The influence of macrolide antibiotics on the uptake...

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The influence of macrolide antibiotics on the uptake of

organic anions and drugs mediated by OATP1B1 and

OATP1B3

Annick Seithel1, Sonja Eberl1, Katrin Singer, Daniel Auge, Georg Heinkele, Nadine B.

Wolf, Frank Dörje, Martin F. Fromm, Jörg König

1contributed equally

Institute of Experimental and Clinical Pharmacology and Toxicology, University of

Erlangen-Nuremberg, Fahrstr. 17, 91054 Erlangen, Germany (AS, SE, KS, DA,

NBW, MFF, JK)

Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Auerbachstr. 112,

70376 Stuttgart, Germany (GH)

Pharmacy Department, Erlangen University Hospital, Palmsanlage 3, 91054

Erlangen, Germany (SE, FD)

DMD Fast Forward. Published on February 12, 2007 as doi:10.1124/dmd.106.014407

Copyright 2007 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 February 12, 2007 as DOI: 10.1124/dmd.106.014407

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Running title: Inhibition of OATP-mediated uptake by macrolides

Address for correspondence:

Dr. Jörg König

Institute of Experimental and Clinical Pharmacology and Toxicology

Friedrich-Alexander-University Erlangen-Nuremberg

Fahrstraße 17

91054 Erlangen

Germany

phone +49 (0)9131 85-22077

Fax +49 (0)9131 85-22773

E-mail: [email protected]

Text pages: 28

Figures: 5

References: 45

Words in Abstract: 250

Words in Introduction: 482

Words in Discussion: 1049

Abbreviations: n.d., not determined; BSP, sulfobromophthalein; SLC, solute carrier;

OATP, organic anion transporting polypeptide; HMG-CoA, 3-hydroxy-3-

methylglutaryl-coenzym A; Km, Michaelis-Menten constant


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Abstract

Macrolides may cause severe drug-interactions due to the inhibition of metabolizing

enzymes. Transporter-mediated uptake of drugs into cells [e.g. by members of the

human organic anion transporting polypeptide (OATP) family] is a determinant of

drug disposition and a prerequisite for subsequent metabolism. However, it has not

been systematically studied, whether macrolides are also inhibitors of uptake

transporters thereby providing an additional mechanism of drug-interactions.

The human OATP family members OATP1B1 and OATP1B3 mediate the uptake of

endogenous substances and drugs like antibiotics and HMG-CoA reductase

inhibitors (statins) into hepatocytes. In this study we investigated the potential role of

these uptake transporters on macrolide-induced drug-interactions. Using

sulfobromophthalein (BSP) and the HMG-CoA reductase inhibitor pravastatin as

substrates, the effect of the macrolides azithromycin, clarithromycin, erythromycin,

roxithromycin, and of the ketolide telithromycin on the OATP1B1- and OATP1B3-

mediated uptake was analyzed. These experiments demonstrated that the

OATP1B1- and OATP1B3-mediated uptake of BSP and pravastatin can be inhibited

by increasing concentrations of all macrolides except azithromycin. The IC50 values

for the inhibition of OATP1B3-mediated BSP uptake were 11 µM for telithromycin,

32 µM for clarithromycin, 34 µM for erythromycin, and 37 µM for roxithromycin. These

IC50 values were lower than the IC50 values for inhibition of OATP1B1-mediated BSP

uptake (96 – 217 µM). These macrolides also inhibited in a concentration-dependent

manner the OATP1B1- and OATP1B3-mediated uptake of pravastatin. In summary,

these results indicate that alterations of uptake transporter function by certain

macrolides / ketolides have to be considered as a potential additional mechanism

underlying drug-drug interactions.


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Introduction

Macrolide antibiotics (e.g. erythromycin, clarithromycin) can cause severe drug

interactions by increasing plasma concentrations of simultaneously administered

compounds. The major mechanism underlying these drug interactions is believed to

be inhibition of the major drug metabolizing enzyme CYP3A4 in small intestine and

liver (Ito et al., 2003; Polasek and Miners, 2006; Wrington and E.Thummel, 2000).

Published data indicate that certain macrolides are also inhibitors of the apically /

luminally localized drug efflux pump P-glycoprotein (Eberl et al., 2005; Kim et al.,

1999; Marzolini et al., 2004). By inhibition of P-glycoprotein function they increase

drug absorption from the gut lumen and decrease biliary elimination and renal

secretion of concomitantly administered drugs such as the cardiac glycoside digoxin

(Rengelshausen et al., 2003). This in turn leads to increased drug concentrations and

drug toxicity.

A newly recognized, additional determinant of drug disposition are uptake

transporters of the OATP (SLCO) family (Hagenbuch and Meier, 2004; König et al.,

2006). Members of the OATP family transport a wide range of drugs including HMG-

CoA reductase inhibitors (cerivastatin, fluvastatin, pitavastatin, pravastatin,

rosuvastatin), benzylpenicillin, digoxin, fexofenadine, methotrexate, and rifampicin

(Hagenbuch and Meier, 2003; König et al., 2006). OATP1B1 and OATP1B3 are

expressed in the basolateral membrane of hepatocytes and mediate the uptake of

endogenous substances and drugs from the portal venous blood into the liver. The

importance of uptake transporters for drug disposition has been demonstrated

analyzing genetic alterations in the SLCO1B1 gene encoding human OATP1B1.

Several polymorphisms or haplotypes have been associated with reduced drug

uptake activity in vitro (Iwai et al., 2004; Kameyama et al., 2005; Michalski et al.,

2002; Tirona et al., 2001). Furthermore, it has been shown in vivo that the basepair-


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exchange T521C, resulting in an amino acid exchange Val174Ala, was related to

increased drug concentrations [e.g. for atrasentan (Katz et al., 2006), fexofenadine

(Niemi et al., 2005b), pitavastatin (Chung et al., 2005), pravastatin (Niemi et al.,

2006; Nishizato et al., 2003), simvastatin acid (Pasanen et al., 2006), repaglinide

(Niemi et al., 2005a), and rosuvastatin (Lee et al., 2005)].

Since alterations in the OATP1B1 protein can be associated with a change in

transport activity for certain drugs, uptake transporters may also be a mechanism for

drug-drug interactions. For instance, it has been demonstrated that the macrolides

clarithromycin and erythromycin significantly increase pravastatin plasma

concentrations (Jacobson, 2004; Product-information, Pravasin® protect, 2005).

Since pravastatin is not metabolized by cytochrome P450 enzymes, uptake

transporters may account for this drug-drug interaction. In spite of the increasingly

recognized role of OATP uptake transporters for drug disposition, it has not been

systematically studied whether macrolides are inhibitors of the uptake of

concomitantly administered drugs mediated by OATPs and thereby providing a new

additional mechanism of macrolide-induced drug interactions.

Therefore, using HEK293 cells stably expressing the human uptake transporters

OATP1B1 or OATP1B3, we tested in the present study the influence of macrolide

antibiotics on the OATP1B1- and OATP1B3-mediated uptake of organic anions and

drugs.


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Materials and Methods

Chemicals and antibodies

[3H]Sulfobromophthalein ([3H]BSP; 7585 GBq/mmol) was obtained from Hartmann

Analytic (Braunschweig, Germany). Unlabeled sulfobromophthalein, erythromycin,

and poly-D-lysine hydrobromide were purchased from Sigma-Aldrich Chemie GmbH

(Taufkirchen, Germany). Unlabeled pravastatin sodium salt was obtained from Tocris

bioscience (Tocris Cookson Inc., Missouri, USA). Unlabeled azithromycin,

clarithromycin, and roxithromycin were obtained from Chemos GmbH (Regenstauf,

Germany). Unlabeled telithromycin was obtained after extraction of Ketek® tablets

(Sanofi-Aventis Deutschland GmbH, Bad Soden, Germany) using ethyl acetate and

crystallization from ethyl acetate : hexane 8:2 (v/v). Purity was assayed by HPLC-UV

to be > 99 %.

The polyclonal antibodies pESL (König et al., 2000b) and pSKT (König et al., 2000a)

were raised in rabbits against human OATP1B1 and OATP1B3, respectively. Both

were kind gifts of Professor Dr. D. Keppler (German Cancer Research Center,

Heidelberg, Germany). The horseradish peroxidase-conjugated goat anti-rabbit IgG

was obtained from Amersham (GE Healthcare Europe GmbH, Munich, Germany).

Methanol (hypergrade quality), n-hexane (p.a.), acetonitrile (hypergrade quality), and

acetic acid (supra pure quality) were purchased from Merck KGaA (Darmstadt,

Germany). Diethyl ether (99.8 % purity), ammonium acetate (p.a.), and ibuprofen

were obtained from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany).

Cell culture and transfection

Human embryonic kidney (HEK293) cells were cultured in minimum essential

medium; containing 10 % heat inactivated fetal bovine serum, 100 U/ml penicillin and

100 µg/ml streptomycin, at 37 °C and 5 % CO2. The cells were routinely sub-

cultivated by trypsinization using trypsin (0.05 %) - EDTA (0.02 %) solution. All cell


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culture media supplements were obtained from Invitrogen GmbH (Karlsruhe,

Germany). HEK293 cells were transfected with the respective plasmid pcDNA3.1(+)-

OATP1B1 (König et al., 2000b) and pcDNA3.1/Hygro(-)-OATP1B3 (Cui et al., 2001a)

using Effectene transfection reagent (Qiagen GmbH, Hilden, Germany). Plasmids

were a generous gift of Professor Dr. D. Keppler (Heidelberg, Germany). After

geneticin (for OATP1B1, 800 µg/ml) or hygromycin (for OATP1B3, 250 µg/ml)

selection, single colonies were characterized for SLCO1B1 (encoding human

OATP1B1) and SLCO1B3 (encoding human OATP1B3) mRNA and OATP1B1 or

OATP1B3 protein expression by real-time PCR and immunoblot analysis. Vector

transfected HEK-control cells were established by the same method using the

respective expression plasmid without insert for transfection.

For BSP uptake and immunoblot experiments HEK cells were seeded in petri dishes

or 6-well plates (PS plate, 6 well, Greiner Bio-One, Frickenhausen, Germany; coated

with 0.1 mg/ml poly-D-lysine), respectively, at an initial density of 125 000 cells

(OATP1B1) and 80 000 cells (OATP1B3) per cm2 growth area.

For pravastatin uptake HEK cells were seeded in poly-D-lysine (0.1 mg/ml)-coated

12-well plates (Cell Culture Multiwell Plate CELLSTAR®, Greiner Bio-One,

Frickenhausen, Germany) at an initial density of 700 000 cells per well.

The cells (HEK-control, HEK-OATP1B1, and HEK-OATP1B3) were grown to

confluence for 3 days and induced with 10 mM sodium butyrate (Merck KGaA,

Darmstadt, Germany) for 24 h prior the uptake and immunoblot experiments to obtain

higher levels of the recombinant proteins (Cui et al., 1999).

Immunoblot Analysis

Pelleted HEK293 cells expressing the respective protein were resuspended in protein

storage buffer (100 mM Tris-HCl, 1 mM EDTA, pH 7.4) containing protease inhibitors

(mini complete protease inhibitor cocktail tablets, Roche Diagnostics-Applied


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Science, Mannheim, Germany) homogenized and sonificated. Protein concentrations

were determined by bicinchonic acid assay (BCATM Protein Assay Kit, Pierce,

Rockford, USA). 20 µg of total protein was diluted with Laemmli buffer and incubated

at 95 °C for 5 min before their separation on 4 % stacking and 10 % resolving SDS-

polyacrylamide gels. Immunoblotting was performed using a tank blotting system

from Bio-Rad (Munich, Germany) and enhanced chemiluminescence detection

(PerkinElmer Life Sciences, Rodgau-Jügesheim, Germany). The primary antibodies

pESL and pSKT were diluted 1:5 000 in TPBS (Dulbecco’s phosphate-buffered

saline, pH 7.4, 0.1 % Tween 20), respectively. The secondary antibody was a

horseradish peroxidase-conjugated goat anti-rabbit IgG from Amersham (GE

Healthcare Europe GmbH, Munich, Germany) used at a 1:10 000 dilution. Human

liver samples and vector transfected HEK293 cells served as positive and negative

controls, respectively.

Uptake assays

Before starting the uptake experiments the cells were washed with pre-warmed

(37 °C) uptake buffer (142 mM NaCl, 5 mM KCl, 1 mM K2HPO4, 1.2 mM MgSO4,

1.5 mM CaCl2, 5 mM glucose, and 12.5 mM HEPES, pH 7.3).

The [3H]BSP was dissolved in uptake buffer and unlabeled BSP was added to the

final concentration of 0.05 µM and 1 µM BSP for studies with HEK-OATP1B1 and

HEK-OATP1B3, respectively. To characterize the macrolides as inhibitors they were

added in increasing concentrations (up to 500 µM). The cells were incubated with the

test solution at 37 °C for 10 minutes as described previously (Letschert et al., 2004;

Michalski et al., 2002). Subsequently, the cells were washed three times with ice-cold

uptake buffer. After lysing the cells with 0.2 % sodium dodecyl sulfate (SDS) the

intracellular accumulation of radioactivity was calculated by liquid scintillation

counting (Perkin Elmer Life Sciences GmbH, Rodgau, Jügesheim, Germany) and the


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appropriate protein concentration was determined by bicinchonic acid assay (BCATM

Protein Assay Kit, Pierce, Rockford, USA).

For experiments with pravastatin as substrate, 50 µM pravastatin was dissolved in

the uptake buffer. In addition, 10 µM or 100 µM of each macrolide was added. The

uptake assay was performed as described above except for the determination of the

intracellular pravastatin accumulation: after lysing the cells with 0.2 % SDS the

amount of intracellular pravastatin was determined by LC/MS/MS.

LC/MS/MS assay for pravastatin

Samples were prepared by adding 100 µl internal standard solution (200 ng/ml

ibuprofen in eluent) to 100 µl of the cell lysates. The injected volume was 30 µl.

LC/MS/MS analysis was performed using a Sciex API 4000TM (Applied Biosystems,

Foster City, USA) triple quadrupole mass spectrometer equipped with an

atmospheric pressure ionization (API) Turbo Ion Spray® interface coupled with a two

position actuator control module (VICI Valco Instruments Co. Inc., Houston, USA) to

separate the cell lysate salts. The HPLC system was an Agilent Series 1100 (Agilent

Technologies Deutschland GmbH, Böblingen, Germany). The HPLC column used

was a Luna 3 µ CN 100 Å (100 X 2.0 mm) with a pre-column VS (Cyano, 4 mm L X 2

mm ID) purchased from Phenomenex Inc. (Phenomenex Ltd. Deutschland,

Aschaffenburg, Germany). A mixture of 12 mM ammonium acetate and methanol

(50/50 v/v) was used as the mobile phase. The flow rate was set at 0.2 ml/min. The

retention time of pravastatin was 1.2 min and 1.4 min for the internal standard. The

peak area ratio of pravastatin to the internal standard was calculated using

Analyst 1.4.2 (Applied Biosystems, Foster City, USA) software. The lower limit of

quantification was 0.5 ng/ml. A calibration curve was constructed using 1/X-weighted

linear regression between spiked cell lysate concentrations and the measured ratios.

The calibration curves were linear over the range 0.5 – 30 ng/ml with the mean


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correlation coefficients (n = 7 analytical runs) between 0.9967 and 0.9989. Cell lysate

calibration standards (0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0, and 30.0 ng/ml),

quality controls, blank, and double blank samples were prepared in the same

manner. The intra-day coefficient of variation was 2.14 % at 0.5 ng/ml, 1.60 % at

1 ng/ml, 6.93 % at 10 ng/ml, 5.32 % at 30 ng/ml (n = 4 to 5).

Data Analysis

The OATP1B1- and OATP1B3-mediated net uptake was obtained by subtracting the

uptake in vector-transfected cells from that in OATP1B1 and OATP1B3 expressing

cells. The percentage of uptake inhibition was calculated from control experiments in

the absence of macrolides (100 % uptake). The corresponding IC50 values for

inhibition of OATP1B1- and OATP1B3-mediated BSP uptake were calculated by

fitting the data to a sigmoidal dose response regression curve (Prism 4.01 2004,

GraphPad Software, San Diego, USA). The IC50 value is the concentration at which

half of the substrate uptake was inhibited.

Statistical Analysis

The experiments were repeated at least 4 times. All data are presented as mean ±

standard error. Multiple comparisons were analyzed by ANOVA with subsequent

Dunett’s or Tukey’s multiple comparison test by using Prism 4.01 2004 (GraphPad

Software, San Diego, USA). A value of P < 0.05 was required for statistical

significance.


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Results

BSP uptake in HEK-OATP1B1 and HEK-OATP1B3 cells

The prerequisite for analyzing the inhibitory potency of drugs on OATP1B1- and

OATP1B3-mediated uptake is the availability of stably transfected cells expressing

the recombinant protein in high amounts. Therefore, HEK293 cells were stably

transfected with the SLCO1B1 cDNA and the SLCO1B3 cDNA and selected for a

high expression of the respective uptake transporter. The protein expression of the

selected cell clones has been analyzed using the OATP1B1-specific antibody pESL

(König et al., 2000a) and the OATP1B3-specific antibody pSKT (König et al., 2000b).

This analysis demonstrated a high protein expression in the HEK-OATP1B1 and

HEK-OATP1B3 cells (Fig. 1A).

Uptake mediated by OATP1B1 or OATP1B3 was analyzed using the prototypic

tritium-labeled substrate sulfobromophthalein (BSP). BSP was shown to be a high

affinity substrate for both OATP1B1 and OATP1B3 with Km values of 140 nM (Cui et

al., 2001b) and 3.3 µM (König et al., 2000a), respectively. The uptake experiments

(Fig. 1B) demonstrated that HEK-OATP1B1 cells as well as HEK-OATP1B3 cells

were able to mediate BSP uptake into cells. The net uptake rates were 2.1 pmol x mg

protein-1 x min-1 for HEK-OATP1B1 cells and 10.8 pmol x mg protein-1 x min-1 for

HEK-OATP1B3 cells.

Inhibition of OATP1B1-mediated BSP uptake by macrolides

Uptake experiments have been carried out as described with adding different

concentrations of the respective macrolide. Interestingly, all investigated macrolides

except azithromycin showed a clear doses-dependent inhibition of OATP1B1-

mediated BSP uptake into HEK-OATP1B1 cells (Fig. 2). Azithromycin has been

analyzed up to a concentration of 500 µM and only at this high concentration a slight

decrease in BSP uptake was observed (Fig. 2A). Erythromycin also has a high IC50


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value of 217 ± 19 µM (Fig. 2C) whereas clarithromycin, telithromycin, and

roxithromycin have IC50 values of 96 ± 5 µM, 121 ± 19 µM, and 153 ± 4 µM,

respectively (Fig. 2B, D, and E). Taken together, clarithromycin, erythromycin,

roxithromycin, and telithromycin were identified to inhibit the OATP1B1-mediated

BSP transport.

Inhibition of OATP1B3-mediated BSP uptake by macrolides

A similar experimental setup was used to analyze the inhibitory effect of macrolides

on OATP1B3-mediated BSP uptake. As shown for the inhibition of OATP1B1-

mediated uptake azithromycin did not inhibit the uptake mediated by the OATP1B3

protein (Fig. 3A). All other investigated macrolides inhibited the OATP1B3-mediated

BSP uptake (Fig. 3B – E). Telithromycin was a potent inhibitor for OATP1B3-

mediated uptake with an IC50 value of 11 ± 0.3 µM (Fig. 3E). The macrolides

erythromycin, clarithromycin, and roxithromycin showed inhibitory potency with IC50

values of 34 ± 14 µM, 32 ± 7 µM, and 37 ± 6 µM, respectively (Fig. 3B – D).

Interestingly, the calculated IC50 values for clarithromycin, erythromycin,

roxithromycin, and telithromycin were determined to be lower than the respective IC50

values for OATP1B1-mediated uptake.

OATP1B1- and OATP1B3-mediated pravastatin uptake and inhibition by

macrolides

To test whether macrolides are also inhibitors for the OATP1B1- and OATP1B3-

mediated uptake of pravastatin, we performed pravastatin uptake and inhibition

experiments. Pravastatin is a known substrate for OATP1B1 (Km value of 34 µM;

(Hsiang et al., 1999). We confirmed that pravastatin is transported by OATP1B1 with

a significantly higher uptake in HEK-OATP1B1 cells (4.5 pmol x mg protein-1 x min-1)

compared to HEK-control cells (0.8 pmol x mg protein-1 x min-1) (Fig. 4A).

Furthermore, we could demonstrate for the first time that pravastatin is also a


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substrate for OATP1B3 (Fig. 4B). The uptake experiments demonstrated also a

significantly higher uptake in HEK-OATP1B3 cells in comparison to HEK-control cells

(3.1 pmol x mg protein-1 x min-1 vs. 0.3 pmol x mg protein-1 x min-1) (Fig. 4B).

Clarithromycin, erythromycin, and roxithromycin significantly inhibited the uptake of

pravastatin in HEK-OATP1B1 cells (Fig. 5A). Addition of 10 µM of clarithromycin or

roxithromycin resulted in a reduced intracellular accumulation of pravastatin to 64 %

and 65 % compared to the control experiments without macrolides (Fig. 5A).

Clarithromycin (100 µM), a potent inhibitor of the OATP1B1-mediated BSP uptake,

led to a reduction to 24 % intracellular accumulation of pravastatin compared to the

control experiments. As for BSP, azithromycin did not inhibit the transporter-mediated

uptake of pravastatin. In contrast, telithromycin which was a moderate inhibitor for

BSP uptake was not significantly affecting the uptake of pravastatin (Fig. 5A) and

showed a moderate uptake inhibition only at the high concentration of 100 µM.

At low macrolide concentrations (10 µM) erythromycin and roxithromycin inhibited

OATP1B3-mediated pravastatin uptake. The addition of 100 µM clarithromycin,

erythromycin, roxithromycin, and telithromycin reduced the pravastatin uptake to

37 %, 36 %, 52 %, and 19 %, respectively (Fig. 5B). Interestingly, telithromycin did

not inhibit the uptake of pravastatin at the low concentration whereas the higher

concentration significantly inhibited the uptake. Moreover, a slight but not significant

transport activation by low clarithromycin and telithromycin concentrations could be

observed. In accordance with the BSP inhibition assay azithromycin was the only

macrolide showing no inhibition among the tested macrolides (Fig. 5B).


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Discussion

In this study we focused on the analysis of the interaction of several macrolide /

ketolide antibiotics with the transport of the organic anion BSP and the HMG-CoA

reductase inhibitor pravastatin mediated by the hepatocellular uptake transporters

OATP1B1 and OATP1B3. Using newly established HEK cells recombinantly

expressing human OATP1B1 and OATP1B3 (Fig. 1) we found a concentration-

dependent inhibition of BSP uptake both in HEK-OATP1B1 and HEK-OATP1B3 cells

for all macrolides (except from azithromycin) and for the ketolide telithromycin. IC50

values were considerably smaller for the uptake inhibition of OATP1B3 than for

OATP1B1 (Figs. 2 and 3). Additionally to the inhibition studies with the prototypic

substrate BSP we have investigated the influence of macrolides and the ketolide

telithromycin on the uptake of the HMG-CoA reductase inhibitor pravastatin. We

demonstrated OATP1B1-mediated pravastatin uptake into HEK-OATP1B1 cells as

described earlier (Hsiang et al., 1999). Furthermore to the best of our knowledge, we

determined for the first time that pravastatin uptake is mediated by the second major

hepatocyte OATP family member OATP1B3 (Fig. 4). This transporter-mediated

pravastatin uptake could be inhibited by co-administration of clarithromycin,

erythromycin, and roxithromycin. In the case of OATP1B3, telithromycin was a potent

inhibitor for BSP uptake, however, a strong inhibition of pravastatin uptake was

observed only at high concentration (100 µM) of telithromycin. Clarithromycin,

erythromycin, and roxithromycin inhibited both OATP1B1- and OATP1B3-mediated

BSP and pravastatin uptake. On the other hand, azithromycin had no effect on BSP

or on pravastatin uptake.

OATP1B1 and OATP1B3 are expressed predominantly in the basolateral membrane

of human hepatocytes mediating the uptake of endogenous substances as well as

several xenobiotics and drugs. Both transporters share an overlapping substrate


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spectrum. Important drugs which are taken up by OATP1B1 and OATP1B3 are

several HMG-CoA reductase inhibitors like fluvastatin and pitavastatin, the antibiotic

rifampicin, and the endothelin receptor antagonist BQ123 (König et al., 2006).

Furthermore, transport of cerivastatin (Shitara et al., 2003), pravastatin (Hsiang et al.,

1999), and rosuvastatin (Schneck et al., 2004) has been shown for OATP1B1

whereas OATP1B3 is able to mediate the uptake of digoxin (Kullak-Ublick et al.,

2001). Due to these substrate spectra and their localization between portal venous

blood and important drug metabolizing enzymes (e.g. CYP3A4) expressed in

hepatocytes, uptake transporters are increasingly recognized as important factors in

the directed elimination of drugs out of the body. Their presence can be a

prerequisite for substances to enter hepatocytes and getting metabolized prior their

elimination over the canalicular membrane into bile. Modification of uptake rates e.g.

by drug competition, therefore, may cause drug-drug interactions by lowering the

uptake rate of one drug followed by increased blood concentrations due to reduced

hepatic metabolism and / or decreased biliary elimination.

Inhibition of cytochrome P450 isoenzymes is one established mechanism of drug-

drug interactions. Multiple studies have demonstrated that macrolides are potent

inhibitors of CYP3A4 and therefore can increase the plasma concentration of co-

administered drugs that are CYP3A4 substrates (Niemi et al., 2001). Drug-

interactions have also been reported between macrolides and some HMG-CoA

reductase inhibitors. Clarithromycin for example increases the plasma concentration

of concomitantly administered simvastatin, atorvastatin, and pravastatin (Jacobson,

2004). In the case of simvastatin and atorvastatin this drug-interaction can be

explained by the inhibition of CYP3A4 which predominantly metabolizes these

statins. Interestingly, pravastatin is one of the statins which is not metabolized by

cytochromes in humans and which is excreted almost unchanged into bile or to a


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small extent into urine (Jacobson, 2004). In this case an interaction of drug

transporting proteins, located in the basolateral hepatocyte membrane may account

for the increased plasma concentration. The data presented in this manuscript

confirmed these in vivo analyses of an interaction between clarithromycin and

pravastatin. Clarithromycin inhibited dose-dependent the OATP1B1- as well as the

OATP1B3-mediated pravastatin uptake in vitro. Therefore transporter-inhibition could

be the underlying mechanism of this pharmacokinetical drug-drug interaction. In

accordance with our findings, Hirano and coworkers very recently demonstrated that

both clarithromycin and erythromycin were inhibitors for the uptake of pitavastatin, an

established OATP1B1 substrate (Hirano et al., 2006) with Ki values of 8.3 and

11.4 µM, respectively.

Interestingly, an in vivo interaction between rosuvastatin, a recently established

HMG-CoA reductase inhibitor, and erythromycin does not appear (Cooper et al.,

2003). Rosuvastatin is a substrate of several OATP-family members (OATP1B1,

OATP1B3, OATP2B1, OATP1A2) and also of the sodium dependent bile salt

transporter NTCP (Ho et al., 2006; Schneck et al., 2004) and therefore, the uptake

inhibition of OATP1B1 or OATP1B3 could be compensated by transport via

alternative transporting proteins.

Published Ki values for inhibition of the metabolizing enzyme CYP3A4 by

clarithromycin, erythromycin, roxithromycin, and telithromycin are 30 µM, 13 µM,

72 µM, and 58 µM, respectively (Aventis-Pharmaceuticals, 2001; Polasek and

Miners, 2006). Interestingly, the determined IC50 values for macrolide-induced

OATP1B3 inhibition are in the same concentration range. In addition, azithromycin,

which has a high Ki value for CYP3A4 (Polasek and Miners, 2006) is the only

macrolide showing neither uptake inhibition of OATP1B1- nor OATP1B3-mediated

uptake.


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As drugs reach the portal vein directly after intestinal absorption, the drug

concentration in portal venous blood is higher than in the systemic circulation. For

calculation of the predicted maximum drug concentration at the inlet to the liver we

used the method of Ito et al. (1998) taking into account the maximum plasma

concentration in the systemic circulation, the single dosage, the absorbed fraction of

the macrolide, the absorption rate and the hepatic blood flow rate [Table 1 (Ito et al.,

1998)]. For clarithromycin, erythromycin, roxithromycin, and telithromycin the

predicted portal venous concentrations are in the same range as the determined IC50

values for inhibition of OATP1B3-mediated uptake. We therefore conclude that

inhibition of drug transporters by macrolides / ketolides could be an additional

mechanism for clinical relevant drug-drug interactions. Further studies are necessary

to gain more inside into the molecular nature of this inhibition mechanism.

Taken together our data demonstrate that macrolides / ketolides can inhibit uptake of

organic anions and drugs mediated by the OATP family members OATP1B1 and

OATP1B3. This modification of uptake rates is a new mechanism of drug-drug

interactions in addition to the hitherto known mechanism of drug-drug interactions

due to the modification of metabolizing enzymes and efflux transporters. Based on

our findings it is therefore of importance to gain more knowledge on the modification

of uptake transporter function as additional mechanism underlying drug-drug

interactions.


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Acknowledgements

We thank Mrs. C. Hoffmann and B. Endress for excellent technical assistance. We

would like to thank Professor Dr. D. Keppler (German Cancer Research Center,

Heidelberg) for providing the polyclonal antibodies pESL and pSKT and the plasmids

pcDNA3.1(+)-OATP1B1 and pcDNA3.1/Hygro(-)-OATP1B3.


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Footnotes

This work was supported by grant DFG Ko 2120/1-3 of the Deutsche

Forschungsgemeinschaft.


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Legends for figures

Fig. 1. Characterization of stably transfected HEK293 cells. A, Immunoblot

analysis of HEK-OATP1B1 and HEK-OATP1B3 cells. Cell homogenates (20 µg)

were separated by SDS-polyacrylamide gel electrophoresis. The left blot shows the

expression of OATP1B1, detected by the polyclonal antibody pESL (diluted 1:5 000).

The expression of OATP1B3 is shown on the right blot, using polyclonal antibody

pSKT (diluted 1:5 000). A human liver sample (Human Liver; 10 µg) and vector-

transfected HEK293 cells (Control) served as positive and negative controls. B,

Intracellular [3H]BSP accumulation in HEK-OATP1B1 (OATP1B1), HEK-OATP1B3

(OATP1B3), and HEK-control (Control) cells after 10 min incubation with 0.05 µM

and 1 µM BSP, respectively. Data are shown as mean value ± standard error (n = 6

to 20). Error bars in control cells are within the borders of the bars.

Fig. 2. Inhibition of BSP uptake by macrolides in HEK-OATP1B1 cells. Inhibitory

effect of A, azithromycin, B, clarithromycin, C, erythromycin, D, roxithromycin, and E,

telithromycin on OATP1B1-mediated BSP (0.05 µM) uptake after 10 minutes

incubation. The OATP1B1-mediated uptake was obtained by subtracting the uptake

in vector-transfected cells from that in OATP1B1-expressing cells. IC50 values were

calculated by fitting the data to a sigmoidal dose responsive regression curve.

OATP1B1-mediated BSP uptake is shown as the percentage of uptake without

macrolides. Each value is the mean value ± standard error (n = 4).

Fig. 3. Inhibition of BSP uptake by macrolides in HEK-OATP1B3 cells. Inhibitory

effect of A, azithromycin, B, clarithromycin, C, erythromycin, D, roxithromycin and E,

telithromycin on OATP1B3-mediated BSP (1 µM) uptake after 10 minutes incubation.

The OATP1B3-mediated uptake was obtained by subtracting the uptake in vector-


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transfected cells from that in OATP1B3-expressing cells. IC50 values were calculated

by fitting the data to a sigmoidal dose responsive regression curve. OATP1B3-

mediated BSP uptake is shown as the percentage of uptake without macrolides.

Each value is the mean value ± standard error (n = 4).

Fig. 4. Pravastatin uptake by HEK-OATP1B1 and HEK-OATP1B3 cells. A,

Intracellular pravastatin accumulation in HEK-OATP1B1 and HEK-control (Control)

cells after 10 min incubation with pravastatin (50 µM). B, Intracellular pravastatin

accumulation in HEK-OATP1B3 and HEK-control (Control) cells after 10 min

incubation with pravastatin (50 µM). Each value is the mean value ± standard error

(n = 4 to 6). *** P < 0.001 vs control.

Fig. 5. Inhibition of the pravastatin uptake by macrolides. Inhibitory effect using

10 µM and 100 µM of the macrolides azithromycin, clarithromycin, erythromycin,

roxithromycin, and telithromycin on A, OATP1B1- and B, OATP1B3-mediated

pravastatin (50 µM) uptake after 10 minutes incubation. The transporter-mediated

uptake was obtained by subtracting the uptake in vector-transfected cells from that in

transporter-expressing cells. OATP-mediated pravastatin uptake is shown as the

percentage of uptake without macrolides. Each value is the mean value ± standard

error (n = 4 to 6). * P < 0.05; ** P < 0.01 vs control.


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Table 1. Comparison of pharmacokinetic data of macrolides in humans with IC50 values of OATP1B1- and OATP1B3-

inhibition obtained in the present study.

dosage

[mg]

cmax

[mg/l]

cmax

[µM]

cport. vn.

[µM]

IC50

OATP1B1

[µM]

IC50

OATP1B3

[µM]

references

Azithromycin 1 x 500a 0.5a 0.7 42 > 250 n.d. a (Product-information, Zithromax®, 2004)

Clarithromycin 2 x 250b 1 - 2b 1.3 - 2.7 23 96 ± 5 32 ± 7 b (Product-information, Klacid®, 2004)

Erythromycin 1 x 1000c 3.0c 4.1 88 217 ± 19 34 ± 14 c (Product-information, Erythrocin®, 2001)

Roxithromycin 1 x 300d 9.7d 11.6 34 153 ± 4 37 ± 6 d (Product-information, Rulid®, 2004)

Telithromycin 1 x 800e 2e 2.5 64 121 ± 20 11 ± 0.3 e (Product-information, Ketek®, 2005)

Note. IC50 data are derived from the measurements shown in Fig. 2 and 3 (each IC50 value is calculated as the mean value of 4 experiments ± standard error). n.d.= not determined, cmax= maximal plasma concentration in the systemic circulation, cport.vn.= predicted concentration in portal venous blood [according to Ito et al. (1998)]

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

DM

D Fast Forw

ard. Published on February 12, 2007 as DO

I: 10.1124/dmd.106.014407

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