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DMD #29967
1
Investigation of the involvement of P-gp and MRP2 in the efflux
of ximelagatran and its metabolites by using short hairpin RNA
knockdown in Caco-2 cells
Malin Darnell, Johan E. Karlsson, Albert Owen, Ismael J. Hidalgo,
Jibin Li, Wei Zhang, Tommy B. Andersson
Clinical Pharmacology & DMPK, AstraZeneca R&D, Mölndal, Sweden (M.D., J.E.K.,
T.B.A.); Absorption Systems LP, Exton, PA, USA (A.O., I.J.H, J.L., W.Z.); and Section of
Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet,
Stockholm, Sweden (M.D., T.B.A.).
DMD Fast Forward. Published on December 18, 2009 as doi:10.1124/dmd.109.029967
Copyright 2009 by the American Society for Pharmacology and Experimental Therapeutics.
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Running title: The involvement of P-gp and MRP2 in ximelagatran transport
Address for correspondence
Tommy B. Andersson, Clinical Pharmacology & DMPK,
AstraZeneca R&D Mölndal, S-431 83 Mölndal, Sweden.
E-mail: [email protected]
Telephone: +46-31-7761534
Fax: +46-31-7763786
Number of text pages: 24
Number of tables: 1
Number of figures: 8
Word count for abstract: 240 (250 max)
Word count for introduction: 457 (750 max)
Number of words in the discussion: 869 (1500 max)
Number of references: 22 (40 max)
Abbreviations: A-to-B, apical-to-basolateral; AhR, aryl hydrocarbon receptor; BCRP, breast
cancer resistance protein; BSP, bromosulfophthalein; B-to-A, basolateral-to-apical; Caco-2,
cancer colon cell line; CEBP, CCAAT/enhancer binding protein; CPS1, carbamoyl phosphate
synthetase I; CYP, cytochrome P450; DDI, drug-drug interaction; DMEM, Dulbecco’s
Modified Eagle’s Medium; ethyl-mel, ethyl-melagatran; FBS, Fetal bovine serum; FXP,
Farnesoid X Receptor; HBSS, Hanks Balanced Salt Solution; hydroxy-mel, hydroxy-
melagatran; HNF4, Hepatocyte Nuclear Factor 4; MDR1, multidrug resistance 1; MRP2,
multidrug resistance-associated protein 2; NEAA, non-essential amino acids; OATP, organic
anion transporting polypeptide; Papp, apparent permeability coefficient; PCR, polymerase
chain reaction; P-gp, P-glycoprotein; RXR, retinoid X receptor; shRNA, short hairpin RNA;
siRNA, Short interference RNA; SLC, Solute Carriers; SULT, sulfotransferase; UGT, UDP-
glucuronosyltransferase; ximel, ximelagatran.
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Abstract
Liver and bile secretion can be an important first pass and clearance route for drug
compounds and also the site of several drug-drug interactions (DDIs). In the clinical program
for ximelagatran development an unexpected effect of erythromycin on the pharmacokinetic
of the direct thrombin inhibitor ximelagatran and its metabolites was detected. This
interaction was believed to be mediated by inhibition of drug transporters, which normally
extrude the drug into the bile. Previous Caco-2 cell experiments indicated the involvement of
an active efflux mechanism for ximelagatran, hydroxy-melagatran and melagatran possibly
mediated by P-glycoprotein (P-gp). However, the inhibitors used may not have been specific
enough and the possibility that transporters other than P-gp were important in the Caco-2 cell
assay cannot be excluded. In this study we used RNA interference, a post-transcriptional gene
silencing mechanism where mRNA is degraded in a sequence-specific manner, to specifically
knockdown P-gp or MRP2 transporters in Caco-2 cells. The data obtained from bi-directional
transport studies in these cells indicate a clear involvement of P-gp but not of MRP2 in the
transport of ximelagatran, hydroxy-melagatran and melagatran across the apical cell
membrane. The present study shows that short hairpin RNA (shRNA) Caco-2 cells are a
valuable tool to investigate the contribution of specific transporters in the trancellular
transport of drug molecules and to predict potential sites of pharmacokinetic interactions. The
results also suggest that inhibition of hepatic P-gp is involved in the erythromycin-
ximelagatran interaction seen in clinical studies.
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The direct thrombin inhibitor ximelagatran was envisaged to have a low potential for
pharmacokinetic drug-drug interactions (DDIs) based on the preclinical information showing
that ximelagatran, its intermediate metabolites and melagatran are neither substrates nor
inhibitors of the major cytochrome P450 isoenzymes (Bredberg et al., 2003). The preclinical
results were supported by the results from three specific healthy volunteer studies showing no
clinically significant DDIs between ximelagatran and diclofenac, diazepam or nifedipin when
co-administered simultaneously (Bredberg et al., 2003). The phase I DDI study showing a
clear interaction with erythromycin, therefore, came as a surprise. Melagatran AUC increased
by 82%, whereas ximelagatran plasma levels appeared to be essentially unchanged
Ximelagatran is bioconverted to melagatran via two intermediates, hydroxy-melagatran and
ethyl-melagatran (Fig. 1) and an elevation in the plasma concentrations was seen for both
intermediates, following coadministration with erythromycin. (Eriksson et al., 2006).
Several in vivo and in vitro studies have been performed to investigate the nature of the
erythromycin - ximelagatran interaction (Eriksson et al., 2006; Sjödin et al., 2008). The
pharmacokinetic profile in humans indicated that the elevation of ximelagatran and its
metabolites in plasma caused by erythromycin occurs during the first pass through the liver
(Eriksson et al., 2006). Loc-I-Gut studies both in pigs and humans (Matsson et al. submitted)
indicate that erythromycin reduced the biliary secretion of ximelagatran and its metabolites
suggesting that the interaction is likely to be due to inhibition of hepatic transporters (Fig. 2)
(Sjödin et al., 2008,). The Loc-I-Gut study in pigs also indicated that the intestinal absorption
of ximelagatran was not affected. Furthermore, Sjödin et al. suggested that the erythromycin
interaction was not likely to be caused by direct inhibition of ximelagatran metabolism
(Sjödin et al., 2008). The hypothesis that the effect of erythromycin on ximelagatran
pharmacokinetic is due to inhibition of biliary transporters is further supported by the Caco-2
cell results showing the presence of an active efflux for ximelagatran, hydroxy-melagatran
and melagatran, possibly mediated by P-glycoprotein (P-gp) (Eriksson et al., 2006). However,
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Caco-2 cells express a number of transporter proteins and the contribution of different biliary
transporters needs to be further investigated.
The aim of the present study was to use the short hairpin RNA (shRNA) Caco-2
knockdowns as a tool to identify biliary efflux transporters and predict potential sites of
transporter-mediated pharmacokinetic interactions. Stable Caco-2 MDR1 (ABCB1)/ P-gp and
MRP2 (ABCC2) knockdowns were established using lentiviral vector expressing shRNA
against MDR1 and MRP2, respectively. The effect of P-gp knockdown on the transport of
ximelagatran and its metabolites was studied to investigate if the interaction seen in vivo
between erythromycin and ximelagatran was mediated by P-gp. Furthermore, MRP2
knockdown Caco-2 cells were used to study the contribution of MRP2 in the bi-directional
transport of ximelagatran and its metabolites.
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Materials and Methods
Chemicals
Lucifer yellow, Hanks Balanced Salt Solution (HBSS), Dulbecco’s Modified Eagle’s
Medium (DMEM), Dulbecco’s phosphate buffered saline (DPBS), and N-[2-
hydroxyethyl]piperazine-N’-[2-ethanesulfonic acid] (HEPES) were obtained from
Gibco/Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was obtained from Omega
(Tarzana, CA). Penicillin-streptomycin, non-essential amino acids (NEAA), L-glutamine, and
trypsin were obtained from CelGro (Herndon, VA). [3H]bromosulfophthalein ([3H]BSP) was
obtained from International Isotopes Clearing House (Leawood, KS). Digoxin was purchased
from Sigma-Aldrich (St. Louis, MO). Ximelagatran, hydroxy-melagatran, ethyl-melagatran,
melagatran and their respective 13C-labelled internal standards were supplied by AstraZeneca
(Mölndal, Sweden). Standard solutions of the analytes and their internal standards were
prepared in 0.01 mol/l hydrochloric acid (HCl). HPLC grade acetonitrile was purchased from
Rathburn (Walkerburn, UK). Ammonium acetate and HCl (HCl[Titrisol]) were purchased
from Merck (Darmstadt, Germany). High purity water was obtained from an ELGA
purification system (High Wycombe, UK).
Transduction of Caco-2 cells with shRNAs
Parental Caco-2 cells were obtained from American Type Culture Collection (Manassas,
VA, ATCC Accession Number CRL-2102). The cells were maintained in DMEM with 4.5
g/L glucose, supplemented with 10% FBS, 1% NEAA, 2 mM L-glutamine, 100 U/mL
penicillin, and 100 μg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO2 and
95% air. Lentivirus plasmid vectors containing shRNA inserts targeting human P-gp
(Accession I.D. NM_000927) and MRP2 (Accession I.D. NM_000392) genes were obtained
from Sigma (St. Louis, MO). The transduction procedure was described previously (Zhang et
al. 2009). Briefly, the cells were plated in 96-well tissue culture plates at a density of 5 x 106
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cell/well and transduced with 1 μg of P-gp- or MRP2-targetting shRNA viral vector.
Transduced cells were selected in culture medium containing 10 µg/mL puromycin. As
transduction control, parental Caco-2 cells were also transduced with a lentivirus plasmid
vector containing shRNA that does not match any known human genes (Cat# SHC002,
Sigma, St. Louis, MO), and the transduction procedures were identical to those used with P-
gp and MRP2 shRNA vectors.
RNA Isolation and cDNA Synthesis
Total RNA was isolated from cells using Trizol reagent (Invitrogen, Carlsbad, CA)
according to the manufacturer’s protocol. cDNA was prepared from 0.25 µg of total RNA by
using SuperScript ІІІ First-Strand Synthesis System for reverse transcription-polymerase
chain reaction (PCR) with random hexamer primers according to the manufacturer’s protocol
(Invitrogen, Carlsbad, CA).
Real-Time PCR
Forty-eight different genes were analyzed with quantitative real-time PCR employing
TaqMan® Custom Array. The 384-well cards were purchased preloaded with TaqMan® Gene
Expression Assays with specific primers and probes and 100 ng of cDNA was added to each
lane. The cDNA quantification was performed using an ABI PRISM® 7900HT and the
thermal cycle conditions comprised 2 min at 50°C, 10 min of polymerase activation at 95°C,
followed by 40 PCR cycles alternating 95°C for 15 s and 60°C for 1 min. Amplification
curves were analyzed using the 7900HT sequence detection software SDS 2.3 and the
expression for all genes was normalized against the expression of Glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) in each sample (Applied Biosystems, Foster City, CA).
Quantification of relative gene expression was performed using the ΔΔCT method. Fold
change were calculated using vector control Caco-2 cells as a calibrator.
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Western blot analysis for P-gp and MRP2 protein detection
Cell monolayers were washed with cold PBS and lysed in ice-cold RIPA Lysis Buffer
(Santa Cruz BioTech, Santa Cruz, CA). Proteins were isolated following the manufacturer’s
protocol. Protein concentrations were determined using Quant-it Protein Assay kit with Qubit
fluorometer (Invitrogen, Carlsbad, CA). Equal amounts of protein were loaded and separated
on 4-20 % SDS- polyacrylamide gels, then transferred to PVDF membranes (Millipore,
Billerica, MA). Membranes blocked with 0.2% I-block solution (Applied Biosystems, Foster
City, CA.) were probed sequentially with anti-MRP2 (Abcam, Cambridge, MA), anti-P-gp
(clone C219 from Abcam, Cambridge, MA) and anti-β-actin antibodies (Sigma Aldrich, MO).
Secondary antibodies were goat anti-mouse IgG linked to horseradish peroxidase (HRP) for
P-gp and β-actin (Zymed, Carlsbad, CA), goat anti-rabbit IgG linked to HRP for MRP2
(Abcam, Cambridge, MA). Protein-antibody complexes were visualized using the Super
Signal West Femto Chemiluminescent Substrate (Pierce, Rockford, IL).
Transport Assays
Cells were seeded at 60,000 cells/ cm2 onto collagen-coated, microporous, polycarbonate
membranes in 12-well Costar Transwell® plates (1.13 cm2 insert area, 0.4 µm pore size;
BioSciences, Bedford, MA). The culture medium was changed 24 h after seeding to remove
cell debris and dead cells; afterwards the medium was changed every other day. The cells
were maintained at 37°C in a humidified atmosphere of 5% CO2 in air for three weeks to form
confluent monolayers. Batches of cell monolayers were certified by measuring the
transepithelial electrical resistance (TEER) values of the monolayers and the apparent
permeability coefficient (Papp) of control compounds: Lucifer yellow (fluorescent low
permeability marker), atenolol (low permeability marker), propranolol (high permeability
marker), and digoxin (P-gp probe substrate). The acceptance criteria for acceptable batches of
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cell monolayers were: TEER > 450 Ω•cm2, Lucifer yellow Papp < 0.4x10-6 cm/s, atenolol Papp
< 0.5 x 10-6 cm/s, propranolol Papp between 15 x 10-6 and 25 x 10-6 cm/s. Prior to the transport
experiment, TEER values were measured for all cell monolayers, and only monolayers with
TEER values that met the acceptance criteria were selected for the transport study. The
transport assay buffer was Hanks’ balance salts solution containing 10 mM HEPES and 15
mM glucose at pH 7.4 (HBSSg buffer). Digoxin as P-gp probe substrate was assayed at 10
μM, [3H]BSP as MRP2 substrate assayed at 0.5 µCi/mL (27 nM), and ximelagatran assayed at
50 μM. Bi-directional transport experiments were conducted at 37°C up to 3 hours. For
apical-to-basolateral (A-to-B) transport, test compound was dosed to the apical chamber
(donor) and blank HBSSg buffer was applied to the basolateral chamber (receiver). For
basolateral-to-apical (B-to-A) transport, test compound was dosed to the basolateral chamber
(donor) and blank HBSSg buffer was added to the apical chambers (receiver). In ximelagatran
assays, 1% bovine serum albumin was included in the receiver HBSSg buffer. Samples were
withdrawn from donor chambers at 1 and 2 h, from the receiver chambers at 1 and 3 h; the
chambers replenished with fresh HBSSg buffer with volumes equal to the sample volumes.
Immediately upon collection, ximelagatran samples were combined with an equal volume of
freshly prepared acetonitrile containing 1% HCl. At the end of ximelagatran transport
experiments, the assay buffer on the cell monolayers was removed by aspiration. The
monolayers were then washed three times with ice cold HBSSg buffer, the insert filters to
which cells attached were removed using a scalpel. They were then submerged in 300 µL of
acetonitrile with 1% HCl in Eppendorf vials. The cells were lysed by means of vortexing,
sonication, and subsequent storage of the Eppendorf vials at -20°C for 1 h to assure complete
lysis. Afterwards, vials were centrifuged at 10,000g for 20 min. The supernatants were
collected for measurement of cellular accumulation of ximelagatran and metabolites.
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Sample Analyses
Lucifer yellow concentrations in the samples were measured using a FLUOstar
fluorescence plate reader (BMG Laboratories, Durham, NC) with excitation and emission
wavelengths of 485 and 538 nm, respectively. Radioactivity of [3H]BSP samples was
measured by liquid scintillation counting (Beckman LSC6500, Fullerton, CA). Concentrations
of atenolol, propranolol and digoxin were measured using reverse phase HPLC with tandem
triple quadruple mass spectrometry (LC-MS/MS) methods. Samples were diluted with 85:15
HBSSg/acetonitrile (v/v) as follows: A-to-B receiver samples by 4-fold, B-to-A receiver
samples by 10-fold, and all donor samples by 24-fold. The HPLC equipment consisted of a
Perkin-Elmer series 200 autosampler and a Perkin-Elmer series 200 micro pump (Waltham,
MA). Chromatography was performed at ambient temperature in the reverse phase mode
using a 30 mm x 2.1 mm i.d. 3 μm Thermo BDS Hypersil C18 analytical column with a guard
column (Thermo Scientific, Waltham, MA). The mobile phase buffer was 40 mM ammonium
formate, pH 3.5; the aqueous phase consisted of 90% deionized water and 10% buffer (v/v);
the organic phase consisted of 90% acetonitrile and 10% buffer (v/v). Typical gradients
started at 100% aqueous phase, linearly changed to 100% organic phase over 1.5 min, held at
100% organic for 0.5 min, then returned back to 100% aqueous during 0.4 min and re-
equilibrated at 100% aqueous for 1.6 min. The flow rate was 300 μL/min and the injection
volume was 5 μL. Mass spectrometry (MS) analyses were performed on a PE Sciex API3000
triple quadrupole mass spectrometer equipped with a TurboIon Spray interface (MDS Sciex,
San Francisco, CA). Instrument settings were optimized for each compound. The multiple
reaction monitoring (MRM) transitions for atenolol, propranolol and digoxin were
267.2/145.1, 260.2/116.1 and 798.8/651.5, respectively. Equipment operation, data
acquisition, and data integration were performed using Analyst version 1.4.2 software
(Applied Biosystems, Foster City, CA).
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Ximelagatran and its metabolites were analysed using LC/MS. The LC system consisted
of a HTS PAL injector (CTC Analytics, Zwingen, Switzerland) combined with an HP 1100
LC binary pump and column oven (Agilent Technologies Deutschland, Waldbronn,
Germany). LC separations were undertaken on a HyPurity C18 analytical column (100×2.1
mm, 5 µm, Thermo, UK) with a HyPurity C18 precolumn at 40 ˚C and with a flow rate of 750
µl/min. The mobile phases consist of 10 mmol/L of ammonium acetate and 5 mmol/L of
acetic acid with 10 % acetonitrile for mobile phase A and 50 % acetonitrile for mobile phase
B. The analytes co-eluted with their respective isotope-labelled internal standards. Detection
was performed with a triple quadrupole mass spectrometer, API4000, equipped with
electrospray interface (Applied Biosystems/MDS Sciex, Concorde, Canada). Instrument
control, data acquisition and data evaluation were performed using Applied Biosystems/MDS
Sciex Analyst 1.4 software.
Calculations
The apparent permeability coefficient, Papp, was calculated for Lucifer yellow, atenolol,
propranolol, [3H]BSP and digoxin using the following equation:
0
/
CA
VdtdCP rr
app ××=
Whereas the rate of appearance, dQ/dt, was calculated for [3H]BSP, digoxin, ximelagatran
and its metabolites.
dCr /dt is the slope of the cumulative concentration in the receiver (Cr) compartment over
time in μM/sec, Vr is the volume of the receiver chamber in cm3, dQ is Cr x Vr, A is the
diffusion area in cm2, and C0 is the measured dosing concentration in μM. Monolayers with
Lucifer yellow Papp > 0.8x10-6 cm/s were considered leaky during transport experiment and
were excluded from further calculations.
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The ratio of appearance rate of [3H]BSP, digoxin, ximelagatran and its metabolites was
defined as:
)(
)(
/
/
BA
AB
dtdQ
dtdQR
−
−=
Statistical Analysis
The significance of the effects of P-gp knockdown on digoxin transport and MRP2
knockdown on [3H]BSP transport compared to vector control cells was calculated with one-
tailed Student t-test. One-way ANOVA followed by one-sided Dunnett’s POST Hoc test was
used to calculate the statistical significances of the increased A-B transport, the decreased B-
A transport and the decreased ratio of ximelagatran and its metabolites in P-gp and MRP2
knockdown cells compared to vector control cells.
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Results
Suppression of MDR1/ P-gp and MRP2 mRNA and protein expression using shRNAs
Stable Caco-2 cell lines with MDR1/ P-gp and MRP2 knockdown were established using
shRNA constructs targeting, respectively, MDR1- and MRP2-encoding mRNA. The mRNA
levels of MDR1 and MRP2 in knockdown cell lines were compared to the levels expressed in
non-coding shRNA vector control cells. MDR1 and MRP2 expression were reduced by 55%
and 65%, respectively, in the corresponding knockdown cell lines cultured on filters for 3
weeks (Fig. 3). Furthermore, the inhibition of P-gp and MRP2 protein expression by
transduction with shRNA was confirmed by Western Blot analysis (Fig. 4).
Gene expression following MDR1 and MRP2 knockdown
The influence of P-gp and MRP2 knockdown on mRNA expression of other transporters,
metabolic enzymes and transcription factors was determined employing quantitative real-time
PCR. Except for the targeted transporters, only 2 of 28 detected genes had more than a 2-fold
reduction or 2-fold increase compared to the vector control cells (Fig. 5). mRNA expression
of UDP-glucuronosyltransferase 2B7 (UGT2B7) increased 3.4-fold in P-gp knockdown cells,
whereas transthyretin increased 2.6-fold in MRP2 knockdown cells. The mRNA expressions
of all other genes studied were not markedly affected by the knockdown of P-gp or MRP2.
The effect of MDR1 and MRP2 knockdown on drug transport
Vector control, P-gp- and MRP2 knockdown cells were cultured on transwell filters
enabling bi-directional transport studies of probe substrates and the study compound,
ximelagatran. Digoxin and [3H]BSP were used as selective substrates for P-gp and MRP2
dependent transport, respectively (Fig. 6 a, b). The efflux ratio of the P-gp probe substrate
digoxin decreased by 83% in P-gp knockdown cells, whereas the efflux ratio of the MRP2
substrate [3H]BSP was reduced by 44% in MRP2 knockdown cells (Table 1), which is
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consistent with the reduction seen in mRNA and protein expression presented above. Further,
the Papp value in the B-to-A direction for digoxin was decreased from 38 to 9.2 x 10-6 cm/s
when knocking down P-gp, whereas MRP2 knockdown reduced the [3H]BSP Papp value from
18 to 9.1 x 10-6 cm/s.
In the cell monolayers ximelagatran was metabolised to its intermediate metabolites,
hydroxy-melagatran and ethyl-melagatran, and its end product melagatran. The rates of
appearance of ximelagatran and its metabolites were greater in the B-to-A direction than in
the A-to-B direction in vector control cells (Table 1), indicating active net efflux across the
apical membrane. Knockdown of P-gp resulted in a significant decreased (p<0.01) appearance
rate on the apical side for ximelagatran, hydroxy-melagatran and melagatran (B-to-A
direction, Fig. 7 a). These results indicate that P-gp is involved in the active efflux of the
parent and its metabolites. In addition, both the cellular accumulation of melagatran and
hydroxy-melagatran (Fig. 8 a, b) and the appearance rate on the basolateral side of
ximelagatran, hydroxy-melagatran and melagatran (A-to-B direction, Fig. 7 b) were increased
due to knockdown of P-gp, further strengthening the conclusion that P-gp is involved in the
transport of these substances. There were no detectable appearance rate of ethyl-melagatran
on the basolateral side and the intracellular concentration was low (Fig. 8 c). Ximelagatran is
rapidly metabolised in the cell and only low levels of ximelagatran was detected
intracellularly (data not shown). Knockdown of MRP2 did not significantly affect the
transport of ximelagatran, hydroxy-melagatran and ethyl-melagatran in either direction.
However, a small significant decrease in the appearance rate of melagatran on the apical side
(B-to-A direction) was observed (Fig. 7 a).
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Discussion
RNA silencing leading to functional inactivation of the target gene is an attractive method
for down regulation of the expression of specific genes. Short interference RNA (siRNA) can
mediate strong and specific suppression of gene expression by sequence-specific cleavage of
mRNA, thus blocking the translation into target protein (Yue et al., 2009). The gene silencing
technique has great potential in drug efflux and transport-mediated drug-drug interactions
studies for elucidating the function of specific transporters in drug disposition.
Caco-2 cells are polarized cells expressing biliary transporters like P-gp and MRP2 in the
apical membrane. These apical expressed transporters are mainly found in excretory organs
(gastrointestinal tract, liver, kidney), counteracting systemic exposure, thus playing an
important role for mediating the bioavailability, distribution and excretion of many
xenobiotics and clinically important drugs. Transporter-mediated interactions can be caused
by co-administration of a compound of interest with other substrates or inhibitors that bind
and/or interact with the same transporter (Choudhuri and Klaassen, 2006). The possible
mechanism(s) of involvement of biliary transporters P-gp and MRP2 in the interaction
produced by erythromycin co-administration on ximelagatran pharmacokinetics was
investigated in this study using shRNA Caco-2 knockdowns. The results indicated a clear
involvment of P-gp but not of MRP2 in the efflux of ximelagatran, hydroxy-melagtran and
melagatran. P-gp-mediated efflux can thus be concluded to be important for the biliary
secretion of ximelagatran and its metabolites. However, this study does not rule out the
possibility that other hepatic transporter e.g. BCRP, BSEP and MATE1 may be of importance
in the disposition of ximelagatran and its metabolites.
Erythromycin and quinidine were previously found to inhibit the in vitro transport of
ximelagatran, hydroxy-melagatran and melagatran across Caco-2 cell monolayers, suggesting
the involvement of P-gp (Eriksson et al., 2006). Several factors may however have
confounded the results from those Caco-2 cell transporter studies using inhibitors. The
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inhibitors may not be selective and the in vitro concentrations used in such experiments are
often high. Thus, it cannot be excluded that these inhibitors blocked the function of several
other transporter proteins expressed in Caco-2 cells e. g. BCRP, MRP3, OATP-B and PEPT1
(Watanabe et al., 2005; Wang et al., 2008, Hilgendorf et al., 2007).
The use of the post-transcriptional gene silencing by RNA interference to specifically
knockdown drug transporters is a rapidly growing field of research. (Yue et al., 2009;
Watanabe et al., 2005; Celius et al., 2004; Hua et al., 2005; Tian et al., 2005; Li et al., 2006;
Zhang et al., 2009). Even though siRNA and shRNA have emerged as powerful tools to study
gene function in a sequence-specific manner, off-target effects have been observed in several
studies (Li et al., 2006; Jackson et al., 2006; Alemán et al., 2007; Tschuch et al., 2008). Non-
specific effects can depend on both knockdown of non-target mRNA (Jackson et al., 2006) or
compensatory effects causing up-regulation of other genes (Chen et al., 2005). As mentioned
previously, the influence of P-gp and MRP2 knockdown on mRNA expression of other
transporters, metabolic enzymes and transcription factors was analysed in this study. Most of
the 28 genes investigated were not markedly affected by the knockdown of P-gp or MRP2.
However, UGT2B7 and transthyretin mRNA expression increased more than 2-fold in P-gp
and MRP2 knockdown cells, respectively. There are several examples showing that loss of
function or downregulation of transporters or metabolic enzymes can be compensated by up-
regulations of other transporters or metabolic enzymes (Chen et al., 2005; Higuchi et al.,
2004; Jeong et al., 2005). Therefore, it is important to investigate changes in the expression of
non-target genes and elucidate if changes affect the disposition of the study compound. The
non-target effects investigated in this study on Caco-2 cells are not likely to be important for
the transport of ximelagatran and its metabolites.
Since in-silico design of siRNA sequences does not entirely eliminate unintended
siRNA:mRNA interactions (Jackson et al., 2006; Tschuch et al., 2008), additional approaches
are advocated. The degree of non-specific effects correlates with concentrations of siRNA
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(Tschuch et al., 2008) and one suggestion is to concentrate the on-target effects while diluting
the off-target effects by using endonuclease-prepared siRNA (esiRNA), a mixture of siRNA
targeting the same mRNA (Li et al., 2006; Kittler et al., 2005; Tan et al., 2007). Further,
Echeverri et al. suggest that at least 2 distinct siRNA or shRNA sequences and/or rescue
experiments should be performed to avoid false positives (Echeverri et al., 2006). Even
though straightforward approaches to achieve the right phenotype and simultaneously avoid
off-targets effects are lacking, above mentioned procedures together with properly designed
control experiments should be a good strategy to generate siRNA knockdowns with low rates
of false positives and negatives.
In conclusion, the results from the Caco-2 cell transport experiments indicate that
ximelagatran, hydroxy-melagatran and melagatran are transported by P-gp but not MRP2
since the efflux across P-gp but not MRP2 knockdown Caco-2 cell monolayers was clearly
reduced. This work, together with previous in vivo and in vitro studies, indicates that
inhibition of hepatic P-gp contributed to the increased plasma levels of melagatran seen in
healthy subjects co-administered erythromycin. Further, this study show that knockdown of
drug transporters using shRNA is a valuable tool to predict potential sites of transporter-
mediated pharmacokinetic interactions in drug discovery and development.
Acknowledgements
We wish to thank Samanth M. Allen, Erica A. Weiskicher, Rebecca A. George, and Yuehua
Huang (Absorption Systems) for their expert technical assistance.
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Footnotes
This study was supported in part by a research grant from The Food and Drug Administration
[1R43FD003482-01].
Part of this work was presented at the ISSX meeting, 2008 October 12-16, San Diego, CA.
Abstract title: “Investigation of transporters involved in biliary clearance of drugs in the
human liver using Caco-2 cells with stable MDR1 and MRP2 knockdown by shRNA”
Requests to receive reprints should be sent to
Tommy B. Andersson
Clinical Pharmacology & DMPK
AstraZeneca R&D Mölndal
S-431 83 Mölndal
Sweden
E-mail: [email protected]
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Legends and figures
Figure 1
The biotransformation of ximelagatran via hydroxy- and ethyl-melagatran to melagatran.
Figure 2
Hepatic clearance of ximelagatran and its metabolites: Uptake of ximelagatran from the blood
into the hepatocyte. Ximelagatran is metabolised to hydroxy-melagatran, ethyl-melagatran
and melagatran. Ximelagatran and its metabolites are secreted into the bile.
Figure 3
Suppression of MDR1- and MRP2 mRNA expression in P-gp- and MRP2 knockdown Caco-2
cell monolayers after 3 weeks culture. The expression is set to 1 in vector control cells. Each
bar represents the results from mRNA harvested from three wells, one experiment.
Figure 4
Western Blot analysis of human P-gp and MRP2 protein expression. Each lane was loaded
with 45 μg of protein extract. Lane A: vector control; lane B: P-gp knockdown; lane C: MRP2
knockdown.
Figure 5
Expression of mRNA for 28 genes in P-gp and MRP2 knockdown Caco-2 cell monolayers
compared to vector control cells. The cells were cultured for 3 weeks and the samples were
pooled from 3 wells, one experiment.
Figure 6
Effects of P-gp and MRP2 knockdown on the rate of appearance of the P-gp and MRP2 probe
substrates digoxin (A) and [3H]BSP (B) in Caco-2 cell monolayers. Each bar represents mean
± S.D., n=3, *p < 0.05, **p < 0.01, and ***p < 0.001.
Figure 7
Effect of MRP2- and P-gp knockdown in Caco-2 cell monolayers on the rate of appearance of
ximelagatran and its metabolites, hydroxy-melagatran, ethyl-melagatran and melagatran (A)
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on the apical side after addition of ximelagatran on the basolateral side (B) on the basolateral
side after addition of ximelagatran on the apical side. Each bar represents mean ± S.D., n=3,
*p < 0.05, **p < 0.01, and ***p < 0.001.
Figure 8
Effect of MRP2 and P-gp knockdown on the intracellular accumulation of (A) melagatran,
(B) hydroxy-melagatran and (C) ethyl-melagatran in Caco-2 cell monolayers, one experiment.
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Tables
Table 1 Appearance ratios of ximelagatran, its metabolites and probe substrates in vector control, P-gp- and MRP2 knockdown Caco-2 cell monolayers.
Vector
control
P-gp
knockdown
MRP2
knockdown
Digoxin 25 ± 11 4.4 ± 0.8 * N.A.
[3H]-BSP 25 ± 8.7 N.A. 14 ± 5.3 *
Ximel. 48 ± 15 5.6 ± 1.2 ** 43 ± 7.5 n.s.
Hydroxy-mel. 6.9 ± 1.1 1.0 ± 0.27 *** 10 ± 1.4 n.s.
Ethyl-mel. N.A. N.A. N.A.
Melagatran 98 ± 24 5.9 ± 1.0 ** 100 ± 23 n.s.
Mean ± S.D., n=3, *p < 0.05, **p < 0.01, and ***p < 0.001.
N.A.= Not applicable
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