Preclinical Mouse Models To Study Human OATP1B1- and OATP1B3

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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

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Molecular Pharmaceutics

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Preclinical mouse models to study human OATP1B1- and OATP1B3-mediated

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drug-drug interactions in vivo

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Authors: Selvi Durmus1, Gloria Lozano-Mena1, 2, Anita van Esch1, Els Wagenaar1, Olaf van Tellingen3,

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Alfred H. Schinkel

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1

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2

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of Barcelona, Barcelona, Spain

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3

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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

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Corresponding author:

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Dr. Alfred H. Schinkel,

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The Netherlands Cancer Institute,

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Division of Molecular Oncology,

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Plesmanlaan 121, 1066 CX

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Amsterdam, The Netherlands,

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E-mail: [email protected],

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Phone: +31 20 5122046,

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Fax: +31 20 669 1383

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Running title: Human OATP1B-mediated drug-drug interactions

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Article category: Research article

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To be submitted to: Molecular Pharmaceutics

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Statement of conflict of interest:

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The research group of A.H. Schinkel receives revenue from commercial distribution of some of the

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mouse strains used in this study. No other conflicts of interest are declared.

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Abstract

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The impact of OATP drug uptake transporters in drug-drug interactions (DDIs) is increasingly

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recognized. OATP1B1 and OATP1B3 are human hepatic uptake transporters that can mediate liver

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uptake of a wide variety of drugs. Recently, we generated transgenic mice with liver-specific

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expression of human OATP1B1 or OATP1B3 in a mouse Oatp1a/1b knockout background. Here, we

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investigated the applicability of these mice in OATP-mediated drug-drug interaction studies using the

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prototypic OATP inhibitor rifampicin and a good OATP substrate, the anticancer drug methotrexate

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(MTX). We next assessed the possibility of OATP-mediated interactions between telmisartan and

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MTX, a clinically relevant drug combination. Using HEK293 cells overexpressing OATP1B1 or

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OATP1B3, we estimated IC50 values for both rifampicin (0.9 or 0.3 µM) and telmisartan (6.7 or 7.9

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µM) in inhibiting OATP-mediated MTX uptake in vitro. Using wild-type, Oatp1a/1b-/- and OATP1B1- or

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OATP1B3-humanized transgenic mice, we found that rifampicin inhibits hepatic uptake of MTX

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mediated by the mouse Oatp1a/1b and human OATP1B1 and OATP1B3 transporters at clinically

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relevant concentrations. This highlights the applicability of these mouse models for DDI studies and

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may be exploited in the clinic to reduce the dose and thus methotrexate-mediated toxicity. On the

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other hand, telmisartan inhibited only human OATP1B1-mediated hepatic uptake of MTX at

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concentrations higher than those used in the clinic; therefore risks for OATP-mediated clinical DDIs for

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this drug combination are likely to be low. Overall, we show here that OATP1B1 and OATP1B3-

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humanized mice can be used as in vivo tools to assess and possibly predict clinically relevant DDIs.

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Keywords: drug-drug interactions, DDI, OATP1B1, OATP1B3, methotrexate, rifampicin, telmisartan,

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hepatic uptake, plasma disposition

49 OATP;

Organic anion-transporting polypeptide, SLCO/Slco; Organic

anion-

50

Abbreviations:

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transporting polypeptide encoding gene name, WT; wild-type, HEK; human embryonic kidney, IC50;

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the half maximal inhibitory concentration required for inhibiting biological or biochemical function, DDI;

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drug-drug interaction, MTX; methotrexate

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Introduction

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Organic anion-transporting polypeptides (OATP/Oatp) are sodium-independent transmembrane

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uptake transporters encoded by SLCO/Slco genes. OATPs are expressed in several organs including

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liver, kidney and small intestine, where they mediate the tissue uptake of substrate compounds . More

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and more studies show that OATPs are important players in disposition of a wide range of drugs

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including statins, cardiac glycosides, antibiotics, and chemotherapeutics . Of the OATP1 family,

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OATP1B1 and -1B3 are highly expressed in the sinusoidal membrane of hepatocytes3, where they

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have key roles in hepatic uptake and/or plasma clearance of many structurally diverse drugs (e.g.

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methotrexate (MTX), paclitaxel, docetaxel, pravastatin, fexofenadine and doxorubicin)4-8. Therefore,

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alterations in OATP1B activity or drug-drug interactions (DDIs) mediated by inhibition of OATP1B

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transporters might have important clinical consequences for the pharmacokinetics, efficacy and toxicity

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of transported therapeutics

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significant changes in the disposition and toxicity of pravastatin, valsartan, MTX and SN-3810, 12, 13. Full

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deficiency of OATP1B1 and -1B3 results in the disruption of hepatic re-uptake and hence increased

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plasma levels of conjugated bilirubin, causing a syndrome called Rotor-type hyperbilirubinemia14.

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Our knowledge of the involvement of OATPs in DDIs is growing rapidly, and the clinical importance of

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such interactions is becoming more and more evident15. FDA and EMA strongly recommend

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investigation of DDIs mediated by OATP1B1 and -1B3 for new molecular entities during drug

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development since 201216. Recently, Shitara et al.2 reported examples of DDIs that are caused by

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inhibition of OATP1B transporters via potent inhibitors, and which affected the pharmacokinetics of

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victim drugs. The inhibitor drugs tested were antibiotics, antiviral drugs and the immunosuppressant

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cyclosporine A, and victim drugs were widely used statins, antidiabetic and hypertension drugs.

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Among the inhibitors, cyclosporine A and rifampicin appeared to be particularly strong inhibitors of

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OATP1B1, which caused increases in the plasma levels of several statins of 2.2- to 23-fold as a result

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of DDIs2. There are only a few recent studies on OATP-mediated DDIs with anticancer drugs, although

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these are widely used and often substrates or inhibitors of OATPs. Hu et al.

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whether inhibition of OATP1B1 by tyrosine kinase inhibitors could explain a decreased docetaxel

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clearance. Sorafenib was selected as a potent inhibitor of OATP1B1 based on in vitro assays, but

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single or multiple doses of sorafenib did not affect docetaxel plasma levels in vivo using Oatp1b2

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knockout and hOATP1B1-expressing transgenic mice . Nieuweboer et al.

1

2

2, 9-11

. For example, genetic variants of OATP1B1 are associated with

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17

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recently investigated

recently showed that



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polysorbate 80 or Kolliphor (Cremophor) EL in the drug formulation can inhibit (mouse) OATP1B2-

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mediated hepatic elimination of paclitaxel. Clearly, DDI studies involving OATP-dependent interactions

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between widely used medicines and anticancer drugs are urgently needed, as such interactions may

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lead to unexpected toxicities and altered efficacy in the treatment of cancers, and anticancer drugs are

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usually administered at high dosages and have a narrow therapeutic window. The antifolate MTX is a

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widely used anticancer and antirheumatic drug that is a good substrate of mouse Oatp1a/1b and

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human OATP1B1 and -1B3 transporters in vivo4. The likelihood of undesired DDIs and thus altered

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pharmacokinetics of MTX might be substantial, when prescribed with other commonly used drugs

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such as antibiotics, hypertension drugs and antidiabetics that are inhibitors/substrates of OATPs.

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Thus, MTX is of great interest for assessment of such interactions and prediction of clinical outcomes.

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To predict clinical DDIs, many studies utilize in vitro cellular uptake assays and in silico11,

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preclinical models

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situation and use of preclinical wild-type and knockout animal models does not necessarily result in

19-22

and

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. However, it remains challenging to translate in vitro data to the human in vivo

24, 25

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optimal prediction due to species differences

. Accordingly, we have generated humanized

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Oatp1a/1b knockout mouse models with liver-specific expression of human OATP1B1 or -1B3, with

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transgenic protein levels roughly comparable to those seen in pooled human liver samples

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study, we investigated whether these mouse models could be used to assess short-term, acute human

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OATP1B1 and -1B3-mediated DDIs in vivo. For this aim, we used the antibiotic rifampicin as a model

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inhibitor of OATP1B transporters and MTX as a victim drug. Secondly, we tested OATP1B-mediated

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DDIs between the antihypertensive drug telmisartan and MTX, which have a higher chance for co-

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prescription in the clinic.

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. In this

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Materials and Methods

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Chemicals

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Methotrexate (100 mg/ml, Pharmachemie, The Netherlands) and cyclosporine A (CsA, 50 mg/ml,

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Novartis, Switzerland) were obtained as parenteral formulations from the pharmacy department of the

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Antonie van Leeuwenhoek Hospital (The Netherlands). Rifampicin was purchased from Sigma-Aldrich

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(USA) and telmisartan was purchased from Sequoia Research Chemicals (UK), both in powder forms.

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7-OH-MTX, rifampicin-d3 and telmisartan-d7 were purchased from Toronto Research Chemicals

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(Canada). All of the chemicals used for HPLC-UV and HPLC-MS/MS analysis were from Sigma-

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Aldrich (USA) or Merck (Germany).

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Drug solutions

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In vitro experiments

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MTX stock solution (100 mg/ml = 220.1 mM) and CsA (50 mg/ml = 41.6 mM) were diluted in Krebs-

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Henseleit buffer to yield 50 µM MTX and 0.5 µM CsA solution, respectively. Rifampicin (50 mM) and

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telmisartan (10 mM) were prepared in DMSO and dimethylformamide (DMF), respectively, and were

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further diluted in Krebs-Henseleit buffer to yield the desired concentrations.

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In vivo experiments

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MTX stock solution (100 mg/ml) was diluted 50 or 250-fold with 0.9% NaCl to yield a concentration of

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2 or 0.4 mg/ml. Rifampicin was dissolved in DMSO (at 80 mg/ml) and further diluted in 0.9% NaCl to

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yield a concentration of 4 mg/ml. Telmisartan was dissolved in DMF (7 mg/ml, maximum solubility) and

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further diluted in PBS to yield a concentration of 1.4 mg/ml. All drugs were administered intravenously

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(i.v.), using a volume of 5 µl/g body weight. All working solutions were prepared on the day of

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experiment.

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Cell culture

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HEK293 cells transduced with vector control, hSLCO1B1 and hSLCO1B3 cDNAs were a kind gift from

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Prof. Werner Siegmund and Dr. Markus Keiser (University of Greifswald, Greifswald, Germany)26. All

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cells were grown in Dulbecco's modified Eagle's medium high glucose (Gibco) supplemented with

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10% fetal bovine serum (Sigma), 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml

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amphotericin B at 37°C with 5% CO2 and 95% humidity. All of these cell lines were authenticated and

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found to be identical to the original cell lines.

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Cellular uptake experiments

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Cellular uptake experiments were performed according to previously described methods

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modified as required. Krebs-Henseleit buffer at pH 7.4, which was adjusted before the experiment was

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started, was used in all the steps of the experiment including dilutions of the drug stock solutions.

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Briefly, cells were preincubated with 1 ml of pre-warmed buffer for 15 min at 37°C. Uptake

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experiments were started by aspirating the preincubation buffer and adding 600 µl of pre-warmed

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buffer containing (inhibitor) drugs; MTX (50 µM) alone or together with rifampicin (0, 1, 2.5, 5, 15, 25,

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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

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positive control for the uptake inhibition; therefore it was also added in the pre-incubation buffer as an

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OATP inhibitor. At designated time points (2.5, 5, 10, 15, 30, 60 or 120 min) experiments were

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terminated by removing the incubation buffer and immediately adding 1 ml of ice-cold buffer. After

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washing twice with ice-cold buffer, cells were lysed with 150 µl of 0.2 N NaOH for a minimum of 15

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min. 10 µl of the cell lysate was used to determine the cellular protein amount by the Bradford method

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using bovine serum albumin as a standard. 60 µl of the cell lysates were used to determine MTX

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levels by HPLC-UV. These were readily detectable when 50 µM MTX was applied. Percentage of

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OATP-mediated uptake of MTX was calculated using the uptake without inhibitor as 100%.

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Animals

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Mice were housed and handled according to institutional guidelines complying with Dutch legislation.

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Male WT, Oatp1a/1b-/-, Oatp1a/1b-/-;1B1tg and Oatp1a/1b-/-;1B3tg with liver-specific expression of

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human SLCO genes, all of a >99% FVB genetic background, were used between 8 and 14 weeks of

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age. Animals were kept in a temperature-controlled environment with a 12 hr light/12 hr dark cycle and

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received a standard diet (AM-II, Hope Farms) and acidified water ad libitum.

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Plasma and liver pharmacokinetic experiments

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Dosages of MTX were 10 and 2 mg/kg body weight, that of rifampicin was 20 mg/kg and that of

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telmisartan was 7 mg/kg body weight. Rifampicin, telmisartan or vehicles (0.9% NaCl or PBS) were

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injected into the tail vein of mice 3 minutes before MTX administration. For all experiments, mice were

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sacrificed at 5 or 15 min after MTX administration by terminal bleeding through cardiac puncture under

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isoflurane anesthesia and organs were rapidly removed. Plasma was isolated from blood samples

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after centrifugation at 2,100 g for 6 min at 4°C, livers were homogenized in 1% bovine serum albumin

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and all the samples were stored at −30°C until analysis.

8, 27

and


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After drug analysis, results were presented as concentrations in the organs (nmol/g), % of the total

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dose (dose corrected for the body weight of each individual mouse being equivalent to 100%) and/or

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organ-to-plasma ratios. Organ-to-plasma ratios were calculated by dividing organ concentration

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expressed as nmol/g by plasma concentration expressed as nmol/ml, assuming 1 ml of plasma is

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roughly equivalent to 1 g of tissue.

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Drug Analyses

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Determination of MTX and 7-OH-MTX

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Levels of MTX and 7-OH-MTX in plasma and liver homogenates were determined by HPLC-UV

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detection as described previously28. For the in vitro experiments, cell lysates in 0.2 N NaOH were

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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

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for 5 min at 4°C, supernatants were injected into the HPLC system directly. Standard curve (4.4 - 2201

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nM) and quality control (22.01, 220.1 and 2201 nM) samples were prepared using blank cell lysates as

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matrix.

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Sample pre-treatment for rifampicin and telmisartan

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Plasma and liver homogenates were thawed at room temperature and diluted 1:50 or 1:100 (for

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rifampicin or telmisartan determinations, respectively) in blank human plasma. A volume of 100 µl of

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plasma or liver homogenate was pipetted into 2 ml polypropylene vials (Eppendorf, Hamburg,

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Germany). Next, volumes of 50 µl of IS working solution (rifampicin-d3 or telmisartan-d3; 1 µg/ml in

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acetonitrile: water 30:70; v/v) and 1000 µl of ethyl acetate were added. After automatic shaking for 15

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min at 480 rpm, the samples were centrifuged for 1 min at 20,000 g. The aqueous layer was snap

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frozen in dry ice/ethanol and the upper organic layer was decanted into a 1.5 ml vial (Brand,

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Wertheim, Germany). The solvent was evaporated to dryness in Speed-Vac SC210A (Savant,

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Farmingdale, NY, USA) and the residue was reconstituted with 100 µl of acetonitrile: water (30:70, v/v)

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by sonicating for 5 min and vortex-mixing for 5 s. Finally, samples were centrifuged for 5 min at 20,000

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g prior to analysis.

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Quantification of rifampicin and telmisartan

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The levels of rifampicin and telmisartan in plasma and liver homogenates were quantified by HPLC

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coupled to tandem mass spectrometry (HPLC-MS/MS). Details of the instrumentation are explained in

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supplemental text.



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The concentrations of rifampicin and telmisartan in plasma and liver homogenates were determined by

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means of the internal standard (I.S.) method, employing the corresponding stable isotope labeled

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drugs (rifampicin-d3 and telmisartan-d3).

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Stock solutions of rifampicin and telmisartan were prepared in DMSO at 10 mM and stored at ‒20ºC.

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For each analytical run, these solutions were diluted in DMSO to obtain working solutions ranging from

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100 nM to 100 µM for the preparation of the above-mentioned calibration standards. A set of

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calibration standards in either blank plasma or liver homogenate was prepared using 100-fold dilution

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of the DMSO stock in blank biological matrix. Final calibration samples contained 1, 3, 10, 30, 100,

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300 and 1000 nM.

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Statistical analysis

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IC50 values for rifampicin and telmisartan for inhibition of MTX uptake were determined using non-

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linear regression (curve fit) analysis in GraphPad Prism 6.01 software after correction for the

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background uptake in control cells. The 2-sided Student's t-test or one-way analysis of variance

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(ANOVA) with post-hoc tests employing Tukey's corrections were used to determine statistical

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significance either between two or multiple groups, respectively. Results were presented as the mean

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± standard deviation (SD). Differences were considered to be statistically significant when P ≤ 0.05.

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Results

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In vitro MTX uptake by OATP1B transporters

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To establish time-dependent uptake of MTX by OATPs, we performed cellular uptake experiments

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with MTX (50 µM) using control and OATP1B1- or OATP1B3-expressing HEK293 cells for various

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time periods between 2.5 and 30 min. 50 µM MTX was used since shortly after i.v. administration (5-

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15 min) of 10 mg/kg MTX in the planned in vivo DDI studies, plasma concentrations of that order are

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achieved (see below). MTX uptake by OATP1B1 or -1B3 was significantly greater compared to control

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cells at all-time points (Figure 1A-B). MTX uptake by OATP1B3 was slightly saturated after 5 min and

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more strongly after 15 min. In subsequent studies we used 15 min uptake, as a clear differential was

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seen at this time point between both the transporter-expressing cells and their controls. The increased

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uptake of MTX in both OATP-expressing cells was inhibited completely by 0.5 µM CsA, which is

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known to inhibit OATPs (Figure 1C); further corroborating that MTX uptake in these cells was OATP-

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mediated. Studies with these cells were performed to estimate the IC50 values of candidate inhibitors

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for subsequent in vivo DDI studies.

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Effect of rifampicin on OATP-mediated MTX uptake in vitro

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To determine whether OATP-mediated MTX uptake could be inhibited by the OATP inhibitor

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rifampicin, we estimated the IC50 of rifampicin for each OATP-expressing cell type. IC50

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concentrations of rifampicin were 0.88 ± 0.18 or 0.31 ± 0.12 µM for OATP1B1- or OATP1B3-mediated

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MTX uptake, respectively (Figure 2). This suggests that rifampicin is a fairly strong inhibitor (low µM

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range) of MTX uptake mediated by these OATPs.

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Effect of rifampicin on OATP-mediated MTX and 7-OH-MTX disposition in vivo

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To investigate whether rifampicin can also inhibit OATP-mediated MTX transport in vivo, we

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administered 20 mg/kg rifampicin or vehicle (i.v.) 3 min before 10 mg/kg MTX dosing (i.v.) to wild-type

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(WT), Oatp1a/1b knockout and transgenic mice with liver-specific expression of OATP1B1 and

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OATP1B3, considering their relevance to human drug disposition.

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In groups receiving vehicle, MTX disposition results were similar to those described previously by van

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de Steeg et al.4. Plasma MTX levels were increased in Oatp1a/1b-/-, Oatp1a/1b-/-;1B1tg and

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Oatp1a/1b-/-;1B3tg mice compared to WT strains, both at 5 and 15 min after MTX administration

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(Figure 3A and B). Liver levels of MTX and liver-to-plasma ratios were profoundly decreased by

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removal of the Oatp1a/1b transporters, and expression of human OATP1B1 or -1B3 in these livers led



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to significant, albeit not complete, rescue of the impaired liver uptake of MTX (Figure 3), suggesting

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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

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each transporter seems to be substantial. Therefore, these findings suggest that MTX hepatic uptake

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is primarily mediated by OATPs.

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In rifampicin pretreated groups, plasma levels of MTX were significantly increased by 4.2-, 1.5- and

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1.9-fold in WT, Oatp1a/1b-/- and Oatp1a/1b-/-;1B3tg mice 5 min after MTX administration (Figure 3A).

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Rifampicin pretreatment significantly increased the plasma levels of MTX in WT and Oatp1a/1b-/-

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;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

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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-

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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.

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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

Preclinical Mouse Models To Study Human OATP1B1- and OATP1B3

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