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Research ArticleSpecial Section on Transporters in Toxicity and Disease

6β-Hydroxycortisol Is an Endogenous Probe for Evaluation of Drug–Drug Interactions Involving a Multispecific Renal Organic Anion Transporter, OAT3/SLC22A8, in Healthy Subjects

Yuichiro Imamura, Yuri Tsuruya, Katja Damme, Dominik Heer, Yuji Kumagai, Kazuya Maeda, Nobuyuki Murayama, Noriko Okudaira, Atsushi Kurihara, Takashi Izumi, Yuichi Sugiyama and Hiroyuki Kusuhara
Drug Metabolism and Disposition April 2014, 42 (4) 685-694; DOI: https://doi.org/10.1124/dmd.113.055475

Abstract

6β-Hydroxycortisol (6β-OHF) is a substrate of the organic anion transporter 3 (OAT3) and the multidrug and toxin extrusion proteins MATE1 and MATE-2K in the corresponding cDNA-transfected cells. This study aimed to examine the contribution of OAT3 and MATEs to the urinary excretion of 6β-OHF in humans using the appropriate in vivo inhibitors, probenecid and pyrimethamine, for OAT3 and MATEs, respectively. Oat3(–/–) mice showed significantly reduced renal clearance of 6β-OHF (CLrenal, 6β-OHF) compared with wild-type mice (18.1 ± 1.5 versus 7.60 ± 1.8 ml/min/kg). 6β-OHF uptake by human kidney slices was inhibited significantly by probenecid to 20–45% of the control values and partly by 1-methyl-4-phenylpyridinium. 6β-OHF plasma concentration and the amount of 6β-OHF excreted into the urine (X6β-OHF) were measured in healthy subjects enrolled in drug-drug interaction studies of benzylpenicillin alone or with probenecid (study 1), adefovir alone or with probenecid (study 2), and metformin alone or with pyrimethamine (study 3). Probenecid treatment caused a 57 and 76% increase in the area under the plasma concentration–time curve for 6β-OHF (AUC6β-OHF) in studies 1 and 2, respectively, but did not affect X6β-OHF. Consequently, CLrenal, 6β-OHF (milliliters per minute) decreased significantly from 231 ± 11 to 135 ± 9 and from 225 ± 26 to 141 ± 12 after probenecid administration in studies 1 and 2, respectively. By contrast, neither AUC6β-OHF nor CLrenal, 6β-OHF was significantly altered by pyrimethamine administration. Taken together, these data suggest that OAT3 plays a significant role in the urinary excretion of 6β-OHF, and that 6β-OHF can be used to investigate the perpetrators of the pharmacokinetic drug interactions involving OAT3 in humans.

Introduction

Pharmacokinetic drug-drug interactions (DDIs) involving drug transporters that cause accumulation of victim drugs in the body can potentiate a pharmacological effect or cause adverse reactions (Giacomini et al., 2010; DeGorter et al., 2012; Konig et al., 2013; Yoshida et al., 2013). Typically, appropriate probe drugs are used when quantitatively evaluating the magnitude of inhibition by test drugs of drug transporters. Apart from drugs, endogenous substances and food-derived compounds have been identified as substrates of drug transporters. These compounds include creatinine, thiamine, and N-methylnicotinamide for the organic cation transporter 2 (OCT2), multidrug and toxin extrusion proteins (MATEs) (Gorboulev et al., 1997; Urakami et al., 2004; Masuda et al., 2006; Tanihara et al., 2007), and various endogenous anionic metabolites for organic anion transporters (OATs) (Deguchi et al., 2004; Wikoff et al., 2011). Plasma or urinary levels of such substances enable the monitoring of drug transporter activities without the need to administer substrate drugs (Fromm, 2012; Kusuhara et al., 2013), for example, when using 6β-hydroxycortisol (6β-OHF) and 4β-cholesterol to evaluate hepatic CYP3A4 activity (Galteau and Shamsa, 2003; Diczfalusy et al., 2011). We have previously reported that the renal clearance of N-methylnicotinamide and thiamine decreased markedly in humans who received an oral dose of pyrimethamine, a potent inhibitor of MATE1/SLC47A1 and MATE2-K/SLC47A2 (Imamura et al., 2011; Ito et al., 2012; Kato et al., 2014).

6β-OHF is produced from cortisol by hepatic CYP3A4 (Abel et al., 1992; Peng et al., 2011), one of the major drug-metabolizing enzymes in the liver and small intestine. Because 6β-OHF is excreted into the urine, the apparent cortisol 6β-hydroxylation clearance (CL6β-OHF) is used as a quantitative index for hepatic CYP3A4 activity in clinical DDI studies (Furuta et al., 2003; Peng et al., 2011). CL6β-OHF is defined as the ratio of the amount of urinary excretion of 6β-OHF to the area under the plasma concentration-time curve of cortisol (AUCcortisol). Cortisol is one endogenous substrate of OAT3 (Asif et al., 2005). During the pharmacokinetic analysis of 6β-OHF in the quantitative assessment of drug interactions involving CYP3A4 using a fluoroquinolone DX-619 (7-[(3R)-3-(1-aminocyclopropyl)pyrrolidin-1-yl]-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-4-oxoquinoline-3-carboxylic acid), we found that the urinary excretion mechanisms of 6β-OHF involved active tubular secretion in humans, and that OAT3/SLC22A8 and MATEs efficiently transported 6β-OHF as a substrate into the corresponding cDNA-transfected cells (Imamura et al., 2013). OAT3 is a multispecific organic anion transporter that plays a major role in the renal uptake of a variety of anionic drugs, zwitterions, and some cationic drugs, as well as endogenous metabolites including uremic toxins in the kidney (Nigam et al., 2007; Burckhardt, 2012; Kusuhara et al., 2013; Wang and Sweet, 2013). It is possible that 6β-OHF is an endogenous probe that may be useful for evaluating the activity of OAT3 and/or MATEs in the kidney.

The primary purpose of this study was to examine the effects of OAT3 and MATE inhibitors on renal clearance of 6β-OHF (CLrenal, 6β-OHF) in healthy subjects to demonstrate directly the sensitivity of 6β-OHF plasma and urine concentrations to the activity of these transporters. We measured 6β-OHF plasma and urinary concentrations in biologic specimens from healthy subjects enrolled in a pharmacokinetic interaction study involving probenecid or pyrimethamine. In study 1, subjects were given benzylpenicillin alone [400,000 U, by mouth (PO)] or concomitantly with probenecid (750 mg, PO); in study 2, they were given adefovir alone (10 mg, PO) or concomitantly with probenecid (750 mg, PO); and in study 3, they were given metformin alone (100 μg, PO) or concomitantly with pyrimethamine (50 mg, PO). Probenecid is a well known, potent inhibitor of OAT1 and OAT3 (Cihlar and Ho, 2000; Tahara et al., 2005; Nozaki et al., 2007b; Takahara et al., 2013) and significantly reduces the renal clearance of various drugs that undergo tubular secretion by OATs (Masereeuw and Russel, 2010; Kusuhara et al., 2013). Pyrimethamine is a potent MATE inhibitor (Ito et al., 2010), and at its therapeutic dose, it significantly reduces the renal clearance of metformin (Kusuhara et al., 2011).

Materials and Methods

Chemicals and Reagents.

Cortisol, 6β-OHF, probenecid, pyrimethamine, and carbenoxolone were purchased from Sigma-Aldrich (St. Louis, MO). d4-Cortisol and d4-6β-OHF were purchased from Toronto Research Chemicals (Toronto, ON, Canada). Pooled human liver microsomes were purchased from BD Gentest (Woburn, MA). All other chemicals and reagents were of analytical grade and are commercially available.

Animals.

Oat3(–/–) mice were obtained from Deltagen, Inc. (San Carlos, CA). Wild-type and Oat3(–/–) mice (female, 23–25 weeks old, and 21–27 g) were used in this study. All animals were maintained under standard conditions with a reverse dark-light cycle. Food and water were available ad libitum. Oat3(–/–) mice do not show any abnormality except lack of the ability to uptake from extracellular space and secrete anionic compounds into the urine (Sweet et al., 2002; Vanwert et al., 2007); they also have 10–15% lower blood pressure than wild-type mice (Vallon et al., 2008). The studies were conducted in accordance with the guidelines provided by the Institutional Animal Care Committee at the Graduate School of Pharmaceutical Sciences (University of Tokyo, Tokyo, Japan).

Determination of Renal Clearance of 6β-Hydroxycortisol in Wild-Type and Oat3(–/–) Mice.

After anesthesia with isoflurane, the urinary bladder was catheterized for urine collection. 6β-OHF (5.8 or 7 nmol/min/kg) was infused via the jugular vein. Blood samples were collected via the jugular vein at 30, 60, and 90 minutes after administration and centrifuged to obtain plasma. Urine specimens were collected at 30–60 and 60–90 minutes. At the end of the experiment, the kidneys and liver were removed. Drug concentration was determined by liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis as described later.

The fractional urinary excretion ratio (Furine), total body clearance (CLtot, 6β-OHF), CLrenal, 6β-OHF, and kidney-to-plasma concentration ratio (Kp,kidney) of 6β-OHF were calculated using the following equations:Embedded Image(1)Embedded Image(2)Embedded Image(3)Embedded Image(4)where V and I represent urinary excretion and infusion rates, respectively, and C represents the concentration in the kidney and plasma (Ckidney and Cp, respectively).

Preparation of Human Kidney Slices and Uptake of 6β-OHF by Human Kidney Slices.

Experimental procedures were performed according to the guidelines of the charitable, state-controlled foundation Human Tissue and Cell Research (Regensburg, Germany), with informed patients consent approved by the local ethics committee of the University of Regensburg, Germany. Human renal samples were obtained from three patients (1 female and 2 male patients) who underwent surgical nephrectomy for cancer, and the intact renal cortical segments were obtained from the specimens.

The human renal sections were provided by Hepacult GmbH (Regensburg, Germany) after being commissioned by Human Tissue and Cell Research and Daiichi Sankyo Europe GmbH (Munich, Germany). Uptake studies with human kidney slices (200 μm thick) were carried out following previous reports (Nozaki et al., 2007a; Matsushima et al., 2009). The buffer for the present study consisted of 120 mM NaCl, 16.2 mM KCl, 1 mM CaCl2, 1.2 mM MgSO4, and 10 mM NaH2PO4/Na2HPO4 (pH 7.5). One slice per tube, weighing 7–23 mg, was selected and incubated at 37°C on a metal block thermostat (Gebr. Liebisch GmbH & Co. KG, Bielefeld, Germany) with oxygenated buffer containing 6β-OHF with or without inhibitors. After 20-minute incubation, slices were washed three times in ice-cold Hanks’ balanced salt solution buffer, blotted on filter paper, weighed, and homogenated with 0.5 ml of Hanks’ balanced salt solution.

For monitoring OAT1, OAT3, and OCT2 activity, uptake of the corresponding reference compounds, 3H-adefovir (0.2 µCi/ml), 14C-benzylpenicillin (0.1 μCi/ml), and 14C-TEA (0.1 μCi/ml), was determined. Washed slices were solubilized with Solvable (Packard BioScience BV, Groningen, The Netherlands) at 37°C overnight and subsequently analyzed in a liquid scintillation counter (Tri-Carb; PerkinElmer, Rodgau, Germany). 6β-OHF in the slices was extracted by acetonitrile with internal standard (d4-6β-OHF), and the specimens were subjected to LC-MS/MS analysis as described later.

Effect of Probenecid and Pyrimethamine on CYP3A4 and 11β-Hydroxysteroid Dehydrogenase 2 Activity.

CYP3A4-mediated midazolam 1′-hydroxylation was determined in human liver microsomes in the absence and presence of probenecid or pyrimethamine (100 µM), and with or without preincubation for 5, 10, 20, and 30 minutes, as reported previously (Imamura et al., 2013). Activity of 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) was determined using microsomes prepared from Sf9 cells expressing 11β-HSD2, as described earlier (Imamura et al., 2013). Briefly, 11β-HSD2–mediated production of cortisone was measured after 20-minute incubation in the presence and absence of probenecid or pyrimethamine (300 µM). Quantification was conducted by LC-MS/MS as described later.

Cell Culture and In Vitro Transport and Inhibition Studies.

Construction of human embryonic kidney 293 (HEK293) cells stably expressing human OAT3 (hOAT3) and human MATE1 and in vitro transport studies were performed as described previously (Enomoto et al., 2003; Ohta et al., 2009). The data obtained from the inhibition study (Fig. 2A) can be fitted to the following equation to calculate the half maximal inhibitory concentration (IC50):Embedded Image(5)where CLuptake (+pyrimethamine) and CLuptake (control) are the uptake clearance determined in the presence or absence of pyrimethamine, and I is the concentration of the inhibitor. Fitting was performed by the nonlinear least-squares method using a MULTI program (Yamaoka et al., 1981), as well as the damping Gauss-Newton method algorithm for fitting.

Clinical Samples for Probenecid/Benzylpenicillin and Probenecid/Adefovir Coadministration in Healthy Subjects.

The following randomly allocated clinical study had been designed to evaluate the effect of probenecid on the urinary excretion of benzylpenicillin and adefovir in healthy Japanese male subjects. The clinical study protocol was approved by the Ethics Review Boards of both the Graduate School of Pharmaceutical Sciences of the University of Tokyo (Tokyo, Japan) and the Clinical Trial Center, Kitasato University East Hospital (Kanagawa, Japan). All participants provided their written informed consent. Determination of cortisol and 6β-OHF in the biologic samples was approved by the Ethics Review Boards of both the Graduate School of Pharmaceutical Sciences of the University of Tokyo and the Clinical Trial Center, Kitasato University East Hospital.

Subjects were between 20 and 35 years old and had a body mass index between 18.5 and 28.5 kg/m2. Twelve healthy subjects were randomly divided into two groups, and six subjects in each group were treated with one of four combinations of substrates (benzylpenicillin or adefovir) and probenecid. The clinical drug interaction study was performed four times in the same subjects [phase 1 (probe drug alone), phase 2 (probe + 500 mg of probenecid), phase 3 (probe + 750 mg of probenecid), and phase 4 (probe + 1500 mg of probenecid) (Supplemental Table 1). Plasma and urine specimens analyzed in this study were from phase 1 (probe drug alone) and phase 3 (probe drug with 750 mg of probenecid). The medical products used in this study were Bicillin G granules 400,000 units (400,000 U benzylpenicillin benzathine hydrate; Banyu Pharmaceutical Co., Ltd, Tokyo, Japan), Hepsera 10-mg tablets (10 mg of adefovir dipivoxil; GlaxoSmithKline plc., Middlesex, UK), and Benecid 250-mg tablets (250 mg of probenecid; Kaken Pharmaceutical Co., Ltd, Tokyo, Japan). The doses of the inhibitors, benzylpenicillin adefovir dipivoxil, and probenecid were 400,000 U (PO), 10 mg (PO), and 750 mg (PO), respectively.

After an overnight fast, each subject received an oral dose of 400,000 U of benzylpenicillin or 10 mg of adefovir with 150 ml of water. Probenecid was administered orally 2 hours before the administration of benzylpenicillin or adefovir. Venous blood samples were collected into heparin-pretreated tubes before and at 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 hours after the administration of the probe drugs and centrifuged. Urine samples were collected in the predose period and periods 0–4 hours, 4–8 hours, and 8–24 hours after treatment with the probe drugs. All of the plasma and urine samples were stored at –80°C until analysis.

Clinical Samples for Metformin/Pyrimethamine Coadministration in Healthy Subjects.

Cortisol and 6β-OHF were quantified in the plasma and urine specimens from subjects who had been enrolled in the aforementioned DDI study with pyrimethamine and metformin (Supplemental Table 1) (Kusuhara et al., 2011). The study protocol was approved by the ethics review boards of both the Graduate School of Pharmaceutical Sciences of the University of Tokyo and the Clinical Investigation Center, Kitasato University East Hospital. All participants provided their written informed consent.

Quantification of Cortisol and 6β-OHF in the Biologic Samples by LC-MS/MS.

Centrifuged plasma or urine samples were mixed with internal standard mixture (d4-cortisol and d4-6β-OHF) and applied to the Oasis HLB µElution SPE plates (Waters Corporation, Milford, MA). Cortisol and 6β-OHF concentrations were measured using a ultra-fast liquid chromatography system (Shimadzu, Kyoto, Japan) coupled with a 5500 QTRAP mass spectrometer (AB-SCIEX, Framingham, MA) using an Accucore RP-MS 2.6 μm 50 × 2.1-mm column (Shiseido Co., Ltd., Tokyo, Japan). The mobile phase comprised solvent A (12 mM ammonium acetate dissolved in 10% methanol) and solvent B (12 mM ammonium acetate dissolved in 90% methanol); the A/B gradient was 80:20 (0–3 minutes) followed by 20:80 (7.5 minutes) at a flow rate of 0.3 ml/min. The analysis was performed in multiple reaction monitoring mode with monitoring of m/z 361.2→331.2 for cortisol, m/z 377.2→347.0 for 6β-OHF, m/z 365.2→335.2 for d4-cortisol, and m/z 381.2→351.0 for d4-6β-OHF. The calibration curve for 6β-OHF was constructed over a concentration range of 0.15–500 for plasma and 0.1–500 ng/ml for urine. The calibration curve for cortisol was constructed over a concentration range of 0.15–500 ng/ml for plasma. The interday variability for the measurement of these compounds was within 15%.

Solubilized kidney slice samples were mixed with internal standard (d4-6β-OHF). 6β-OHF concentrations were measured using a Waters Acquity I-Class ultra-performance liquid chromatography system coupled with Waters Xevo TQ-S triple quadruple mass spectrometer (Waters Corporation) using a BEH C18 1.7 µm, 100 × 2.1 mm column (Waters Corporation), and the mobile phase comprised solvent A (10 mM ammonium acetate and 0.1% formic acid dissolved in 10% acetonitrile) and solvent B (10 mM ammonium acetate and 0.1% formic acid dissolved in 90% acetonitrile); the A/B gradient was 100:0 (0–0.5 minute) at a flow rate of 0.4 ml/min followed by 40:60 (0.5–3 minutes) at 0.35 ml/min. The analysis was performed in multiple reaction monitoring mode with monitoring of m/z 379.4→325.2 for 6β-OHF and m/z 383.4→329.2 for d4-6β-OHF. The calibration curve for 6β-OHF was constructed over the concentration range of 0.15–320 pmol/ml within a 15% coefficient of variation and accuracy margin.

Calculation of CLrenal, 6β-OHF and CL6β-OHF.

CLrenal, 6β-OHF and CL6β-OHF were calculated as follows:Embedded Image(6)Embedded Image(7)where X6β-OHF is the amount of 6β-OHF excreted into the urine and AUC6β-OHF and AUCcortisol are the area under the time-concentration profiles (AUC) of plasma 6β-OHF and cortisol during the designated time intervals, respectively.

Statistical Analysis.

Statistically significant differences in this study were determined using two-tailed unpaired t tests. Differences were considered to be significant at P < 0.05 and < 0.01.

Results

Effect of Impaired Oat3 on the Urinary Excretion Rate of 6β-OHF in Mice.

The endogenous level of 6β-OHF was below the lower limit of quantification in both the plasma and urine specimens. Plasma concentrations and urinary excretions of 6β-OHF were determined in mice given 6β-OHF by constant intravenous infusion. Pharmacokinetic parameters are summarized in Table 1. The urinary excretion rate of 6β-OHF accounts for 33% ± 2% of the infusion rate in wild-type mice (Table 1). The urinary excretion rate was significantly lower in Oat3(–/–) than in wild-type mice, although there was no difference in the plasma concentrations (Fig. 1). Consequently, the CLrenal,6β-OHF was significantly lower in Oat3(–/–) mice, whereas nonrenal clearance was unchanged between wild-type and Oat3(–/–) mice (34.8 ± 3.1 and 38.1 ± 3.1 ml/min/kg, respectively) (Table 1). The kidney-to-plasma concentration ratio of 6β-OHF was decreased by 23% in Oat3(–/–) mice (P < 0.05).

View this table:
TABLE 1

Pharmacokinetic parameters of 6β-OHF in wild-type and Oat3(−/−) mice.

Pharmacokinetic parameters of 6β-OHF were determined as described in Materials and Methods. Data were taken from Fig. 1.

Fig. 1.
Fig. 1.

Plasma concentration (A) and urinary excretion rates (B) of 6β-OHF in wild-type and Oat3(–/–) mice. Wild-type and Oat3(–/–) mice were given 6β-OHF intravenously (7 nmol/min/kg). Plasma concentrations were determined at designated times, and urinary excretion rates were determined during intervals from 30 to 60 and 60 to 90 minutes. Each point and bar represents the mean and S.E. (n = 6–7). **P < 0.01.

Effects of Probenecid and 1-Methyl-4-Phenylpyridinium on the Uptake of 6β-OHF by Human Kidney Slices.

Human kidney slices used in the transport studies were obtained from three subjects. The activities of OAT1, OAT3, and OCT2 in the human kidney slices were monitored routinely using their typical substrates, 3H-adefovir, 14C-benzylpenicillin, and 14C-TEA, respectively. The uptake of 3H-adefovir was reduced to 7.2–13% of the control value by 1 mM probenecid, the uptake of 14C-benzylpenicillin was reduced to 29–36% by 1 mM probenecid, and the uptake of 14C-TEA was reduced to 4.6–7.9% by 1 mM MPP+ (1-methyl-4-phenylpyridinium) (Supplemental Table 2).

The concentration of endogenous 6β-OHF in the blank kidney slices was less than 1% of the amount of 6β-OHF that accumulated in the kidney slices after incubating the kidney slices in the presence of 20 µM exogenous 6β-OHF. 6β-OHF uptake was decreased to 41% (P < 0.01), 25% (P < 0.01), and 20% (P < 0.01) of the corresponding control values in the presence of probenecid, and to 68% (P < 0.01), 58%, and 58% of the corresponding control values in the presence of MPP+ (Fig. 2).

Fig. 2.
Fig. 2.

Effects of probenecid and MPP+ on the uptake of 6β-OHF by human kidney slices. The uptake of 6β-OHF (20 µM) by human kidney slices from three different volunteers [male 70 years (A), female 51 years (B), and male 75 years (C)] for 20 minutes was determined at 37°C. Three slices were used in each batch of human kidney. Each point and bar represent the mean ± S.E. (n = 3). *P < 0.05; **P < 0.01.

Effects of Probenecid and Pyrimethamine on CYP3A4 and 11β-HSD2 Activities.

The inhibition potency of probenecid and pyrimethamine for CYP3A4 and 11β-HSD2, which are involved in the production of 6β-OHF from cortisol and conversion of 6β-OHF to 6β-hydroxycortisone, respectively, was examined in vitro. Midazolam 1′-hydroxylation in human liver microsomes, which is catalyzed predominantly by CYP3A4, was decreased to 29.6% ± 1.1% of the control value by ketoconazole (0.1 µM, direct inhibitor). Probenecid and pyrimethamine (100 µM) had no effect on midazolam 1′-hydroxylation in human liver microsomes.

To examine whether probenecid and pyrimethamine are irreversible inhibitors of CYP3A4, human liver microsomes were preincubated in the presence of inhibitors for 30 minutes prior to the addition of midazolam. Midazolam 1′-hydroxylation in human liver microsomes was decreased to 52.7% ± 1.2% after preincubation in the presence of troleandomycin (30 µM), which is the irreversible-type inhibitor of CYP3A4 (Chan et al., 1998), whereas neither probenecid nor pyrimethamine affected the activity (data not shown).

The activity of 11β-HSD2 (14.8 ± 0.3 pmol/h/mg protein) decreased to 0.426 ± 0.021 pmol/h/mg protein by carbenoxolone (1 µM), which is an inhibitor of 11β-HSD2 (Brown et al., 1996), whereas neither probenecid nor pyrimethamine affected 11β-HSD2 activity by 300 µM (data not shown).

Effect of Pyrimethamine and Probenecid on OAT3- and MATE1-Mediated Transport of 6β-OHF in Their cDNA-Transfected Cells.

OAT3-mediated 6β-OHF uptake in OAT3-HEK293 cells (17.1 ± 1.6 μl/min/mg protein) was reduced to 7.8% ± 2.0% of control (1.3 ± 0.3 μl/min/mg protein) by probenecid (1 mM) (Fig. 3A). Pyrimethamine inhibited OAT3-mediated 6β-OHF uptake with an IC50 of 32.0 ± 6.2 µM (Fig. 3A). Since the concentration of 6β-OHF in the uptake buffer (5 µM) was low enough compared with its Km value for OAT3 (183 µM; Imamura et al., 2013), the IC50 of 6β-OHF for OAT3 is theoretically close to the corresponding Ki value.

Fig. 3.
Fig. 3.

Effects of probenecid and pyrimethamine on OAT3- and MATE1-mediated 6β-OHF uptake. (A) The uptake of 6β-OHF (5 μM) by hOAT3 for 5 minutes was determined in the presence of probenecid (1 mM) and pyrimethamine at designated concentrations at 37°C. The solid line represents the fitted line obtained by nonlinear regression analysis. (B) The uptake of 6β-OHF (10 μM) by human MATE1 for 1 minute was determined in the presence of pyrimethamine (100 μM) and probenecid at designated concentrations at 37°C. Each value represents the mean ± S.E. (n = 3). **P < 0.01.

MATE1-mediated 6β-OHF uptake of 16.2 ± 0.3 μl/min/mg protein in MATE1-HEK293 cells was reduced to 1.1 ± 0.1 μl/min/mg protein (6.5% ± 0.9% of control) by pyrimethamine (100 μM) (Fig. 3B). Only a weak inhibition of MATE1 was detected in the presence of probenecid (300 µM) (Fig. 3B).

Effects of Pyrimethamine and Probenecid on the Plasma and Urinary Excretion of 6β-OHF in Healthy Subjects.

The maximum unbound plasma concentration of probenecid was calculated as the product of the unbound fraction of probenecid in the plasma, which ranges from 0.05 to 0.17 (Selen et al., 1982), and the maximum plasma concentration observed in the study. They ranged from 12.2 ± 0.9 to 41.6 ± 3.0 μM in the probenecid-benzylpenicillin–treated groups (study 1) and 11.6 ± 0.9 to 39.4 ± 3.0 μM in the probenecid-adefovir–treated groups (study 2) (Supplemental Table 1). The plasma concentrations of 6β-OHF were significantly increased when coadministered with probenecid (Figs. 4A and 5A). AUC6β-OHF values from 0 to 24 hours were increased 1.7- and 1.8-fold in the probenecid-treated groups (studies 1 and 2, respectively) (Figs. 4B and 5B; Table 2), whereas the amount of 6β-OHF excreted into the urine was unchanged (Figs. 4C and 5C). Consequently, CLrenal, 6β-OHF was decreased significantly in the probenecid-treated groups (Figs. 4D and 5D). Compared with the corresponding values in the corresponding control groups, CLrenal, 6β-OHF 0-24h was decreased by 42 and 37%, and the renal secretion clearance of 6β-OHF (CLrenal sec, 6β-OHF) was decreased 46 and 41% by probenecid (studies 1 and 2, respectively; Table 2). The absolute value of CLrenal, 6β-OHF was lower in the interval from 8 to 24 hours compared with other intervals, and the effect of probenecid was moderate or weak in this interval (Figs. 4D and 5D).

Fig. 4.
Fig. 4.

Effect of probenecid on the plasma concentration and urinary excretion of 6β-OHF and on plasma concentration of cortisol in healthy subjects. Plasma concentration-time profile (A), AUC (B), and the amount excreted into the urine of 6β-OHF (X6β-OHF) (C) were determined in healthy subjects who received benzylpenicillin alone (control) or concomitantly with probenecid (probenecid-treated group). Open and closed symbols in A–C represent the control and probenecid-treated groups, respectively. (D) CLrenal, 6β-OHF was calculated in the designated time intervals in control and probenecid-treated groups (PRB) as described in Materials and Methods. Each point and bar represent the mean ± S.E. (n = 6). *P < 0.05; **P < 0.01.

Fig. 5.
Fig. 5.

Effect of probenecid on the plasma concentration and urinary excretion of 6β-OHF and on plasma concentration of cortisol in healthy subjects. Plasma concentration-time profile (A), AUC (B), and X6β-OHF (C) were determined in healthy subjects who received benzylpenicillin with or without pretreatment of probenecid. Open and closed symbols in A–C represent the control and probenecid-treated groups, respectively. (D) CLrenal, 6β-OHF was calculated in the designated time intervals in control and probenecid-treated groups (PRB) as described in Materials and Methods. Each point and bar represent the mean ± S.E. (n = 6). *P < 0.05; **P < 0.01.

View this table:
TABLE 2

Summary of kinetic indices of 6β-OHF and cortisol after coadministration of benzylpenicillin/probenecid, adefovir/probenecid, or metformin/pyrimethamine in healthy subjects

Each parameter represents the mean ± S.E.

The maximum unbound plasma concentrations of pyrimethamine were determined in the previous study, which were ∼300 nM (Kusuhara et al., 2011). Plasma 6β-OHF was unaffected by pyrimethamine (Fig. 6A), and AUC values of 6β-OHF from 0 to 12 hours in control and pyrimethamine-treated groups were unchanged (Fig. 6B; Table 2). X6β-OHF 0-12h was decreased by 19% in the pyrimethamine-treated group than that of the control group (Fig. 6C; Table 2), although the difference did not reach statistical significance. CLrenal, 6β-OHF and CLrenal sec, 6β-OHF were slightly decreased by 23 and 25%, respectively, by pyrimethamine without statistical significance (Table 2).

Fig. 6.
Fig. 6.

Effect of pyrimethamine on the plasma concentration and urinary excretion of 6β-OHF and on plasma concentration of cortisol in healthy subjects. Plasma concentration-time profile (A), AUC (B), and X6β-OHF (C) were determined in healthy subjects who received metformin with or without pyrimethamine treatment. Open and closed symbols in A–C represent the control and pyrimethamine-treated groups, respectively. (D) CLrenal, 6β-OHF was calculated in the designated time intervals in control and pyrimethamine-treated group (PYR) as described in Materials and Methods. Each point and vertical bar represent the mean ± S.E. (n = 8).

Discussion

6β-OHF was found to be a substrate of OAT3 and MATEs in the corresponding cDNA-transfected cells (Imamura et al., 2013). We speculate that the tubular secretion of 6β-OHF in the kidney involves these transporters, and that 6β-OHF may be an endogenous in vivo probe that can be used to monitor the activities of these transporters. The present study examined the effect of in vivo inhibitors of these transporters on the plasma concentrations and urinary excretion of 6β-OHF to demonstrate its usefulness as an in vivo probe for OAT3 and MATEs in humans.

To examine the contribution of Oat3 to the urinary excretion of 6β-OHF, an in vivo study using Oat3(–/–) mice was conducted. CLrenal, 6β-OHF was significantly lower in Oat3(–/–) mice compared with wild-type mice (Table 1). Because CLrenal, 6β-OHF accounted for, at most, 34% of the systemic elimination in wild-type mice (Table 1), it is reasonable that the reduction in the urinary excretion did not cause significant accumulation of 6β-OHF in the plasma in Oat3(–/–) mice.

To evaluate the contribution of drug transporters to the renal uptake process, an inhibition study was conducted using human kidney slices, which are used with appropriate inhibitors to investigate the contribution of uptake transporters in the kidney (Nozaki et al., 2007b; Matsushima et al., 2009). The uptake of 6β-OHF was inhibited significantly by probenecid and partly by MPP+ (Fig. 2). The marked inhibition of the uptake by probenecid in kidney slices is consistent with our previous prediction that OAT3 makes a greater contribution to the renal uptake of 6β-OHF than does OCT2 (Imamura et al., 2013). Inhibition of the uptake by MPP+ also suggests that OCT2 contributes to the uptake. Whether 6β-OHF is an OCT2 substrate remains controversial. Human OCT2-HEK cells showed significantly greater uptake of 6β-OHF than did mock-vector transfected cells; however, this uptake was not inhibited by the representative inhibitor (Imamura et al., 2013). We also found that MPP+ inhibited OAT3-mediated uptake of estrone-3-sulfate and 6β-OHF by 58 and 42%, respectively, in hOAT3-HEK cells at 1 mM (data not shown). The effect of MPP+ in kidney slices partly involved inhibition of OAT3, and the actual contribution of OCT2 was smaller than that for the inhibition by MPP+.

To confirm the clinical data, the specificity of probenecid and pyrimethamine was measured in vitro. These drugs had no effect on CYP3A4 and 11β-HSD2 activity, suggesting that these inhibitors affect neither 6β-OHF production from cortisol nor 6β-OHF metabolism to 6β-hydroxycortisone. Pyrimethamine inhibited OAT3 in vitro (Fig. 3), but the inhibition of OAT3 by pyrimethamine was negligible in vivo because its Ki value (32 μM) was greater than the clinical unbound concentration (∼300 nM; Kusuhara et al., 2011). Probenecid had no effect on MATE1. Therefore, among the known enzymes and transporters that might be involved in the disposition of 6β-OHF, probenecid and pyrimethamine are potential inhibitors of OAT3 and MATEs, respectively (Table 3). The plasma 6β-OHF concentration was significantly higher in the probenecid-treated group, whereas the amount excreted into the urine did not differ between groups (Fig. 4). This was also observed in another group (Fig. 5). CLrenal, 6β-OHF decreased significantly in these two independent studies (Table 2). Taken together, the in vivo data obtained from Oat3(–/–) mice (Fig. 1) and in vitro data from human kidney slices (Fig. 2) suggest that the reduction in the CLrenal, 6β-OHF was probably attributable to inhibition of OAT3-mediated uptake of 6β-OHF by probenecid.

View this table:
TABLE 3

Summary of unbound plasma concentration of probenecid and pyrimethamine and their inhibitory potential for 6β-OHF–related transporters and enzymes

Because the control subjects received a therapeutic dose of benzylpenicillin or adefovir, the possibility that benzylpenicillin or adefovir modifies the effect of probenecid needs further discussion. The Km value of benzylpenicillin for OAT3 was reported as 52 μM in cDNA-transfected cells (Tahara et al., 2005) and as 14–90 μM in human kidney slices (Nozaki et al., 2007a). We confirmed that, at a concentration of 300 nM, adefovir had no inhibitory effect on OAT3-mediated 6β-OHF uptake (data not shown). Considering the total plasma concentrations of benzylpenicillin and adefovir in the clinical studies (at most 1 μM and 100 nM, respectively), their effect on OAT3 (i.e., saturation) can be excluded. To provide concrete evidence, a clinical study in which subjects receive only probenecid should be conducted. We note that the tubular secretion of 6β-OHF was still greater than its glomerular filtration even in the probenecid-treated groups. The Ki value of probenecid was greater for the OAT3-mediated 6β-OHF transport than for that determined using other substrates, although it remained below the unbound plasma concentration of probenecid during the experiments (Table 3). Higher doses of probenecid may be needed to achieve complete inhibition of the tubular secretion of 6β-OHF.

The present study also showed that the magnitude of inhibition of CLrenal, 6β-OHF by probenecid depends on the time after probenecid treatment. CLrenal sec, 6β-OHF was decreased by probenecid to 37% ± 5% (0–4 hours, P < 0.01), 42% ± 6% (4–8 hours, P < 0.01), and 72% ± 8% (8–24 hours, P < 0.05) of the corresponding control values in study 1, and to 45% ± 2% (0–4 hours, P < 0.01), 47% ± 3% (4–8 hours, P < 0.01), and 78% ± 11% (8–24 hours, P = 0.2128) of the corresponding control values in study 2 (Figs. 4C and 5C). This may be explained partly by a reduction in probenecid concentration with time. In addition, the absolute value of CLrenal, 6β-OHF in the control group was lower during the 8–24-hour interval compared with the other intervals, suggesting that OAT3 is under regulation by circadian rhythm, which would lower its contribution to the net urinary excretion.

Unlike probenecid, the effect of pyrimethamine on CLrenal, 6β-OHF was not changed, although 6β-OHF is a MATE1 substrate in vitro (Imamura et al., 2013). The significant inhibition of MATEs by pyrimethamine (>70%) is supported by the significant reduction in the renal clearance of N-methylnicotinamide and thiamine (Kusuhara et al., 2011; Ito et al., 2012; Kato et al., 2014). In addition to the lower-than-expected contribution to the efflux of MATEs, there is another possible reason for this discrepancy. When the luminal efflux is far greater than the basolateral efflux, the influx clearance on the basolateral membrane approximates the overall tubular secretion clearance (Kusuhara and Sugiyama, 2009). Under such conditions, as far as the assumption is satisfied, the inhibition of luminal efflux hardly affects the overall tubular secretion clearance. In fact, the magnitude of inhibition of the tubular secretion clearance of metformin by pyrimethamine was at most 20%–30% (Kusuhara et al., 2011). Therefore, the importance of MATE1 in the luminal efflux of 6β-OHF cannot be excluded based only on these results; however, it is evident that 6β-OHF is inferior to the MATE substrates mentioned earlier in terms of their sensitivity to MATE activities.

The present study highlights the importance of OAT3 in the urinary excretion of 6β-OHF. Both the plasma concentration and renal clearance of 6β-OHF are highly sensitive to OAT3 activity. Caution is needed when the plasma concentrations alone are used to evaluate OAT3 activity because the plasma concentration and amount of 6β-OHF excreted into the urine are also influenced by CYP3A4 activity. DX-619 decreased CLrenal, 6β-OHF in healthy subjects; however, the plasma concentration of 6β-OHF was unchanged because of the reduced production rate by mechanism-based inhibition of CYP3A4 (Imamura et al., 2013). It is recommended that CLrenal, 6β-OHF should be determined when evaluating OAT3 activity with drugs that potentially inhibit or induce CYP3A4 in the liver.

The apparent cortisol 6β-hydroxylation clearance (CL6β-OHF) is used as a quantitative index of hepatic CYP3A4 activity. Theoretically, CL6β-OHF involves the total body and renal clearance of 6β-OHF (CLtot, 6β-OHF and CLrenal, 6β-OHF, respectively) (Imamura et al., 2013), as shown in eq. 8:Embedded Image(8)where CLCYP3A4 is the metabolic clearance of 6β-OHF by CYP3A4. Therefore, unless urinary excretion is the major elimination pathway, pharmacokinetic interaction could influence CL6β-OHF and thereby affect the estimation of the magnitude of DDI involving hepatic CYP3A4. To address this issue, we calculated CL6β-OHF in the control and probenecid- or pyrimethamine-treated groups. Probenecid and pyrimethamine had no effect on the plasma cortisol concentrations (Supplemental Fig. 1) and CL6β-OHF (Supplemental Table 3), indicating that CLrenal, 6β-OHF/CLtot, 6β-OHF is near unity, supporting the idea that CL6β-OHF can be used to investigate hepatic CYP3A4 irrespective of the interaction with OAT3. Species differences in the effect of OAT3 inhibition or impairment on the plasma concentrations of 6β-OHF might be explained by the contribution of renal clearance to total body clearance. In our previous report, we could not conclude whether DX-619, a CYP3A4 mechanism-based inhibitor, significantly inhibited hepatic CYP3A4 based on CL6β-OHF because CLrenal, 6β-OHF was also significantly decreased during DX-619 administration (Imamura et al., 2013). The present study supports the idea that DX-619 administration caused significant inhibition of CYP3A4 (∼72% inhibition) at that dose.

These data suggest that OAT3 plays a significant role in the urinary excretion of 6β-OHF, and that 6β-OHF can be used to investigate the perpetrators of the pharmacokinetic drug interactions involving OAT3, but not MATEs, in humans.

Acknowledgments

The authors thank Sumito Ito and Tomoko Kito (Graduate School of Pharmaceutical Sciences, the University of Tokyo) and Christian Lechner (Nippon Boehringer Ingelheim Co., Ltd.) for their technical assistance.

Authorship Contributions

Participated in research design: Imamura, Kusuhara.

Conducted experiments: Imamura, Tsuruya, Murayama, Okudaira, Kurihara, Maeda, Kumagai, Damme, Heer.

Performed data analysis: Imamura, Damme, Heer.

Wrote or contributed to the writing of the manuscript: Imamura, Izumi, Sugiyama, Kusuhara.

Footnotes

    • Received October 17, 2013.
    • Accepted January 30, 2014.

  • The study was sponsored by Daiichi-Sankyo Co., Ltd, Tokyo, Japan. This study was also supported by Grants-in-Aid for Public-Private Sector Joint Research on Publicly Essential Drugs (KHB 1208) from the Japan Health Sciences Foundation, Grant-in-Aid for Scientific Research (S) [Grant 24229002], for Scientific Research (B) [Grant 23390034], and for Challenging Exploratory Research [Grant 24659071] from the Japan Society for the Promotion of Science, Japan.

  • Y.I., N.M., N.O., A.K., and T.I. are employees of Daiichi Sankyo Co., Ltd. K.D. and D.H. are employees of Daiichi Sankyo Europe GmbH. The remaining authors declare no conflict of interest.

  • dx.doi.org/10.1124/dmd.113.055475.

  • ↵Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

Abbreviations

AUC
area under the concentration-time curve
AUCcortisol
area under the plasma concentration-time curve of cortisol
AUC6β-OHF
area under the concentration-time curve of plasma 6β-OHF
CL6β-OHF
apparent cortisol 6β-hydroxylation clearance
CLrenal
6β-OHF, renal clearance of 6β-OHF
CLrenal sec
6β-OHF, renal secretion clearance of 6β-OHF
CLtot
6β-OHF, total body clearance
DDI
drug-drug interaction
DX-619
7-[(3R)-3-(1-aminocyclopropyl)pyrrolidin-1-yl]-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-4-oxoquinoline-3-carboxylic acid
HEK
human embryonic kidney
hOAT
human OAT
11β-HSD
11β-hydroxysteroid dehydrogenase
LC-MS/MS
liquid chromatography–tandem mass spectrometry
MATE
multidrug and toxin extrusion protein
MPP+
1-methyl-4-phenylpyridinium
OAT
organic anion transporter
OCT
organic cation transporter
6β-OHF
6β-hydroxycortisol
PO
by mouth
X6β-OHF
amount of 6β-OHF excreted into the urine

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Research ArticleSpecial Section on Transporters in Toxicity and Disease

6β-Hydroxycortisol as Endogenous In Vivo Probe for OAT3

Yuichiro Imamura, Yuri Tsuruya, Katja Damme, Dominik Heer, Yuji Kumagai, Kazuya Maeda, Nobuyuki Murayama, Noriko Okudaira, Atsushi Kurihara, Takashi Izumi, Yuichi Sugiyama and Hiroyuki Kusuhara
Drug Metabolism and Disposition April 1, 2014, 42 (4) 685-694; DOI: https://doi.org/10.1124/dmd.113.055475
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