1 expression in pretreatment biopsy specimens was immunohistochemically determined and correlated with histological response to preoperative chemotherapy. RESULTS: p53 with wild-type (p53WT-ex) and mutant-type (p53MT-ex) expression patterns was identified in 40.9% and 59.1% of patients, respectively. High expression of OCT1 (OCT1High) was detected in 45.5%, and the remaining 54.5% showed low expression (OCT1Low). In a univariate analysis of the entire cohort, p53MT-ex was significantly correlated with poor response (P = 0.026), whereas OCT1Low showed marginal significance (P = 0.091). In a combined analysis, tumors with either p53MT-ex or OCT1Low showed a significant correlation with poor response compared with tumors with both p53WT-ex and OCT1High (P < 0.001). The sensitivity, specificity, and accuracy of combined p53/OCT1 were 93.9%, 47.1%, and 81.8%, respectively. Multivariate analysis identified p53 (P = 0.017), OCT1 (P = 0.032), and combined p53/OCT1 (P < 0.001) as independent predictors of histological response. When samples were stratified according to chemotherapy regimen in the univariate analysis, combined p53/OCT1 was the only significant factor for poor response in the CF (P = 0.011) and DCF (P = 0.021) groups, whereas p53 showed no statistical significance. CONCLUSIONS: Our results suggest that either p53MT-ex or OCT1Low expression in pretreatment biopsy specimens may be a potential predictor of poor response to preoperative chemotherapy with the CF-based regimens in ESCC, although the specificity needs to be improved.

Interaction of Masitinib with Organic Cation Transporters.

Tyrosine kinase inhibitors (TKI) such as Masitinib were reported to be useful as therapeutic options in malignant disorders and nonmalignant diseases, like coronavirus disease 2019 (COVID-19). Most kinases must be translocated into targeted cells by the action of specific transport proteins, as they are hydrophilic and not able to cross cell membranes freely. Accordingly, the efficacy of TKI in target cells is closely dependent on the expression of their transporters. Specifically, Masitinib is an organic cation and is expected to interact with organic cation transporters (OCT and Multidrug and Toxin Extrusion proteins-MATE-). The aim of this work was to characterize the interaction of Masitinib with different OCTs. Human embryonic kidney 293 cells stably transfected with murine or human OCT were used for the experiments. The interaction of Masitinib with OCTs was investigated using quenching experiments. The intracellular accumulation of this drug was quantified using high performance liquid chromatography. Our results identified interactions of Masitinib with almost all investigated mouse (m) and human (h) OCTs and hMATE1 and indicated OCT1 and hOCT2 to be especially potent Masitinib translocators across cell membranes. Interestingly, some important differences were observed for the interaction with murine and human OCTs. In the future, investigations concerning further in vitro and in vivo properties of Masitinib and its efficacy related to transporter-related uptake mechanisms under pathophysiological conditions should be performed. Clinical trials in humans and other animals with Masitinib have already shown promising results. However, further research is necessary to understand the disease specific transport mechanisms of Masitinib to contribute to a successful and responsible therapy employment.

Exacerbation of Cisplatin Cellular Toxicity by Regulation of the Human Organic Cation Transporter 2 through Angiotensin II.

Cisplatin (CDDP) is an efficient chemotherapeutic drug, whose use is associated with the development of serious undesired toxicities, such as nephrotoxicity. The human organic cation transporter 2 (hOCT2), which is highly expressed in the basolateral membrane domain of renal proximal tubules seems to play an important role in the development of CDDP nephrotoxicity. The role of angiotensin II (AII) signaling by binding to the AII receptor type 1 (AT1R) in the development and/or progression of CDDP nephrotoxicity is debated. Therefore, in this work, the regulation of hOCT2 activity by AII and its role in the development of CDDP cellular toxicity was investigated. To do this, hOCT2 was overexpressed by viral transduction in Madin-Darby Canine Kidney (MDCK) cells which were cultivated on a filter. This approach allows the separation of an apical and a basolateral membrane domain, which are easily accessible for experimentation. In this system, hOCT2 was mainly localized on the basolateral plasma membrane domain of the cells. The transporter was functional since a specific uptake of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) with an affinity (Km) of 35 µM was only detectable by the addition of ASP+ to the basolateral compartment of hOCT2 expressing MDCK (hOCT2-MDCK) cells. Similarly, CDDP toxicity was evident mainly by CDDP addition to the basolateral compartment of hOCT2-MDCK cells cultivated on a filter. The addition of 1 nM AII stimulated hOCT2 function via PKC activation and worsened CDDP cytotoxicity via binding to AT1R. Therefore, the AII signaling pathway may be implicated in the development and/or progression of CDDP nephrotoxicity. This signaling pathway may be a target for protective interventions for example by blocking AT1R in the kidneys. However, it should be further investigated whether these findings obtained in a cell culture system may have translational relevance for the clinical situation. For toxicity experiments, a 100 µM CDDP concentration was used, which is high but allows us to identify clearly toxic effects due to hOCT2. In summary, down-regulation of hOCT2 activity by the inhibition of the AII signaling pathway may protect against CDDP nephrotoxicity.

Substrates of the Human Brain Proton-Organic Cation Antiporter and Comparison with Organic Cation Transporter 1 Activities.

Many organic cations (OCs) may be transported through membranes by a genetically still uncharacterized proton-organic cation (H + OC) antiporter. Here, we characterized an extended substrate spectrum of this antiporter. We studied the uptake of 72 drugs in hCMEC/D3 cells as a model of the human blood-brain barrier. All 72 drugs were tested with exchange transport assays and the transport of 26 of the drugs was studied in more detail concerning concentration-dependent uptake and susceptibility to specific inhibitors. According to exchange transport assays, 37 (51%) drugs were good substrates of the H + OC antiporter. From 26 drugs characterized in more detail, 23 were consistently identified as substrates of the H + OC antiporter in six different assays and transport kinetic constants could be identified with intrinsic clearances between 0.2 (ephedrine) and 201 (imipramine) mL × minute-1 × g protein-1. Excellent substrates of the H + OC antiporter were no substrates of organic cation transporter OCT1 and vice versa. Good substrates of the H + OC antiporter were more hydrophobic and had a lower topological polar surface area than non-substrates or OCT1 substrates. These data and further research on the H + OC antiporter may result in a better understanding of pharmacokinetics, drug-drug interactions and variations in pharmacokinetics.

Cloning and Functional Characterization of Dog OCT1 and OCT2: Another Step in Exploring Species Differences in Organic Cation Transporters.

OCT1 and OCT2 are polyspecific membrane transporters that are involved in hepatic and renal drug clearance in humans and mice. In this study, we cloned dog OCT1 and OCT2 and compared their function to the human and mouse orthologs. We used liver and kidney RNA to clone dog OCT1 and OCT2. The cloned and the publicly available RNA-Seq sequences differed from the annotated exon-intron structure of OCT1 in the dog genome CanFam3.1. An additional exon between exons 2 and 3 was identified and confirmed by sequencing in six additional dog breeds. Next, dog OCT1 and OCT2 were stably overexpressed in HEK293 cells and the transport kinetics of five drugs were analyzed. We observed strong differences in the transport kinetics between dog and human orthologs. Dog OCT1 transported fenoterol with 12.9-fold higher capacity but 14.3-fold lower affinity (higher KM) than human OCT1. Human OCT1 transported ipratropium with 5.2-fold higher capacity but 8.4-fold lower affinity than dog OCT1. Compared to human OCT2, dog OCT2 showed 10-fold lower transport of fenoterol and butylscopolamine. In conclusion, the functional characterization of dog OCT1 and OCT2 reported here may have implications when using dogs as pre-clinical models as well as for drug therapy in dogs.

Fast and Durable Intraoperative Near-infrared Imaging of Ovarian Cancer Using Ultrabright Squaraine Fluorophores.

The residual tumor after surgery is the most significant prognostic factor of patients with epithelial ovarian cancer. Near-infrared (NIR) fluorescence-guided surgery is actively utilized for tumor localization and complete resection during surgery. However, currently available contrast-enhancing agents display low on-target binding, unfavorable pharmacokinetics, and toxicity, thus not ideal for clinical use. Here we report ultrabright and stable squaraine fluorophores with optimal pharmacokinetics by introducing an asymmetric molecular conformation and surface charges for rapid transporter-mediated cellular uptake. Among the tested, OCTL14 shows low serum binding and rapid distribution into cancer tissue via organic cation transporters (OCTs). Additionally, the charged squaraine fluorophores are retained in lysosomes, providing durable intraoperative imaging in a preclinical murine model of ovarian cancer up to 24 h post-injection. OCTL14 represents a significant departure from the current bioconjugation approach of using a non-targeted fluorophore and would provide surgeons with an indispensable tool to achieve optimal resection.

Rutaecarpine enhances the anti-diabetic activity and hepatic distribution of metformin via up-regulation of Oct1 in diabetic rats.

Diabetes mellitus is a chronic metabolic disorder with multiple complications, patients who receive metformin may have a simultaneous intake of herbal medicine containing rutaecarpine due to cardiovascular protection and hypolipidemic effects of rutaecarpine. There might be drug interactions between metformin and rutaecarpine. This study aimed to investigate the effects of rutaecarpine on the pharmacodynamics and pharmacokinetics of metformin in diabetic rats.The diabetic rat model was induced with high-fat diet and low dose streptozotocin. Metformin with or without rutaecarpine was administered by oral gavage for 42 days. Pharmacodynamics and pharmacokinetics parameters were evaluated.The pharmacodynamics results revealed that co-administration of rutaecarpine with metformin resulted in a remarkable reduction of serum glucose and lipid profiles in diabetic rats compared to metformin treated alone. The pharmacokinetics results showed that co-treatments of rutaecarpine with metformin did not affect the systemic exposure and renal distribution of metformin, but increased metformin concentration in liver. Furthermore, rutaecarpine increased Oct1-mediated metformin uptake into hepatocytes by upregulation of Oct1 expression in the liver.The above data indicate that rutaecarpine enhanced the anti-diabetic effect of metformin, which may be associated with the increased hepatic distribution of metformin through up-regulation of Oct1 in response to rutaecarpine.

Organic Cation Transporters and Nongenomic Glucocorticoid Action.

Corticosteroid hormones exert powerful influences on neuronal physiology and behavior by activating intracellular glucocorticoid receptors (GR) and mineralocorticoid receptors (MR), which act as ligand-gated transcription factors, altering gene expression. In addition to these genomic effects on physiology and behavior, which are usually delayed by minutes to hours, corticosteroid hormones also initiate rapid effects through diverse nongenomic mechanisms. One such mechanism involves the direct inhibition by corticosteroid hormones of monoamine transport mediated by the "uptake2" transporter, organic cation transporter 3 (OCT3), a high-capacity, low-affinity transporter for norepinephrine, epinephrine, dopamine, serotonin, and histamine. In this review we describe studies that demonstrate OCT3 expression and corticosterone-sensitive monoamine transport in the brain and present evidence supporting the hypothesis that corticosterone exerts rapid, nongenomic actions on glia and neurons, ultimately modulating physiology and behavior, by inhibiting OCT3-mediated monoamine clearance. We also describe the corticosteroid sensitivity of the other members of the uptake2 family and examine their potential contributions to nongenomic effects of corticosteroids in the brain.