Scintillation proximity assay for measuring uptake by the human drug transporters hOCT1, hOAT3, and hOATP1B1

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 366 (2007) 117–125 www.elsevier.com/locate/yabio

Scintillation proximity assay for measuring uptake by the human drug transporters hOCT1, hOAT3, and hOATP1B1 Christina Lohmann a, Birgitta Gelius b, Jeanette Danielsson b, Ulrica Skoging-Nyberg b, Evelyn Hollnack a, Adam Dudley c, Johanna Wahlberg b, Janet Hoogstraate d, Lena Gustavsson a,* b

a Discovery DMPK&BA, AstraZeneca R&D Lund, 22187 Lund, Sweden Global Protein Science & Supply, DECS, AstraZeneca R&D So¨derta¨lje, 15185 So¨derta¨lje, Sweden c Discovery DMPK, AstraZeneca R&D, Wilmington, USA d Research DMPK, AstraZeneca R&D So¨derta¨lje, 15185 So¨derta¨lje, Sweden

Received 17 August 2006 Available online 27 April 2007

Abstract Increasing evidence suggests a key role of transport proteins in the pharmacokinetics of drugs. Within the solute carrier (SLC) family, various organic cation transporters (OCTs), organic anion transporters (OATs), and organic anion transporting polypeptides (OATPs) that interact with drug molecules have been identified. Traditionally, cellular uptake assays require multiple steps and provide low experimental throughput. We here demonstrate the use of a scintillation proximity approach to detect substrate uptake by human drug transporters in real time. HEK293 cells stably transfected with hOCT1, hOATP1B1, or hOAT3 were grown directly in Cytostar-T scintillating microplates. Confluent cell monolayers were incubated with 14C- or 3H-labeled transporter substrates. Cellular uptake brings the radioisotopes into proximity with the scintillation plate base. The resulting light emission signals were recorded on-line in a microplate scintillation counter. Results show time- and concentration-dependent uptake of 14C-tetraethylammonium, 3H-methylphenylpyridinium (HEK-hOCT1), 3H-estradiol-17b-D-glucuronide (HEK-hOATP1B1), and 3H-estrone-3-sulfate (HEK-hOAT3), while no respective uptake was detected in empty vector-transfected cells. Km of 14C-tetraethylammonium and 3H-estrone-3-sulfate uptake and hOAT3 inhibition by ibuprofen and furosemide were similar to conventional dish uptake studies. The scintillation proximity approach is high throughput, amenable to automation and allows for identification of SLC transporter substrates and inhibitors in a convenient and reliable fashion, suggesting its broad applicability in drug discovery.  2007 Elsevier Inc. All rights reserved. Keywords: Transporter; Drug; Scintillation proximity; hOCT1; hOAT3; hOATP1B1

Increasing evidence suggests a key role of cellular plasma membrane transport proteins in the pharmacokinetics of drugs. Transporters of the solute carrier (SLC)1 and ATP binding cassette families are expressed in the *

intestine, liver, brain, kidney, and various tissues, with probable impact on drug absorption, distribution, and elimination [1–3]. With regard to the SLC family, various organic cation transporters (OCTs), organic anion

Corresponding author. Fax: +46 46 337383. E-mail address: [email protected] (L. Gustavsson). 1 Abbreviations used: SLC, solute carrier; OCT, organic cation transporter; OAT, organic anion transporter; OATP, organic anion transporting polypeptide; hOCT1, human organic cation transporter 1; hOATP1B1, human organic anion transporting polypeptide 1B1; hOAT3, human organic anion transporter 3; 14C-TEA, [1-14C]tetraethylammonium bromide; 3H-E17bG, [6,7-3H]estradiol-17b-D-glucuronide; 3H-E3S, [6,7-3H]estrone-3-sulfate; 3 H-MPP, N-[methyl-3H]-4-phenylpyridinium acetate; HEK, human embryonic kidney; PBS, phosphate-buffered saline; SPA, scintillation proximity assay; FCS, fetal calf serum; HBSS, Hanks balanced salt solution. 0003-2697/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.04.038

118

Cellular drug uptake measured by scintillation proximity / C. Lohmann et al. / Anal. Biochem. 366 (2007) 117–125

transporters (OATs), and organic anion transporting polypeptides (OATPs) that interact with drug molecules have been identified (reviewed in [1,4]). Human organic cation transporter 1 (hOCT1; SLC22A1) within the SLC22 family is primarily expressed in liver and mediates the first step in the hepatic excretion of many cationic drugs, which is uptake into hepatocytes [5]. Drugs such as aciclovir, ganciclovir, metformin, and desipramin are transported by hOCT1 as are the model cations tetraethylammonium (TEA) and methyl-4-phenylpyridinium (MPP) [5]. Human organic anion transporter 3 (hOAT3; SLC22A8) is mainly expressed in the kidney, where it locates to the basolateral membrane of proximal tubule cells, and in the epithelial cells of the choroid plexus [6]. hOAT3 mediates renal excretion and reabsorption of anionic drugs [5] and has been shown to transport ibuprofen, indomethacin, salicylate, methotrexate, cimetidine, and estrone-3-sulfate [4]. Human organic anion transporting polypeptide 1B1 (hOATP1B1) belongs to the former OATP/SLC21 family, which recently has been reclassified within the OATP/ SLCO superfamily [7], in agreement with the Human Genome Organisation Nomenclature Committee Database. hOATP1B1 (SLCO1B1, formerly called OATP-C or SLC21A6) is predominantly expressed in the liver at the sinusoidal (basolateral) cell membrane of hepatocytes and is suggested to play a crucial role in hepatic drug clearance [1,2,7]. hOATP1B1 substrates comprise pravastatin, penicillin G, methotrexate, rifampicin, and estradiol-17b-D-glucuronide [4,7]. In recent years, it has become widely accepted that optimizing the pharmacokinetic properties of drug candidates during the early stages of drug development is essential [8,9]. Consequently, a need for adequate screening assays to identify transporter substrates/inhibitors among drug candidates is emerging. SLC transporters can be heterologously expressed in mammalian cells [10]. Traditionally, uptake transport assays are performed by incubating transporter-transfected cells in petri dishes or multiwell plates (6-well, 12-well, 24-well) with the test compound. Cellular uptake of the compound then is terminated and surplus compound removed by washing the cells several times with ice-cold buffer. Subsequently, the cells are lysed, samples are transferred for analysis, for example, into scintillation vials, scintillation cocktail is added, and the amount of test compound in the cell lysate is determined by liquid scintillation counting. Alternatively, mass spectrometry is applied for compound analysis. In either case, throughput of the assay is significantly hampered by the multiple washing, pipetting, and transferring steps required for every sample and thus by the rather limited number of cell samples that can be handled in parallel. Additionally, conventional uptake measurement is restricted to only one time point per well, due to the fact that cell lysis is required. This study was aimed at establishing an alternative assay format for detecting SLC transporter interactions in real time, providing enhanced throughput. The Cytostar-T scin-

tillation proximity assay by Amersham Biosciences has previously been used to monitor intracellular concentrations of [14C]thymidine to study cell growth [11] and multidrug resistance in SKMES-1/ADR cells [12], of [45Ca] to study glutamate receptors [13], of 14C-glycocholate to examine bile acid transport [14] or to detect [14C]glycine and [14C]taurine uptake in JAR cells [15]. We have applied this SPA technology to investigate substrate uptake by the human SLC transporter proteins hOCT1, hOATP1B1, and hOAT3 in stably transfected human cell lines. For this purpose, mammalian cells transfected with SLC transporters were grown directly in Cytostar-T scintillating microplates, the base of which consists of scintillant-containing transparent plastic. Uptake of radiolabeled substrates into the adherent cell layer brings the radioisotopes into proximity with the scintillation plate base, thus resulting in light emission signals that can be recorded on-line and at multiple time points per well in a microplate scintillation counter. We here demonstrate the use of a scintillation proximity approach to detect substrate uptake by the human drug transporters hOCT1, hOAT3 and hOATP1B1, each overexpressed in HEK293 cells. Furthermore, the new assay approach was applied to select transfected clonal cell lines, to determine Km values of substrate– transporter interactions, and to identify inhibitors of the SLC transporter hOAT3. Materials and methods Materials [1-14C]tetraethylammonium bromide (14C-TEA; 2.4 mCi/ mmol), [6,7-3H]estradiol-17b-D-glucuronide (3H-E17bG; 45 Ci/mmol), and [6,7-3H]estrone-3-sulfate (3H-E3S; 57.3 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). N-[methyl-3H]-4-phenylpyridinium acetate (3H-MPP; 80 Ci/mmol) was from Biotrend Chemikalien GmbH (Koeln, Germany). Unlabeled estrone-3-sulfate, ibuprofen and furosemide were obtained from Sigma– Aldrich (Schnelldorf, Germany). Cytostar-T scintillating microplates were from Amersham Biosciences (Buckinghamshire, England). Cell culture Human embryonic kidney (HEK293; ATCC CRL 1573) cells were grown as adherent monolayer cultures in T75 flasks (Corning Inc., New York, USA) in Dulbecco’s modified Eagle’s medium/Ham’s F12 medium/Iscove’s modified Dulbecco’s medium (DMEM/Ham’s F12/IMDM) containing 10% (v/v) fetal calf serum (FCS), 2 mM L-glutamine, 1.8 mM calcium chloride and 500 lg/ml geneticin (G-418; Invitrogen Ltd., Paisley, Scotland) at 37 C in a humidified 5% CO2 atmosphere, if not otherwise indicated. For subculturing or harvesting, cells at 70–80% confluence were washed with phosphate-buffered saline (PBS) (Ca2+/Mg2+-free) (Invitrogen), and 3 ml trypsin/EDTA (0.05% trypsin, 0.53 mM EDTA; Invitrogen) was added for 1–2 min. The cell

Cellular drug uptake measured by scintillation proximity / C. Lohmann et al. / Anal. Biochem. 366 (2007) 117–125

suspension was collected in 5 ml growth medium, centrifuged for 4 min at 115 g, and resuspended in growth medium for seeding into new T75 flasks (ca. 1 · 106 cells per T75 flask) or into assay plates as described below.

119

Cytostar-T scintillation proximity assay — uptake, Km, and inhibition studies

For transfection, HEK293 cells were grown in suspension in calcium-free DMEM/Ham’s F12/IMDM containing 2% (v/v) FCS and 2 mM L-glutamine. Transfection with the above plasmids was done using Ro-1539 (XTremeGene) transfection reagent (Roche Applied Science, Rotkreuz, Switzerland), resulting in mixed clone populations. At 48 h after transfection, cells were seeded to grow as adherent monolayers in culture flasks in DMEM/Ham’s F12/IMDM containing 10% FCS, 2 mM glutamine, 1.8 mM calcium chloride, and 500 lg/ml G-418, as described above. Clonal selection was performed from the clone mixes by serial dilution and subsequent propagation of clonal cell lines.

For SPA uptake, Km, and inhibition experiments, confluent cell monolayers grown in 96-well scintillating microplates were used. Growth medium was removed and replaced by 100 ll assay buffer (Hanks’ balanced salt solution (HBSS), Invitrogen, additionally buffered with 10 mM Hepes, pH 7.4) containing the radiolabeled substrates 14CTEA, 3H-E17bG, 3H-E3S, or 3H-MPP in concentrations as indicated. Real-time uptake measurements were performed by immediate scintillation counting in a Wallac MicroBeta 1450 Trilux plate reader (PerkinElmer) equipped with 12 detectors. The detection windows for 14C and 3H were set according to the manufacturer’s instructions. Counting time was 30 s per well, resulting in a total counting time of about 5 min per plate and counting cycle. 2–4 wells were used for every substrate or inhibitor concentration. For inhibition studies, radiolabeled 3H-E3S in a given concentration (100 nM) and unlabeled inhibitors (furosemide, ibuprofen) in various concentrations in assay buffer were added to the cells. Incubation and uptake measurements were performed as described above. For Km determination, 14C-TEA in various concentrations (0, 50, 100, 150, 200, 250, 350, 500, and 750 lM) in assay buffer was added to confluent HEK-hOCT1 cell layers in 96-well SPA plates. Scintillation signals were recorded on-line in a MicroBeta 1450 Trilux counter as described above. Uptake was linear up to 30 min. Unspecific uptake of 14C-TEA into control cells (empty vectortransfected) was subtracted from the uptake values obtained for hOCT1-transfected cells. The resulting initial uptake rates (cpm/min/well) were plotted versus 14C-TEA concentration, and apparent Km values were determined using XLfit (4.1.1) without weighting of the data, assuming Michaelis–Menten kinetics. Triplicate wells were used for each substrate concentration.

Growing of cells in Cytostar-T scintillating microplates

Growing of cells in 12-well plates

To improve cell adherence, Cytostar-T scintillating microplates were coated with poly-D-lysine (MW 30,000– 70,000) (Sigma–Aldrich) before plating of cells. Coating was performed by adding 100 ll of a 0.1 mg/ml solution of poly-D-lysine in PBS to each well and incubating for 30 min at room temperature. The wells were subsequently washed three times with PBS and air-dried in the laminar flow hood. Cells were harvested from T75 flasks at 70–80% confluence by trypsinization as described above. 5 · 104 cells in 150–200 ll growth medium were plated into each well of the poly-D-lysine coated microplates and cultured at 37 C in a humidified 5% CO2 atmosphere for 2–3 days to reach confluence. Routinely, expression of recombinant proteins was stimulated by incubation with 10 mM sodium butyrate in normal growth medium for 24 h before experiments [16].

For conventional dish uptake assays, cells were plated in poly-D-lysine-coated 12-well plates (Becton–Dickinson, Stockholm, Sweden) at a density of 5 · 105 cells/well and grown to confluence at 37 C in a humidified 5% CO2 atmosphere. At 24 h before experiments, 10 mM sodium butyrate was added to the medium to stimulate expression of recombinant proteins [16].

hOCT1, hOATP1B1, hOAT3, and empty vector expression plasmids cDNA for hOCT1 (SLC22A1) and hOAT3 (SLC22A8) were subcloned into pT-REx-DEST30 vectors using the Gateway technology by Invitrogen, and hOATP1B1 (SLCO1B1, formerly SLC21A6) was expressed from pcDNA3.1 (Invitrogen). To generate empty vectors for mock transfection, pT-REx/GW30/lacZ (Invitrogen) was digested with MIuI and XhoI, and pcDNA/V5-GW-CAT (Invitrogen) was digested with ApaI and SacI to remove the attB recombination sites and the lacZ or the CAT gene, respectively. The two empty vector-transfected cell lines (pT-REx, pcDNA) and untransfected HEK293 cells showed no difference in substrate uptake in initial background profiling experiments. For further studies, the pT-REx empty vector-transfected cell line was used to determine unspecific background uptake. Generation of stable cell lines

Uptake/inhibition assay in 12-well plates using liquid scintillation counting Culture medium was removed from confluent cell monolayers grown in 12-well plates. After washing with assay buffer (HBSS/Hepes, pH 7.4), cells were incubated with 0.5 ml of substrate solution in assay buffer, with or without inhibitor in concentrations as indicated, for appropriate

120

Cellular drug uptake measured by scintillation proximity / C. Lohmann et al. / Anal. Biochem. 366 (2007) 117–125

time at 37 C. In the OAT3 inhibition assay, 3H-E3S was diluted 1:10 with non-labeled estrone-3-sulfate to give a total estrone-3-sulfate concentration of 100 nM. Uptake was stopped by washing the cells three times with ice-cold assay buffer. Subsequently, cells were lysed in 0.5 ml 1% Triton X-100. Cell lysates were transferred to scintillation vials and mixed with 10 ml scintillation cocktail (Ultima Gold, PerkinElmer). Liquid scintillation counting was performed in a Packard Tri-Carb 2500 TR liquid scintillation counter (PerkinElmer). Results and discussion Applicability of the SPA for detecting hOCT1-, hOATP1B1and hOAT3-mediated transport of various 14C- and 3 H-labeled substrates HEK-hOCT1 (clone mix) and HEK control cells were incubated with the hOCT1 substrate, 14C-tetraethylammonium, in 96-well Cytostar-T plates, and scintillation signals were recorded. Fig. 1A shows time- and concentrationdependent uptake of 14C-TEA into hOCT1-transfected

Uptake (cpm/well)

A

2000 1800 1600 1400 1200 1000 800 600 400 200 0

500 μM - w1 500 μM - w2 500 μM - w3 500 μM - w4 250 μM - w1 250 μM - w2 250 μM - w3 250 μM - w4 100 μM - w1 100 μM - w2 100 μM - w3 100 μM - w4 40 μM - w1 40 μM - w2 40 μM - w3 40 μM - w4

0

50

100

TIME (min)

UPTAKE (cpm/well)

B

2000 1800 1600 1400 1200 1000 800 600 400 200 0

500 μM - w1 500 μM - w2 250 μM - w1 250 μM - w2 100 μM - w1 100 μM - w2 40 μM - w1 40 μM - w2

0

50

100

TIME (min)

Fig. 1. Time course of 14C-TEA uptake by HEK293 cells transfected with hOCT1 (A) and HEK293 control cells (B) measured by scintillation proximity. 14C-TEA in various concentrations (circle, 500 lM, triangle, 250 lM, diamond, 100 lM, square, 40 lM) was added to confluent layers of HEK-hOCT1 cells (A) and HEK control cells (B) in 96-well SPA plates. Scintillation signals were recorded on-line in a MicroBeta 1450 Trilux counter as described under Materials and methods. For each concentration, 2–4 wells were measured in parallel (w1–w4). Four independent experiments yielded similar results (n = 4). Results of one representative experiment are shown. The initial uptake rates are given in Table 1.

cells detected by scintillation signals significantly increasing over time, whereas no uptake of 14C-TEA was seen in the control cells, where the signals remained unchanged over time (Fig. 1B). These data clearly demonstrate the applicability of the SPA to detect specific hOCT1-mediated uptake of 14C-TEA in adherent mammalian cells. To examine the variation between the SPA recordings in single wells, for each 14C-TEA concentration, uptake was monitored using 4 wells in parallel on the same 96-well Cytostar-T plate (w1–w4 in Fig. 1A). The initial uptake rates including standard deviation and coefficient of variation were calculated separately for every well (Table 1). The R2 values for the regression lines ranged from 0.86 to 0.99 (mean R2 being 0.95) indicating that the uptake followed first order kinetics during the initial time period from which the initial rate was calculated. Results show only little scatter and high reproducibility between wells (Fig. 1, Table 1). Background scintillation signals obtained with control cells were remarkably constant over time (Fig. 1B) and linearly dependent on the 14C-TEA concentration in the incubation buffer (Fig. 2), indicating that background signals result from 14C-TEA molecules in the buffer above the cell layer rather than from unspecific uptake processes. Accordingly, no significant uptake of 14C-TEA into control cells was observed by conventional liquid scintillation counting (data not shown). Background scintillation signals obtained in blank wells without cells were also constant over time and linearly dependent on the 14C-TEA concentration in the incubation buffer (Fig. 2). Notably, the SPA background scintillation signal in wells with a layer of control cells was even lower than that in blank wells not containing a cell layer, indicating that the confluent cell layer acts as a spacer between the scintillation base plate and the 14C-TEA molecules in the buffer, thus further minimizing background noise (Fig. 2). To investigate the applicability of the SPA technology for uptake measurements in SLC-transfected cells not only with 14C-labeled but also with tritium-labeled compounds, Table 1 Initial rates and within-assay variation for hOCT1 cells (clone mix) 14

C-TEA concentration [lM]

40 100 250 500

14

C-TEA uptake in HEK-

Initial uptake rate [cpm/well/min] Mean

SD

CV%

5.4 10 18 22

0.41 0.47 1.8 0.92

7.7 4.7 9.9 4.3

14 C-TEA in concentrations as indicated was added to confluent layers of HEK-hOCT1 cells (clone mix) in 96-well SPA plates. Scintillation signals were recorded on-line in a MicroBeta 1450 Trilux counter. For each 14CTEA concentration, uptake was measured in 4 wells in parallel. Uptake was linear up to 30 min. The initial rates of uptake were calculated from the initial linear phase of 14C-TEA uptake by linear regressions, individually for all 4 wells per substrate concentration. The correlation coefficients for the linear regressions ranged from R2 = 0.86 to R2 = 0.99. The mean initial rates of uptake (n = 4), standard deviations (SD), and coefficients of variation (CV%) are given.

A

1400 no cells R2 = 0.9972

1200 1000 800

control cells R2 = 0.9983

600 400

hOATP1B1 - w1 hOATP1B1 - w2 hOATP1B1 - w3 hOATP1B1 - w4 mean hOATP1B1 control cells - w1 control cells - w2 control cells - w3 control cells - w4 mean control cells

200 150 100 50

200 0 0

121

300 250

UPTAKE (cpm/well)

SCINTILLATION SIGNAL (cpm/well)

Cellular drug uptake measured by scintillation proximity / C. Lohmann et al. / Anal. Biochem. 366 (2007) 117–125

200

400

0

600

0

10

14

C-TEA (µM)

B UPTAKE (cpm/well)

Fig. 2. SPA background scintillation signal for 14C-TEA in empty wells (open circles) and wells with adherent confluent monolayers of control cells (filled squares). 14C-TEA in different concentrations (40, 100, 250, 500 lM) in assay buffer (100 ll/well) was added to blank wells of a SPA plate (open circles) and to wells containing confluent monolayers of HEK control cells (filled squares). The resulting scintillation signals were constant over 120 min (see Fig. 1B, control cells). Average scintillation signals ±standard deviation of 23 measurements during 120 min, in two wells per 14C-TEA concentration, are given. Linear regression yielded correlation coefficients of R2 = 0.997 (no cells) and R2 = 0.998 (control cells).

30

600 500 hOAT3 - w1 hOAT3 - w2 hOAT3 - w3 hOAT3 - w4 mean hOAT3 control - w1 control - w2 control - w3 control - w4 mean control

400 300 200 100 0

the hOCT1 substrate, 3H-MPP, was added to HEKhOCT1 and control cells in SPA uptake experiments. The time-dependent uptake of 3H-MPP into hOCT1-transfected cells could readily be detected in the SPA, while no uptake of the substrate was detected in the empty vectortransfected control cells (data not shown) demonstrating that the SPA is suitable for measuring uptake of 3H-labeled substrates in hOCT1-transfected cells also. This is an important finding as, for many research chemicals and drugs in development, 3H-labeling is more easily available than 14C-labeling. Moreover, to test the applicability of the SPA technology for measuring substrate uptake by human SLC transporters other than hOCT1, SPA uptake experiments with HEK-hOAT3 cells and HEK-hOATP1B1 cells were performed. A time-dependent uptake of the hOATP1B1 substrate 3H-E17bG by HEK-hOATP1B1 cells was detected in the SPA, while very little uptake into empty vectortransfected control cells was detected (Fig. 3A). Similar results were obtained for uptake of the hOAT3 substrate 3 H-E3S by hOAT3-transfected cells (Fig. 3B). Thus, we showed that specific uptake of hOCT1, hOATP1B1, and hOAT3 substrates in the respective transfected cell lines could be measured readily using the SPA. From these findings, we suggest a broad applicability of the SPA for detecting SLC transporter-mediated substrate uptake into, and possibly efflux from, adherent mammalian cells. As the contribution of SLC transporters to drug pharmacokinetics is increasingly being recognized lately [1–3], the assay presented here will provide a useful and efficient tool in drug discovery screening for identifying SLC transporter interactions of pharmaceutical development compounds.

20 TIME (min)

0

5

10

15

20

TIME (min)

Fig. 3. Time course of 3H-E17bG uptake by HEK-hOATP1B1 cells (A) and [3H]-E3S uptake by HEK-hOAT3 cells (B) compared to uptake into control cells measured by scintillation proximity. (A) 3H-E17bG (0.5 lM) or (B) 3H-E3S (0.2 lM) was added to confluent cell layers of (A) HEKhOAT3 cells and (B) HEK-hOATP1B1 cells or (A) and (B) empty vectortransfected cells (control cells) in 96-well SPA plates. Scintillation signals were recorded on-line in a MicroBeta 1450 Trilux counter. In addition to values of all individual wells (w1–w4), average values ±SD of the four wells each are shown.

Use of the SPA for selection of transporter-transfected clonal cell lines according to uptake functionality To test whether the SPA could be used in simplifying the procedure of selecting functional clones after transfection, a population of HEK293 cells was transfected with hOCT1 DNA, resulting in a HEK-hOCT1 clone mix comprising cells with diverse expression levels of hOCT1. Individual clonal cell lines were obtained from this clone mix by serial dilution and subsequent propagation of single clones. Uptake of 14C-TEA by individual clonal HEK-hOCT1 cell lines was measured using the SPA approach. As expected, there was a large variation in the functional uptake activity of the individual clonal cell lines. When exemplarily screening six different clonal cell lines for 14C-TEA uptake functionality (Fig. 4) two of the cell lines tested (symbols + and j; cf. control cells with symbol s) did not show any uptake of 14C-TEA at all, one cell line (symbol ) showed reduced uptake compared to the clone mix ( ), one cell line (–) showed uptake comparable to the clone mix, and two clonal cell lines (symbols · and m) showed increased

•

122

Cellular drug uptake measured by scintillation proximity / C. Lohmann et al. / Anal. Biochem. 366 (2007) 117–125

1200 UPTAKE RATE (cpm/min/well)

UPTAKE (cpm/well)

50

1000 800 600 400 200 0

0

20

40 60 TIME (min)

80

100

40 30 20 10 0

0

14

Fig. 4. Uptake of C-TEA (100 lM) by individual HEK-hOCT1 clonal cell lines, the hOCT1 clone mix, and control cells. 14C-TEA (100 lM) was added to confluent cell layers grown in 96-well Cytostar SPA plates. Scintillation signals were recorded on-line in a MicroBeta 1450 Trilux counter. Solid circles d, hOCT1 clone mix; open circles s, control cells; triangles, crosses, bars, diamonds, squares (m, ·, +, –, , j, j), six different clonal cell lines derived from the hOCT1 clone mix. Two independent experiments using 4–8 wells per cell line yielded similar results. The data of one experiment are shown.

uptake of 14C-TEA compared to the clone mix. The cell lines showing optimal functionality could easily be identified in the SPA. This approach was used to select clonal cell lines with best functional transport levels for further studies. Thus, the SPA was successfully applied to assess the transport properties of individual clonal hOCT1 cell lines displaying different functional expression levels of hOCT, indicating that the SPA is a convenient and powerful tool in the assessment of clonal cell lines transfected with SLC uptake transporters. So far, the selection of a clonal cell line displaying optimal functionality usually is based on dish uptake studies, which require washing steps, cell lysis, and liquid scintillation counting for each clonal cell line. The SPA omits the need for washing steps, lysis, sample transfer, and scintillation fluid, thus allowing numerous cell lines to be tested simultaneously on the same assay plate, which significantly enhances throughput. Use of the SPA for determining Km values The apparent Km value for hOCT1-mediated uptake of 14C-TEA was determined using the SPA technology for selected hOCT1 clonal cell lines (Fig. 5 and Table 2). For Km determination, transfected and control cells in triplicates were incubated in SPA plates with multiple substrate concentrations and scintillation signals were recorded. Background signals in control cells (empty vector-transfected) were subtracted from the signals obtained for transfected cells. Km values were determined from the initial slopes as described under Materials and methods, assuming Michaelis–Menten kinetics. As shown in Table 2, results obtained for hOCT1 clonal cell lines were similar across selected cellular clones. Most important, the values obtained in the SPA correspond very well to literature data (229 ± 78.4 lM [17]), thus under-

450

900

CONC.14C-TEA (µM) Fig. 5. Km values of 14C-TEA uptake in HEK-hOCT1 cells, as determined in the scintillation proximity assay. 14C-TEA in various concentrations (0, 50, 100, 150, 200, 250, 350, 500, and 750 lM) was added to confluent cell layers in 96-well SPA plates. Scintillation signals were recorded on-line in a MicroBeta 1450 Trilux counter. Uptake was linear up to 30 min. For Km determination, unspecific uptake of 14C-TEA into control cells (empty vector-transfected) was subtracted from the uptake values obtained for hOCT1-transfected cells. Triplicate wells were used for each substrate concentration. Km values were determined from the initial slopes as described under Materials and methods, assuming Michaelis–Menten kinetics. One representative experiment on one hOCT1 clone is shown in Fig. 4. Three independent experiments were carried out as described for each of the hOCT1 clones a, b, c, d. The resulting Km values for clones a–d are given in Table 2.

Table 2 Km and Vmax values of 14C-TEA uptake in various clones of HEK-hOCT1 cells Clone

Km (14C-TEA) [lM]

Vmax [cpm/min/well]

a b c d

206 ± 23 217 ± 27 208 ± 51 237 ± 20

52 ± 10 61 ± 9 60 ± 6 67 ± 6

14 C-TEA in various concentrations (0, 50, 100, 150, 200, 250, 350, 500, and 750 lM) was added to confluent cell layers of various HEK-hOCT1 clones in 96-well SPA plates. Scintillation signals were recorded on-line in a MicroBeta 1450 Trilux counter. Uptake was linear up to 30 min. For Km determination, unspecific uptake of 14C-TEA into control cells (mocktransfected) was subtracted from the uptake values obtained for hOCT1transfected cells. Km values were determined as described under Materials and methods, assuming Michaelis–Menten kinetics. Triplicate wells were used for each substrate concentration. Three independent experiments were carried out as described for each of the hOCT1 clones a, b, c, d. The resulting Km values for clones a–d are given in Table 1 (average ± SD; n = 3).

lining the fitness of the SPA approach for valid Km determinations on SLC transporters/substrates. Additionally, the Km for hOAT3-mediated uptake of 3H-E3S was determined using the SPA and HEK-hOAT3 cells to be 4 lM (data not shown), which is in agreement with data reported in the literature [18]. The apparent Km values of SLC transporter substrate uptake could be determined in a rapid, convenient, and reliable fashion using the SPA approach.

Cellular drug uptake measured by scintillation proximity / C. Lohmann et al. / Anal. Biochem. 366 (2007) 117–125

Use of the SPA for identifying transporter inhibitors

When calculating the precision for percentage inhibition at one concentration in the SPA, a format that is useful for screening of new chemical entities, the CV% was 612% within one plate and the between-assay CV% was below 15%. The between-assay variation for IC50 values was also very low. Although the precision of the 12-well assay was higher than that for the SPA, the SPA shows an excellent within- and between-assay variability for screening purposes. This clearly demonstrates the applicability of the SPA approach to identify hOAT3 inhibitors, and possibly further SLC transporter inhibitors, in a quick and convenient way, making it a promising tool in pharmaceutical research, particularly in screening for potential transporter-based drug–drug interactions. The SPA approach provides the additional advantage that, due to small incubation volumes, it requires only small amounts of test compounds. By measuring uptake at various inhibitor concentrations, this assay could be used to calculate IC50 and/or Ki values of SLC inhibition for candidate drugs.

To test the suitability of the SPA approach for identifying SLC transport inhibitors, the known inhibitory effects of ibuprofen [19] and furosemide [20] on hOAT3-mediated uptake of 3H-E3S were studied in the SPA and compared with the respective effects observed in a conventional dish uptake assay. As shown in Fig. 6, addition of both ibuprofen (Fig. 6A) and furosemide (Fig. 6C) in concentrations ranging from 10 to 200 lM resulted in a concentrationdependent decrease of 3H-E3S uptake into HEK-hOAT3 cells measured by SPA. Importantly, similar results were obtained in a conventional dish assay by liquid scintillation counting, where comparable inhibition of hOAT3-mediated uptake of 3H-E3S was detected for both ibuprofen and furosemide (Figs. 6B and D). The extent of hOAT3 inhibition observed corresponded well between the assays (Table 3). The within and between assay variations were also assessed for both the SPA and the dish assays (Table 3).

A

20

123

B

12

18 10 H-E3S UPTAKE (cpm/min/well)

14 12 10 8 6

8 6 4

3

3

H-E3S UPTAKE (cpm/min/well)

16

4

2

2 0 0

50

100

150

200

0

250

0

50

C

100

150

200

250

IBUPROFEN (µM)

IBUPROFEN (µM)

D

18

10

H-E3S UPTAKE (cpm/min/well)

14 12 10 8 6

8 6 4

3

3

H-E3S UPTAKE (cpm/min/well)

16

4

2

2 0

0

50

100

150

200

FUROSEMIDE (µM)

250

0

0

50

100

150

200

250

FUROSEMIDE (µM)

Fig. 6. Inhibition of hOAT3-mediated uptake of [3H]-E3S by ibuprofen (A, B) and furosemide (C, D) measured by scintillation proximity and conventional dish assay.(A, C) Scintillation proximity assay. [3H]-E3S (100 nM) and inhibitors (A) ibuprofen and (C) furosemide in various concentrations as indicated were added to confluent cell layers in 96-well SPA plates. Scintillation signals of 3H-E3S uptake were recorded on-line in a MicroBeta 1450 Trilux counter. The initial rates of uptake were calculated and plotted vs inhibitor concentration. Average values ±SD of 4 wells each are shown. (B, D) Dish assay (12-well assay). [3H]-E3S (100 nM) and inhibitors (B) ibuprofen, (D) furosemide in various concentrations as indicated were added to confluent cell layers in 12-well cell culture plates. After 10 s of incubation, uptake was terminated by washing the cells with ice-cold buffer. 3H-E3S in cell lysates was quantified by liquid scintillation counting. The uptake rates were plotted vs inhibitor concentration. Average values ±SD (n = 3 for each inhibitor concentration) are given. Similar profiles were obtained when uptake was terminated after 5 min of incubation.

124

Cellular drug uptake measured by scintillation proximity / C. Lohmann et al. / Anal. Biochem. 366 (2007) 117–125

Table 3a Precision and comparison of SPA and 12-well assays for hOAT3 inhibition: ibuprofen Within-assay precision; % inhibition at one single concentration SPA 12-well assay % inhibition at 100 lM % inhibition at 120 lM (n = 4) (n = 3) M ± SD Exp 1 Exp 2 Exp 3

71.9 ± 6.8 74.8 ± 6.2 58.2 ± 5.6

CV% 9.5 8.2 9.6

M ± SD

CV%

83.2 ± 1.3 83.4 ± 0.8 85.8 ± 0.4

1.6 1.0 0.5

Between-assay precision; IC50 and % inhibition at one single concentration SPA (n = 3) 12-well assay (n = 3)

% Inhibition IC50 [lM]

M ± SD

CV%

M ± SD

CV%

68.3 ± 8.8 51.5 ± 2.8

12 5.4

84.1 ± 1.4 11.9 ± N.D.

1.7 N.D.

HEK-hOAT3 cells were incubated with [3H]-E3S (100 nM) and ibuprofen in different concentrations (see Fig. 6), using 4 wells per concentration in the SPA and 3 wells per concentration in the 12-well assay. In the SPA, % inhibition and IC50 were calculated from the slopes of the initial linear rates of the uptake curves. In the 12-well assay, incubations were stopped after 10 s and the uptake rates determined. Similar IC50 values were obtained in the 12-well assay when the uptake was terminated after 5 min of incubation. Data are given as mean ± SD.

Table 3b Precision and comparison of SPA and 12-well assays for hOAT3 inhibition: furosemide Within-assay precision; % inhibition at one single concentration SPA 12-well assay % inhibition at 100 lM % inhibition at 120 lM (n = 4) (n = 3)

Exp 1 Exp 2 Exp 3

M ± SD

CV%

M ± SD

CV%

76.0 ± 8.7 77.2 ± 4.0 68.9 ± 4.3

12 5.2 6.3

82.6 ± 2.9 75.8 ± 0.4 89.3 ± 0.8

3.6 0.5 0.9

Between-assay precision; IC50 and % inhibition at one single concentration SPA (n = 3) 12-well assay (n = 3) M ± SD % Inhibition IC50 [lM]

74.0 ± 4.5 45.9 ± 2.3

CV% 6.1 5.0

M ± SD

CV%

82.6 ± 6.7 21.5 ± N.D.

8.2 N.D.

HEK-hOAT3 cells were incubated with [3H]estrone-3-sulfate (100 nM) and furosemide in different concentrations (see Fig. 6), using 4 wells per concentration in the SPA and 3 wells per concentration in the 12-well assay. In the SPA, % inhibition and IC50 were calculated from the slopes of the initial linear rates of the uptake curves. In the 12-well assay, incubations were stopped after 10 s and the uptake rates determined. Similar IC50 values were obtained in the 12-well assay when the uptake was terminated after 5 min of incubation. Data are given as mean ± SD.

Moreover, the SPA inhibition assay described here provides high compound throughput and may be run in an automated fashion. Conclusion This study demonstrates the applicability of a scintillation proximity assay for detecting compound interactions

with human SLC transporters in real time. The assay is high throughput and amenable to automation and allows for determination of Km values, for selection of clonal cell lines by their level of transporter function, and for identification of SLC transporter inhibitors in a convenient and reliable fashion, suggesting its successful use in future drug discovery. Acknowledgments We thank Stefan Halle´n and Bjo¨rn Johansson for excellent technical advice on multiplate scintillation counting. References [1] H. Kusuhara, Y. Sugiyama, Role of transporters in the tissueselective distribution and elimination of drugs: transporters in the liver, small intestine, brain and kidney, J. Controlled Release 78 (2002) 43–54. [2] N. Mizuno, T. Niwa, Y. Yotsumoto, Y. Sugiyama, Impact of drug transporter studies on drug discovery and development, Pharmacol. Rev. 55 (2003) 425–461. [3] A. Ayrton, P. Morgan, Role of transport proteins in drug absorption, distribution and excretion, Xenobiotica 31 (2001) 469–497. [4] J.E. van Montfoort, B. Hagenbuch, G.M. Groothuis, H. Koepsell, P.J. Meier, D.K. Meijer, Drug uptake systems in liver and kidney, Curr. Drug Metab. 4 (2003) 185–211. [5] H. Koepsell, H. Endou, The SLC22 drug transporter family, Pfluegers Arch.-Eur. J. Physiol. 447 (2004) 666–676. [6] G. Burckhardt, N.A. Wolff, Structure of renal organic anion and cation transporters, Am. J. Physiol. 278 (2000) F853–F866. [7] B. Hagenbuch, P.J. Meier, Organic anion transporting polypeptides of the OATP/ SLC21 family: phylogenetic classification as OATP/ SLCO superfamily, new nomenclature and molecular/ functional properties, Pfluegers Arch. - Eur. J. Physiol. 447 (2004) 653–665. [8] S.A. Roberts, High-throughput screening approaches for investigating drug metabolism and pharmacokinetics, Xenobiotica 31 (2001) 557–589. [9] R.E. White, High-throughput screening in drug metabolism and pharmacokinetic support of drug discovery, Annu. Rev. Pharmacol. Toxicol. 40 (2000) 133–157. [10] J.B. Pritchard, D.S. Miller, Expression systems for cloned xenobiotic transporters, Toxicol. Appl. Pharmacol. 204 (2005) 256–262. [11] R. Graves, R. Davies, G. Brophy, G. O’Beirne, N. Cook, Noninvasive, real-time method for the examination of thymidine uptake events–application of the method to V-79 cell synchrony studies, Anal. Biochem. 248 (1997) 251–257. [12] R. Graves, R. Davies, P. Owen, M. Clynes, I. Cleary, G. O’Beirne, An homogeneous assay for measuring the uptake and efflux of radiolabelled drugs in adherent cells, J. Biochem. Biophys. Methods. 34 (1997) 177–187. [13] A. Cushing, M.J. Price-Jones, R. Graves, A.J. Harris, K.T. Hughes, D. Bleakman, D. Lodge, Measurement of calcium flux through ionotropic glutamate receptors using Cytostar-T scintillating microplates, J. Neurosci. Methods. 90 (1999) 33–36. [14] H. Bonge, S. Hallen, J. Fryklund, J.E. Sjostrom, Cytostar-T scintillating microplate assay for measurement of sodium-dependent bile acid uptake in transfected HEK-293 cells, Anal. Biochem. 282 (2000) 94–101. [15] J.B. Williams, P.J. Mallorga, W. Lemaire, D.L. Williams, S. Na, S. Patel, J.P. Conn, D.J. Pettibone, C. Austin, C. Sur, Development of a scintillation proximity assay for analysis of Na+/Cl- -dependent neurotransmitter transporter activity, Anal. Biochem. 321 (2003) 31–37.

Cellular drug uptake measured by scintillation proximity / C. Lohmann et al. / Anal. Biochem. 366 (2007) 117–125 [16] J. Grunberg, K. Knogler, R. Waibel, I. Novak-Hofer, High-yield production of recombinant antibody fragments in HEK-293 cells using sodium butyrate, BioTechniques 34 (2003) 968–972. [17] L. Zhang, M.E. Schaner, K.M. Giacomini, Functional characterization of an organic cation transporter (hOCT1) in a transiently transfected human cell line (HeLa), J. Pharmacol. Exp. Ther. 286 (1998) 354–361. [18] S.H. Cha, T. Sekine, J-I. Fukushima, Y. Kanai, Y. Kobayashi, T. Goya, H. Endou, Identification and characterisation of human organic anion transporter 3 expressing predominantly in the kidney, Mol. Pharmacol. 59 (2001) 1277–1286.

125

[19] S. Khamdang, M. Takeda, R. Noshiro, S. Narikawa, A. Enomoto, N. Anzai, P. Piyachaturawat, H. Endou, Interactions of human organic anion transporters and human organic cation transporters with nonsteroidal anti-inflammatory drugs, J. Pharmacol. Exp. Ther. 303 (2002) 534–539. [20] H. Hasannejad, M. Takeda, K. Taki, H.J. Shin, E. Babu, P. Jutabha, S. Khamdang, M. Aleboyeh, M.L. Onozato, A. Tojo, A. Enomoto, N. Anzai, S. Narikawa, X.L. Huang, T. Niwa, H. Endou, Interactions of human organic anion transporters with diuretics, J. Pharmacol. Exp. Ther. 308 (2004) 1021–1029.