investigation of the involvement of p-gp and mrp2 in the ......dec 18, 2009 · gibco/invitrogen...

DMD #29967

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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.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on December 18, 2009 as DOI: 10.1124/dmd.109.029967

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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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