Organic Cation Transporters OCT1 Research Articles

Preclinical assessment of drug-drug interaction of fb-PMT, a novel anti-cancer thyrointegrin αvβ3 antagonist.

The objective of the current study was to identify potential drug-drug interactions (DDIs) with the drug candidate fb-PMT, a novel anticancer thyrointegrin αvß3 antagonist. This was accomplished by using several in vitro assays to study interactions of fb-PMT with both cytochrome P450 (CYP) enzymes and drug transporters, two common mechanisms leading to adverse drug effects. In vitro experiments showed that fb-PMT exhibited weak reversible inhibition of CYP2C19 and CYP3A4. In addition, fb-PMT did not show time-dependent inhibition with any of the seven CYP isoforms tested, including 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4. Human liver microsomal incubations demonstrated that fb-PMT is stable. Potential transporter-mediated DDIs with fb-PMT were assessed with two ATP binding cassette (ABC) family transporters (P-glycoprotein and breast cancer resistance protein) using Caco2 cells and seven solute carrier family (SLC) transporters (organic cation transporter OCT2, organic anion transporters OAT1 and OAT3, organic anion transporter peptides OATP1B1 and OATP1B3, and the multidrug and toxic extrusion proteins MATE1 and MATE2-K using transfected HEK293 cells). Fb-PMT was not a substrate for any of the nine transporters tested in this study, nor did it inhibit the activity of seven of the transporters tested. However, fb-PMT inhibited the uptake of rosuvastatin by both OATP1B1 and OATP1B3 with half-maximal inhibitory concentrations greater than 3 and less than 10μM. In summary, data suggest that the systemic administration of fb-PMT is unlikely to lead to DDIs through CYP enzymes or ABC and SLC transporters in humans.

Determination and disposition of meta-iodobenzylguanidine in plasma and heart of transporter-deficient mice by UPLC-MS/MS

A simple LC-MS/MS method for the quantitative determination of the norepinephrine analogue meta-iodobenzyl-guanidine (mIBG) was developed and validated for mouse plasma and tissues, including salivary gland and heart. The assay procedure involved a one-step solvent extraction of mIBG and the internal standard N-(4-fluorobenzyl)-guandine from plasma or tissue homogenates with acetonitrile. An Accucore aQ column was used to separate analytes using a gradient elution with a total run time of 3.5 min. Validation studies with quality control samples processed on consecutive days revealed values for intra-day and inter-day precision of < 11.3%, with values for accuracy ranging 96.8–111%. Linear responses were observed over the entire calibration curves range (up to 100 ng/mL), and the lower limit of quantification was 0.1 ng/mL, using sample volumes of 5 µL. The developed method was successfully applied to evaluate the plasma pharmacokinetics and tissue distribution of mIBG in wild-type mice and animals lacking the organic cation transporters OCT1, OCT2, OCT3, and/or MATE1 to further understand mechanisms contributing to drug distribution and elimination and causes of inter-individual pharmacokinetic variability.

Organelle-Selective Membrane Labeling through Phospholipase D-Mediated Transphosphatidylation.

The specialized functions of eukaryotic organelles have motivated chemical approaches for their selective tagging and visualization. Here, we develop chemoenzymatic tools using metabolic labeling of abundant membrane lipids for selective visualization of organelle compartments. Synthetic choline analogues with three N-methyl substituents replaced with 2-azidoethyl and additional alkyl groups enabled the generation of corresponding derivatives of phosphatidylcholine (PC), a ubiquitous and abundant membrane phospholipid. Subsequent bioorthogonal tagging via the strain-promoted azide-alkyne cycloaddition (SPAAC) with a single cyclooctyne-fluorophore reagent enabled differential labeling of the endoplasmic reticulum, the Golgi complex, mitochondria, and lysosomes depending upon the substitution pattern at the choline ammonium center. Key to the success of this strategy was the harnessing of both the organic cation transporter OCT1 to enable cytosolic delivery of these cationic metabolic probes and endogenous phospholipase D enzymes for rapid, one-step metabolic conversion of the choline analogues to the desired lipid products. Detailed analysis of the trafficking kinetics of both the SPAAC-tagged fluorescent PC analogues and their non-fluorescent, azide-containing precursors revealed that the latter exhibit time-dependent differences in organelle selectivity, suggesting their use as probes for visualizing intracellular lipid transport pathways. By contrast, the stable localizations of the fluorescent PC analogues will allow applications not only for organelle-selective imaging but also for local modulation of physiological events with organelle-level precision by tethering of bioactive small molecules, via click chemistry, within defined subcellular membrane environments.

Active Pharmaceutical Ingredient Uptake by Zebrafish (Danio rerio) Oct2 (slc22a2) Transporter Expressed in Xenopus laevis Oocytes.

Uptake of active pharmaceutical ingredients (APIs) across the gill epithelium of fish is via either a passive or facilitated transport process, with the latter being more important at the lower concentrations more readily observed in the environment. The solute carrier (SLC) 22A family, which includes the organic cation transporter OCT2 (SLC22A2), has been shown in mammals to transport several endogenous chemicals and APIs. Zebrafish oct2 was expressed in Xenopus oocytes and the uptake of ranitidine, propranolol, and tetraethylammonium characterized. Uptake of ranitidine and propranolol was time- and concentration-dependent with a km and Vmax for ranitidine of 246 µM and 45 pmol/(oocyte × min) and for propranolol of 409 µM and 190 pmol/(oocyte × min), respectively. Uptake of tetraethylammonium (TEA) was inhibited by propranolol, amantadine, and cimetidine, known to be human OCT2 substrates, but not quinidine or ranitidine. At external media pH 7 and 8 propranolol uptake was 100-fold greater than at pH 6; pH did not affect ranitidine or TEA uptake. It is likely that cation uptake is driven by the electrochemical gradient across the oocyte. Uptake kinetics parameters, such as those derived in the present study, coupled with knowledge of transporter localization and abundance and API metabolism, can help derive pharmacokinetic models. Environ Toxicol Chem 2022;41:2993-2998. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

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.

The Function and Surface Expression of the Organic Cation Transporter 1 (OCT1) is Affected by Free Cholesterol

BackgroundThe function of the major renal organic cation transporter OCT2, is affected by plasma membrane cholesterol. OCT1, its liver counterpart, is important for the uptake of cationic drugs such as metformin into human hepatocytes. Fatty liver disease where free cholesterol accumulates in the liver can be a consequence of type 2 diabetes, which is treated with metformin. Thus, understanding how altered membrane cholesterol content impacts the function of OCT1 is important for the disposition of the drugs it transports.AimWe wanted to investigate to what extent the addition and the removal of free cholesterol from the plasma membrane would alter the function and expression of OCT1.MethodsHEK293 Flp‐In cells stably expressing OCT1 were used for all experiments. To add cholesterol, cells were treated with a cholesterol‐MCD complex in serum‐free media for 30 minutes. To remove cholesterol from the membrane cells were incubated for 30 minutes in serum‐free media containing methyl‐b‐cyclodextrin (MCD). Uptake of the radiolabeled model substrate MPP+ (1‐methyl‐4‐phenylpyridinium) was measured at 37°C. Michaelis‐Menten kinetics were determined under initial linear rate conditions (15 second uptake). OCT1 expression at the plasma membrane was determined using a surface biotinylation assay.ResultsTreatment of the cells with increasing concentrations of both cholesterol and MCD resulted in a concentration‐dependent decrease of OCT1 transport. Kinetics of OCT1‐mediated MPP+ uptake following cholesterol treatment demonstrated that the Km values increased from 35 ± 6.7 μM to 73 ± 10.2 μM while the Vmax values were essentially unchanged: 7.5 ± 0.6 pmol/mg x min and 8.4 ± 0.3 pmol/mg x min, respectively. As a consequence, the capacity of OCT1 decreased by 45%. Treatment of cells with MCD slightly decreased the Km value to 26 ± 4.9 μM while the Vmax value stayed the same at 7.7 ± 0.5 pmol/mg x min. With respect to surface expression, treatment with 20 mM MCD treatment decreased surface expression of OCT1 by about 65% while cholesterol treatment resulted in a 30% decrease.Summary and ConclusionBoth the addition and removal of free cholesterol from the plasma membrane impacts OCT1 transport and expression at the plasma membrane. These results suggest that drug disposition will be different in patients with either higher or lower free cholesterol in the liver, which can occur in fatty liver disease or type 2 diabetes.

Molecular basis for stereoselective transport of fenoterol by the organic cation transporters 1 and 2

Stereoselectivity is important in many pharmacological processes but its impact on drug membrane transport is scarcely understood. Recent studies showed strong stereoselective effects in the cellular uptake of fenoterol by the organic cation transporters OCT1 and OCT2. To provide possible molecular explanations, homology models were developed and the putative interactions between fenoterol enantiomers and key residues explored in silico through computational docking, molecular dynamics simulations, and binding free energy calculations as well as in vitro by site-directed mutagenesis and cellular uptake assays. Our results suggest that the observed 1.9-fold higher maximum transport velocity (vmax) for (R,R)- over (S,S)-fenoterol in OCT1 is because the enantiomers bind to two distinct binding sites. Mutating PHE355 and ILE442, predicted to interact with (R,R)-fenoterol, reduced the vmax ratio to 1.5 and 1.3, respectively, and to 1.2 in combination. Mutating THR272, predicted to interact with (S,S)-fenoterol, slightly increased stereoselectivity (vmax ratio of 2.2), while F244A resulted in a 35-fold increase in vmax and a lower affinity (29-fold higher Km) for (S,S)-fenoterol. Both enantiomers of salbutamol, for which almost no stereoselectivity was observed, were predicted to occupy the same binding pocket as (R,R)-fenoterol. Unlike for OCT1, both fenoterol enantiomers bind in the same region in OCT2 but in different conformations. Mutating THR246, predicted to interact with (S,S)-fenoterol in OCT2, led to an 11-fold decreased vmax. Altogether, our mutagenesis results correlate relatively well with our computational predictions and thereby provide an experimentally-corroborated hypothesis for the strong and contrasting enantiopreference in fenoterol uptake by OCT1 and OCT2.

Development, validation, and application of an LC-MS/MS method for the determination of the AXL/FLT3 inhibitor gilteritinib in mouse plasma

A simple, fast and precise LC-MS/MS method for the quantitation of the tyrosine kinase inhibitor gilteritinib was developed and validated for micro-volumes of mouse plasma. The assay procedure involved a one-step extraction of gilteritinib and the internal standard [2H5]-gilteritinib with acetonitrile. An Accucore aQ column was used to separate analytes using a gradient elution delivered at a flow rate of 0.4 mL/min, and a total run time of 2.5 min. Validation studies with quality control samples processed on consecutive days revealed that values for intra-day and inter-day precision were <7.04%, with an accuracy of 101–108%. Linear responses were observed over the entire calibration curve range (up to 500 ng/mL), and the lower limit of quantification was 5 ng/mL. The developed method was successfully used to examine the pharmacokinetics of oral gilteritinib in wild-type mice and mice lacking the organic cation transporters OCT1, OCT2, and MATE1 to further understand mechanisms contributing to drug-drug interactions and causes of inter-individual pharmacokinetic variability.

General Overview of Organic Cation Transporters in Brain.

Inhibitors of Na+/Cl- dependent high affinity transporters for norepinephrine (NE), serotonin (5-HT), and/or dopamine (DA) represent frequently used drugs for treatment of psychological disorders such as depression, anxiety, obsessive-compulsive disorder, attention deficit hyperactivity disorder, and addiction. These transporters remove NE, 5-HT, and/or DA after neuronal excitation from the interstitial space close to the synapses. Thereby they terminate transmission and modulate neuronal behavioral circuits. Therapeutic failure and undesired central nervous system side effects of these drugs have been partially assigned to neurotransmitter removal by low affinity transport. Cloning and functional characterization of the polyspecific organic cation transporters OCT1 (SLC22A1), OCT2 (SLC22A2), OCT3 (SLC22A3) and the plasma membrane monoamine transporter PMAT (SLC29A4) revealed that every single transporter mediates low affinity uptake of NE, 5-HT, and DA. Whereas the organic transporters are all located in the blood brain barrier, OCT2, OCT3, and PMAT are expressed in neurons or in neurons and astrocytes within brain areas that are involved in behavioral regulation. Areas of expression include the dorsal raphe, medullary motoric nuclei, hypothalamic nuclei, and/or the nucleus accumbens. Current knowledge of the transport of monoamine neurotransmitters by the organic cation transporters, their interactions with psychotropic drugs, and their locations in the brain is reported in detail. In addition, animal experiments including behavior tests in wildtype and knockout animals are reported in which the impact of OCT2, OCT3, and/or PMAT on regulation of salt intake, depression, mood control, locomotion, and/or stress effect on addiction is suggested.

Trospium Chloride Transport by Mouse Drug Carriers of the Slc22 and Slc47 Families.

Background: The muscarinic receptor antagonist trospium chloride (TCl) is used for pharmacotherapy of the overactive bladder syndrome. TCl is a hydrophilic positively charged drug. Therefore, it has low permeability through biomembranes and requires drug transporters for distribution and excretion. In humans, the organic cation transporters OCT1 and OCT2 and the multidrug and toxin extrusion MATE1 and MATE2-K carriers showed TCl transport. However, their individual role for distribution and excretion of TCl is unclear. Knockout mouse models lacking mOct1/mOct2 or mMate1 might help to clarify their role for the overall pharmacokinetics of TCl. Method: In preparation of such experiments, TCl transport was analyzed in HEK293 cells stably transfected with the mouse carriers mOct1, mOct2, mMate1, and mMate2, respectively. Results: Mouse mOct1, mOct2, and mMate1 showed significant TCl transport with Km values of 58.7, 78.5, and 29.3 µM, respectively. In contrast, mMate2 did not transport TCl but showed MPP+ transport with Km of 60.0 µM that was inhibited by the drugs topotecan, acyclovir, and levofloxacin. Conclusion: TCl transport behavior as well as expression pattern were quite similar for the mouse carriers mOct1, mOct2, and mMate1 compared to their human counterparts.

Inhibitory effects of vandetanib on creatinine transport via renal organic cation transporter OCT2

Vandetanib (ZD6474, Zactima®, Caprelsa®) is a newly developed dual tyrosine kinase inhibitor of vascular endothelial growth factor and epidermal growth factor receptor. Recently, several reports have indicated the interaction of vandetanib with tyrosine kinase inhibitors and transporters. However, these characteristics of vandetanib remain unclear. We examined the interaction of vandetanib with the human organic cation transporter 2 (hOCT2) stably expressed in human embryonic kidney (HEK) 293 cells. The specific uptake of vandetanib was not observed in hOCT2-expressing HEK293 cells. Vandetanib inhibited the uptake of creatinine mediated by hOCT2 in a dose-dependent manner. The IC50 value for vandetanib inhibition of creatinine uptake by hOCT2 was 3.7 ± 1.0 μM (average ± SE of three separate experiments). The IC50 value of cimetidine and trimethoprim for hOCT2 were 100 ± 13.5 and 52.1 ± 8.0 μM, respectively. Vandetanib showed markedly higher affinity for hOCT2 than cimetidine and trimethoprim. These results suggest that hOCT2 may play a crucial role in elevating the serum creatinine levels, as well as increasing the risk of renal impairment during vandetanib administration.

Transporter tandems: precise tools for normalizing active transporter in the plasma membrane.

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.

Insight into the Anticancer Activity of Copper(II) 5-Methylenetrimethylammonium-Thiosemicarbazonates and Their Interaction with Organic Cation Transporters.

A series of four water-soluble salicylaldehyde thiosemicarbazones with a positively charged trimethylammonium moiety ([H2LR]Cl, R = H, Me, Et, Ph) and four copper(II) complexes [Cu(HLR)Cl]Cl (1–4) were synthesised with the aim to study (i) their antiproliferative activity in cancer cells and, (ii) for the first time for thiosemicarbazones, the interaction with membrane transport proteins, specifically organic cation transporters OCT1–3. The compounds were comprehensively characterised by analytical, spectroscopic and X-ray diffraction methods. The highest cytotoxic effect was observed in the neuroblastoma cell line SH-5YSY after 24 h exposure and follows the rank order: 3 > 2 > 4 > cisplatin > 1 >> [H2LR]Cl. The copper(II) complexes showed marked interaction with OCT1–3, comparable to that of well-known OCT inhibitors (decynium 22, prazosin and corticosterone) in the cell-based radiotracer uptake assays. The work paves the way for the development of more potent and selective anticancer drugs and/or OCT inhibitors.

Targeting OCT2 to ameliorate cisplatin‐induced ototoxicity in zebrafish and mice

BackgroundThe organic cation transporter OCT2 is expressed in renal tubular cells, dorsal root ganglia, and outer hair cells, where it regulates uptake of xenobiotic substrates such as the widely used anticancer drug, cisplatin. We previously reported that various tyrosine kinase inhibitors (TKIs) with YES1‐kinase inhibitory ability, including dasatinib, can potently reduce OCT2 function (Sprowl et al, Nat Commun 2016). Against this background, we here explored the utility of zebrafish (Danio rerio), compared with mice, as in vivo screening tools to identify effective modulators of OCT2‐dependent, debilitating side effect associated with cisplatin treatment, such as ototoxicity (hearing loss).MethodsIn vitro uptake studies were performed with HeLa cells expressing human OCT2, mouse Oct2 or the single zebrafish OCT (zfOct) with high genomic similarity to mammalian OCTs. HEK293T cells were used for transfection of mutated zfOct using different substrates (ASP+, cisplatin) with or without inhibitors. In vivo studies were done in ABxLF zebrafish (5 dpf) and wild‐type or OCT2‐deficient mice on an FVB background. Cisplatin‐induced hearing loss in zebrafish was evaluated by hair cell death in the lateral line after treatment with YO‐PRO1, and rheotaxis movement was captured by video camera. Auditory brainstem responses (8, 16, 32 kHz) were measured to assess hearing loss associated with cisplatin (10 mg/kg, i.p.) in mice with and without dasatinib (15 mg/kg, p.o.) pretreatment.ResultsA comparative in vitro screen of OCT2 and zfOct using the prototypical fluorescent substrate ASP showed that both transporters could be inhibited by the same TKIs, including dasatinib (63.6% vs 66.5%) and nilotinib (53.6% vs 68.8%), with comparable IC50 values. Hela cells expressing OCT2 or zfOct accumulated cisplatin by &gt;2‐fold compared to control cells, and these processes were sensitive to inhibition by the same TKIs (inhibition from 40 to 100% of the uptake). Tyr to Phe mutagenesis in zfOct at previously confirmed YES1‐regulated tyrosine phosphorylation sites in OCT2 was associated with impaired transport function. In addition, silencing YES1 expression with siRNA impaired zfOct function, suggesting that a similar regulatory mechanism exists for both the mammalian and zebrafish transporters. In vivo, neuromasts, consisting of receptive hair cells, accumulated OCT2/zfOct specific substrates such as ASP+, and this process could be inhibited by pretreatment with cisplatin (100 μM, 6‐h pre‐incubation) or dasatinib (10 μM, 6 h). In addition, cisplatin treatment was associated with a disruption of neuromast‐related control of hearing and balance, as determined from a seeker response assay. In mice, pretreatment with dasatinib protected against cisplatin‐induced high frequency hearing loss in a manner similar to that observed in OCT2‐deficient mice.ConclusionThese studies provide new insights into the regulatory mechanism and role of OCT2 in cisplatin‐induced toxicities in zebrafish and mice, and support the further translational exploration of OCT2 inhibitors as a preventative strategy to ameliorate a debilitating toxicity associated with a commonly used chemotherapeutic agent.

Functional Characterization of SLC Transporters Using Solid Supported Membranes.

Here, we present a protocol for the functional characterization of the H+-coupled human peptide transporter PepT1 and sufficient notes to transfer the protocol to the Na+-coupled sugar transporter SGLT1, the organic cation transporter OCT2, the Na+/Ca2+ exchanger NCX, and the neuronal glutamate transporter EAAT3.The assay was developed for the commercially available SURFE2R N1 instrument (Nanion Technologies GmbH) which applies solid supported membrane (SSM)-based electrophysiology. This technique is widely used for the functional characterization of membrane transporters with more than 100 different transporters characterized so far. The technique is cost-effective, easy to use, and capable of high-throughput measurements.SSM-based electrophysiology utilizes SSM-coated gold sensors to physically adsorb membrane vesicles containing the protein of interest. A fast solution exchange provides the substrate and activates transport. For the measurement of PepT1 activity, we applied a peptide concentration jump to activate H+/peptide symport. Proton influx charges the sensor. A capacitive current is measured reflecting the transport activity of PepT1 . Multiple measurements on the same sensor allow for comparison of transport activity under different conditions. Here, we determine EC50 for PepT1-mediated glycylglycine transport and perform an inhibition experiment using the specific peptide inhibitor Lys[Z(NO2)]-Val.

Organic Cation Transporters in Health and Disease.

The organic cation transporters (OCTs) OCT1, OCT2, OCT3, novel OCT (OCTN)1, OCTN2, multidrug and toxin exclusion (MATE)1, and MATE kidney-specific 2 are polyspecific transporters exhibiting broadly overlapping substrate selectivities. They transport organic cations, zwitterions, and some uncharged compounds and operate as facilitated diffusion systems and/or antiporters. OCTs are critically involved in intestinal absorption, hepatic uptake, and renal excretion of hydrophilic drugs. They modulate the distribution of endogenous compounds such as thiamine, L-carnitine, and neurotransmitters. Sites of expression and functions of OCTs have important impact on energy metabolism, pharmacokinetics, and toxicity of drugs, and on drug-drug interactions. In this work, an overview about the human OCTs is presented. Functional properties of human OCTs, including identified substrates and inhibitors of the individual transporters, are described. Sites of expression are compiled, and data on regulation of OCTs are presented. In addition, genetic variations of OCTs are listed, and data on their impact on transport, drug treatment, and diseases are reported. Moreover, recent data are summarized that indicate complex drug-drug interaction at OCTs, such as allosteric high-affinity inhibition of transport and substrate dependence of inhibitor efficacies. A hypothesis about the molecular mechanism of polyspecific substrate recognition by OCTs is presented that is based on functional studies and mutagenesis experiments in OCT1 and OCT2. This hypothesis provides a framework to imagine how observed complex drug-drug interactions at OCTs arise. Finally, preclinical in vitro tests that are performed by pharmaceutical companies to identify interaction of novel drugs with OCTs are discussed. Optimized experimental procedures are proposed that allow a gapless detection of inhibitory and transported drugs.

Stereoselective cell uptake of adrenergic agonists and antagonists by organic cation transporters

Stereoselectivity is well described for receptor binding and enzyme catalysis, but so far has only been scarcely investigated in carrier-mediated membrane transport. We thus studied transport kinetics of racemic (anti)adrenergic drugs by the organic cation transporters OCT1 (wild-type and allelic variants), OCT2, OCT3, MATE1, and MATE2-K with a focus on stereospecificity.OCT1 showed stereoselective uptake with up to 2-fold higher vmax over their corresponding counterpart enantiomers for (R,R)-fenoterol, (R,R)-formoterol, (S)-salbutamol, (S)-acebutolol, and (S)-atenolol. Orciprenaline and etilefrine were also transported stereoselectively. The Km was 2.1-fold and 1.5-fold lower for the (S,S)-enantiomers of fenoterol and formoterol, while no significant difference in Km was seen for the other aforementioned drugs. Common OCT1 variants showed similar enantiopreference to wild-type OCT1, with a few notable exceptions (e.g. a switch in enantiospecificity for fenoterol in OCT1*2 compared to the wild-type). Other cation transporters showed strong differences to OCT1 in stereoselectivity and transport activity: The closely related OCT2 displayed a 20-fold higher vmax for (S,S)-fenoterol compared to (R,R)-fenoterol and OCT2 and OCT3 showed 3.5-fold and 4.6-fold higher vmax for the pharmacologically active (R)-salbutamol over (S)-salbutamol. MATE1 and MATE2-K generally mediated transport with a higher capacity but lower affinity compared to OCT1, with moderate stereoselectivity.Our kinetic studies showed that significant stereoselectivity exists in solute carrier-mediated membrane transport of racemic beta-adrenergic drugs with surprising, and in some instances even opposing, preferences between closely related organic cation transporters. This may be relevant for drug therapy, given the strong involvement of these transporters in hepatic and renal drug elimination.

Highly Variable Pharmacokinetics of Tyramine in Humans and Polymorphisms in OCT1, CYP2D6, and MAO-A.

Tyramine, formed by the decarboxylation of tyrosine, is a natural constituent of numerous food products. As an indirect sympathomimetic, it can have potentially dangerous hypertensive effects. In vitro data indicated that the pharmacokinetics of tyramine possibly depend on the organic cation transporter OCT1 genotype and on the CYP2D6 genotype. Since tyramine is a prototypic substrate of monoamine oxidase A (MAO-A), genetic polymorphisms in MAO-A may also be relevant. The aims of this study were to identify to what extent the interindividual variation in pharmacokinetics and pharmacodynamics of tyramine is determined by genetic polymorphisms in OCT1, CYP2D6, and MAO-A. Beyond that, we wanted to evaluate tyramine as probe drug for the in vivo activity of MAO-A and OCT1. Therefore, the pharmacokinetics, pharmacodynamics, and pharmacogenetics of tyramine were studied in 88 healthy volunteers after oral administration of a 400 mg dose. We observed a strong interindividual variation in systemic tyramine exposure, with a mean AUC of 3.74 min*µg/ml and a high mean CL/F ratio of 107 l/min. On average, as much as 76.8% of the dose was recovered in urine in form of the MAO-catalysed metabolite 4-hydroxyphenylacetic acid (4-HPAA), confirming that oxidative deamination by MAO-A is the quantitatively most relevant metabolic pathway. Systemic exposure of 4-HPAA varied only up to 3-fold, indicating no strong heritable variation in peripheral MAO-A activity. Systolic blood pressure increased by more than 10 mmHg in 71% of the volunteers and correlated strongly with systemic tyramine concentration. In less than 10% of participants, individually variable blood pressure peaks by >40 mmHg above baseline were observed at tyramine concentrations of >60 µg/l. Unexpectedly, the functionally relevant polymorphisms in OCT1 and CYP2D6, including the CYP2D6 poor and ultra-rapid metaboliser genotypes, did not significantly affect tyramine pharmacokinetics or pharmacodynamics. Also, the MOA-A genotypes, which had been associated in several earlier studies with neuropsychiatric phenotypes, had no significant effects on tyramine pharmacokinetics or its metabolism to 4-HPAA. Thus, variation in tyramine pharmacokinetics and pharmacodynamics is not explained by obvious genomic variation, and human tyramine metabolism did not indicate the existence of ultra-low or -high MAO-A activity.

Opioids as Substrates and Inhibitors of the Genetically Highly Variable Organic Cation Transporter OCT1

Genetic variants in the hepatic uptake transporter OCT1, observed in 9% of Europeans and white Americans, are known to affect pharmacokinetics and efficacy of tramadol, morphine, and codeine. Here, we report further opioids to be substrates and inhibitors of OCT1. Methylnaltrexone, hydromorphone, oxymorphone, and meptazinol were identified as OCT1 substrates. Methylnaltrexone is the strongest OCT1 substrate currently reported. It showed 86-fold higher accumulation in OCT1-overexpressing cells compared to control cells. We observed substantial differences in the inhibitory potency among structurally highly similar morphinan opioids (IC50 ranged from 6.4 μM for dextrorphan to 2 mM for oxycodone). The ether linkage of C4-C5 in the morphinan ring leads to a strong reduction of inhibitory potency. In conclusion, although polyspecific, OCT1 possesses a strong selectivity for its ligands. In contrast to methylnaltrexone and hydromorphone, oxycodone and hydrocodone do not interact with OCT1 and may be safer for use in individuals with genetic OCT1 deficiency.

Metformin has a direct effect on ovarian cells that is dependent on organic cation transporters

Metformin (MET) is the most widely prescribed hypoglycemic drug in type 2 diabetes and Polycystic Ovary Syndrome. Besides its effects on glucose metabolism, MET exerts beneficial effects on these patients’ fertility. However, the exact mechanisms of action of MET on female fertility are still unclear. In this work, we analyzed a possible direct effect of MET on ovarian cells. We found expression of the organic cation transporters OCT1, OCT2 and OCT3, responsible for MET uptake into the cells, in rat granulosa cells and human cumulus cells. Furthermore, MET increased pAMPK and decreased VEGF levels both in vivo and in rat granulosa cells in culture. These last effects were reversed when OCTs were inhibited. Our results suggest that MET acts directly on ovarian cells regulating cell metabolism and VEGF expression. Our findings are relevant to optimize PCOS fertility treatment and to explore ovarian MET actions in other female pathologies.