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Research ArticleArticle

Cynomolgus Monkey as a Clinically Relevant Model to Study Transport Involving Renal Organic Cation Transporters: In Vitro and In Vivo Evaluation

Hong Shen, Tongtong Liu, Hao Jiang, Craig Titsch, Kristin Taylor, Hamza Kandoussi, Xi Qiu, Cliff Chen, Sunil Sukrutharaj, Kathy Kuit, Gabe Mintier, Prasad Krishnamurthy, R. Marcus Fancher, Jianing Zeng, A. David Rodrigues, Punit Marathe and Yurong Lai
Drug Metabolism and Disposition February 2016, 44 (2) 238-249; DOI: https://doi.org/10.1124/dmd.115.066852

Abstract

Organic cation transporter (OCT) 2, multidrug and toxin extrusion protein (MATE) 1, and MATE2K mediate the renal secretion of various cationic drugs and can serve as the loci of drug-drug interactions (DDI). To support the evaluation of cynomolgus monkey as a surrogate model for studying human organic cation transporters, monkey genes were cloned and shown to have a high degree of amino acid sequence identity versus their human counterparts (93.7, 94.7, and 95.4% for OCT2, MATE1, and MATE2K, respectively). Subsequently, the three transporters were individually stably expressed in human embryonic kidney (HEK) 293 cells and their properties (substrate selectivity, time course, pH dependence, and kinetics) were found to be comparable to the corresponding human form. For example, six known human cation transporter inhibitors, including pyrimethamine (PYR), showed generally similar IC50 values against the monkey transporters (within sixfold). Consistent with the in vitro inhibition of metformin (MFM) transport by PYR (IC50 for cynomolgus OCT2, MATE1, and MATE2K; 1.2 ± 0.38, 0.17 ± 0.04, and 0.25 ± 0.04 µM, respectively), intravenous pretreatment of monkeys with PYR (0.5 mg/kg) decreased the clearance (54 ± 9%) and increased in the area under the plasma concentration-time curve of MFM (AUC ratio versus control = 2.23; 90% confidence interval of 1.57 to 3.17). These findings suggest that the cynomolgus monkey may have some utility in support of in vitro-in vivo extrapolations (IVIVEs) involving the inhibition of renal OCT2 and MATEs. In turn, cynomolgus monkey-enabled IVIVEs may inform human DDI risk assessment.

Introduction

It is widely appreciated that renal elimination of cations, drug, toxin, or endogenous metabolite related, is achieved not only by glomerular filtration but also by active transport processes that facilitate their tubular secretion and reabsorption. Indeed, the mechanisms governing the renal elimination of organic cations have been studied in detail, and the transporters facilitating their tubular uptake and extrusion have been identified (Okuda et al., 1996; Otsuka et al., 2005; Masuda et al., 2006).

Secretion of organic cations in renal proximal tubules involves at least two distinct transporters, located in the basolateral and apical membranes of proximal tubule cells (Motohashi et al., 2002, 2013; Masuda et al., 2006). Specifically, organic cations, like metformin (MFM), 1-methyl-4-phenylpyridinium (MPP+), tetraethylammonium (TEA), and cimetidine (CMD), are taken up from the circulation by organic cation transporter 2 (OCT2) expressed on the basolateral domain of renal proximal tubular cells. In turn, uptake is followed by efflux into the tubular fluid by multidrug and toxin extrusion protein (MATE) 1 and MATE2K expressed on the apical domain of the same cells. Therefore, OCT2 and MATEs function coordinately to mediate vectorial transport of certain cationic drugs, from blood to tubular fluid, which represents the tubular secretion clearance component of total renal clearance.

Perturbation of cation transport function, because of modulation by drugs or polymorphisms, can lead to drug-drug interactions (DDIs) and exacerbated drug-induced renal toxicity (Fisel et al., 2014). Although less than a threefold change in systemic exposure of victim drug is typically reported, the concentrations of drug in the kidney can be dramatically increased because of inhibition of efflux. It is possible to envision altered efficacy and toxicity in such a scenario. Consequently, in vitro studies using various transporter expression systems are routinely performed to evaluate whether a new molecular entity is an inhibitor or substrate of human renal organic cation transporters. Such studies can be performed preclinically, or after dosing of the new molecular entity to human subjects, and are widely accepted by pharmaceutical companies and regulatory authorities in support of DDI and drug-induced nephrotoxicity risk assessment (Okuda et al., 1999; Giacomini, 2010; US FDA, 2012; European Medicines Agency, 2012; Morrissey et al., 2013). In fact, there is a need to integrate, understand, and translate the in vitro data in support of risk assessment prior to human dosing or prioritize specific types of clinical DDI studies. To date, however, examples of IVIVEs for DDIs involving renal transporters, such as OCTs and MATEs, are few in number.

Increasingly, investigators are leveraging a combination of in vitro and in vivo animal (e.g., humanized rodents and nonhuman primates) data to support IVIVE involving the inhibition of drug-metabolizing enzymes (e.g., cytochrome P450 3A4) and transporters (e.g., organic anion transporting polypeptide) (Tang and Prueksaritanont, 2010; Shen et al., 2013; Jaiswal et al., 2014). When successful, appropriately characterized and validated animal models can provide mechanistic insight, support modeling, and enable IVIVE exercises. Obviously, one has to consider species differences in genetics, substrate specificity, tissue distribution, and abundance of transporters and enzymes. For example, OCT2 expression in human and monkey kidney tissue is similar. In comparison, both Oct1 and Oct2 are expressed in mouse and rat kidney, although the expression levels relative to that of human OCT2 are unknown (Urakami et al., 1998; Motohashi et al., 2002; Alnouti et al., 2006; Bleasby et al., 2006). Conversely, MATE1 and MATE2K are known to be highly expressed in human kidney, but the counterpart of human MATE2K has not been identified in rats and mice (Otsuka et al., 2005; Masuda et al., 2006). This has led some to question the utility of rodent models. On the other hand, the cynomolgus monkey has been considered a more appropriate animal model to assess DDIs involving renal clearance. However, monkey OCT2, MATE1, and MATE2 have not been cloned and characterized.

The aim of the present study was to assess the applicability of cynomolgus monkey as a suitable surrogate preclinical model for studying OCT2-, MATE1-, and MATE2K-mediated DDIs. It was possible to clone full-length cynomolgus monkey OCT2, MATE1, and MATE2K (cOCT2, cMATE1, and cMATE2K) and establish stably transfected cell lines for each of the three transporters. In turn, the in vitro profile of each transporter (substrate specificity, time course, pH dependence, transport kinetics, and inhibition profile) was characterized and compared with that of the corresponding human form. The effort was extended to include an in vivo monkey DDI study, employing pyrimethamine (PYR) as perpetrator and MFM as victim, and the results were compared with those of literature reports involving human subjects.

Materials and Methods

Chemicals and Materials.

[14C]metformin ([14C]MFM; 98 mCi/mmol) and [3H]methotrexate ([3H]MTX 25.9 Ci/mmol) were purchased from Moravek Biochemicals, Inc. (Brea, CA). [3H]1-Methyl-4-phenylpyridinium ([3H]MPP+; 80 Ci/mmol), [14C]tetraethylammonium ([14C]TEA; 3.2 mCi/mmol), and [3H]estrone-3-sulfate ([3H]E3S; 45.6 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA). [3H]cimetidine ([3H]CMD; 24.4 Ci/mmol) was obtained from American Radiolabeled Chemicals (St. Louis, MO). Unlabeled MFM, PYR, CMD, quinidine (QD), vandetanib (VDN), ketoconazole (KCZ), and imipramine (IPM) were purchased from Toronto Research Chemicals Inc. (North York, Ontario). All other chemicals were of reagent grade and purchased from Sigma-Aldrich (St. Louis, MO). Human embryonic kidney (HEK 293) Flip-In cells and Lipofectamine 2000 transfection system were purchased from Invitrogen-Life Technologies (Carlsbad, CA). All cell culture media and reagents were obtained from Mediatech, Inc (Manassas, VA) or Life Technologies (Carlsbad, CA). Poly-d-lysine-coated 24-well plates were obtained from BD Biosciences (Bedford, MA). Sources of other materials are stated in individual method sections.

Cloning of cOCT2, cMATE1 and cMATE2K, Cell Culture, and Uptake Transport Studies.

The cloning of cOCT2, cMATE1 and cMATE2K, and stable transfection in HEK 293 cells are described in detail in the Supplemental Material. The monkey transporter cDNA gene sequences were deposited in National Center for Biotechnology Information GenBank with accession numbers of KP731382, KP731383, and KP731384 for cOCT2, cMATE1, and cMATE2K, respectively.

All cells were grown in Dulbeccos’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, 0.1 mM nonessential amino acids, 2 mM l-glutamine, and 0.1 mg/ml Hygromycin B at 37°C in an incubator supplied with 5% CO2. Subculture was performed every week, and passage numbers between 5 and 30 were used throughout the study to keep the transporter expression level and functional activity consistent.

The protocol for uptake experiments has been previously described (Shen et al., 2013). Briefly, HEK 293 cells were seeded into poly-d-lysine-coated 24-well plates at a density of 0.5 × 106 cells per well. Two to three days after seeding, cells were grown to confluence, and uptake experiments were performed. Cells were washed twice with 1.5 ml Hanks' balanced salt solution (HBSS) prewarmed at 37°C. The uptake study was initiated by adding 0.2 ml of prewarmed standard buffer (HBSS containing 10 mM HEPES, pH 7.4 for OCT2, and pH 8.4 for MATE1 and MATE2K, respectively) containing radiolabeled compounds ([14C]MFM or others). At the end of the incubation period, the buffer was removed and the cells in each well were rinsed three times with 1 ml ice-cold HBSS (4°C). For a time course uptake study, the uptake of 2 µM [14C]MFM was terminated at specific times by aspirating and rinsing. To measure the pH-dependent transport of MFM, the uptake of 1 µM [14C]MFM in buffers at different pH (HBSS with 10 mM HEPES, and HCl and NaOH for adjusting to pH 5.0, 6.0, 7.0, 7.4, 8.0, 9.0, 10.0, and 11.0) was determined over 2 minutes. For the assessment of MFM transport kinetics, a constant amount of radiolabel with varying amounts of unlabeled substrate was used.

To assess the inhibitory potency of PYR, CMD, QD, VDN, KCZ, and IPM toward uptake of 2 µM [14C]MFM by monkey and human organic cation transporters, the test compound at various concentrations was added simultaneously with MFM. At 2 minutes (within the linear time range; Fig. 2), the buffer was removed to terminate the reaction and the cells were washed three times with ice-cold HBSS. After being air-dried for at least 30 minutes in the fume hood, the cells were lysed with 0.3 ml of 0.1% Triton X-100, and the radioactivity was determined by liquid scintillation counting. The protein concentrations in cell experiments were determined by the bicinchoninic acid method using bovine serum albumin as a standard (Pierce Chemical, Rockfold, IL).

Liquid Chromatography Coupled with Tandem Mass Spectrometry (LC-MS/MS) Quantification of OCT2, MATE1, and MATE2K Proteins in Transporter-Overexpressing Cells.

Membrane protein fraction from transporter-overexpressing cells was extracted using ProteoExtract Native Membrane Protein Extraction kit, as per the manufacturer’s instructions (Calbiochem, San Diego, CA). Membrane protein fraction samples were digested as described previously (Qiu et al., 2013). In brief, all samples were adjusted to a maximum protein concentration of 2 mg/ml. Samples containing 40 to 200 µg membrane proteins were reduced with 10 mM dithiothreitol at 95°C for 5 minutes in 25 mM ammonium bicarbonate buffer with 1% deoxycholate. After cooling down, the membrane protein was then alkylated with 15 mM iodoacetamide in the dark for 30 minutes, followed by trypsin (trypsin/protein ratio: 1/50) digestion at 37°C for 16 hours with shaking. To prepare calibration curves, the same amount of human serum albumin was digested followed the same procedure. The digestion was terminated by addition of an equal volume of water (with 0.2% formic acid) containing a fixed concentration of the synthetic stable isotope-labeled peptides (final concentration: 20 nM) that serves as an internal standard (IS). For construction of the calibration curve, an equal volume of water (with 0.2% formic acid) containing the IS (20 nM) and the synthetic unlabeled peptide standard (0.1–200 nM each). The samples were centrifuged at 14,000 g for 10 minutes, and the supernatant was filtered through Millipore filtration plate (Billerica, MA), dried, and reconstituted in 100 µl 0.1% formic acid in water.

Peptide quantification was performed using a Shimadzu Nexera UHPLC system (LC-30A; Columbia, MD) coupled with an API-6500 triple quadrupole mass spectrometer (AB Sciex, Foster City, CA). A sample volume of 20 µl was injected onto the column and the UHPLC separation was achieved using Waters BEH300 C18 2.1 × 100 mm Peptide Separation Technology column with a 1.7 µm particle size and 300 Å pore size (Milford, MA), and peptides were eluted with 0.3 ml/min of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) using the following gradient. The separation of peptides was achieved by the following linear gradients: 1 minute, 5% solvent B; 8 minutes, 30% solvent B; 8.2 minutes, 90% solvent B; 10.2 minutes, 5% solvent B; 14 minutes, controller stopped. Quality controls for the method were conducted in a sample spiked with a fixed concentration of a representative synthetic unlabeled peptide (14 nM). The accuracy (relative error) was less than 10% and precision (coefficient of variation) was less than 5% for all quality control samples (Qiu et al., 2013). All selected peptides and their optimized mass transitions with the highest sensitivity are specified in Table 1.

View this table:
TABLE 1

Summary of proteospecific peptides and their respective MRM transitions used for renal organic cation transporter protein quantification with LC-MS/MS

Pharmacokinetic Experiments Employing Cynomolgus Monkeys.

All experiments with cynomolgus monkeys were performed in accordance with the National Institutes of Health guidelines and approved by Bristol-Myers Squibb Animal Care and Use Committee. The animals were housed in a temperature- and humidity-controlled room with a 12-hour light/dark cycle. Pharmacokinetic experiments with MFM, either alone or in combination with PYR, were performed in male cynomolgus monkeys weighing between 5 to 7 kg (n = 3 and 2 animals per group for intravenous and oral administration, respectively) in a crossover study design with a 1-week washout between treatments and the MFM alone treatment ahead of the coadministration treatment. Monkeys were fasted beginning the night before each oral but not intravenous dose of MFM or PYR. For intravenous application, MFM was dissolved in physiologic saline and given by intravenous infusion via a femoral vein at 3.9 m/kg over 15 minutes (5 ml/kg). In the intravenous coadministration group, PYR dissolved in saline with 10% ethanol to obtain a concentration of 0.1 mg/ml, was also given by femoral vein infusion for 15 minutes at a dose of 0.5 mg/kg (7.5 ml/kg), 60 minutes before the administration of MFM. Animals receiving MFM alone were injected with an equivalent volume of vehicle (i.e., 7.5 ml/kg). For oral application, monkeys were given a single oral dose of 8.6 mg/kg MFM that was dissolved in saline by gavage (5 ml/kg). In oral coadministration group, each monkey also received an oral dose of 2.5 mg/kg PYR (5 ml/kg) as a suspension in saline with 10% ethanol, 60 minutes before the administration of MFM. Animals receiving MFM alone were orally administered with an equivalent volume of vehicle (i.e., 5 ml/kg). Approximately 500 μl of arterial blood was collected in tubes containing potassium (K2) EDTA with vascular access port at 0, 0.25, 0.5, 0.45, 1, 2, 3, 5, 7, 24, and 48 hours (intravenous) after MFM administration. Plasma was generated by centrifugation at 10,000 g for 3 minutes. Urine samples were collected during the following intervals: 0–7 hours, 7–24 hours, and 24–48 hours after intravenous administration of 3.9 mg/kg MFM and 0–3 hours, 3–6 hours, and 6–24 hours after oral administration of 8.6 mg/kg MFM. The volume of urine obtained was recorded at the end of each collection interval. The plasma and urine samples were stored at −70°C until analysis within the known period of stability.

LC-MS/MS Measurement of Pharmacokinetic Samples.

The plasma and urine specimens (100 µl) were mixed with 300 µl of acetonitrile containing 1% formic acid and 1 ng/ml of d6-metformin (internal standard). The mixed solutions were centrifuged at 2,000 g for 5 minutes, and 300 µl of the supernatant was transferred to a clean 96-well plate. The supernatant was dried under nitrogen at 40°C until dry, reconstituted with 100 µl of 50 mM ammonium formate in acetonitrile/water (50/50, v/v, pH 3.2), and then injected (10 µl) to LC-MS/MS. A Triple Quad 5500 mass spectrometer (AB Sciex) tandem with a Nexera LC-20 UHPLC system (Shimadzu Scientific Instruments Inc.) was used for the LC-MS/MS analysis in the positive electrospray ionization and multiple reaction monitoring (MRM) mode. The chromatographic separation was achieved on a Zorbax 300-SCX column (3.0 mm × 50 mm, 5 µm; Agilent Technologies, Santa Clara, CA) under 40°C at a flow rate of 0.4 ml/min using an isocratic method. The mobile phase consisted of 50 mM ammonium formate in acetonitrile/water (50/50, v/v, pH 3.2). MFM, PYR, and d6-metformin were detected at MRM transitions of m/z 130.0 → 60.0, 249.1 → 177.1, and 136.1 → 60.0, respectively. The calibration standards and quality control samples were prepared in charcoal-stripped plasma and urine that were absent of the analytes. The curves were linear in the range of 0.05–10 ng/ml (weighting 1/×2). Assay performance was evaluated using the quality controls in terms of assay selectivity, precision (≤6%), and accuracy (≤15%).

Data Analysis.

The data represent the results from a single in vitro study run in triplicate and a minimum of two experiments were performed. For example, Supplemental Fig. 4 shows two experiments conducted for a single inhibition study. To estimate transport kinetics parameters of MFM into cynomolgus monkey and human transporter-expressing HEK 293 cells, the transporter-mediated uptake was calculated by subtracting the uptake in mock-transfected cells from that in transporter-expressing HEK 293 cells. The following equation was used to estimate the parameters:Embedded Imagewhere V is the rate of uptake measured at the given concentration; Vmax is the maximal rate of uptake; Kt represents the Michaelis-Menten constant at which the transport rate is half its maximal value; [S] is the substrate concentration, and CLint is the intrinsic clearance.

For transporter inhibition studies, IC50 values, the concentration of inhibitor required for 50% inhibition of uptake, were calculated using the following equation:Embedded Imagewhere y is percent of control (MFM uptake in the absence of inhibitor) at the given inhibitor concentration (I); γ is the Hill coefficient that describes steepness of inhibition curve. Vmax, Kt, and IC50 were determined by fitting data to the equations using Phoenix WinNonlin 6.3 (Certara, L.P., St. Louis, MO).

For pharmacokinetic analysis, the noncompartmental analyses of MFM and PYR plasma concentration-time data were performed to estimate the area under the plasma concentration-time curve (AUC), clearance (CL), steady-state volume of distribution (Vss), apparent terminal half-life (T1/2), maximal plasma concentration (Cmax), and the time to reach maximal plasma concentration (Tmax) using Kinetica (Thermo Fisher Scientific, Waltham, MA). Renal clearance (CLR) was calculated as the ratio of the cumulative urinary excretion (Ae; amount recovered in urine) to plasma AUC:Embedded ImageData are also reported as geometric mean ratio of MFM pharmacokinetic parameter in the presence of PYR versus absence of PYR. In each case, a two-sided 90% confidence interval (90% CI) for the geometric mean ratio between the treatments was calculated. Unpaired or paired Student’s t test was applied according to the nature of each data set. Data were analyzed using Prism (GraphPad Software, Inc., San Diego, CA). A P value of less than 0.05 was considered to be statistically significant (*P < 0.05, **P < 0.01, and ***P < 0.001).

Results

Cloning of cOCT2, cMATE1, and cMATE2K.

PCR primers based on the conserved nucleotide sequences just outside the coding frames of human, chimpanzee, and rhesus monkey OCT2, MATE1, and MATE2K were used to amplify full-length fragments from pooled cynomolgus monkey kidney cDNA. The nucleotide sequences of cOCT2, cMATE1, and cMATE2K are reported as GenBank Accession Numbers KP731382, KP731383, and KP731384, respectively. The derived amino acid sequences of cOCT2, cMATE1, and cMATE2K were found to be 93.7, 94.7, and 95.4% identical and 96.9, 96.0, and 97.4% similar (i.e., substitution of residues with similar chemical and functional properties) to hOCT2, hMATE1, and hMATE2K reference sequences, respectively (Supplemental Fig. 1). In addition to the splice form that is orthologous to human MATE2K transcript (NM_001099646), a different splice isoform that omits exon 6, resulting in a loss of 15 amino acids (GenBank KP731385) , was found in 2 of 8 cMATE2K clones. No human equivalent for this splice form currently has been reported in National Center for Biotechnology Information GenBank. There was a single nucleotide discrepancy in 6 of 7 cOCT2 clones, resulting in an amino acid difference (M194T) compared with the published human, chimpanzee, and rhesus monkey OCT2 sequences.

To characterize transport activity, the recombinant Flp-In expression vectors were prepared using the fragments of full-length cOCT2, cMATE1, and cMATE2K. The vectors were coexpressed with one containing Flp recombination target-integrating enzyme in HEK 293 cells by stable transfection. Positively transfected cells were selected by hygromycin B resistance and screening uptake of [3H]MPP+ and [14C]MFM. Increased expression of the monkey transporter was shown by dramatically enhanced expression of mRNA levels compared with the mock cells transfected with empty vector using reverse transcription polymerase chain reaction method (data not shown). The transporter-overexpressing cell lines selected for further studies were then subjected to LC-MS/MS analysis to determine transporter protein content, and the results revealed comparable expression levels between cynomolgus monkey and human transporter transfected cell lines (monkey versus human: 39.4 ± 1.4 versus 58.7 ± 1.4, 347 ± 31.1 versus 329 ± 30.8, 41.7 ± 3.7 versus 18.6 ± 1.1 pmol/mg membrane protein for OCT2, MATE1, and MATE2K, respectively).

Transporter Activity in the Presence of cOCT2, cMATE1, and cMATE2K.

To confirm that cOCT2, cMATE1, and cMATE2K proteins in the transfected HEK 293 cells were functionally active, uptake studies were conducted with four cationic drugs and two anionic drugs. Cellular uptake of the radiolabeled drugs was determined after 5 minutes of incubation and it was found that all three monkey transporters were active (versus mock cells) with MFM, MPP+, TEA, and CMD (P < 0.001; Fig. 1, A–D), similar to hOCT2 and hMATEs (Okuda et al., 1999; Kimura et al., 2005; Otsuka et al., 2005; Tahara et al., 2005; Tanihara et al., 2007; Zolk et al., 2009b). With the exception of E3S for hMATE2K (∼2-fold versus mock cells), both monkey and human OCT2 and MATEs exhibited low uptake rates with E3S and MTX although some uptake rates are significantly greater than that in mock cells (Fig. 1).

Fig. 1.
Fig. 1.

Comparison of uptake of [14C]MFM (A), [3H]MPP+ (B), [14C]TEA (C), [3H]CMD, [3H]E3S (E), and [3H]MTX (F) into HEK 293 cells overexpressing cynomolgus monkey and human OCT2, MATE1, or MATE2K and into vector control cells (mock). Cells were incubated for 5 minutes with the radiolabeled compounds. Data are shown as mean ± S.D. (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001, when the uptake with transporter-expressing cells was compared with that in mock cell.

cOCT2, cMATE1, and cMATE2K Exhibit Similar Time Course and pH-Dependent Transport of MFM Compared with Human Organic Cation Transporters.

To further examine functional activity of cOCT2, cMATE1, and cMATE2K, the time and pH dependence of MFM uptake was also assessed. In cOCT2-HEK and cMATE1-HEK cells, the uptake of [14C]MFM was almost identical to that in hOCT2-HEK and hMATE1-HEK cells, respectively, was linear over 2 minutes, and displayed a plateau after 5 minutes (Fig. 2, A and B). Likewise, OCT2 and MATE1 protein expression levels are similar (less than 2-fold difference) between monkey and human transporter transfected cell lines (i.e., monkey versus human: 39.4 ± 1.4 versus 58.7 ± 1.4 and 347 ± 31.1 versus 329 ± 30.8 pmol/mg membrane protein for OCT2 and MATE1, respectively). In contrast, [14C]MFM uptake rate by cMATE2K was approximately 2-fold higher than that by hMATE2K (Fig. 2C). In agreement, the transporter protein level was found to be about 2-fold greater in cMATE2K-HEK cells than in hMATE2K-HEK cells (41.7 ± 3.7 versus 18.6 ± 1.1 pmol/mg membrane protein). [14C]MFM uptake was shown to be linear for both cMATE2K and hMATE2K during the first 2 minutes (Fig. 2C). Mock-HEK cells transported substantially low quantities of MFM compared with the transporter-expressing cells, showing a nonsaturable and linear uptake (Fig. 2). A 2-minute uptake window was selected for use in subsequent pH-dependent, kinetic, and inhibition studies because it covered the initial rate of MFM uptake into the transporter-expressing cells.

Fig. 2.
Fig. 2.

Time course for the uptake of [14C]MFM into HEK 293 cells overexpressing cynomolgus monkey and human OCT2 (A), MATE1 (B), or MATE2K (C) compared with vector control cells (mock). Cells were incubated with 2 µM [14C]MFM up to 10 minutes. Data are shown as mean ± S.D. (n = 3).

MATE1- and MATE2K-mediated transport is stimulated by an oppositely directed proton gradient (Otsuka et al., 2005; Masuda et al., 2006; Konig et al., 2011). Therefore, specific uptake of 1 µM [14C]MFM into the monkey transporter-expressing cells was measured in the presence of different extracellular pH conditions (5.0, 6.0, 7.0, 7.4, 8.0, 9.0, 10.0, and 11.0) (Fig. 3). The pH-dependence experiments have been done with the buffer containing HEPES. HEPES has a pKa of 7.5 at 25°C and provides robust buffering capacity in the pH range of 6.8 to 8.2. Although HEPES and HCl or NaOH can be blended to produce a buffer at any pH between 5 and 11 in the experiment, the buffering capacity may not be very good at pH beyond the range of 6.8 to 8.2. In the presence of stably transfected HEK cells, cMATE1- and hMATE1-mediated MFM uptake was comparable at various extracellular pH values. Both cMATE1- and hMATE1-mediated uptake was increased with increasing extracellular pH and was maximal at pH 9.0 and then decreased when pH was raised from 9.0 to 11.0 (Fig. 3B). The cMATE2K- and hMATE2K-mediated MFM uptake also demonstrated pH dependence. At low extracellular pH (less than 7.4), the MFM uptake is negligible, whereas the uptake increased significantly when pH was raised from pH 7.4 with maximum uptake at pH 10.0 (Fig. 3C). In contrast, cOCT2- and hOCT2-mediated uptake appeared considerably less sensitive to the increase of pH (less than 2.5-fold difference) (Fig. 3A). This observation is consistent with that reported in the literature (Urakami et al., 1998; Okuda et al., 1999). The assessment of pH dependence of transport includes the measurement of [14C]MFM uptake at high pH values that are physiologically irrelevant (greater than 8.0).

Fig. 3.
Fig. 3.

pH-dependent uptake of [14C]MFM into HEK 293 cells overexpressing cynomolgus monkey and human OCT2 (A), MATE1 (B), or MATE2K (C). Cells were incubated with 1 µM [14C]MFM for 2 minutes. Extracellular pH was varied between 5.0 and 11.0. Data are shown as mean ± S.D. (n = 3).

cOCT2, cMATE1, and cMATE2K Show Similar MFM Transport Kinetics Compared with Human Organic Cation Transporters.

To evaluate MFM transport kinetics by monkey and human organic cation transporters and compare the efficacy of transport and affinity for the substrate, the initial rates of MFM uptake, measured after 2-minute incubation, were determined for MFM concentrations ranging from 4.6 to 10,000 μM. The uptake into transporter-overexpressing cells was corrected for nonspecific binding and passive diffusion components (i.e., uptake into mock-HEK cells at each concentration), as described under Materials and Methods, to obtain the saturable component (Fig. 4, open triangles and circles). All three cynomolgus monkey organic transporters showed similar saturable transport kinetics and followed a Michaelis-Menten curve for MFM uptake in comparison with the corresponding human transporters, with Kt and Vmax less than 2.3-fold difference and only slight differences in CLint values when normalized to total protein concentration in cell lysates (Table 2). To be specific, cMATE1 and hMATE1 showed the highest affinity for MFM among transporters in each species, with an estimated Kt of 340 ± 29.4 versus 228 ± 15.3 µM, respectively, followed by OCT2 and MATE2K. Moreover, cOCT2 and hOCT2 showed the highest efficiency of transport, with a CLint of 105 versus 104 μl/min/mg protein, that is, 6- to 12-fold higher than MATE1 and MATE2-K, which showed the same transport efficiency (17.4 versus 19.7 and 10.2 versus 8.7 μl/min/mg protein, respectively). Furthermore, when normalized to the expression of the individual transporter protein in each cell line, the monkey-to-human ratios of Vmax and CLint are less than twofold (Table 2). It should be noted that the Vmax/Kt ratio (CLint) determined for each transporter, employing the full range of substrate concentrations chosen, could be estimated based on the initial uptake rate measured at the low substrate concentration of 2 µM (<< Kt) (cOCT2: 44.2 versus 105; hOCT2: 51.0 versus 104; cMATE1: 17.2 versus 17.4; hMATE1: 15.3 versus 19.7; cMATE2K: 12.0 versus 10.2; and hMATE2K: 6.0 versus 8.7 µl/min per mg protein). Therefore, it is assumed that uptake rates across the range of substrate concentrations were linear with time.

Fig. 4.
Fig. 4.

Concentration-dependent uptake of [14C]MFM into HEK 293 cells overexpressing cynomolgus monkey and human OCT2 (A), MATE1 (B), or MATE2K (C). Cells were incubated with [14C]MFM (4.6 to 10,000 µM) for 2 minutes (linear range). Transporter-mediated [14C]MFM transport was determined as the difference in uptake into the transporter overexpressing cells versus mock cells at each substrate concentration. The curves represent the best fit of the Michaelis-Menten equation (mock subtracted net-active uptake component). Data are shown as mean ± S.D. (n = 3).

View this table:
TABLE 2

Parameters describing the kinetics of MFM transport by cynomolgus monkey and human OCT2, MATE1, and MATE2K

PYR, CMD, QD, VDN, KCZ, and IPM inhibit cOCT2-, cMATE1-, and cMATE2K-Mediated MFM Uptake at Similar Concentrations Compared with Human Organic Cation Transporters.

To characterize species difference in inhibitory potencies of the standard organic cation inhibitors, a concentration-dependent effect of these compounds was evaluated in the transporter-overexpressing HEK 293 cells, and the concentrations of inhibitors rendering 50% inhibition of MFM uptake (IC50) were evaluated by fitting the data as described under Materials and Methods. PYR, a known organic cation transporter inhibitor, equally inhibited MFM uptake by monkey and human transporters in a concentration-dependent manner (Fig. 5 and Table 3), with similar IC50 values observed for OCT2 (cOCT2 versus hOCT2: 1.2 ± 0.38 versus 4.1 ± 0.58 µM), MATE1 (cMATE1 versus hMATE1: 0.17 ± 0.04 versus 0.11 ± 0.04 µM), and MATE2K (cMATE2K versus hMATE2K: 0.25 ± 0.04 versus 0.15 ± 0.01 µM) (Table 3). This was also observed for other known inhibitors, with the exception of IPM against OCT2 (monkey versus human: 12.3 ± 1.8 versus 2.1 ± 0.10 µM) (Table 3; Supplemental Fig. 3). In all cases, because the MFM concentrations used to generate the IC50 values were significantly lower than the Kt for monkey and human OCT2, MATE1, and MATE2K, the Ki values are likely equal to IC50 values if the transport inhibition is competitive in nature.

Fig. 5.
Fig. 5.

Inhibition of OCT2 (A)-, MATE1 (B)-, and MATE2K (C)-mediated uptake of [14C]MFM by PYR. Increasing concentrations of PYR (0.21 to 150 µM for OCT2, 0.01 to 5.6 µM for MATEs) were added simultaneously with 2 µM [14C]MFM for 2-minute incubation (linear range). Extracellular pH was 7.4 or 8.4 for OCT2 or MATEs, respectively. The extent of inhibition of transporter-mediated uptake is expressed as a percentage of the uptake in the absence of inhibitor. Nonlinear regression analysis of the data was used to determine apparent IC50 values of PYR (IC50s reported in Table 3). Data are shown as mean ± S.D. (n = 3).

View this table:
TABLE 3

Inhibition of cynomolgus monkey and human organic cation transporters by six selected inhibitors

Effect of Intravenous PYR on the Pharmacokinetics of MFM.

To examine the in vivo inhibitory effect of PYR on the MFM uptake mediated by renal organic cation transporters, a single intravenous dose DDI study was conducted with three male cynomolgus monkeys. The intravenous dose of PYR (0.5 mg/kg) increased the area under the concentration-time curve from time zero to infinity (AUC0-inf) of intravenous MFM (Fig. 6A). The AUC0-inf of MFM was significantly increased by 123% (46.4 ± 9.0 versus 102 ± 2.3 µM⋅h; P < 0.01); the 90% CI of the MFM AUC ratio was 1.57 to 3.17 (Table 4). There was a significant 54% (± 9%) decrease in MFM clearance (CL) (P < 0.05) and no change in steady-state volume of distribution (Vss) between treatments (0.98 ± 0.16 versus 0.88 ± 0.21 l/kg), resulting in a significant 98% increase in apparent terminal half-life (T1/2) between treatments (7.1 ± 1.3 versus 13.9 ± 1.8 hours; P < 0.01). In addition, the monkey urine samples were collected for up to 48 hours after intravenous administration of MFM, and approximately 80% of the dose was excreted in the urine within 7 hours irrespective of PYR treatment (Fig. 7A). The decrease in total CL can be solely attributed to the decrease in renal clearance of MFM (CLR) when coadministered with PYR (from 11.2 ± 2.4 to 5.0 ± 0.1 ml/min/kg versus from 10.7 ± 3.1 to 5.3 ml/min/kg) (Table 4). Consistently almost complete MFM dose was excreted unchanged in the urine after intravenous administration with or without PYR (Fig. 7A).

Fig. 6.
Fig. 6.

Mean plasma concentrations of MFM [intravenous dose (A) and oral dose (B)] and PYR (C) in cynomolgus monkeys after a single intravenous dose of 3.9 mg/kg MFM with and without PYR given as an intravenous dose (0.5 mg/kg; n = 3) and after a single oral dose of 8.6 mg/kg MFM with and without PYR given as an oral dose (2.5 mg/kg; n = 2). Insets depict the same data over a 7-hour period.

View this table:
TABLE 4

Summary of pharmacokinetic parameters for MFM and PYR in cynomolgus monkeys (n = 3) after a single intravenous dose of MFM (3.9 mg/kg) with and without an intravenous dose of PYR (0.5 mg/kg)

Fig. 7.
Fig. 7.

Effects of PYR on urinary exertion of MFM after a single intravenous dose of MFM (3.9 mg/kg) alone or with PYR (0.5 mg/kg i.v.) (A) and after single oral dose of MFM (8.6 mg/kg) alone or with PYR (2.5 mg/kg by mouth) (B) in cynomolgus monkeys. The amount of MFM excreted in urine in monkeys was determined for the MFM alone (▪) and PYR-treated (□) groups. PYR was dosed 1 hour ahead of MFM.

The pharmacokinetics of PYR in monkeys was evaluated also. At a 15-minute intravenous infusion dose of 0.5 mg/kg, the PYR plasma concentration at 1.25 hour postdose (C1.5 hour) was 571 ± 23.6 nM. The PYR plasma level at 49 hours after PYR dosing (C49 hour) was 82.9 ± 22.0 nM (Fig. 6C and Table 4).

Effect of PYR on the Pharmacokinetics of Oral MFM.

It was also possible to conduct a single-dose DDI study after an MFM oral dose of 8.6 mg/kg and an oral 2.5 mg/kg dose of PYR (2 male cynomolgus monkeys). The AUC0-inf of MFM decreased by 54% with concomitant PYR compared with MFM alone (65.7 versus 30.6 µM⋅h) (Fig. 6B; Table 5). Furthermore, the urinary recovery over the 0- to 24-hour collection period also reduced by 42% (Fig. 7B). As a result, there is no difference in CLR between treatments (9.3 versus 11.2 ml/min/kg) (Table 5). After an oral dose of 2.5 mg/kg, PYR plasma levels reached a peak of 246 nM, with a Tmax of 4.0 hours (Fig. 6C). PYR systemic exposure after the oral dose of 2.5 mg/kg was significantly less than after the intravenous dose of 0.5 mg/kg (Fig. 6C). For PYR, a second peak plasma concentration was apparent after intravenous and oral administration (Fig. 6C). Because enterohepatic recirculation of PYR has been demonstrated in rat, dog, and human (Cavallito et al., 1978; Coleman et al., 1985), it is hypothesized that the same process is operative in the monkey.

View this table:
TABLE 5

Summary of pharmacokinetic parameters for MFM and PYR in cynomolgus monkeys (n = 2) after a single oral dose of MFM (8.6 mg/kg) with and without an oral PYR dose (2.5 mg/kg)

Discussion

Recently, an increasing number of investigators have used the cynomolgus monkey as a model to study the inhibition of drug transporters in vivo (Tahara et al., 2006; Shen et al., 2013, 2015b; Takahashi et al., 2013; Uchida et al., 2014; Chu et al., 2015; Karibe et al., 2015). Such a model offers advantages in drug development, when the transporter inhibition potential of a clinical candidate requires more extensive evaluation of changes in victim drug pharmacokinetics and organ toxicity. However, compared with other transporters, there is very limited information related to cOCT2, cMATE1, and cMATE2K. All three are known to be expressed in the kidney and, like their human counterparts, likely function to mediated the secretion of substrates such as MFM, creatinine, and cisplatin (Urakami et al., 2004; Tanihara et al., 2007, 2009; Imamura et al., 2011; Shen et al., 2015a). It is worth noting that although the impact of transporters on systemic exposure is relatively modest (less than threefold), their effects on drug distribution or tissue exposure could be more dramatic.

For the first time, the cloning of full-length cOCT2, cMATE1, and cMATE2K cDNAs is reported. The three cDNAs were shown to have a high derived amino acid sequence homology to hOCT2, hMATE1, and hMATE2K (96.9, 96.0, and 97.4%, respectively) (Supplemental Fig. 1). It is notable that the counterparts of hMATE2K have not been cloned in rodents (Terada and Inui, 2008; Motohashi and Inui, 2013), and rabbit MATE2K has only 74% amino acid identity to hMATE2K (Zhang et al., 2007), although both hMATE1 and hMATE2K are highly expressed in kidney at similar mRNA levels. Two spliced isoforms of cMATE2K were identified that differ from each other by 15 residues. However, no human equivalent spliced deletion isoform has been reported. It is unknown which isoform represents the true wild type, because cDNAs representing each form were derived from a pooled kidney RNA prepared from three animals and the frequency of these forms in a larger population was not determined. However, the deleted isoform unlikely encoded functional transporter because no increased activity was observed (versus mock cells) with [3H]MPP+ and [14C]MFM as substrates (Supplemental Fig. 2).

Functionally, monkey and human renal organic cation transporters were found to be very similar. For example, cOCT2, cMATE1, and cMATE2K transported MFM, MPP+, TEA, and CMD into the transporter-overexpressing cells at similar rates comparable to hOCT2, hMATE1, and hMATE2K. In contrast, E3S and MTX, two organic anions, are unlikely substrates for monkey and human organic cation transporters, with the exception of E3S for hMATE2K [approximately twofold increased uptake into cMATE2K-HEK cells compared with Mock-HEK cells (Fig. 1)]. In addition, monkey and human renal organic transporters transported MFM into HEK 293 cells in similar time- and concentration-dependent manner (Figs. 2 and 4), resulting in a comparable Kt between two species (Table 2). The Kt values generated in the present studies are in agreement with those of human organic transporters reported previously (Kimura et al., 2005; Masuda et al., 2006; Choi et al., 2007; Tanihara et al., 2007; Chen et al., 2009; Zolk et al., 2009a,b; Meyer zu Schwabedissen et al., 2010). Moreover, the monkey-to-human ratios of Vmax and CLint are less than twofold when normalized to the individual transporter protein level in each cell line (Table 2), suggesting a similar transport rate and efficiency for OCT2, MATE1, and MATE2K between species. It has been reported that both hMATE1 and hMATE2K act as proton/substrate antiporters (Otsuka et al., 2005; Masuda et al., 2006; Muller et al., 2011), and we found that both cMATE1- and cMATE2K-mediated MFM transport were stimulated by an oppositely directed proton gradient with maximal uptake occurring at pH 9.0 and 10.0, respectively (Fig. 3). These results support the hypothesis that the transport functions of hOCT2, hMATE1, and hMATE2K are similar to those of the corresponding monkey forms.

Although several compounds (PYR, CMD, QD, VDN, KCZ, and IPM) were tested as inhibitors of MFM uptake by cOCT2, cMATE1, and cMATE2K in vitro (Table 3), PYR was selected for in vivo evaluation as an inhibitor, because it is a well-established potent inhibitor of MATE1 and MATE2K (Ito et al., 2010; Kusuhara et al., 2011). In 8 healthy volunteers, the concomitant oral administration of 50 mg PYR (as antimalarial) is known to inhibit renal MFM clearance by 23 and 35% at the microdose (100 µg) and therapeutic dose (250 mg), respectively, and increase MFM plasma AUC by 39% at the therapeutic dose but not after a microdose (Kusuhara et al., 2011). The interaction is likely the result of the inhibition of both basolateral OCT2 and apical MATEs. The plasma concentrations of PYR after a single oral dose of 50 mg PYR were not reported in the clinical study, and it was not possible to compare the total and unbound plasma drug levels to its IC50 values. In the present study, intravenous infusion of 0.5 mg/kg PYR in cynomolgus monkeys reduced MFM clearance by 54 ± 9%, resulting in an increase in MFM AUC by 123% (Table 4). This is in line with the in vivo inhibitory effect of PYR in humans (Kusuhara et al., 2011). In contrast, an intravenous infusion of 2 µmol/kg (or approximately 0.5 mg/kg) PYR in mice has been shown not to affect MFM plasma concentrations and urinary excretion rates, although the PYR pretreatment significantly increases the kidney-to-plasma ratios of MFM (Ito et al., 2010).

In the present study, oral coadministration of 2.5 mg/kg PYR to cynomolgus monkeys decreased MFM AUC by 54% (Table 5). Such a decrease in MFM AUC appears to contradict PYR’s potent inhibition of the luminal efflux (Tables 3 and Fig. 5). This discrepancy can be explained as follows. First, the blood and renal proximal tubular intracellular concentrations of PYR after oral administration are unlikely sufficient to affect the vectorial transport of MFM across renal proximal tubules. This view is supported by the fact that the CLR of MFM was not changed at all after oral treatment of PYR (Table 5). Indeed, the Cmax and AUC values in monkeys after oral administration of 2.5 mg/kg PYR are estimated to be 7- and 41-fold less than those in humans when dosed orally at 50 mg (Weidekamm et al., 1982) and approximately 3-fold less than those in monkeys after intravenous dose of 0.5 mg/kg PYR (Tables 4 and 5). The absolute oral bioavailability (F) of PYR in monkeys is approximately 7%, which is less than that reported for human subjects (∼17%) (Weidekamm et al., 1982; Karibe et al., 2015). Given the fact that intravenous CL of PYR is low compared with monkey hepatic blood flow (2.4 versus 44 ml/min/kg), liver extraction cannot explain the low plasma levels after PYR oral administration. For PYR, therefore, incomplete absorption and gut first pass is implicated. One or both can render a lower Cmax,u/IC50 ratio after an oral dose. As described above, consistent with reports employing hOCT2, hMATE1, and hMATE2K (Takano et al., 1984; Ito et al., 2010; Kusuhara et al., 2011), PYR was shown to be an cOCT2, cMATE1, and cMATE2K inhibitor with IC50 values of 1.2 ± 0.38, 0.17 ± 0.04, and 0.25 ± 0.04 μM, respectively (Table 3). Assuming that the binding of PYR to monkey plasma proteins is equal to that of human plasma proteins (i.e., 94%) (Hsyu and Giacomini, 1987), the Cmax,u/IC50 ratios after intravenous infusion of 0.5 mg/kg PYR are greater than the cutoff level of 0.1 for renal transporter inhibition (0.03, 0.21, and 0.15 for cOCT2, cMATE1, and cMATE2K, respectively), necessitating the conduct of a clinical DDI study based on the proposed US Food and Drug Administration guidelines (US FDA, 2012). Furthermore, the Cmax,u/IC50 ratios after oral administration of 2.5 mg/kg PYR are less than the cutoff level (0.01, 0.09, and 0.06 for cOCT2, cMATE1, and cMATE2K, respectively).

Second, it is hypothesized that oral treatment of PYR affects MFM absorption in cynomolgus monkeys. MFM is a hydrophilic base that exists as a cationic species under physiologic pH conditions and has poor passive membrane permeability. The intestinal absorption of MFM is incomplete and dose dependent (bioavailability of 86 to 42% as the dose increases from 0.25 to 2.0 g), and it has been suggested that MFM absorption is mediated by an active, saturable absorption process (Scheen, 1996; Bell and Hadden, 1997; Klepser and Kelly, 1997). In this study, the F values in monkeys after oral administration of 8.6 mg/kg MFM in the absence and presence of PYR are estimated to be 64% and 30%, respectively. Consistently, the accumulative urinary excretion of MFM dose over 24 hours in the absence and presence of PYR are 65.7% and 30.6%, respectively (Table 5; Fig. 7). However, the transporters mediating active uptake of MFM in the intestine have not been well defined; OCT3 (Muller et al., 2005; Wright, 2005), plasma membrane monoamine transporter (Zhou et al., 2007), and OCTN1 (Nakamichi et al., 2013) have been implicated. Therefore, additional studies may be warranted to evaluate more comprehensively the transporters governing the disposition of MFM in monkeys and humans.

In summary, based on the results of the present study, it is concluded that the cynomolgus monkey may serve as a surrogate animal model to more accurately assess pharmacokinetic changes and toxicity potential occurring as a result of DDIs involving inhibition of renal OCT2, MATE1, and MATE2K.

Acknowledgments

The authors thank Sanjith Kallipatti and Sabariya Selvam (Bristol-Myers Squibb Biocon R&D Center, Bangalore, India) for designing cloning primers for cynomolgus monkey OCT2, MATE1, and MATE2K. We also thank Sumit Gupta for synthesizing cDNA from cynomolgus monkey kidney RNA.

Authorship Contributions

Participated in research design: Shen, Mintier, Fancher, Zeng, Rodrigues, Marathe, and Lai.

Conducted experiments: Shen, Liu, Jiang, Titsch, Taylor, Qiu, Chen, Sukrutharaj, Kuit, Mintier, and Krishnamurthy.

Contributed new reagents or analytic tools: Shen, Liu, Jiang, Titsch, and Mintier.

Performed data analysis: Shen, Liu, Jiang, Kandoussi, Mintier, and Lai.

Wrote or contributed to the writing of the manuscript: Shen, Jiang, Qiu, Mintier, Rodrigues, Marathe, and Lai.

Footnotes

Abbreviations

AUC
area under the concentration-time curve
Cmax
maximum plasma concentration
cMATE1
cynomolgus multidrug and toxin extrusion protein 1
cMATE2K
cynomolgus kidney-specific multidrug and toxin extrusion protein 2
CMD
cimetidine
cOCT2
cynomolgus organic cation transporter 2
CI
confidence interval
CL
clearance
CLR
renal clearance
DDI
drug-drug interaction
E3S
estrone-3-sulfate
HBSS
Hank’s balanced salt solution
HEK 293
human embryonic kidney 293 cells
hMATE1
human multidrug and toxin extrusion protein 1
hMATE2K
human kidney-specific multidrug and toxin extrusion protein 2
hOCT2
human organic cation transporter 2
IC50
concentration of inhibitor required for 50% inhibition of transport
IPM
imipramine
IVIVE
in vitro-in vivo extrapolation
Kt
Michaelis-Menten constant that corresponds to the substrate concentration at which the transport rate is half of Vmax
KCZ
ketoconazole
LC-MS/MS
liquid chromatography coupled with tandem mass spectrometry
MFM
metformin
MPP+
1-methyl-4-phenylpyridinium
MRM
multiple reaction monitoring
MTX
methotrexate
OCT
organic cation transporter
PYR
pyrimethamine
QD
quinidine
T1/2
apparent terminal half-life
Tmax
time to reach maximal plasma concentration
TEA
tetraethylammonium
VDN
vandetanib
Vmax
maximum uptake rate
Vss
steady-state volume of distribution

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Use of Monkey to Assess Transport by OCT2 and MATEs

Hong Shen, Tongtong Liu, Hao Jiang, Craig Titsch, Kristin Taylor, Hamza Kandoussi, Xi Qiu, Cliff Chen, Sunil Sukrutharaj, Kathy Kuit, Gabe Mintier, Prasad Krishnamurthy, R. Marcus Fancher, Jianing Zeng, A. David Rodrigues, Punit Marathe and Yurong Lai
Drug Metabolism and Disposition February 1, 2016, 44 (2) 238-249; DOI: https://doi.org/10.1124/dmd.115.066852
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