Subscriber access provided by FLORIDA STATE UNIV
Article
Preclinical mouse models to study human OATP1B1and OATP1B3-mediated drug-drug interactions in vivo Selvi Durmus, Gloria Lozano-Mena, Anita van Esch, Els Wagenaar, Olaf van Tellingen, and Alfred H. Schinkel Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00453 • Publication Date (Web): 16 Oct 2015 Downloaded from http://pubs.acs.org on October 27, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 31
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Molecular Pharmaceutics
1
Preclinical mouse models to study human OATP1B1- and OATP1B3-mediated
2
drug-drug interactions in vivo
3
Authors: Selvi Durmus1, Gloria Lozano-Mena1, 2, Anita van Esch1, Els Wagenaar1, Olaf van Tellingen3,
4
Alfred H. Schinkel
5
1
6
2
7
of Barcelona, Barcelona, Spain
8
3
1
Division of Molecular Oncology, The Netherlands Cancer Institute, Amsterdam, The Netherlands, Department of Physiology and Nutrition and Institute of Food Safety Research (INSA-UB), University
Department of Clinical Chemistry, The Netherlands Cancer Institute, Amsterdam, The Netherlands
9 10
Corresponding author:
11
Dr. Alfred H. Schinkel,
12
The Netherlands Cancer Institute,
13
Division of Molecular Oncology,
14
Plesmanlaan 121, 1066 CX
15
Amsterdam, The Netherlands,
16
E-mail:
[email protected],
17
Phone: +31 20 5122046,
18
Fax: +31 20 669 1383
19 20
Running title: Human OATP1B-mediated drug-drug interactions
21
Article category: Research article
22
To be submitted to: Molecular Pharmaceutics
23
Statement of conflict of interest:
24
The research group of A.H. Schinkel receives revenue from commercial distribution of some of the
25
mouse strains used in this study. No other conflicts of interest are declared.
26 1 ACS Paragon Plus Environment
Molecular Pharmaceutics
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 31
27
Abstract
28
The impact of OATP drug uptake transporters in drug-drug interactions (DDIs) is increasingly
29
recognized. OATP1B1 and OATP1B3 are human hepatic uptake transporters that can mediate liver
30
uptake of a wide variety of drugs. Recently, we generated transgenic mice with liver-specific
31
expression of human OATP1B1 or OATP1B3 in a mouse Oatp1a/1b knockout background. Here, we
32
investigated the applicability of these mice in OATP-mediated drug-drug interaction studies using the
33
prototypic OATP inhibitor rifampicin and a good OATP substrate, the anticancer drug methotrexate
34
(MTX). We next assessed the possibility of OATP-mediated interactions between telmisartan and
35
MTX, a clinically relevant drug combination. Using HEK293 cells overexpressing OATP1B1 or
36
OATP1B3, we estimated IC50 values for both rifampicin (0.9 or 0.3 µM) and telmisartan (6.7 or 7.9
37
µM) in inhibiting OATP-mediated MTX uptake in vitro. Using wild-type, Oatp1a/1b-/- and OATP1B1- or
38
OATP1B3-humanized transgenic mice, we found that rifampicin inhibits hepatic uptake of MTX
39
mediated by the mouse Oatp1a/1b and human OATP1B1 and OATP1B3 transporters at clinically
40
relevant concentrations. This highlights the applicability of these mouse models for DDI studies and
41
may be exploited in the clinic to reduce the dose and thus methotrexate-mediated toxicity. On the
42
other hand, telmisartan inhibited only human OATP1B1-mediated hepatic uptake of MTX at
43
concentrations higher than those used in the clinic; therefore risks for OATP-mediated clinical DDIs for
44
this drug combination are likely to be low. Overall, we show here that OATP1B1 and OATP1B3-
45
humanized mice can be used as in vivo tools to assess and possibly predict clinically relevant DDIs.
46 47
Keywords: drug-drug interactions, DDI, OATP1B1, OATP1B3, methotrexate, rifampicin, telmisartan,
48
hepatic uptake, plasma disposition
49 OATP;
Organic anion-transporting polypeptide, SLCO/Slco; Organic
anion-
50
Abbreviations:
51
transporting polypeptide encoding gene name, WT; wild-type, HEK; human embryonic kidney, IC50;
52
the half maximal inhibitory concentration required for inhibiting biological or biochemical function, DDI;
53
drug-drug interaction, MTX; methotrexate
54 55 56
2 ACS Paragon Plus Environment
Page 3 of 31
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Molecular Pharmaceutics
57
Introduction
58
Organic anion-transporting polypeptides (OATP/Oatp) are sodium-independent transmembrane
59
uptake transporters encoded by SLCO/Slco genes. OATPs are expressed in several organs including
60
liver, kidney and small intestine, where they mediate the tissue uptake of substrate compounds . More
61
and more studies show that OATPs are important players in disposition of a wide range of drugs
62
including statins, cardiac glycosides, antibiotics, and chemotherapeutics . Of the OATP1 family,
63
OATP1B1 and -1B3 are highly expressed in the sinusoidal membrane of hepatocytes3, where they
64
have key roles in hepatic uptake and/or plasma clearance of many structurally diverse drugs (e.g.
65
methotrexate (MTX), paclitaxel, docetaxel, pravastatin, fexofenadine and doxorubicin)4-8. Therefore,
66
alterations in OATP1B activity or drug-drug interactions (DDIs) mediated by inhibition of OATP1B
67
transporters might have important clinical consequences for the pharmacokinetics, efficacy and toxicity
68
of transported therapeutics
69
significant changes in the disposition and toxicity of pravastatin, valsartan, MTX and SN-3810, 12, 13. Full
70
deficiency of OATP1B1 and -1B3 results in the disruption of hepatic re-uptake and hence increased
71
plasma levels of conjugated bilirubin, causing a syndrome called Rotor-type hyperbilirubinemia14.
72
Our knowledge of the involvement of OATPs in DDIs is growing rapidly, and the clinical importance of
73
such interactions is becoming more and more evident15. FDA and EMA strongly recommend
74
investigation of DDIs mediated by OATP1B1 and -1B3 for new molecular entities during drug
75
development since 201216. Recently, Shitara et al.2 reported examples of DDIs that are caused by
76
inhibition of OATP1B transporters via potent inhibitors, and which affected the pharmacokinetics of
77
victim drugs. The inhibitor drugs tested were antibiotics, antiviral drugs and the immunosuppressant
78
cyclosporine A, and victim drugs were widely used statins, antidiabetic and hypertension drugs.
79
Among the inhibitors, cyclosporine A and rifampicin appeared to be particularly strong inhibitors of
80
OATP1B1, which caused increases in the plasma levels of several statins of 2.2- to 23-fold as a result
81
of DDIs2. There are only a few recent studies on OATP-mediated DDIs with anticancer drugs, although
82
these are widely used and often substrates or inhibitors of OATPs. Hu et al.
83
whether inhibition of OATP1B1 by tyrosine kinase inhibitors could explain a decreased docetaxel
84
clearance. Sorafenib was selected as a potent inhibitor of OATP1B1 based on in vitro assays, but
85
single or multiple doses of sorafenib did not affect docetaxel plasma levels in vivo using Oatp1b2
86
knockout and hOATP1B1-expressing transgenic mice . Nieuweboer et al.
1
2
2, 9-11
. For example, genetic variants of OATP1B1 are associated with
17
17
18
recently investigated
recently showed that
3 ACS Paragon Plus Environment
Molecular Pharmaceutics
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 31
87
polysorbate 80 or Kolliphor (Cremophor) EL in the drug formulation can inhibit (mouse) OATP1B2-
88
mediated hepatic elimination of paclitaxel. Clearly, DDI studies involving OATP-dependent interactions
89
between widely used medicines and anticancer drugs are urgently needed, as such interactions may
90
lead to unexpected toxicities and altered efficacy in the treatment of cancers, and anticancer drugs are
91
usually administered at high dosages and have a narrow therapeutic window. The antifolate MTX is a
92
widely used anticancer and antirheumatic drug that is a good substrate of mouse Oatp1a/1b and
93
human OATP1B1 and -1B3 transporters in vivo4. The likelihood of undesired DDIs and thus altered
94
pharmacokinetics of MTX might be substantial, when prescribed with other commonly used drugs
95
such as antibiotics, hypertension drugs and antidiabetics that are inhibitors/substrates of OATPs.
96
Thus, MTX is of great interest for assessment of such interactions and prediction of clinical outcomes.
97
To predict clinical DDIs, many studies utilize in vitro cellular uptake assays and in silico11,
98
preclinical models
99
situation and use of preclinical wild-type and knockout animal models does not necessarily result in
19-22
and
2, 23
. However, it remains challenging to translate in vitro data to the human in vivo
24, 25
100
optimal prediction due to species differences
. Accordingly, we have generated humanized
101
Oatp1a/1b knockout mouse models with liver-specific expression of human OATP1B1 or -1B3, with
102
transgenic protein levels roughly comparable to those seen in pooled human liver samples
103
study, we investigated whether these mouse models could be used to assess short-term, acute human
104
OATP1B1 and -1B3-mediated DDIs in vivo. For this aim, we used the antibiotic rifampicin as a model
105
inhibitor of OATP1B transporters and MTX as a victim drug. Secondly, we tested OATP1B-mediated
106
DDIs between the antihypertensive drug telmisartan and MTX, which have a higher chance for co-
107
prescription in the clinic.
4, 13
. In this
108
4 ACS Paragon Plus Environment
Page 5 of 31
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Molecular Pharmaceutics
109
Materials and Methods
110
Chemicals
111
Methotrexate (100 mg/ml, Pharmachemie, The Netherlands) and cyclosporine A (CsA, 50 mg/ml,
112
Novartis, Switzerland) were obtained as parenteral formulations from the pharmacy department of the
113
Antonie van Leeuwenhoek Hospital (The Netherlands). Rifampicin was purchased from Sigma-Aldrich
114
(USA) and telmisartan was purchased from Sequoia Research Chemicals (UK), both in powder forms.
115
7-OH-MTX, rifampicin-d3 and telmisartan-d7 were purchased from Toronto Research Chemicals
116
(Canada). All of the chemicals used for HPLC-UV and HPLC-MS/MS analysis were from Sigma-
117
Aldrich (USA) or Merck (Germany).
118
Drug solutions
119
In vitro experiments
120
MTX stock solution (100 mg/ml = 220.1 mM) and CsA (50 mg/ml = 41.6 mM) were diluted in Krebs-
121
Henseleit buffer to yield 50 µM MTX and 0.5 µM CsA solution, respectively. Rifampicin (50 mM) and
122
telmisartan (10 mM) were prepared in DMSO and dimethylformamide (DMF), respectively, and were
123
further diluted in Krebs-Henseleit buffer to yield the desired concentrations.
124
In vivo experiments
125
MTX stock solution (100 mg/ml) was diluted 50 or 250-fold with 0.9% NaCl to yield a concentration of
126
2 or 0.4 mg/ml. Rifampicin was dissolved in DMSO (at 80 mg/ml) and further diluted in 0.9% NaCl to
127
yield a concentration of 4 mg/ml. Telmisartan was dissolved in DMF (7 mg/ml, maximum solubility) and
128
further diluted in PBS to yield a concentration of 1.4 mg/ml. All drugs were administered intravenously
129
(i.v.), using a volume of 5 µl/g body weight. All working solutions were prepared on the day of
130
experiment.
131
Cell culture
132
HEK293 cells transduced with vector control, hSLCO1B1 and hSLCO1B3 cDNAs were a kind gift from
133
Prof. Werner Siegmund and Dr. Markus Keiser (University of Greifswald, Greifswald, Germany)26. All
134
cells were grown in Dulbecco's modified Eagle's medium high glucose (Gibco) supplemented with
135
10% fetal bovine serum (Sigma), 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml
136
amphotericin B at 37°C with 5% CO2 and 95% humidity. All of these cell lines were authenticated and
137
found to be identical to the original cell lines.
138
5 ACS Paragon Plus Environment
Molecular Pharmaceutics
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 31
139
Cellular uptake experiments
140
Cellular uptake experiments were performed according to previously described methods
141
modified as required. Krebs-Henseleit buffer at pH 7.4, which was adjusted before the experiment was
142
started, was used in all the steps of the experiment including dilutions of the drug stock solutions.
143
Briefly, cells were preincubated with 1 ml of pre-warmed buffer for 15 min at 37°C. Uptake
144
experiments were started by aspirating the preincubation buffer and adding 600 µl of pre-warmed
145
buffer containing (inhibitor) drugs; MTX (50 µM) alone or together with rifampicin (0, 1, 2.5, 5, 15, 25,
146
50 or 100 µM) or telmisartan (0, 1, 2.5, 5, 25, 50, 100 or 250 µM) or CsA (0.5 µM). CsA was used as a
147
positive control for the uptake inhibition; therefore it was also added in the pre-incubation buffer as an
148
OATP inhibitor. At designated time points (2.5, 5, 10, 15, 30, 60 or 120 min) experiments were
149
terminated by removing the incubation buffer and immediately adding 1 ml of ice-cold buffer. After
150
washing twice with ice-cold buffer, cells were lysed with 150 µl of 0.2 N NaOH for a minimum of 15
151
min. 10 µl of the cell lysate was used to determine the cellular protein amount by the Bradford method
152
using bovine serum albumin as a standard. 60 µl of the cell lysates were used to determine MTX
153
levels by HPLC-UV. These were readily detectable when 50 µM MTX was applied. Percentage of
154
OATP-mediated uptake of MTX was calculated using the uptake without inhibitor as 100%.
155
Animals
156
Mice were housed and handled according to institutional guidelines complying with Dutch legislation.
157
Male WT, Oatp1a/1b-/-, Oatp1a/1b-/-;1B1tg and Oatp1a/1b-/-;1B3tg with liver-specific expression of
158
human SLCO genes, all of a >99% FVB genetic background, were used between 8 and 14 weeks of
159
age. Animals were kept in a temperature-controlled environment with a 12 hr light/12 hr dark cycle and
160
received a standard diet (AM-II, Hope Farms) and acidified water ad libitum.
161
Plasma and liver pharmacokinetic experiments
162
Dosages of MTX were 10 and 2 mg/kg body weight, that of rifampicin was 20 mg/kg and that of
163
telmisartan was 7 mg/kg body weight. Rifampicin, telmisartan or vehicles (0.9% NaCl or PBS) were
164
injected into the tail vein of mice 3 minutes before MTX administration. For all experiments, mice were
165
sacrificed at 5 or 15 min after MTX administration by terminal bleeding through cardiac puncture under
166
isoflurane anesthesia and organs were rapidly removed. Plasma was isolated from blood samples
167
after centrifugation at 2,100 g for 6 min at 4°C, livers were homogenized in 1% bovine serum albumin
168
and all the samples were stored at −30°C until analysis.
8, 27
and
6 ACS Paragon Plus Environment
Page 7 of 31
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Molecular Pharmaceutics
169
After drug analysis, results were presented as concentrations in the organs (nmol/g), % of the total
170
dose (dose corrected for the body weight of each individual mouse being equivalent to 100%) and/or
171
organ-to-plasma ratios. Organ-to-plasma ratios were calculated by dividing organ concentration
172
expressed as nmol/g by plasma concentration expressed as nmol/ml, assuming 1 ml of plasma is
173
roughly equivalent to 1 g of tissue.
174
Drug Analyses
175
Determination of MTX and 7-OH-MTX
176
Levels of MTX and 7-OH-MTX in plasma and liver homogenates were determined by HPLC-UV
177
detection as described previously28. For the in vitro experiments, cell lysates in 0.2 N NaOH were
178
pretreated with 1.5 M HClO4 in a ratio of 1:1.6 v/v. After vortexing 5 s and centrifugation at 16,873 g
179
for 5 min at 4°C, supernatants were injected into the HPLC system directly. Standard curve (4.4 - 2201
180
nM) and quality control (22.01, 220.1 and 2201 nM) samples were prepared using blank cell lysates as
181
matrix.
182
Sample pre-treatment for rifampicin and telmisartan
183
Plasma and liver homogenates were thawed at room temperature and diluted 1:50 or 1:100 (for
184
rifampicin or telmisartan determinations, respectively) in blank human plasma. A volume of 100 µl of
185
plasma or liver homogenate was pipetted into 2 ml polypropylene vials (Eppendorf, Hamburg,
186
Germany). Next, volumes of 50 µl of IS working solution (rifampicin-d3 or telmisartan-d3; 1 µg/ml in
187
acetonitrile: water 30:70; v/v) and 1000 µl of ethyl acetate were added. After automatic shaking for 15
188
min at 480 rpm, the samples were centrifuged for 1 min at 20,000 g. The aqueous layer was snap
189
frozen in dry ice/ethanol and the upper organic layer was decanted into a 1.5 ml vial (Brand,
190
Wertheim, Germany). The solvent was evaporated to dryness in Speed-Vac SC210A (Savant,
191
Farmingdale, NY, USA) and the residue was reconstituted with 100 µl of acetonitrile: water (30:70, v/v)
192
by sonicating for 5 min and vortex-mixing for 5 s. Finally, samples were centrifuged for 5 min at 20,000
193
g prior to analysis.
194
Quantification of rifampicin and telmisartan
195
The levels of rifampicin and telmisartan in plasma and liver homogenates were quantified by HPLC
196
coupled to tandem mass spectrometry (HPLC-MS/MS). Details of the instrumentation are explained in
197
supplemental text.
7 ACS Paragon Plus Environment
Molecular Pharmaceutics
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 8 of 31
198
The concentrations of rifampicin and telmisartan in plasma and liver homogenates were determined by
199
means of the internal standard (I.S.) method, employing the corresponding stable isotope labeled
200
drugs (rifampicin-d3 and telmisartan-d3).
201
Stock solutions of rifampicin and telmisartan were prepared in DMSO at 10 mM and stored at ‒20ºC.
202
For each analytical run, these solutions were diluted in DMSO to obtain working solutions ranging from
203
100 nM to 100 µM for the preparation of the above-mentioned calibration standards. A set of
204
calibration standards in either blank plasma or liver homogenate was prepared using 100-fold dilution
205
of the DMSO stock in blank biological matrix. Final calibration samples contained 1, 3, 10, 30, 100,
206
300 and 1000 nM.
207
Statistical analysis
208
IC50 values for rifampicin and telmisartan for inhibition of MTX uptake were determined using non-
209
linear regression (curve fit) analysis in GraphPad Prism 6.01 software after correction for the
210
background uptake in control cells. The 2-sided Student's t-test or one-way analysis of variance
211
(ANOVA) with post-hoc tests employing Tukey's corrections were used to determine statistical
212
significance either between two or multiple groups, respectively. Results were presented as the mean
213
± standard deviation (SD). Differences were considered to be statistically significant when P ≤ 0.05.
214 215 216
8 ACS Paragon Plus Environment
Page 9 of 31
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Molecular Pharmaceutics
217
Results
218
In vitro MTX uptake by OATP1B transporters
219
To establish time-dependent uptake of MTX by OATPs, we performed cellular uptake experiments
220
with MTX (50 µM) using control and OATP1B1- or OATP1B3-expressing HEK293 cells for various
221
time periods between 2.5 and 30 min. 50 µM MTX was used since shortly after i.v. administration (5-
222
15 min) of 10 mg/kg MTX in the planned in vivo DDI studies, plasma concentrations of that order are
223
achieved (see below). MTX uptake by OATP1B1 or -1B3 was significantly greater compared to control
224
cells at all-time points (Figure 1A-B). MTX uptake by OATP1B3 was slightly saturated after 5 min and
225
more strongly after 15 min. In subsequent studies we used 15 min uptake, as a clear differential was
226
seen at this time point between both the transporter-expressing cells and their controls. The increased
227
uptake of MTX in both OATP-expressing cells was inhibited completely by 0.5 µM CsA, which is
228
known to inhibit OATPs (Figure 1C); further corroborating that MTX uptake in these cells was OATP-
229
mediated. Studies with these cells were performed to estimate the IC50 values of candidate inhibitors
230
for subsequent in vivo DDI studies.
231
Effect of rifampicin on OATP-mediated MTX uptake in vitro
232
To determine whether OATP-mediated MTX uptake could be inhibited by the OATP inhibitor
233
rifampicin, we estimated the IC50 of rifampicin for each OATP-expressing cell type. IC50
234
concentrations of rifampicin were 0.88 ± 0.18 or 0.31 ± 0.12 µM for OATP1B1- or OATP1B3-mediated
235
MTX uptake, respectively (Figure 2). This suggests that rifampicin is a fairly strong inhibitor (low µM
236
range) of MTX uptake mediated by these OATPs.
237
Effect of rifampicin on OATP-mediated MTX and 7-OH-MTX disposition in vivo
238
To investigate whether rifampicin can also inhibit OATP-mediated MTX transport in vivo, we
239
administered 20 mg/kg rifampicin or vehicle (i.v.) 3 min before 10 mg/kg MTX dosing (i.v.) to wild-type
240
(WT), Oatp1a/1b knockout and transgenic mice with liver-specific expression of OATP1B1 and
241
OATP1B3, considering their relevance to human drug disposition.
242
In groups receiving vehicle, MTX disposition results were similar to those described previously by van
243
de Steeg et al.4. Plasma MTX levels were increased in Oatp1a/1b-/-, Oatp1a/1b-/-;1B1tg and
244
Oatp1a/1b-/-;1B3tg mice compared to WT strains, both at 5 and 15 min after MTX administration
245
(Figure 3A and B). Liver levels of MTX and liver-to-plasma ratios were profoundly decreased by
246
removal of the Oatp1a/1b transporters, and expression of human OATP1B1 or -1B3 in these livers led
9 ACS Paragon Plus Environment
Molecular Pharmaceutics
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 10 of 31
247
to significant, albeit not complete, rescue of the impaired liver uptake of MTX (Figure 3), suggesting
248
human OATP-mediated transport. Considering the presence of only one of each of the human OATPs
249
in the transgenic strains instead of both together as in humans, the extent of liver uptake rescue by
250
each transporter seems to be substantial. Therefore, these findings suggest that MTX hepatic uptake
251
is primarily mediated by OATPs.
252
In rifampicin pretreated groups, plasma levels of MTX were significantly increased by 4.2-, 1.5- and
253
1.9-fold in WT, Oatp1a/1b-/- and Oatp1a/1b-/-;1B3tg mice 5 min after MTX administration (Figure 3A).
254
Rifampicin pretreatment significantly increased the plasma levels of MTX in WT and Oatp1a/1b-/-
255
;1B3tg mice to similar concentrations as found in Oatp1a/1b-/- mice. This suggests that with the 20
256
mg/kg i.v. rifampicin dose, we could strongly inhibit the OATP-mediated MTX uptake. Indeed,
257
rifampicin treatment led to significant decreases in liver MTX levels in the wild-type and both
258
OATP1B1- and OATP1B3-transgenic strains, but not the Oatp1a/1b-/- mice (Figure 3C and
259
Supplementary Figure 1A), indicating that rifampicin specifically inhibits OATP-mediated uptake of
260
MTX. When corrected for plasma levels, substantial decreases were observed in liver-to-plasma ratios
261
after rifampicin treatment. Although not reaching the level of knockout mice, WT mice showed a highly
262
significant decrease by a factor of 8 in liver-to-plasma ratios of MTX (P < 0.001, Figure 3E),
263
suggesting a very strong inhibition of Oatp1a/1b-mediated hepatic uptake of MTX in this strain. Liver-
264
to-plasma ratios of MTX in Oatp1a/1b-/-;1B1tg and Oatp1a/1b-/-;1B3tg mice were also decreased by a
265
factor of 3.5 and 11.4, respectively, and more or less back to the levels in knockout mice (Figure 3E).
266
Furthermore, Oatp1a/1b-/- mice did not show any significant difference in liver-to-plasma ratios of MTX
267
upon rifampicin treatment, indicating that the hepatic uptake of MTX that was inhibited by rifampicin
268
was OATP-specific.
269
To assess whether the rifampicin effect also applied at a later time point, we tested the disposition of
270
MTX at 15 min. The disposition of MTX between the strains and conditions followed the same trend as
271
observed at 5 min. At 15 min, plasma levels of MTX were increased in WT (4.7-fold, P < 0.01) and
272
Oatp1a/1b-/-;1B3tg (2.2-fold, P < 0.05) mice by rifampicin treatment but not in Oatp1a/1b-/- and
273
Oatp1a/1b-/-;1B1tg mice (Figure 3B). Decreases in liver MTX levels were significant in Oatp1a/1b-/-
274
;1B1tg (~3.5-fold, P < 0.01) and Oatp1a/1b-/-;1B3tg (~7.6-fold, P < 0.01) mice (Figure 3D and
275
Supplementary Figure 1B). Liver-to-plasma ratios clearly showed that hepatic uptake of MTX was
276
substantially decreased by rifampicin in WT (5.8-fold, P < 0.001) and Oatp1a/1b-/-;1B1tg (3.7-fold, P