Abstract

Multidrug and toxin extrusion protein (MATE/SLC47A) secretes metabolites and xenobiotics into the urine in the proximal tubules of the kidney. Uptake assays have been commonly used for evaluating MATE-mediated transport of new chemical entities in drug development. The purpose of this study was to examine the relationship between in vitro uptake activities by MATEs and the impact of MATE-mediated transport in in vivo renal secretion. In vitro uptake in mouse Mate1 (mMate1)-expressing human embryonic kidney 293 (HEK293) cells and several in vivo parameters from mMate1 knockout and wild-type mice were compared using nine cationic compounds (almotriptan, naratriptan, talinolol, sumatriptan, alogliptin, sitagliptin, rivaroxaban, saxagliptin, and vildagliptin). Compounds that showed statistically significant decrease in secretory clearances with respect to kidney concentrations (CLR,kidney) in mMate1 knockout mice were categorized as in vivo substrates in this study. A good correlation (R2 = 0.637) was observed between the in vitro uptake ratio and the in vivo ratio of CLR,kidney of mMate1 knockout mice and wild-type mice. This study supported the rationale of using an uptake assay to determine whether investigational compounds are the substrate of MATEs and to predict drug-drug interaction risk via renal secretion by MATE from the viewpoint of drug development in pharmaceutical companies.

SIGNIFICANCE STATEMENT We revealed that substrates judged by in vitro experiments using mouse multidrug and toxin extrusion (mMate)1-expressing cells were excreted in urine via mMate1 in vivo, and a good correlation (R2 = 0.637) was observed between in vitro uptake ratio and in vivo ratio of secretory clearance with respect to the kidney concentrations (CLR,kidney) of mMate1 knockout and wild-type mice. This study supported the rationale of using an uptake assay to predict potential human MATE1–mediated drug-drug interaction as a victim.

Introduction

Renal elimination is one of the major clearance pathways of xenobiotics from the body. Human multidrug and toxin extrusion proteins hMATE1 and hMATE2-K are expressed on the brush border membrane (BBM) of kidney proximal tubule cells and work as organic cations/H+ antiporters (Yonezawa and Inui, 2011; Motohashi and Inui, 2013). hMATE1 and hMATE2-K are characterized by their broad substrate specificities toward metabolites and xenobiotics. Several drugs in the market [pyrimethamine (Kusuhara et al., 2011; Miyake et al., 2021), cimetidine (Somogyi et al., 1987; Wiebe et al., 2020), and trimethoprim (Müller et al., 2015)] serve as in vivo relevant inhibitors of hMATE1/2-K, which causes a drug interaction with metformin; this inhibition results in a significant reduction in the renal clearance of metformin, an in vivo probe substrate for hMATE1/2-K. Cumulative clinical evidence supports the importance of hMATE1/2-K in drug metabolism and disposition. Therefore, it is important to examine whether new chemical entities are hMATE1/2-K substrates to elucidate their drug-drug interaction (DDI) risk as victims as well as perpetrators.

Since secretion clearance can be estimated by subtracting the product of glomerular filtration rate (GFR) and unbound fraction plasma (fp) from renal clearance assuming no reabsorption, clearance ratio (CR) is defined as renal clearance divided by the product of fp and GFR. When CR is greater than one, it suggests relevant contribution of active secretion to the urinary excretion. According to the guidelines published from health authorities, a sponsor is recommended to evaluate whether their candidate drug is a substrate of renal transporters when its secretion clearance exceeds 25% of the total clearance (European Medicines Agency, 2012; MHLW of Japan, 2018; US Food and Drug Administration, 2020). An uptake assay using recombinant cells overexpressing either hMATE1 or hMATE2-K, in which a proton gradient is imposed to render active transport, is one of the commonly used in vitro methods. This analysis assumes bidirectional transport of MATEs depending on the concentration gradient of substrates and protons across the plasma membrane. Provided that the physiologically relevant transport direction of MATEs is efflux in the kidney, we have reported the equivalence of IC50 determination of hMATE1-mediated uptake and efflux in vitro (Saito et al., 2020); however, no investigation has been carried out to determine whether the substrate recognition is identical in both directions.

There is a concern about in vitro–in vivo correlation (IVIVC) of pure hMATE1-mediated renal secretion in humans. According to the extended clearance concept (Yoshida et al., 2013; Varma et al., 2015), MATE activities are associated with the intrinsic clearance for efflux with respect to the kidney concentrations (CLR,kidney) but not blood concentrations of the test compounds (CLR). Hence, MATE-mediated transport could correlate with this parameter. Clinically, only limited information is available to identify in vivo relevant hMATE1 substrate drugs based on pharmacogenomics or DDI studies: metformin (Nies et al., 2016), N-methylnicotinamide (Ito et al., 2012), and gefapixant (Nussbaum et al., 2022). In this study we focused on the mouse as an animal model for this purpose. Mice have two orthologs of hMATE: mouse Mate1 and mouse Mate2 (mMate1 and mMate2). mMate1 is a predominant isoform in the kidney since it is expressed on the BBM of kidney proximal tubule cells, whereas mMate2 is not (Otsuka et al., 2005). Indeed, mMate1 knockout (KO) mice are available to characterize the pharmacokinetic role of mMate1 in vivo (Tsuda et al., 2009). Deletion of the mMate1 gene delayed systemic elimination of metformin, as indicated by a marked reduction in the renal clearance. There has been no report about species differences in substrate specificity using many compounds.

In this study, we conducted in vitro studies to analyze substrate recognition and uptake clearance and in vivo studies to determine the plasma concentrations, urinary excretion rates, kidney concentration, and the intrinsic excretion clearance across the BBM of nine cationic compounds after intravenous infusion in mMate1 KO mice and wild-type (WT) mice. The substrate judgment based on in vitro uptake and in vivo were compared; further, a correlation was assessed between the mMate1-mediated secretory clearance obtained in vivo and those in vitro mMate1-overexpressing human embryonic kidney 293 (HEK293) cells.

Materials and MethodsChemicals and Reagents

Unlabeled metformin was purchased from Wako Pure Chemical Industries (Osaka, Japan), and [14C]metformin (100 mCi/mmol) was purchased from Moravek Biochemicals (Brea, CA). Unlabeled almotriptan malate was purchased from Sigma (St. Louis, MO). Unlabeled alogliptin, naratriptan hydrochloride, rivaroxaban, saxagliptin, sitagliptin phosphate, sumatriptan succinate, talinolol, and vildagliptin were purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). All other chemicals and reagents were of analytical grade and are commercially available.

Animals

Wild-type male C57BL/6J mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). mMate1 knockout male mice (Tsuda et al., 2009) were provided by Prof. Ken-ichi Inui (Kyoto University) and Prof. Satohiro Masuda (Kyushu University).

Cloning of mMate1, Cell Culture, and Transfection

Mouse MATE1 (mMate1) cDNA (NM_026183) was synthesized and subcloned into pcDNA3.1(−) (Invitrogen, Carlsbad, CA). Cell culture and transfection were done as described in our previous study (Lechner et al., 2016). Validity of mMate1-expressing HEK293 cells used in this study was confirmed by results of time- and pH-dependent uptake of typical MATE substrates, 1-methyl-4-phenylpyridinium (MPP+) and metformin, as published previously (Ito et al., 2010). Inhibitory effects of several MATE inhibitors (pyrimethamine, cimetidine, and trimethoprim) were also confirmed (Supplemental Fig. 1; Supplemental Table 2).

Uptake Experiments Using Transiently Transfected HEK293 Cells

Uptake experiments were done as described in our previous study (Saito et al., 2020). The detailed procedure of uptake experiments, radioactivity measurement, and the determination of protein concentration are described in Supplemental Method 1. For test substrate measurement, cells were collected using a rubber scraper after the addition of 200 μl water. Cell suspensions (20 μl) were precipitated with 100 μl of acetonitrile containing 0.1% formic acid. Mixed solutions were centrifuged at 1000 g for 5 minutes. The supernatants were mixed with an equal volume of 1% formic acid, and aliquots were used for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis.

Infusion Study Compounds in Wild-Type and mMate1 Knockout Mice

Infusion studies were done as described in our previous study (Kito et al., 2019). The detailed procedure of anesthesia, catheterize, and sample collection are described in Supplemental Method 2. The supernatant of plasma, urine specimens, and homogenized kidney were subjected to LC-MS/MS analysis after precipitation with acetonitrile.

Determination of the Unbound Fraction in Plasma

The plasma unbound fractions (fp) of test substrates were determined as described in our previous study (Kito et al., 2019) (Supplemental Method 2). The concentrations were determined by LC-MS/MS analysis.

LC-MS/MS Analysis for Test Substrates

The concentrations of test substrates from in vitro experiments were quantified using an LC-MS/MS system consisting of an API 5000TM triple quadruple mass spectrometer (AB SCIEX, Foster City, CA) coupled with a Prominence UFLC XR system (Shimadzu, Kyoto, Japan). An AB SCIEX QTRAP 5500 mass spectrometer (AB SCIEX) coupled with a Prominence liquid chromatography (LC) system (Shimadzu, Kyoto, Japan) was used for quantification of test substrates from in vivo experiments. Detailed LC conditions, mass-to-charge ratios, and validation results are shown in Supplemental Table 1.

Data Analysis

mMate1- or hMATE1-mediated uptake clearance was calculated by normalizing the amount of substrate inside the cells to that in the buffer and the protein concentration in each well using the following equation: where Uptake CL is the uptake clearance (μl/designated time point/mg), Xcell is the amount in the cells (pmol/designated time per well), and Cmedium is the concentration of test substrates in the medium (μM). Uptake CL was normalized by the amount of total cellular protein (mg per well). mMate1- or hMATE1-mediated net uptake was calculated by subtracting the mean value of uptake into mock vector-transfected cells from that into mMate1- or hMATE1-transfected cells. Uptake ratio (UR) was calculated by dividing the mean value of uptake into vector-transfected cells from that into mMate1- or hMATE1-transfected cells.

The fractional urinary ratio (Furine), the total body clearance (CLtot), the renal clearance with respect to the plasma concentration (CLR,plasma), the apparent kidney-to plasma concentration ratio (Kp,kidney), and the secretion clearance with respect to the concentration in the kidney (CLR,kidney) were calculated using the following equations: where Vurine,ave represents the urinary excretion rate from 90 to 120 minutes. The Vurine was calculated as the averaged rate by dividing 30 minutes as urine collection interval from the amount excreted into urine at 30–60, 60–90, and 90–120 minutes. Cp,ave represents the mean value of plasma concentrations at 90 and 120 minutes. Ckidney represents tissue concentrations at 120 minutes. CLR,kidney and CR were calculated by using 14 μl/min/kg as GFR (Davies and Morris, 1993).

Statistical Analysis

Unpaired t test after comparing variances with F-test was used for the analysis between two groups using Microsoft Excel 365, and one-way ANOVA followed by Dunnett’s test was used for the analysis among three or more groups using R version 4.1.2.

ResultsIn Vitro Uptake of Cationic Compounds in mMate1-Expressing HEK293 Cells

Time-dependent uptake (0.5–10 minutes) of nine cationic compounds into mMate1-expressing HEK293 cells and the effect mMate1 inhibitor pyrimethamine were examined (Fig. 1). Almotriptan, naratriptan, talinolol, sumatriptan, alogliptin, and sitagliptin at 0.3–1 μM substrate concentration showed substantially higher uptake in mMate1-expressing HEK293 cells compared with that in control cells. A time of 0.25 minutes was selected as an operable minimum incubation time to investigate individual initial uptakes at 0.3–1 μM substrate concentration in mMate1-expressing HEK293 cells (Supplemental Fig. 2). No inhibition toward mMate1-mediated metformin uptake up to 100 μM of each compound was also confirmed, suggesting a low possibility of saturation at the concentration used in the initial uptake evaluation (data not shown). Net uptake clearances and uptake ratios versus mock cells were calculated for almotriptan (20, 2.8), naratriptan (24, 9.4), talinolol (15, 4.0), sumatriptan (15, 5.1), alogliptin (11, 2.8), sitagliptin (8.2, 2.7), rivaroxaban (4.0, 1.4), saxagliptin (0.86, 1.3), and vildagliptin (0.63, 1.2), respectively. Six out of nine compounds were judged as mMate1 substrates from in vitro uptake experiments according to the criteria specified in the regulatory DDI guidelines (UR > 2).

Fig. 1.

Time-dependent uptake of cationic compounds in mMate1-expressing HEK293 cells. Uptake of 0.3 μM of cationic compounds (almotriptan, naratriptan, talinolol, sumatriptan, alogliptin, sitagliptin, rivaroxaban, saxagliptin, and vildagliptin) and the effect of mMate1 inhibitor pyrimethamine were determined at pH 7.4 in mMate1-expressing (●, 〇) and mock vector-transfected (▪, □) HEK293 cells after intracellular preacidification with 20 mM ammonium chloride (NH4CL). Each bar represents the mean ± S.D. (n = 3). Statistical analysis was conducted using one-way ANOVA with Dunnett’s post-test. *P < 0.05.

In Vivo Renal Elimination of Cationic Compounds in mMate1 Knockout Mice and WT Mice

Nine cationic compounds were administered to mMate1 knockout and WT mice by constant intravenous infusion. Plasma concentrations, kidney concentrations, and urinary excretion rate were determined (Fig. 2; Table 1). Since plasma time-profiles of compounds did not reach a plateau even at 120 minutes, Cp,ave, which is the mean value of plasma concentrations at 90 and 120 minutes, was used for further calculation.

Fig. 2.

Comparison of the renal elimination of cationic compounds between mMate1 knockout mice and wild-type mice. Plasma concentrations, urinary excretion rates, and kidney concentrations were determined in control (〇) and Mate1 knockout mice (▪) whose bladders were cannulated for the collection of urine. Almotriptan (4.0 nmol/min per kg), naratriptan (4.4 nmol/min per kg), talinolol (2.3 nmol/min per kg), sumatriptan (5.1 nmol/min per kg), alogliptin (1.8 nmol/min per kg), sitagliptin (2.2 nmol/min per kg), rivaroxaban (2.3 nmol/min per kg), saxagliptin (2.4 nmol/min per kg), and vildagliptin (3.2 nmol/min per kg) were administered to male C57BL/6 mice by intravenous infusion. Blood samples were collected at designated times, and urine samples were collected at 30-minute intervals. At the end of the experiment, the kidneys were removed. Each point represents the mean value, and error bars represent the S.D. (n = 4). Statistical analysis was conducted using unpaired t test. *P < 0.05.

TABLE 1

Pharmacokinetic parameters for cationic compounds in mMate1 knockout mice and wild-type mice

Each value was determined from the data shown in Fig. 2. The equations to determine the kinetic parameters are described in the Materials and Methods section. Each value represents the mean ± S.D. (n = 4) Statistical analysis was conducted using unpaired t test.

A change of in vivo parameters was observed for sumatriptan (40%) and talinolol (27%) in Cp,ave increase and for almotriptan (41%), naratriptan (28%), talinolol (45%), sumatriptan (33%), and alogliptin (30%) in Vurine decrease. An increase in kidney concentrations was observed in naratriptan (116%), talinolol (123%), sumatriptan (102%), alogliptin (37%), saxagliptin (103%), and sitagliptin (42%). Even in the case that the plasma exposure did not change, the urinary excretion rate and/or increased kidney concentrations of the six compounds as a substrate from in vitro uptake experiments were decreased, resulting in statistically significant reduction in CLR,kidney (<52% of control). Thus, sumatriptan, talinolol, alogliptin, alogliptin, saxagliptin, and sitagliptin are categorized as substrates of mMate1 from in vivo experiments, resulting in no false-positive results compared with in vitro substrate judgment. Only saxagliptin showed a discrepancy between in vivo and in vitro substrate judgment.

Correlation between Renal Secretory Elimination and In Vitro Uptake Data

The differences (Fig. 3, A and B) and ratios (Fig. 3, C and D) of in vivo parameters (CLR,plasma and CLR,kidney) between WT mice and mMate1 KO mice were calculated and compared with their in vitro net uptake clearances and uptake ratios. The R2 values were 0.32 (CLR,plasma, P = 0.114) and 0.43 (CLR,plasma, P = 0.0555) in the difference analysis, and in the ratio analysis, the R2 values were 0.29 (CLR,plasma, P = 0.136) and 0.66 (CLR,kidney, P = 0.00737). Thus, only the ratio analysis of CLR,kidney showed statistically significant correlation.

Fig. 3.

Comparison between in vitro uptake and in vivo renal secretory elimination of cationic compounds. In vitro net uptake and uptake ratio were compared with the difference (A and B) or the ratio (C and D) in either plasma concentration-based renal clearance [CLR,plasma: (A and C)] or kidney concentration-based renal clearance [CLR,kidney: (B and D)] between WT and mMate1 KO mice (data taken from Table 1). Closed squares, closed triangles, and open circle represent substrates (both in vitro and in vivo), nonsubstrate (both in vitro and in vivo), and false-negative substrate, respectively.

Species Difference between Mice and Human MATE-Mediated Uptake

To compare interspecies differences between mouse and human, the initial uptake of nine cationic compounds into hMATE1-expressing HEK293 cells were also examined. Judging from the criteria (UR > 2), almotriptan (3.6), naratriptan (9.4), talinolol (4.0), sumatriptan (8.2), and sitagliptin (2.6) were in vitro hMATE1 substrates. The uptake ratios of tested compounds were well correlated (Fig. 4) between species, although alogliptin showed a statistically significant uptake only in mMate1-expressing cells and rivaroxaban showed a statistically significant uptake only in hMATE1-expressing cells (Supplemental Fig. 1; Supplemental Table 3).

Fig. 4.

Comparison of uptake ratio of cationic compounds between mMate1- and hMATE1-expressing HEK293 cells. Initial uptake of almotriptan (0.3 μM), naratriptan (0.3 μM), talinolol (1 μM), sumatriptan (0.3 μM), alogliptin (1 μM), sitagliptin (1 μM), rivaroxaban (1 μM), saxagliptin (1 μM), and vildagliptin (1 μM) was determined for 0.25 min at pH 7.4 in mMate1-, hMATE1-expressing HEK293, and mock vector-transfected HEK293 cells after intracellular preacidification with 20 mM ammonium chloride (NH4CL). Uptake ratios were calculated by dividing the mean value of uptake in mock vector-transfected cells from that in transporter-expressing cells.

Discussion

Inhibition of MATEs in the kidney can elevate kidney concentration of its substrates without substantial reflection in plasma concentrations, leading to nephrotoxicity and/or altered efficacy in some cases. However, it is currently difficult to estimate the intrinsic hMATE1 clearance from human clinical trial data and hence to accurately verify the correlation between in vitro and in vivo clearances in human. mMate1 KO mice were the preferred animal model to understand the magnitude of pure loss of MATE-mediated renal eliminations. Therefore, in this study we examined MATE-mediated urinary excretion in mouse ortholog mMate1 KO mice and explored whether the substrate recognition from the physiologically opposite in vitro uptake results is qualitatively and quantitatively the same as in vivo using nine cationic compounds. Then, we tried to investigate whether in vitro substrate recognition and in vitro uptake ratios are similar between mMate1 and hMATE1.

We successfully expanded the knowledge about the importance of mMate1 in the urinary excretion of drugs in mice. For six cationic compounds identified as a novel substrate of mMate1 in vitro study, one or several in vivo parameters were changed (Fig. 2). In vivo relevance was examined by an in vivo pharmacokinetic study using mMate1 KO mice. The effect of mMate1 dysfunction on the net renal clearance depends on the rate-determining step of renal excretion of each test compound according to the extended clearance concept. When uptake is a rate-determining process, mMate1 dysfunction likely causes an increase in the kidney concentration without affecting the urinary excretion rate. Therefore, CLR,kidney change between WT and mMate1 KO mice is considered to be a better indicator for mMate1-mediated renal secretion. Seven out of nice compounds that showed statistically significant decrease in CLR,kidney were categorized as in vivo substrates in this study (Table 1). Note that four mice were used in this study. Consequently, we could detect significant differences in CLR,kidney for seven compounds. However, the conclusion that the compounds are not in vivo substrates requires careful design of in vivo study, considering the power.

Saxagliptin was an exceptional test drug: there was no mMate1-specific uptake in vitro even in the presence of outward proton gradient, whereas there was a significant impact of mMate1 gene knockout on the pharmacokinetic parameter (Figs. 1 and 2; Table 1). There is no reasonable explanation of the inconsistency between in vitro and in vivo data. Practically speaking, such discrepancies can be a problem in drug development, especially in drug-drug interaction prediction. It is required to find better in vitro systems with higher in vivo predictability. Cells coexpressing both organic cation transporter 2 (OCT2) and hMATE1 are often used to investigate the involvement of OCT2 and hMATE transporters in renal elimination, and metformin secretory transport was nicely demonstrated by the double transfected cells (König et al., 2011). Some compounds (pramipexole, lamivudine, memantine, trimethylamine-N-oxide), which are considered not to be substrates in uptake assay using single hMATE1-expressing cells, showed directional transcellular transport across cell monolayers coexpressing both OCT2 and hMATE1 (Müller et al., 2013, 2017; Knop et al., 2015; Gessner et al., 2018). IVIVC between pure hMATE1-mediated urinary excretion and the clearance obtained from double transfected cells has not been well established. Considering the results obtained in this study, it is likely that clearance should be defined with regard to the cellular accumulation.

To consider a quantitative relationship between in vitro uptake and in vivo MATE-mediated renal secretions, CLR,kidney was calculated with respected to the kidney concentrations in WT and KO mice (Table 1). In this study, we found a statistically significant correlation only in the ratio analysis of CLR,kidney (R2 = 0.664, P = 0.00737) among four different analyses (Fig. 3). This R2 value was improved from 0.664 to 0.847 (P = 0.00398) when the only false-negative case of saxagliptin was excluded. CLR,plasma is a routinely obtained in vivo parameter that comprises basolateral influx and efflux clearances and clearance for the efflux across the BBM in the proximal tubular cells. Therefore, CLR,plasma could correlate with CLR,kidney only under a limited condition (i.e., basolateral efflux is greater than the efflux across the brush border side, and the ratio of basolateral influx to efflux is constant across the test compounds). Furthermore, it is considered to be reasonable that the CLR,kidney ratio between WT and KO is an independent parameter from the fraction unbound in the kidney (fkidney), but the CLR,kidney difference could be affected by the absolute value of this parameter. The good correlation observed in the CLR,kidney ratio also supported that the substrate recognition of mMate1 was identical regardless of the in vitro uptake (outward facing) or in vivo secretion (inward facing). Thus, the uptake ratio is likely a promising in vitro metric to suggest the importance of mMate1 in the kidney.

As expected, based on the high homology of 78% between mMate1 and hMATE1, a good correlation of uptake ratio between mMate1 and hMATE1 was observed (Fig. 4), suggesting that the uptake ratio of hMATE1 is also correlated with hMATE1-mediated CLR,kidney in human. It should be noted that the renal clearance of these drugs involves significant tubular secretion (Supplemental Table 4). However, given that it is still difficult to estimate pure human MATE-mediated intrinsic clearance in the kidney, the following challenges should be considered. First is the limitation in the methodology to define CLR,kidney. Positron emission tomography is the only method to obtain the time profiles of tissue concentrations in humans (Shingaki et al., 2015; Gormsen et al., 2016). However, it is not practical to routinely conduct such imaging study in drug development because it requires a specialized facility and development of the chemical reaction to introduce the positron emitting nucleus to the test probe. Second, there is no MATE inhibitor that completely inhibits hMATE activity at clinical concentrations. For instance, the effects of the potent MATE inhibitor pyrimethamine (20 μmol/kg, bolus injection 15 minutes prior to start of infusion) on CLR,kidney of the nine test compounds were less potent than those obtained from mMate1 KO mice studies (Supplemental Fig. 3). The dose-dependent effect of pyrimethamine was examined in healthy subjects where the highest dose of pyrimethamine was 75 mg, at which renal clearance (CLR) of metformin and other MATE substrates such as N-methylnicotinamide and N-methyladenosine was decreased 55%, 58%, and 48% (Miyake et al., 2021). Pyrimethamine is a potent inhibitor to cause significant inhibition at its therapeutic dose; however, the hMATE1/2-K–mediated transporter was unlikely completely inhibited. The third is that unlike in mice, hMATE2-K is expressed in human kidney as efflux transporter in the kidney (Prasad et al., 2016; Fukuda et al., 2017; Ishiguro et al., in preparation;). Provided that hMATE1 and hMATE2-K show an overlapped substrate specificity (Damme et al., 2011), pan MATE inhibitors are required to investigate the activities of hMATE1 and hMATE2-K in the human kidney, and the magnitude of hMATE inhibition on human pharmacokinetics needs to be clarified using such inhibitors. To estimate quantitative hMATE1-mediated clearances more precisely, further investigation is necessary using a mathematical modeling approach that takes into consideration the time profiles (Mathialagan et al., 2017; Nakada et al., 2018).

This is the first study to explore the correlation between physiologically opposite in vitro uptake and physiologically forward in vivo mMate1-mediated renal secretion. Substrates judged as in vitro substrates were confirmed to be excreted in urine via mMate1, indicating that the substrate recognitions were identical between outward facing and inward facing regardless of direction of transport. The IVIVC in mMate1 and the good correlation of the in vitro uptake ratios between mice and humans suggest the good correlation between uptake ratio and hMATE1-mediated CLR,kidney also in human. Our findings support the rationale of an uptake assay to assess the possibility of hMATE1-mediated DDI as victim also in humans.

Acknowledgments

The authors would like to acknowledge Prof. Inui at Kyoto University and Prof Masuda at Kyushu University for providing mMate1 knockout mice. The excellent technical assistance of Michiru Miyake in performing the in vitro experiments at Nippon Boehringer Ingelheim is gratefully acknowledged. The authors also thank Dr. Wataru Kishimoto at Nippon Boehringer Ingelheim Co., Ltd. for helpful advice in drafting this manuscript.

Authorship Contributions

Participated in research design: Saito, Kito, Takatani, Ishiguro, Bister, Kusuhara.

Conducted experiments: Saito, Kito, Takatani, Kudo.

Performed data analysis: Saito, Kito, Takatani, Kudo, Kusuhara.

Wrote or contributed to the writing of the manuscript: Saito, Ishiguro, Kusuhara.

FootnotesReceived September 19, 2022.Accepted January 11, 2023.

This work received no external funding.

No author has an actual or perceived conflict of interest with the contents of this article.

dx.doi.org/10.1124/dmd.122.001115.

↵This article has supplemental material available at dmd.aspetjournals.org.

AbbreviationsBBMbrush border membraneCkidneytissue concentrations at 120 minutesCLclearanceCLR, kidneyrenal clearance with respect to kidney concentrationCLR, plasmarenal clearance with respect to plasma concentrationCLtottotal body clearanceCp, avemean value of plasma concentrations at 90 and 120 minutesCRclearance ratioDDIdrug-drug interactionfpunbound fraction plasmaFurinefractional urinary ratioGFRglomerular filtration rateHEK293human embryonic kidney 293hMatehuman MateIVIVCin vitro–in vivo correlationKOknockoutKp, kidneyapparent kidney-to plasma concentration ratioLCliquid chromatographyLC-MS/MSliquid chromatography–tandem mass spectrometryMATEmultidrug and toxin extrusion proteinmMatemouse MateOCT2organic cation transporter 2URuptake ratioVurine, aveurinary excretion rate from 90 to 120 minutesWTwild-typeCopyright © 2023 by The Author(s)References↵↵

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