The influence of macrolide antibiotics on the uptake of organic

Transcription

DMD Fast Forward. Published on February 12, 2007 as DOI: 10.1124/dmd.106.014407
DMD Fast
onand
February
2007
as doi:10.1124/dmd.106.014407
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DMD #14407
The influence of macrolide antibiotics on the uptake of
organic anions and drugs mediated by OATP1B1 and
Annick Seithel1, Sonja Eberl1, Katrin Singer, Daniel Auge, Georg Heinkele, Nadine B.
Wolf, Frank Dörje, Martin F. Fromm, Jörg König
1
contributed 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)
1
Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics.
Downloaded from dmd.aspetjournals.org at ASPET Journals on October 12, 2016
OATP1B3
This article has not been copyedited and formatted. The final version may differ from this version.
DMD #14407
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: Joerg.Koenig@pharmakologie.med.uni-erlangen.de
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;
methylglutaryl-coenzym A; Km, Michaelis-Menten constant
2
HMG-CoA,
3-hydroxy-3-
DMD #14407
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.
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 OATP1B3mediated 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.
3
The human OATP family members OATP1B1 and OATP1B3 mediate the uptake of
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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 /
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 HMGCoA
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 basepair4
luminally localized drug efflux pump P-glycoprotein (Eberl et al., 2005; Kim et al.,
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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
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.
5
drug-drug interactions. For instance, it has been demonstrated that the macrolides
DMD #14407
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,
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 subcultivated by trypsinization using trypsin (0.05 %) - EDTA (0.02 %) solution. All cell
6
clarithromycin, and roxithromycin were obtained from Chemos GmbH (Regenstauf,
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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
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
7
OATP1B1) and SLCO1B3 (encoding human OATP1B3) mRNA and OATP1B1 or
DMD #14407
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 SDSpolyacrylamide 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
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
8
pESL and pSKT were diluted 1:5 000 in TPBS (Dulbecco’s phosphate-buffered
DMD #14407
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.
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
9
LC/MS/MS assay for pravastatin
DMD #14407
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
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.
10
uptake in vector-transfected cells from that in OATP1B1 and OATP1B3 expressing
DMD #14407
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
(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 OATP1B1mediated 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
11
selected cell clones has been analyzed using the OATP1B1-specific antibody pESL
DMD #14407
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
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 OATP1B3mediated 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 OATP1B3mediated 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
12
on OATP1B3-mediated BSP uptake. As shown for the inhibition of OATP1B1-
DMD #14407
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).
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).
13
Clarithromycin (100 µM), a potent inhibitor of the OATP1B1-mediated BSP uptake,
DMD #14407
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 concentrationdependent inhibition of BSP uptake both in HEK-OATP1B1 and HEK-OATP1B3 cells
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
14
for all macrolides (except from azithromycin) and for the ketolide telithromycin. IC50
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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
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 drugdrug interactions. Multiple studies have demonstrated that macrolides are potent
inhibitors of CYP3A4 and therefore can increase the plasma concentration of coadministered drugs that are CYP3A4 substrates (Niemi et al., 2001). Druginteractions 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
15
blood and important drug metabolizing enzymes (e.g. CYP3A4) expressed in
DMD #14407
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
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.
16
accordance with our findings, Hirano and coworkers very recently demonstrated that
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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
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.
17
predicted portal venous concentrations are in the same range as the determined IC50
DMD #14407
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.
18
DMD #14407
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DMD #14407
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24
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DMD #14407
Footnotes
This work was supported by grant DFG Ko 2120/1-3 of the Deutsche
Forschungsgemeinschaft.
25
DMD #14407
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-
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 vector26
transfected HEK293 cells (Control) served as positive and negative controls. B,
DMD #14407
transfected cells from that in OATP1B3-expressing cells. IC50 values were calculated
by fitting the data to a sigmoidal dose responsive regression curve. OATP1B3mediated 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)
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.
27
cells after 10 min incubation with pravastatin (50 µM). B, Intracellular pravastatin
DMD #14407
Table 1. Comparison of pharmacokinetic data of macrolides in humans with IC50 values of OATP1B1- and OATP1B3inhibition obtained in the present study.
cmax
cmax
cport. vn.
[mg]
[mg/l]
[µM]
[µM]
IC50
IC50
OATP1B1
OATP1B3
[µM]
[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)]
28
dosage

The influence of macrolide antibiotics on the uptake of organic

Transcription

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