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.

Effects of a Common Eight Base Pairs Duplication at the Exon 7-Intron 7 Junction on Splicing, Expression, and Function of OCT1.

Organic cation transporter 1 (OCT1, SLC22A1) is localized in the sinusoidal membrane of human hepatocytes and mediates hepatic uptake of weakly basic or cationic drugs and endogenous compounds. Common amino acid substitutions in OCT1 were associated with altered pharmacokinetics and efficacy of drugs like sumatriptan and fenoterol. Recently, the common splice variant rs35854239 has also been suggested to affect OCT1 function. rs35854239 represents an 8 bp duplication of the donor splice site at the exon 7-intron 7 junction. Here we quantified the extent to which this duplication affects OCT1 splicing and, as a consequence, the expression and the function of OCT1. We used pyrosequencing and deep RNA-sequencing to quantify the effect of rs35854239 on splicing after minigene expression of this variant in HepG2 and Huh7 cells and directly in human liver samples. Further, we analyzed the effects of rs35854239 on OCT1 mRNA expression in total, localization and activity of the resulting OCT1 protein, and on the pharmacokinetics of sumatriptan and fenoterol. The 8 bp duplication caused alternative splicing in 38% (deep RNA-sequencing) to 52% (pyrosequencing) of the minigene transcripts when analyzed in HepG2 and Huh7 cells. The alternatively spliced transcript encodes for a truncated protein that after transient transfection in HEK293 cells was not localized in the plasma membrane and was not able to transport the OCT1 model substrate ASP+. In human liver, however, the alternatively spliced OCT1 transcript was detectable only at very low levels (0.3% in heterozygous and 0.6% in homozygous carriers of the 8 bp duplication, deep RNA-sequencing). The 8 bp duplication was associated with a significant reduction of OCT1 expression in the human liver, but explained only 9% of the general variability in OCT1 expression and was not associated with significant changes in the pharmacokinetics of sumatriptan and fenoterol. Therefore, the rs35854239 variant only partially changes splicing, causing moderate changes in OCT1 expression and may be of only limited therapeutic relevance.

Isobutyrylcarnitine as a Biomarker of OCT1 Activity and Interspecies Differences in its Membrane Transport.

Genome-wide association studies have identified an association between isobutyrylcarnitine (IBC) and organic cation transporter 1 (OCT1) genotypes. Higher IBC blood concentrations in humans with active OCT1 genotypes and experimental studies with mouse OCT1 suggested an OCT1-mediated efflux of IBC. In this study, we wanted to confirm the suggested use of IBC as an endogenous biomarker of OCT1 activity and contribute to a better understanding of the mechanisms behind the association between blood concentrations of carnitine derivatives and OCT1 genotype. Blood and urine IBC concentrations were quantified in healthy volunteers regarding intra- and interindividual variation and correlation with OCT1 genotype and with pharmacokinetics of known OCT1 substrates. Furthermore, IBC formation and transport were studied in cell lines overexpressing OCT1 and its naturally occurring variants. Carriers of high-activity OCT1 genotypes had about 3-fold higher IBC blood concentrations and 2-fold higher amounts of IBC excreted in urine compared to deficient OCT1. This was likely due to OCT1 function, as indicated by the fact that IBC correlated with the pharmacokinetics of known OCT1 substrates, like fenoterol, and blood IBC concentrations declined with a 1 h time delay following peak concentrations of the OCT1 substrate sumatriptan. Thus, IBC is a suitable endogenous biomarker reflecting both, human OCT1 (hOCT1) genotype and activity. While murine OCT1 (mOCT1) was an efflux transporter of IBC, hOCT1 exhibited no IBC efflux activity. Inhibition experiments confirmed this data showing that IBC and other acylcarnitines, like butyrylcarnitine, 2-methylbutyrylcarnitine, and hexanoylcarnitine, showed reduced efflux upon inhibition of mOCT1 but not of hOCT1. IBC and other carnitine derivatives are endogenous biomarkers of hOCT1 genotype and phenotype. However, in contrast to mice, the mechanisms underlying the IBC-OCT1 correlation in humans is apparently not directly the OCT1-mediated efflux of IBC. A plausible explanation could be that hOCT1 mediates cellular concentrations of specific regulators or co-substrates in lipid and energy metabolism, which is supported by our in vitro finding that at baseline intracellular IBC concentration is about 6-fold lower alone by OCT1 overexpression.

Differences in Metformin and Thiamine Uptake between Human and Mouse Organic Cation Transporter 1: Structural Determinants and Potential Consequences for Intrahepatic Concentrations.

The most commonly used oral antidiabetic drug, metformin, is a substrate of the hepatic uptake transporter OCT1 (gene name SLC22A1). However, OCT1 deficiency leads to more pronounced reductions of metformin concentrations in mouse than in human liver. Similarly, the effects of OCT1 deficiency on the pharmacokinetics of thiamine were reported to differ between human and mouse. Here, we compared the uptake characteristics of metformin and thiamine between human and mouse OCT1 using stably transfected human embryonic kidney 293 cells. The affinity for metformin was 4.9-fold lower in human than in mouse OCT1, resulting in a 6.5-fold lower intrinsic clearance. Therefore, the estimated liver-to-blood partition coefficient is only 3.34 in human compared with 14.4 in mouse and may contribute to higher intrahepatic concentrations in mice. Similarly, the affinity for thiamine was 9.5-fold lower in human than in mouse OCT1. Using human-mouse chimeric OCT1, we showed that simultaneous substitution of transmembrane helices TMH2 and TMH3 resulted in the reversal of affinity for metformin. Using homology modeling, we suggest several explanations, of which a different interaction of Leu155 (human TMH2) compared with Val156 (mouse TMH2) with residues in TMH3 had the strongest experimental support. In conclusion, the contribution of human OCT1 to the cellular uptake of thiamine and especially of metformin may be much lower than that of mouse OCT1. This may lead to an overestimation of the effects of OCT1 on hepatic concentrations in humans when using mouse as a model. In addition, comparative analyses of human and mouse orthologs may help reveal mechanisms of OCT1 transport. SIGNIFICANCE STATEMENT: OCT1 is a major hepatic uptake transporter of metformin and thiamine, but this study reports strong differences in the affinity for both compounds between human and mouse OCT1. Consequently, intrahepatic metformin concentrations could be much higher in mice than in humans, impacting metformin actions and representing a strong limitation of using rodent animal models for predictions of OCT1-related pharmacokinetics and efficacy in humans. Furthermore, OCT1 transmembrane helices TMH2 and TMH3 were identified to confer the observed species-specific differences in metformin affinity.

Pharmacokinetic Drug-Drug Interactions Between Trospium Chloride and Ranitidine Substrates of Organic Cation Transporters in Healthy Human Subjects.

Trospium chloride, a muscarinic receptor blocker, is poorly absorbed with different rates from areas in the jejunum and the cecum/ascending colon. To evaluate whether organic cation transporter (OCT) 1, OCT2 and multidrug and toxin extrusion (MATE) 1 and MATE2-K are involved in pharmacokinetics, competitions with ranitidine, a probe inhibitor of the cation transporters, were evaluated in transfected HEK293 cells. Furthermore, a drug interaction study with trospium chloride after intravenous (2 mg) and oral dosing (30 mg) plus ranitidine (300 mg) was performed in 12 healthy subjects and evaluated by noncompartmental analysis and population pharmacokinetic modeling. Ranitidine inhibited OCT1, OCT2, MATE1, and MATE2-K with half maximal inhibitory concentration values of 186 ± 25 µM, 482 ± 105 µM, 134 ± 37 µM, and 35 ± 11 µM, respectively. In contrast to our hypothesis, coadministration of ranitidine did not significantly decrease oral absorption of trospium. Instead, renal clearance was lowered by ∼15% (530 ± 99 vs 460 ± 120 mL/min; P < .05). It is possible that ranitidine was not available in competitive concentrations at the major colonic absorption site, as the inhibitor is absorbed in the small intestine and undergoes degradation by microbiota. The renal effects apparently result from inhibition of MATE1 and/or MATE2-K by ranitidine as predicted by in vitro to in vivo extrapolation. However, all pharmacokinetic changes were not of clinical relevance for the drug with highly variable pharmacokinetics. Intravenous trospium significantly lowered mean absorption time and relative bioavailability of ranitidine, which was most likely caused by muscarinic receptor blocking effects on intestinal motility and water turnover.

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.

Assay Conditions Influence Affinities of Rat Organic Cation Transporter 1: Analysis of Mutagenesis in the Modeled Outward-Facing Cleft by Measuring Effects of Substrates and Inhibitors on Initial Uptake.

The effects of mutations in the modeled outward-open cleft of rat organic cation transporter 1 (rOCT1) on affinities of substrates and inhibitors were investigated. Human embryonic kidney 293 cells were stably transfected with rOCT1 or rOCT1 mutants, and uptake of the substrates 1-methyl-4-phenylpyridinium+ (MPP+) and tetraethylammonium+ (TEA+) or inhibition of MPP+ uptake by the nontransported inhibitors tetrabutylammonium+ (TBuA+), tetrapentylammonium+ (TPeA+), and corticosterone was measured. Uptake measurements were performed on confluent cell layers using a 2-minute incubation or in dissociated cells using incubation times of 1, 5, or 10 seconds. With both methods, different apparent Michaelis-Menten constant (Km) values, different IC50 values, and varying effects of mutations were determined. In addition, varying IC50 values for the inhibition of MPP+ uptake and varying effects of mutations were obtained when different MPP+ concentrations far below the apparent Km value were used for uptake measurements. Eleven mutations were investigated by measuring initial uptake in dissociated cells and employing 0.1 µM MPP+ for uptake during inhibition experiments. Altered affinities for substrates and/or inhibitors were observed when Phe160, Trp218, Arg440, Leu447, and Asp475 were mutated. The mutations resulted in changes of apparent Km values for TEA+ and/or MPP+ Mutation of Trp218 and Asp475 led to altered IC50 values for TBuA+, TPeA+, and corticosterone, whereas the mutation of Phe160 and Leu447 changed the IC50 values for two inhibitors. Thereby amino acids in the outward-facing conformation of rOCT1 could be identified that interact with structurally different inhibitors and probably also with different substrates.