The OCT2/MATE1 Interaction Between Trifluridine, Metformin and Cimetidine: A Crossover Pharmacokinetic Study.

BACKGROUND AND OBJECTIVES: Trifluridine/tipiracil, registered for the treatment of patients with metastatic gastric and colorectal cancer, is a substrate and inhibitor for the organic cation transporter 2 (OCT2) and the multidrug and toxin extrusion protein 1 (MATE1), which raises the potential for drug-drug interactions with other OCT2/MATE1 modulators. Therefore, we prospectively examined the effect of an OCT2/MATE1 inhibitor (cimetidine) and substrate (metformin) on the pharmacokinetics of trifluridine. METHODS: In this three-phase crossover study, patients with metastatic colorectal or gastric cancer were sequentially treated with trifluridine/tipiracil alone (phase A), trifluridine/tipiracil concomitant with metformin (phase B) and trifluridine/tipiracil concomitant with cimetidine (phase C). The primary endpoint was the relative difference in exposure of trifluridine assessed by the area under the curve from timepoint zero to infinity. A > 30% change in exposure was considered clinically relevant. A p-value of < 0.025 was considered significant because of a Bonferroni correction. RESULTS: Eighteen patients were included in the analysis. Metformin did not significantly alter the exposure to trifluridine (- 12.6%; 97.5% confidence interval - 25.0, 1.8; p = 0.045). Cimetidine did alter the exposure to trifluridine significantly (+ 18.0%; 97.5% confidence interval 4.5, 33.3; p = 0.004), but this increase did not meet our threshold for clinical relevance. Metformin trough concentrations were not influenced by trifluridine/tipiracil. CONCLUSIONS: Our result suggests that the OCT2/MATE1 modulators cimetidine and metformin can be co-administered with trifluridine/tipiracil without clinically relevant effects on drug exposure. CLINICAL TRIAL REGISTRATION: NL8067 (registered 04-10-2019).

No dose adjustment of metformin or substrates of organic cation transporters (OCT)1 and OCT2 and multidrug and toxin extrusion protein (MATE)1/2K with fostemsavir coadministration based on modeling approaches.

Fostemsavir is an approved gp120-directed attachment inhibitor and prodrug for the treatment of human immunodeficiency virus type 1 infection in combination with other antiretrovirals (ARVs) in heavily treatment-experienced adults with multi-drug resistance, intolerance, or safety concerns with their current ARV regimen. Initial in vitro studies indicated that temsavir, the active moiety of fostemsavir, and its metabolites, inhibited organic cation transporter (OCT)1, OCT2, and multidrug and toxin extrusion transporters (MATEs) at tested concentration of 100 uM, although risk assessment based on the current Food and Drug Administration in vitro drug-drug interaction (DDI) guidance using the mechanistic static model did not reveal any clinically relevant inhibition on OCTs and MATEs. However, a DDI risk was flagged with EMA static model predictions. Hence, a physiologically based pharmacokinetic (PBPK) model of fostemsavir/temsavir was developed to further assess the DDI risk potential of OCT and MATEs inhibition by temsavir and predict changes in metformin (a sensitive OCT and MATEs substrate) exposure. No clinically relevant impact on metformin concentrations across a wide range of temsavir concentrations was predicted; therefore, no dose adjustment is recommended for metformin when co-administered with fostemsavir.

Ergothioneine and mitochondria: An important protective mechanism?

Mitochondrial dysfunction is implicated in a wide range of human disorders including many neurodegenerative and cardiovascular diseases, metabolic diseases, cancers, and respiratory disorders. Studies have suggested the potential of l-ergothioneine (ET), a unique dietary thione, to prevent mitochondrial damage and improve disease outcome. Despite this, no studies have definitively demonstrated uptake of ET into mitochondria. Moreover, the expression of the known ET transporter, OCTN1, on the mitochondria remains controversial. In this study, we utilise mass spectrometry to demonstrate direct ET uptake in isolated mitochondria as well as its presence in mitochondria isolated from ET-treated cells and animals. Mitochondria isolated from OCTN1 knockout mice tissues, have impaired but still detectable ET uptake, raising the possibility of alternative transporter(s) which may facilitate ET uptake into the mitochondria. Our data confirm that ET can enter mitochondria, providing a basis for further work on ET in the prevention of mitochondrial dysfunction in human disease.

[Uric Acid Metabolism, Uric Acid Transporters and Dysuricemia].

Serum urate levels are determined by the balance between uric acid production and uric acid excretion capacity from the kidneys and intestinal tract. Dysuricemia, including hyperuricemia and hypouricemia, develops when the balance shifts towards an increase or a decrease in the uric acid pool. Hyperuricemia is mostly a multifactorial genetic disorder involving several disease susceptibility genes and environmental factors. Hypouricemia, on the other hand, is caused by genetic abnormalities. The main genes involved in dysuricemia are xanthine oxidoreductase, an enzyme that produces uric acid, and the urate transporters urate transporter 1/solute carrier family 22 member 12 (URAT1/SLC22A12), glucose transporter 9/solute carrier family 2 member 9 (GLUT9/SLC2A9) and ATP binding cassette subfamily G member 2 (ABCG2). Deficiency of xanthine oxidoreductase results in xanthinuria, a rare disease with marked hypouricemia. Xanthinuria can be due to a single deficiency of xanthine oxidoreductase or in combination with aldehyde oxidase deficiency as well. The latter is caused by a deficiency in molybdenum cofactor sulfurase, which is responsible for adding sulphur atoms to the molybdenum cofactor required for xanthine oxidoreductase and aldehyde oxidase to exert their action. URAT1/SLC22A12 and GLUT9/SLC2A9 are involved in urate reabsorption and their deficiency leads to renal hypouricemia, a condition that is common in Japanese due to URAT1/SLC22A12 deficiency. On the other hand, ABCG2 is involved in the secretion of urate, and many Japanese have single nucleotide polymorphisms that result in its reduced function, leading to hyperuricemia. In particular, severe dysfunction of ABCG2 leads to hyperuricemia with reduced extrarenal excretion.

Characterization of Organic Anion and Cation Transport in Three Human Renal Proximal Tubular Epithelial Models.

The polarised expression of specific transporters in proximal tubular epithelial cells is important for the renal clearance of many endogenous and exogenous compounds. Thus, ideally, the in vitro tools utilised for predictions would have a similar expression of apical and basolateral xenobiotic transporters as in vivo. Here, we assessed the functionality of organic cation and anion transporters in proximal tubular-like cells (PTL) differentiated from human induced pluripotent stem cells (iPSC), primary human proximal tubular epithelial cells (PTEC), and telomerase-immortalised human renal proximal tubular epithelial cells (RPTEC/TERT1). Organic cation and anion transport were studied using the fluorescent substrates 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP) and 6-carboxyfluorescein (6-CF), respectively. The level and rate of intracellular ASP accumulation in PTL following basolateral application were slightly lower but within a 3-fold range compared to primary PTEC and RPTEC/TERT1 cells. The basolateral uptake of ASP and its subsequent apical efflux could be inhibited by basolateral exposure to quinidine in all models. Of the three models, only PTL showed a modest preferential basolateral-to-apical 6-CF transfer. These results show that organic cation transport could be demonstrated in all three models, but more research is needed to improve and optimise organic anion transporter expression and functionality.

Atomistic description of the OCTN1 recognition mechanism via in silico methods.

The Organic Cation Transporter Novel 1 (OCTN1), also known as SLC22A4, is widely expressed in various human tissues, and involved in numerous physiological and pathological processes remains. It facilitates the transport of organic cations, zwitterions, with selectivity for positively charged solutes. Ergothioneine, an antioxidant compound, and acetylcholine (Ach) are among its substrates. Given the lack of experimentally solved structures of this protein, this study aimed at generating a reliable 3D model of OCTN1 to shed light on its substrate-binding preferences and the role of sodium in substrate recognition and transport. A chimeric model was built by grafting the large extracellular loop 1 (EL1) from an AlphaFold-generated model onto a homology model. Molecular dynamics simulations revealed domain-specific mobility, with EL1 exhibiting the highest impact on overall stability. Molecular docking simulations identified cytarabine and verapamil as highest affinity ligands, consistent with their known inhibitory effects on OCTN1. Furthermore, MM/GBSA analysis allowed the categorization of substrates into weak, good, and strong binders, with molecular weight strongly correlating with binding affinity to the recognition site. Key recognition residues, including Tyr211, Glu381, and Arg469, were identified through interaction analysis. Ach demonstrated a low interaction energy, supporting the hypothesis of its one-directional transport towards to outside of the membrane. Regarding the role of sodium, our model suggested the involvement of Glu381 in sodium binding. Molecular dynamics simulations of systems at increasing levels of Na+ concentrations revealed increased sodium occupancy around Glu381, supporting experimental data associating Na+ concentration to molecule transport. In conclusion, this study provides valuable insights into the 3D structure of OCTN1, its substrate-binding preferences, and the role of sodium in the recognition. These findings contribute to the understanding of OCTN1 involvement in various physiological and pathological processes and may have implications for drug development and disease management.

Illuminating the function of the orphan transporter, SLC22A10, in humans and other primates.

SLC22A10 is an orphan transporter with unknown substrates and function. The goal of this study is to elucidate its substrate specificity and functional characteristics. In contrast to orthologs from great apes, human SLC22A10, tagged with green fluorescent protein, is not expressed on the plasma membrane. Cells expressing great ape SLC22A10 orthologs exhibit significant accumulation of estradiol-17ß-glucuronide, unlike those expressing human SLC22A10. Sequence alignments reveal a proline at position 220 in humans, which is a leucine in great apes. Replacing proline with leucine in SLC22A10-P220L restores plasma membrane localization and uptake function. Neanderthal and Denisovan genomes show proline at position 220, akin to modern humans, indicating functional loss during hominin evolution. Human SLC22A10 is a unitary pseudogene due to a fixed missense mutation, P220, while in great apes, its orthologs transport sex steroid conjugates. Characterizing SLC22A10 across species sheds light on its biological role, influencing organism development and steroid homeostasis.

SLC22A17 as a Cell Death-Linked Regulator of Tight Junctions in Cerebral Ischemia.

BACKGROUND: Beyond neuronal injury, cell death pathways may also contribute to vascular injury after stroke. We examined protein networks linked to major cell death pathways and identified SLC22A17 (solute carrier family 22 member 17) as a novel mediator that regulates endothelial tight junctions after ischemia and inflammatory stress. METHODS: Protein-protein interactions and brain enrichment analyses were performed using STRING, Cytoscape, and a human tissue-specific expression RNA-seq database. In vivo experiments were performed using mouse models of transient focal cerebral ischemia. Human stroke brain tissues were used to detect SLC22A17 by immunostaining. In vitro experiments were performed using human brain endothelial cultures subjected to inflammatory stress. Immunostaining and Western blot were used to assess responses in SLC22A17 and endothelial tight junctional proteins. Water content, dextran permeability, and electrical resistance assays were used to assess edema and blood-brain barrier (BBB) integrity. Gain and loss-of-function studies were performed using lentiviral overexpression of SLC22A17 or short interfering RNA against SLC22A17, respectively. RESULTS: Protein-protein interaction analysis showed that core proteins from apoptosis, necroptosis, ferroptosis, and autophagy cell death pathways were closely linked. Among the 20 proteins identified in the network, the iron-handling solute carrier SLC22A17 emerged as the mediator enriched in the brain. After cerebral ischemia in vivo, endothelial expression of SLC22A17 increases in both human and mouse brains along with BBB leakage. In human brain endothelial cultures, short interfering RNA against SLC22A17 prevents TNF-α (tumor necrosis factor alpha)-induced ferroptosis and downregulation in tight junction proteins and disruption in transcellular permeability. Notably, SLC22A17 could repress the transcription of tight junctional genes. Finally, short interfering RNA against SLC22A17 ameliorates BBB leakage in a mouse model of focal cerebral ischemia. CONCLUSIONS: Using a combination of cell culture, human stroke samples, and mouse models, our data suggest that SLC22A17 may play a role in the control of BBB function after cerebral ischemia. These findings may offer a novel mechanism and target for ameliorating BBB injury and edema after stroke.

How PBPK Can Help to Understand Old Drugs and Inform their Dosing in Elderly: Amantadine Case Study.

Amantadine, despite being on the market for 55 years, has several unknown aspects of its pharmacokinetics especially related to the influence of covariates such as age, disease, or interactions linked to amantadine's renal elimination. As amantadine is used in Parkinson's disease and is considered a potential candidate in COVID treatment and other diseases, there is an unmet need for thorough understanding of its pharmacokinetic in special populations, such as the elderly. We aimed to mechanistically describe amantadine pharmacokinetics in healthy subjects and shed some light on the differences in drug behavior between healthy volunteers (18-65 years) and an elderly/geriatric population (65-98 years) using PBPK modeling and simulation. The middle-out PBPK model includes mechanistic description of drug renal elimination, specifically an organic cation transporter (OCT)2-mediated electrogenic bidirectional transport (basolateral) and multidrug and toxic compound extrusion (MATE)1-mediated efflux (apical). The model performance was verified against plasma and urine data reported after single and multiple dose administration in healthy volunteers and elderly patients from 18 independent studies. The ratios of predicted vs. observed maximal plasma concentration and area under the concentration-time curve values were within 1.25-fold. The model illustrates that renal transporter activity is expected to decrease in healthy elderly compared to healthy volunteers, which is in line with literature proteomic data for OCT2. The model was applied to assess the potential of reaching toxicity-related plasma concentrations in different age groups of geriatric subjects.

Inflammation and Organic Cation Transporters Novel (OCTNs).

Inflammation is a physiological condition characterized by a complex interplay between different cells handled by metabolites and specific inflammatory-related molecules. In some pathological situations, inflammation persists underlying and worsening the pathological state. Over the years, two membrane transporters namely OCTN1 (SLC22A4) and OCTN2 (SLC22A5) have been shown to play specific roles in inflammation. These transporters form the OCTN subfamily within the larger SLC22 family. The link between these proteins and inflammation has been proposed based on their link to some chronic inflammatory diseases such as asthma, Crohn's disease (CD), and rheumatoid arthritis (RA). Moreover, the two transporters show the ability to mediate the transport of several compounds including carnitine, carnitine derivatives, acetylcholine, ergothioneine, and gut microbiota by-products, which have been specifically associated with inflammation for their anti- or proinflammatory action. Therefore, the absorption and distribution of these molecules rely on the presence of OCTN1 and OCTN2, whose expression is modulated by inflammatory cytokines and transcription factors typically activated by inflammation. In the present review, we wish to provide a state of the art on OCTN1 and OCTN2 transport function and regulation in relationships with inflammation and inflammatory diseases focusing on the metabolic signature collected in different body districts and gene polymorphisms related to inflammatory diseases.

Unique Binding Sites of Uricosuric Agent Dotinurad for Selective Inhibition of Renal Uric Acid Reabsorptive Transporter URAT1.

Dotinurad was developed as a uricosuric agent, inhibiting urate (UA) reabsorption through the UA transporter URAT1 in the kidneys. Due to its high selectivity for URAT1 among renal UA transporters, we investigated the mechanism underlying this selectivity by identifying dotinurad binding sites specific to URAT1. Dotinurad was docked to URAT1 using AutoDock4, utilizing the AlphaFold2-predicted structure. The inhibitory effects of dotinurad on wild-type and mutated URAT1 at the predicted binding sites were assessed through URAT1-mediated [14C]UA uptake in Xenopus oocytes. Nine amino acid residues in URAT1 were identified as dotinurad-binding sites. Sequence alignment with UA-transporting organic anion transporters (OATs) revealed that H142 and R487 were unique to URAT1 among renal UA-transporting OATs. For H142, IC50 values of dotinurad increased to 62, 55, and 76 nM for mutated URAT1 (H142A, H142E, and H142R, respectively) compared with 19 nM for the wild type, indicating that H142 contributes to URAT1-selective interaction with dotinurad. H142 was predicted to interact with the phenyl-hydroxyl group of dotinurad. The IC50 of the hydroxyl group methylated dotinurad (F13141) was 165 µM, 8420-fold higher than dotinurad, suggesting the interaction of H142 and the phenyl-hydroxyl group by forming a hydrogen bond. Regarding R487, URAT1-R487A exhibited a loss of activity. Interestingly, the URAT1-H142A/R487A double mutant restored UA transport activity, with the IC50 value of dotinurad for the mutant (388 nM) significantly higher than that for H142A (73.5 nM). These results demonstrate that H142 and R487 of URAT1 determine its selectivity for dotinurad, a uniqueness observed only in URAT1 among UA-transporting OATs. SIGNIFICANCE STATEMENT: Dotinurad selectively inhibits the urate reabsorption transporter URAT1 in renal urate-transporting organic ion transporters (OATs). This study demonstrates that dotinurad interacts with H142 and R487 of URAT1, located in the extracellular domain and unique among OATs when aligning amino acid sequences. Mutations in these residues reduce affinity of dotinurad for URAT1, confirming their role in conferring selective inhibition. Additionally, the interaction between dotinurad and URAT1 involving H142 is found to mediate hydrogen bonding.

Targeted mutagenesis of negatively charged amino acids outlining the substrate translocation path within the human organic cation transporter 3.

Recently published cryo-EM structures of human organic cation transporters of the SLC22 family revealed seven, sequentially arranged glutamic and aspartic acid residues, which may be relevant for interactions with positively charged substrates. We analyzed the functional consequences of removing those negative charges by creating D155N, E232Q, D382N, E390Q, E451Q, E459Q, and D478N mutants of OCT3. E232Q, E459Q, and D478N resulted in a lack of localization in the outer cell membrane and no relevant uptake activity. However, D155N and E451Q showed a substrate-specific loss of transport activity, whereas E390Q had no remaining activity despite correct membrane localization. In contrast, D382N showed almost wild-type-like uptake. D155 is located at the entrance to the substrate binding pocket and could, therefore be involved in guiding cationic substrates towards the inside of the binding pocket. For E390, we confirm its critical function for transporter function as it was recently shown for the corresponding position in OCT1. Interestingly, E451 seems to be located at the bottom of the binding pocket in the outward-open confirmation of the transporter. Substrate-specific loss of transport activity of the E451Q variant suggests an essential role in the transport cycle of specific substances as part of an opportunistic binding site. In general, our study highlights the impact of the cryo-EM structures in guiding mutagenesis studies to understand the molecular level of transporter-ligand interactions, and it also confirms the importance of testing multiple substrates in mutagenesis studies of polyspecific OCTs.

Discovery of digallic acid as XOD/URAT1 dual target inhibitor for the treatment of hyperuricemia.

The development of XOD/URAT1 dual target inhibitors has emerged as a promising therapeutic strategy for the management of hyperuricemia. Here, through virtual screening, we have identified digallic acid as a novel dual target inhibitor of XOD/URAT1 and subsequently evaluated its pharmacological properties, pharmacokinetics, and toxicities. Digallic acid inhibited URAT1 with an IC50 of 5.34 ± 0.65 µM, which is less potent than benzbromarone (2.01 ± 0.36 µM) but more potent than lesinurad (10.36 ± 1.23 µM). Docking and mutation analysis indicated that residues S35, F241 and R477 of URAT1 confer a high affinity for digallic acid. Digallic acid inhibited XOD with an IC50 of 1.04 ± 0.23 µM. Its metabolic product, gallic acid, inhibited XOD with an IC50 of 0.91 ± 0.14 µM. Enzyme kinetic studies indicated that both digallic acid and gallic acid act as mixed-type XOD inhibitors. It shares the same binding mode as digallic acid, and residues E802, R880, F914, T1010, N768 and F1009 contribute to their high affinity. The anion group (carboxyl) of digallic acid contribute significantly to its inhibition activity on both XOD and URAT1 as indicated by docking analysis. Remarkably, at a dosage of 10 mg/kg in vivo, digallic acid exhibited a stronger urate-lowering and uricosuric effect compared to the positive drug benzbromarone and lesinurad. Pharmacokinetic study indicated that digallic acid can be hydrolyzed into gallic acid in vivo and has a t1/2 of 0.77 ± 0.10 h. Further toxicity evaluation indicated that digallic acid exhibited no obvious renal toxicity, as reflected by CCK-8, biochemical analysis (CR and BUN) and HE examination. The findings of our study can provide valuable insights for the development of XOD/URAT1 dual target inhibitors, and digallic acid deserves further investigation as a potential anti-hyperuricemic drug.

Overexpression of Slc22a18 facilitates fat accumulation in mice.

We previously reported that solute carrier family 22 member 18 (Slc22a18) regulates lipid accumulation in 3T3-L1 adipocytes. Here, we provide additional evidence derived from experiments with adenoviral vector expression and genetic manipulation of mice. In primary cultured rat hepatocytes, adenoviral overexpression of mouse Slc22a18 increased triglyceride accumulation and triglyceride synthetic activity, which was decreased in an adenoviral knockdown experiment. Adenoviral overexpression of mouse Slc22a18 in vivo caused massive fatty liver in mice, even under normal dietary conditions. Conversely, adenoviral knockdown of mouse Slc22a18 reduced hepatic lipid accumulation induced by a high-glucose and high-sucrose diet. We created Slc22a18 knockout mice, which grew normally and showed no obvious spontaneous phenotypes. However, compared with control littermates, the knockout mice exhibited decreased hepatic triglyceride content under refeeding conditions, significantly reduced epididymal fat mass, and tended to have lower liver weight in conjunction with leptin deficiency. Finally, we created transgenic mice overexpressing rat Slc22a18 in an adipose-specific manner, which had increased body weight and epididymal fat mass primarily because of increased adipocyte cell volume. In these transgenic mice, a positive correlation was observed between adiposity and the expression levels of the rat Slc22a18 transgene. Taken together, these results indicate that Slc22a18 has positive effects on lipid accumulation in vivo.

Design, synthesis, and biological evaluation of 3-phenyl substituted pyridine derivatives as potential dual inhibitors of XOR and URAT1.

Xanthine oxidoreductase (XOR) and uric acid transporter 1 (URAT1) are two most widely studied targets involved in production and reabsorption of uric acid, respectively. Marketed drugs almost target XOR or URAT1, but sometimes, single agents might not achieve aim of lowering uric acid to ideal value in clinic. Thus, therapeutic strategies of combining XOR inhibitors with uricosuric drugs were proposed and implemented. Based on our initial work of virtual screening, A and B were potential hits for dual-targeted inhibitors on XOR/URAT1. By docking A/B with XOR/URAT1 respectively, compounds I1-7 were designed to get different degree of inhibition effect on XOR and URAT1, and I7 showed the best inhibitory effect on XOR (IC50 = 0.037 ± 0.001 µM) and URAT1 (IC50 = 546.70 ± 32.60 µM). Further docking research on I7 with XOR/URAT1 led to the design of compounds II with the significantly improved inhibitory activity on XOR and URAT1, such as II11 and II15. Especially, for II15, the IC50 of XOR is 0.006 ± 0.000 µM, superior to that of febuxostat (IC50 = 0.008 ± 0.000 µM), IC50 of URAT1 is 12.90 ± 2.30 µM, superior to that of benzbromarone (IC50 = 27.04 ± 2.55 µM). In acute hyperuricemia mouse model, II15 showed significant uric acid lowering effect. The results suggest that II15 had good inhibitory effect on XOR/URAT1, with the possibility for further investigation in in-vivo models of hyperuricemia.

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.

Identification and characterization of an endogenous biomarker of the renal vectorial transport (OCT2-MATE1).

The renal tubular organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1) mediate the vectorial elimination of many drugs and toxins from the kidney, and endogenous biomarkers for vectorial transport (OCT2-MATE1) would allow more accurate drug dosing and help to characterize drug-drug interactions and toxicity. Human serum uptake in OCT2-overexpressing cells and metabolomics analysis were carried out. Potential biomarkers were verified in vitro and in vivo. The specificity of biomarkers was validated in renal transporter overexpressing cells and the sensitivity was investigated by Km . The results showed that the uptake of thiamine, histamine, and 5-hydroxytryptamine was significantly increased in OCT2-overexpressing cells. In vitro assays confirmed that thiamine, histamine, and 5-hydroxytryptamine were substrates of both OCT2 and MATE1. In vivo measurements indicated that the serum thiamine level was increased significantly in the presence of the rOCT2 inhibitor cimetidine, and the level in renal tissue was increased significantly by the rMATE1 inhibitor pyrimethamine. There were no significant changes in the uptake or efflux of thiamine in cell lines overexpressed OAT1, OAT2, OAT3, MRP4, organic anion transporting polypeptide 4C1, P-gp, peptide transporter 2, urate transporter 1, and OAT4. The Km for thiamine with OCT2 and MATE1 were 71.2 and 10.8 µM, respectively. In addition, the cumulative excretion of thiamine at 2 and 4 h was strongly correlated with metformin excretion (R2  > 0.6). Thus, thiamine is preferentially secreted by the OCT2 and MATE1 in renal tubules and can provide a reference value for evaluating the function of the renal tubular OCT2-MATE1.

Use of a Double-Transfected System to Predict hOCT2/hMATE1-Mediated Renal Drug-Drug Interactions.

Accurate predictions of renal drug-drug interactions (DDIs) mediated by the human organic cation transporter 2 (hOCT2) and multidrug and toxin extrusion proteins (hMATEs) remain challenging. Current DDI evaluation using plasma maximal unbound inhibitor concentrations (Imax,u) and IC50 values determined in single transporter-transfected cells frequently leads to false or overprediction especially for hMATE1. Emerging evidence suggests intracellular unbound inhibitor concentration may be more relevant for hMATE1 inhibition in vivo. However, determination of intrarenal inhibitor concentrations is impractical. Here, we explored the use of hOCT2/hMATE1 double-transfected Madin-Darby canine kidney (MDCK) cells as a new in vitro tool for DDI risk assessment. Our results showed that potent in vitro hMATE1 inhibitors (hydroxychloroquine, brigatinib, and famotidine) failed to inhibit metformin B-to-A flux in the double-transfected system. On the other side, the classic hOCT2/hMATE1 inhibitors, pyrimethamine and cimetidine, dose-dependently inhibited metformin apparent B-to-A permeability (Papp). The different behaviors of these hMATE1 inhibitors in the double-transfected system can be explained by their different ability to gain intracellular access either via passive diffusion or transporter-mediated uptake. A new parameter (IC50,flux) was proposed reflecting the inhibitor's potency on overall hOCT2/hMATE1-mediated tubular secretion. The IC50,flux values significantly differ from the IC50 values determined in single transporter-transfected cells. Importantly, the IC50,flux accurately predicted in vivo DDIs (within 2-fold) when used in a static model. Our data demonstrated that the IC50,flux approach circumvents the need to measure intracellular inhibitor concentrations and more accurately predicted hOCT2/hMATE1-mediated renal DDIs. This system represents a new approach that could be used for improved DDI assessment during drug development. SIGNIFICANCE STATEMENT: This study demonstrated that flux studies in double-transfected MDCK cells and the IC50,flux represents a better approach to assess in vivo DDI potential for the renal organic cation secretion system. This study highlights the importance of inhibitor intracellular accessibility for accurate prediction of hMATE1-mediated renal DDIs. This approach has the potential to identify in vitro hMATE1 inhibitors that are unlikely to result in in vivo DDIs, thus reducing the burden of unnecessary and costly clinical DDI investigations.

Interaction and Transport of Benzalkonium Chlorides by the Organic Cation and Multidrug and Toxin Extrusion Transporters.

Humans are chronically exposed to benzalkonium chlorides (BACs) from environmental sources. The U.S. Food and Drug Administration (FDA) has recently called for additional BAC safety data, as these compounds are cytotoxic and have great potential for biochemical interactions. Biodistribution studies revealed that BACs extensively distribute to many tissues and accumulate at high levels, especially in the kidneys, but the underlying mechanisms are unclear. In this study, we characterized the interactions of BACs of varying alkyl chain length (C8 to C14) with the human organic cation transporters (hOCT1-3) and multidrug and toxin extrusion proteins (hMATE1/2K) with the goal to identify transporters that could be involved in BAC disposition. Using transporter-expressing cell lines, we showed that all BACs are inhibitors of hOCT1-3 and hMATE1/2K (IC50 ranging 0.83-25.8 µM). Further, the short-chain BACs (C8 and C10) were identified as substrates of these transporters. Interestingly, although BAC C8 displayed typical Michaelis-Menten kinetics, C10 demonstrated a more complex substrate-inhibition profile. Transwell studies with transfected Madin-Darby canine kidney cells revealed that intracellular accumulation of basally applied BAC C8 and C10 was substantially higher (8.2- and 3.7-fold, respectively) in hOCT2/hMATE1 double-transfected cells in comparison with vector-transfected cells, supporting a role of these transporters in mediating renal accumulation of these compounds in vivo. Together, our results suggest that BACs interact with hOCT1-3 and hMATE1/2K as both inhibitors and substrates and that these transporters may play important roles in tissue-specific accumulation and potential toxicity of short-chain BACs. Our findings have important implications for understanding human exposure and susceptibility to BACs due to environmental exposure. SIGNIFICANCE STATEMENT: Humans are systemically exposed to benzalkonium chlorides (BACs). These compounds broadly distribute through tissues, and their safety has been questioned by the FDA. Our results demonstrate that hOCT2 and hMATE1 contribute to the renal accumulation of BAC C8 and C10 and that hOCT1 and hOCT3 may be involved in the tissue distribution of these compounds. These findings can improve our understanding of BAC disposition and toxicology in humans, as their accumulation could lead to biochemical interactions and deleterious effects.

A Drug-Drug Interaction Study to Evaluate the Impact of Rimegepant on OCT2- and MATE1-Mediated Transport of Metformin in Healthy Participants.

Rimegepant is a calcitonin gene-related peptide receptor antagonist approved for migraine treatment. This phase 1, open-label, single-center, fixed-sequence study evaluated the effect of rimegepant on the pharmacokinetics (PK) of metformin. Twenty-eight healthy participants received metformin 500 mg twice daily from Days 1 to 4 and Days 7 to 10, and once daily on Days 5 and 11. Rimegepant, 75 mg tablet, was administered once daily from Days 9 to 12. At pre-specified time points, plasma metformin concentration, serum glucose levels, and safety and tolerability were evaluated. A 16% increase in the area under the plasma metformin concentration-time curve (AUC) for 1 dosing interval (AUC0-τ,ss), a statistically insignificant increase in maximum and minimum steady-state metformin concentration (Cmax,ss and Cmin,ss), and a decrease in metformin renal clearance were observed on Day 11 following metformin-rimegepant coadministration compared with metformin alone; however, the changes were not clinically relevant. Additionally, coadministration of rimegepant with metformin did not induce clinically meaningful change in the maximum observed glucose concentration (Gmax) or AUCgluc compared with metformin alone. Overall, rimegepant and metformin coadministration did not result in clinically relevant changes in metformin PK, renal clearance, or the antihyperglycemic effects of metformin. Rimegepant is considered safe for use with metformin.