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TRANSCRIPTIONAL AND POSTTRANSCRIPTIONAL REGULATION OF DUODENAL P-GLYCOPROTEIN AND MRP2 IN HEALTHY HUMAN SUBJECTS AFTER CHRONIC TREATMENT WITH RIFAMPIN AND CARBAMAZEPINE Susanne Brueck, Henrike Bruckmueller, Danilo Wegner, Diana Busch, Paul Martin, Stefan Oswald, Ingolf Cascorbi, and Werner Siegmund Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00458 • Publication Date (Web): 30 Jul 2019 Downloaded from pubs.acs.org on July 31, 2019

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Molecular Pharmaceutics

TRANSCRIPTIONAL AND POSTTRANSCRIPTIONAL REGULATION OF DUODENAL P-GLYCOPROTEIN AND MRP2 IN HEALTHY HUMAN SUBJECTS AFTER CHRONIC TREATMENT WITH RIFAMPIN AND CARBAMAZEPINE Susanne Brueck1, Henrike Bruckmueller2, Danilo Wegner1, Diana Busch1, Paul Martin2, Stefan Oswald1, Ingolf Cascorbi2 and Werner Siegmund1* 1Department

of Clinical Pharmacology, Center of Drug Absorption and Transport, University

Medicine of Greifswald, Department of Clinical Pharmacology, Felix-Hausdorff-Straße. 3, 17487 Greifswald, Germany 2Institute

of Clinical and Experimental Pharmacology, University Hospital Schleswig-Holstein,

Arnold-Heller-Straße, 24105 Kiel, Germany

ACS Paragon Plus Environment

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KEYWORDS: healthy subjects, intestinal drug transporters, induction, rifampin, carbamazepine, nuclear receptors, microRNA, ezetimibe, talinolol

ABSTRACT

To predict the outcome of intestinal drug transporter induction on pharmacokinetics, signaling of the DNA message along with mRNA transcription and protein translation leading to transporter function must be understood. We quantified gene expression of PXR and CAR, gene expression and protein abundance of P-gp, MRP2 and BCRP, the content of 754 microRNAs in human duodenal biopsy specimens and pharmacokinetics of talinolol and ezetimibe before and after treatment with rifampin and carbamazepine, respectively. Rifampin significantly induced transcription of ABCB1 and ABCC2 and protein abundance of P-gp but not of MRP2. P-gp abundance significantly correlated to plasma exposure of ezetimibe and its glucuronide. Carbamazepine induced mRNA expression of CAR, ABCB1 and ABCC2 but did not elevate protein abundance. Using in silico prediction tools and luciferase reporter assays, microRNAs were identified which can contribute to ligand-specific regulation of intestinal drug transporters, and different changes in drug disposition after induction with rifampin and carbamazepine.


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DNA-Properties Nuclear Receptor • Tissue expression and regulation • Ligand concentration

mRNATranscription

Protein Translation and Function

Pharmacokinetics of Ligands

Posttranscriptional Regulation

• • • •

• microRNA • Histone deacetylation

Rifampicin Carbamazepine Efavirenz St. John´s Wort

Pharmacokinetics of Probe Drugs

Efficacy and Safety of Probe Drugs

Abstract Graphic


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INTRODUCTION Hepatic and intestinal metabolism and transport are the major physiological mechanisms behind the pharmacokinetics of drugs.1,2 Therefore, genetic polymorphisms, hepatic and intestinal diseases as well as drug-drug interactions (DDI) can influence clinical efficacy and safety of many drugs as it is well known that these factors influence the function of drug transporters and drug metabolizing enzymes.1,2 To predict the quantitative effects of these factors on the pharmacokinetics of drugs, the complex signaling and regulatory pathways involved in transcription, translation, and post-translational processes of metabolizing enzymes and transporter proteins must be known, as many confounders including nuclear receptors and epigenetic factors are involved.3,4 It has been shown in healthy subjects by quantification of mRNA expression and protein abundance in different tissues, evaluations with biomarkers (e.g. 4-OH-cholesterol/cholesterol ratio, glucaric acid excretion, metabolic cortisol clearance), and by using pharmacokinetic outcome characteristics (e.g. bioavailability, metabolic or renal clearance) of probe drugs, that nuclear receptor ligands can induce drug transporting proteins and drug metabolizing enzymes in a compartment-specific manner. For example, rifampin and Saint John’s wort regulate both processes in the small intestine5–9 and liver5,7, but likely not in the kidney6–9 as distinguishable by increase of renal clearance probe drugs (e.g. digoxin, talinolol). In contrast to rifampin, carbamazepine acts as inducer of P-glycoprotein (P-gp) and the multidrug resistance associated protein 2 (MRP2) in the intestine, liver and kidney. In the small intestine, however, only the ABCB1 mRNA expression, but not P-gp abundance was up-regulated.10 Efavirenz does not influence drug metabolism and transport neither in the intestine nor the kidneys but, however, is an inducer in peripheral mononuclear blood cells and the liver.11,12


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Here, we hypothesize on reasons for compartment-specific gene regulation of nuclear receptor ligands. Firstly, due to different nuclear receptor preference, for example, rifampin and Saint John’s wort are pregnane X receptor (PXR) ligands, whereas efavirenz and carbamazepine bind predominantly to the constitutive androstane receptor (CAR).13,14 Secondly, there is a compartment-specific expression of transcription factors.15 Thirdly, mRNA expression and protein synthesis of transporters and drug metabolizing enzymes can be modified by epigenetic factors (e.g. DNA methylation, histone deacetylation) and posttranscriptional regulation, e.g. via microRNAs (miRNAs).4 Fourthly, local availability of the inducers in the nuclear receptor compartments may vary between tissues. For instance, carbamazepine occurs in the small intestine in active concentrations only during the short time of oral absorption whereas rifampin recirculates via the entero-hepatic circle. In the systemic circulation, rifampin reaches PXRactivating concentrations only for a short time (T½ ~ 3 hours) that may not be transporterinducing, e.g. in the kidneys, whereas CAR-activating plasma levels of carbamazepine are almost constant during the entire dosing intervals.10,16,17 To describe some aspects of the complex signaling from mRNA transcription into protein translation and pharmacokinetic outcome, we re-evaluated mRNA expression of PXR and CAR, mRNA expression and protein abundance of P-gp, MRP2 and breast cancer resistance protein (BCRP) and the pharmacokinetic outcome characteristics of ezetimibe and talinolol (probe drugs for MRP2 and P-gp, respectively).7,18–20 Duodenal biopsy specimens and the pharmacokinetic data from our biobank/databank were used which were obtained in two previously performed DDI studies from healthy subjects before and after enzyme induction with rifampin and carbamazepine, respectively.7,10 Furthermore, we


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evaluated the potential influence of miRNAs on post-transcriptional regulation of the transporters.


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EXPERIMENTAL SECTION Clinical study protocol For our study, we re-evaluated the pharmacokinetic data and duodenal mucosa biopsy specimens of two previously performed DDI studies with ezetimibe (10 mg po, single dose) or talinolol (100 mg po, repeated dosing for 19 days). The data and tissue specimens were obtained in the ezetimibe study before and after chronic treatment with rifampin (600 mg for 8 days), and in the talinolol study before and after concomitant treatment with 600 mg carbamazepine for 14 - 18 days.7,10 The data and tissue were stored in our databank and biobank, respectively, and were used for the additional evaluations strictly within the scope of the previous study objectives, and with the written informed consent as given by the healthy subjects which were included in the rifampin-ezetimibe study (11 males, 1 female; age 21 - 31 years; body mass index 19.2 - 26.4 kg/m2), and carbamazepine-talinolol study (4 males, 4 females; age, 23 - 35 years; body mass index 20.8 - 24.7 kg/m2). The re-evaluation study was approved by the Independent Ethics Committee of the University Medicine of Greifswald (BB 086/17). Duodenal mRNA and miRNA expression and protein abundance Duodenal biopsies were disrupted using the Tissue Lyser II (Qiagen, Hilden, Germany). After isolation of total RNA with a NucleoSpin® miRNA kit (Macherey-Nagel, Düren, Germany), purity and concentration of the isolated RNA were measured by means of the NanoDrop™ 1000 spectrophotometer (Thermo Fisher Scientific, Darmstadt, Germany), and integrity was evaluated using the analyzer Agilent® 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). For expression analysis, mRNA was reversely transcribed by means of a high-capacity cDNA reverse transcription kit with RNAse inhibitor (Thermo Fisher Scientific, Darmstadt, Germany),


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and analyzed with the 7900 HT Fast Real-Time PCR System using custom TaqMan® array micro fluidic cards (Thermo Fisher Scientific, Darmstadt, Germany) with 500 ng cDNA per reaction for 11 drug metabolizing enzymes and enzyme families, 6 transporters of the ATPbinding cassette (ABC) transporter family, 21 transporters of the solute carrier (SLC) family, 5 nuclear receptors, and 5 reference genes (Supplemental Table S1). The mRNA expression of UGT1A1, 1A3, 1A4, 1A6 and 1A9 was quantified using TaqMan® gene expression assays (Thermo Fisher Scientific, Darmstadt, Germany) with 50 ng of cDNA per reaction. Three reference genes were selected according to the results of the BestKeeper tool (http://www.genequantification.de/bestkeeper.html).21 Relative gene expression was analyzed using the 2^-ΔΔCt method including the geometric mean of three reference genes (18s, GAPDH, GUSB).22 Two technical replicates of one biopsy specimen was measured. The miRNA expression analysis was conducted using the TaqMan® array with the human microRNA A+B cards set v3.0 including 754 miRNAs (Thermo Fisher Scientific, Darmstadt, Germany). Reverse transcription was performed with 450 ng of total RNA using the TaqMan® microRNA reverse transcription kit (Thermo Fisher Scientific, Darmstadt, Germany). The Megaplex RT product was pre-amplified using a 12-cycle PCR reaction according to the protocol of the manufacturer. Array cards ran on the sequence detection system 7900 HT with default cycling conditions (Thermo Fisher Scientific, Darmstadt, Germany). miRNA expression was quantified using the 2^-ΔΔCt method with RNU 48 as the endogenous control.22 Protein abundance of P-gp, MRP2 and BCRP was quantified in membrane fractions enriched by differential centrifugation using a validated LC-MS/MS-based targeted proteomics assays as recently described.23 One technical replicate of one biopsy was measured. The between-sample variability (CV) as assessed by independent sample preparation, digestion and LC–MS/MS


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analysis of six different specimens from an identical tissue sample of a donor was 14.8 %, 34.5 % and 22.1 % for P-gp, MRP2 and BCRP.23 In silico miRNA target prediction Correlation analysis between miRNA expression and protein abundance of drug transporting proteins was performed to identify inversely correlated miRNA-drug transporting protein pairs with significance p < 0.05. The Target Scan Human Release 7.0, MicroCosm Targets Version 5, DIANA-microT-CDS v5.0 and microRNA.org August 2010 Release were selected to evaluate potential miRNA-binding sites on the mRNAs of drug transporting proteins.24–26 Luciferase assay Reporter gene vectors containing the 3’-UTR of ABCB1, ABCC2, ABCG2 and the empty vectorplasmid construct were purchased from GeneCopoeia (Rockville, MD) (for details Supplemental Table S2). For reporter gene experiments, HepG2 cells (DMSZ, Braunschweig, Germany) were cultured as recently described.27 The transfection complex consisting of siPORT™ NeoFX™ transfection agent, plasmid DNA (70 ng/well) of either the empty vector control, or vector constructs containing the transporter 3’-UTR and the respective miRNA precursor, or precursor negative control diluted in Opti-MEM I medium, was prepared according to manufacturer’s instructions. 20 µl transfection complex were dispensed and overlayed with 80 µl cell suspension (1.5 x 105 cells, passage 11 - 13) per well in a 96-well plate format. 24 hours after transfection, the medium was replaced by culture medium. Activities of firefly and Renilla reniformis luciferases were evaluated 48 hours after transfection using the Dual-Luciferase® reporter assay system (Promega, Mannheim, Germany) and the VeritasTM microplate luminometer (Tuner Biosystems, Sunnyvale, CA) according to the manufacturer’s instructions. Firefly luciferase


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activity of each sample was normalized to the activity of Renilla reniformis luciferase serving as the transfection control. Subsequently, luciferase activities of the investigated miRNAtransporter interactions were normalized to the empty vector control and negative control precursor miRNA. The entity of predicted mRNA-miRNA interactions was subjected to a prescreening applying 10 nM and 30 nM of precursor miRNAs (7 biological replicates of each transfection condition). For repeated measurements, we selected miRNAs, which in the prescreening showed marked effects on reporter gene activity. Determination of glucaric acid Urinary D-glucaric acid excretion, a biomarker for hepatic PXR-type enzyme induction was determined with a modified assay according to Jung et al. using the spectrophotometer Hitachi U-2900 (Hitachi High Technologies, Krefeld, Germany).28–30 Statistical analysis Samples were characterized by arithmetic means and standard deviations (M ± SD), and evaluated for differences using the Wilcoxon signed rank test and Mann Whitney test as appropriate. Evaluation of correlations was performed according to Spearman and by multivariate analysis using the SPSS 21 program package (IBM Corporation, Armonk, NY).


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RESULTS We re-evaluated the mRNA expression and protein abundance of P-gp, MRP2, and BCRP and expression of 754 miRNAs in duodenal biopsy specimens (3-5 mg wet weight) from healthy subjects, as well as the pharmacokinetic characteristics of ezetimibe after single dose administration and talinolol after multiple-dose administrations which were obtained in two DDI studies before and after chronic treatment with rifampin and carbamazepine together with talinolol and which were stored in our biobank and databank, respectively.7,10 Expression of mRNAs was also evaluated for additional 24 drug transporter proteins, 16 drug metabolizing enzymes/enzyme families and 5 nuclear receptors including PXR and CAR. The following results were obtained: Duodenal expression of nuclear receptors, drug metabolizing enzymes and transporters in non-induced subjects In the human duodenum of our 20 healthy subjects, PXR mRNA was ~ 20-fold higher expressed than the CAR mRNA. MRP2 (0.87 ± 0.22 pmol/mg protein) was the major efflux carrier in the duodenal tissue followed by P-gp (0.67 ± 0.25 pmol/mg protein) and BCRP (0.62 ± 0.30 pmol/mg protein). The mRNA expression of certain efflux transporters in the non-induced state was significantly correlated to PXR mRNA (ABCC2: r = 0.762; p < 0.0001; ABCG2: r = 0.677, p = 0.011) and CAR mRNA (ABCC2: r = 0.495; p = 0.027; ABCG2: r = 0.555, p = 0.011). On the contrary, protein abundance of the transporters was not significantly correlated to mRNA expression (P-gp: r = 0.229, p = 0.332; MRP2: r = 0.322, p = 0.166; BCRP: r = -0.347, p = 0.133). 335 of the screened miRNAs (n = 754) were detected in our duodenal tissue specimens.


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In the Supplemental Figure S1, additional data on duodenal mRNA expression are provided for drug transporter proteins and drug metabolizing enzymes for which protein data were not available.

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2

0

** PXR

CAR

rifampin

Figure 1:

before after

*

12

mRNA expression (2^-ΔCtx103)

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

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PXR

CAR

carbamazepine

mRNA expression (normalized to 18s/GAPDH/GUSB) of nuclear receptors in duodenal biopsies before and after chronic oral administration of rifampin (n = 12) or carbamazepine (n = 8). Data are represented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001, Wilcoxon signed rank-test


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Expression of nuclear receptors, microRNAs and drug transporters after rifampin Treatment with rifampin had no influence on mRNA expression of duodenal PXR and CAR (Figure 1). The mRNA transcription of ABCB1 and ABCC2 but not of ABCG2 was significantly upregulated. The increased ABCC2 mRNA transcription was not translated into a higher MRP2 protein abundance. The P-gp content significantly increased, however, to a much lower extent compared to the increase in ABCB1 mRNA (Figure 2). P-gp protein abundance was significantly correlated to mRNA-expression (r = 0.604, p = 0.002). After treatment with rifampin, 17 miRNAs were up-regulated > 1.5-fold to baseline (Supplemental Table S3). Expression of 20 miRNAs correlated inversely to P-gp, of 11 miRNAs to MRP2 and of one miRNA to BCRP protein abundance or the protein/mRNA ratio (p < 0.05) (Supplemental Table S4). Using in silico tools, one miRNA was predicted to bind to the ABCB1-3'UTR mRNA (miR-485-3p) and one to ABCG2-3'UTR (miR-577) (Supplemental Table S2). Expression of nuclear receptors, microRNAs and drug transporters after carbamazepine Treatment with carbamazepine significantly induced the mRNA expression of duodenal PXR and CAR (Figure 1). Furthermore, it significantly induced mRNA transcription of ABCB1, ABCC2 and ABCG2. In contrast to transcription, however, protein abundance of the transporters did not change (Figure 2).


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*** fold change in expression

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mRNA protein

5

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*** 2

1

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Figure 2:

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ABCB1 ABCC2 ABCG2

ABCB1 ABCC2 ABCG2

rifampin

carbamazepine

Fold change in mRNA expression and protein abundance in duodenal biopsies after chronic oral administration of rifampin (n = 12, left) or carbamazepine (n = 8, right). Data are represented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001, Wilcoxon signed rank-test

After treatment with carbamazepine, thirty two miRNAs were up-regulated > 1.5 fold and two were down-regulated < 0.5 fold by carbamazepine (Supplemental Table S3). Twenty miRNAs showed a negative correlation (p < 0.05) to P-gp, 32 to MRP2 and 20 to BCRP protein abundance or the protein/mRNA ratio (Supplemental Table S4).


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In silico miRNA target prediction In silico investigation predicted the binding of three miRNAs to ABCB1-3'UTR (miR-9-3p; miR148b-5p, miR-485-3p), five miRNAs to ABCC2-3'UTR (let-7f-2-3p; miR-26a-5p; miR-155-5p; miR-218-5p; miR-362-3p) and nine miRNAs to ABCG2-3'UTR (miR-10a-3p; miR-19b-1; miR30e-3p; miR-141-5p; miR-340-5p; miR-374b-3p; miR-378a-3p; miR-378a-5p, miR-577) (Supplemental Table S2). Transporter protein induction of P-gp, MRP2 and BCRP with rifampin and carbamazepine was in some cases weakly but significantly paired to the respective changes in expression certain miRNAs (Supplemental Table S4). After treatment with rifampin, miR-485-3p was correlated to P-gp (r = -0.452, p = 0.027) and miR-577 to BCRP (r = -0.437, p = 0.033). After carbamazepine treatment, miR26a-5p was correlated with MRP2 (r = -0.587, p = 0.027). The binding of these miRNAs to the respective mRNAs has been predicted in silico (see above) and evaluated using a reporter gene assay (see below). Translation of transporter expression in pharmacokinetics of ezetimibe and talinolol The area under the curves (AUC) of the ezetimibe glucuronide and of the total ezetimibe (ezetimibe plus glucuronide) were significantly correlated to protein abundance of P-gp (r = 0.560, p = 0.004 and r = -0.512, p = 0.011). MRP2 abundance was not correlated to exposure of ezetimibe and its glucuronide. The correlations along the signaling chain for translation of P-gp into the AUC of ezetimibe glucuronide after treatment with rifampin are shown in Figure 3.


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P-glycoprotein (pmol/mg)

ABCB1 mRNA (2^-Ct x 103)

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4 3 2 1 0

r = 0.368 p = 0.077 0

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r = 0.604 p = 0.002 0

PXR mRNA (2^-Ct x 103)

AUC ezetimibe + glucuronide (ngxh/ml)

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0.4 r = - 0.452 p = 0.027 0.0 0.00

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-Ct

miRNA 485-3p (2^

Figure 3:

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0.10

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1200

800

400 r = - 0.512 p = 0.011 0 0.0

3

x 10 )

0.3

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1.2

1.5


Correlations along the signaling chain for translation of P-glycoprotein into the AUC of ezetimibe + glucuronide after treatment with rifampin.

There was no correlation between bioavailability of talinolol and protein abundance of any efflux transporter. Urinary excretion of glucaric acid, a biomarker for PXR activation, significantly increased (2.8-fold) after chronic treatment with rifampin.


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Luciferase Assay To further investigate the role of miRNAs identified in in vivo studies, in vitro studies were subsequently carried out. The identified pairs from both studies were subjected to a luciferase reporter gene assay. After a prescreening, 10 miRNA - target gene pairs were selected for further investigation (Figure 4).

140% 120%

relative reporter gene activity

100%

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***

80%

***

60% 40% 20%

ABCB1

Figure 4:

ABCC2

miR-577

miR-378-3p

miR-374b-3p

miR-340-5p

miR-19b-1-5p

miR-10a-3p

miR-155-5p

miR-26a-5p

miR-485-3p

miR-148b-5p

0% miR-NC

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


ABCG2

Relative reporter gene activity normalized to empty vector and negative control miRNA precursor for ABCB1, ABCC2 and ABCG2. Data are represented as mean ± SD of 4 (ABCB1, ABCC2) or 3 (ABCG2) independent transfections comprehending each 7 biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001, Mann Whitney test


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Finally, co-transfection with the ABCB1 3’-UTR target clone and its miR-485-3p (10 nM) precursor led to a significant decrease in relative reporter gene activity (75.5 %; p < 0.01), while no significant changes were found for co-transfection with the miR-148b-5p precursor. In terms of ABCC2 3’-UTR, co-transfection with the miR-26a-5p precursor (10 nM), but not with miR-155-5p, significantly reduced relative reporter gene activity (72.4%; p < 0.001). Cotransfection with the ABCG2 3’-UTR target clone and miR-577 precursor led to a marked decrease of relative reporter gene activity (46.5 %; p < 0.001). Interference of the ABCG2 3’-UTR target with miR-10a-3p, miR-19b-1-5p, miR-378-3p, miR-340-5p or miR-374b-3p precursor only induced weak, or no significant reduction, of reporter gene activity.

DISCUSSION In our study, we gained a more extensive insight into the complex changes in the cellular signaling cascade which controls mRNA transcription and translation into the function of the duodenal efflux transporter proteins P-gp and MRP2 by the nuclear receptor ligands rifampin and carbamazepine in humans. For the research project, we used previously collected pharmacokinetic data and biomaterial from two former DDI studies involving ezetimibe and talinolol, which are probe drugs for MRP2 and P-gp, respectively.7,10 We used currently existing quantitative assays for molecular biology and targeted proteomics, which were not available at the time of the previous clinical studies, and investigated major genetic and epigenetic variables such as nuclear receptors and miRNAs. Using this approach, we could detect a significant up-regulation of the intestinal mRNA expression of ABCB1 and ABCC2 but not of ABCG2 following chronic oral administration of rifampin. In contrast to the anticipated up-regulation in mRNA expression of ABCB1 and


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ABCC2, protein abundance only significantly increased in the case of P-gp, but not MRP2. In contrast to rifampin, carbamazepine led to a higher expression of PXR and CAR, but similar to rifampin, to significantly higher mRNA-expression of ABCB1, ABCC2 and ABCG2. Surprisingly, translation into protein abundance of all transporters remained unchanged (Figure 2). To explain the discrepancies between mRNA transcription after treatment with rifampin or carbamazepine, various factors must be taken into consideration. Firstly, we clearly demonstrated that PXR is the major nuclear receptor in the human duodenum. Therefore, it is likely that rifampin, a potent activator of PXR, causes stronger induction of the PXR-regulated intestinal transporters than carbamazepine, which binds preferentially to CAR.13 It must be taken into account that carbamazepine, but not rifampin also regulates the mRNA transcription of PXR and CAR, which was shown for the first time in our clinical study. Secondly, the different pharmacokinetic properties of rifampin and carbamazepine could contribute to differences in induction. It can be assumed that the effective concentrations as were applied in in vitro assays (10 µM for rifampin, 25 µM for carbamazepine) were distinctly lower compared to the concentrations reached at receptor sites of PXR and CAR in our healthy subjects, because the concentrations of rifampin and carbamazepine in the gut lumen were at least 3 mM and 10 mM, respectively, as derived from the molar doses/250 ml water for administration ([I]2-value).13,31 However, rifampin but not carbamazepine undergoes major entero-hepatic circulation making it available in gut lumen for an extended period of time.32 Therefore, the intestinal nuclear receptors are obviously exposed for a considerably longer time to inducing concentrations of rifampin as compared to carbamazepine, which is only available in the gut for a short time frame for absorption.


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Thirdly, after treatment with rifampin, we found a significant induction of the mRNA expression of ABCB1 and ABCC2, whereas the P-gp abundance increased to a relatively lower extent and the MRP2 content did not significantly increase at all (Figure 2). Similar results were found after carbamazepine treatment for ABCB1, ABCC2 and ABCG2 with a significant induction of mRNA expression, but no significant changes in protein abundance. miRNAs are short noncoding RNAs that are involved in the post-transcriptional regulation of gene expression either by promoting of mRNA degradation or by repression of translational repression.33,34 When considering ABCB1 and ABCC2 in our study, it is most likely that the miRNAs caused translational repression rather then mRNA degradation. This is supported by mRNA and protein data after induction that exhibit the absence of increased protein abundance while mRNA levels are elevated as expected. As exemplified for three miRNAs, we were able to confirm the in silico predicted interactions and thereby illustrate the potential functional relevance of miRNAs in regulation of intestinal drug transporters. The newly identified interactions of ABCB1-3'-UTR – miR-485-3p and ABCC2-3'-UTR – miR-26a-5p provide new insights into the regulation of intestinal drug transporters as they contribute to the explanation of the observed discrepancy between mRNA and protein level of both transporters. In addition, we newly identified the interaction of miR-577 with ABCG2 3’-UTR using correlation analysis followed by reporter gene experiments. However, when considering the functional relevance of this interaction, the ABCG2 mRNA and protein data showing no significant differences after induction reveal that a regulation of ABCG2 by miR-577 plays most likely no role in our study. Various mechanisms, like miRNA turnover, sub-cellular localization of miRNA and target gene or modification of the miRNA target site, can prevent the functional


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consequences of miRNAs and need to be further investigated in particular in tissue specific context.35 The observed discrepancy between ABCC2 mRNA expression and MRP2 protein abundance following chronic treatment with rifampin was in contrast to the results of two previous studies in which an increase of the mRNA and protein content was found.7,36 As in one previous study, however, the MRP2 abundance was only slightly increased (1.4-fold versus 1.2-fold) and the inter-subject variability was high in both studies.36 To investigate the influence of regulatory mechanisms on pharmacokinetics, we re-evaluated our new protein data for P-gp and MRP2 with the previously assessed pharmacokinetic outcome criteria of ezetimibe and talinolol.7,10 Our new correlation analysis indicated that systemic exposure of ezetimibe and ezetimibe glucuronide can be predicted by duodenal P-gp abundance rather than MRP2. Therefore, on the contrary to our previous discussion, intestinal MRP2 seems not to be the rate-determining key factor for bioavailability of ezetimibe. According to our new understanding, oral absorption of ezetimibe after chronic treatment with rifampin is likely lowered by an up-regulation of intestinal P-gp for which ezetimibe has high affinity.7 Previously, we have also shown that chronic treatment with carbamazepine leads to a lowering of the systemic exposure with talinolol. As the protein abundance of intestinal P-gp remains unchanged, however, the decrease in systemic exposure must have been caused by reasons other than by lowering the bioavailability. Considering the increase of renal clearance of talinolol by carbamazepine, it is very likely that the rationale behind this phenomenon is an upregulation of renal P-gp which is in line with the high mRNA expression of CAR in kidney, and with the finding that carbamazepine acts as strong inducer of CAR.13,15 In addition, the plasma


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concentrations of carbamazepine during chronic treatment are higher than concentrations, which were found to be enzyme inducing in in vitro experiments.13,17 Conclusions: To our knowledge, this is the first study which has evaluated PXR/CAR-type induction of rifampin and carbamazepine on mRNA expression and protein abundance of P-gp and MRP2, as well as on PXR/CAR mRNA expression and miRNA expression in the human duodenum in relation to pharmacokinetic outcome criteria of the probe drugs ezetimibe and talinolol. After treatment with rifampin, which binds predominantly to PXR, the transcriptional signal is significantly transmitted into an increase in the duodenal ABCB1 mRNA expression and protein abundance of P-gp. This in turn leads to a lowering of the bioavailability of the probe drug ezetimibe in healthy subjects. Rifampin itself did not regulate the PXR mRNA expression. There is evidence, however, that the magnitude of the ABCB1 mRNA expression is controlled by a specific miRNA up-regulation. Our previous assumption that intestinal MRP2 is the key factor in lowering systemic exposure with ezetimibe and ezetimibe glucuronide after PXR-type induction with rifampin must be revised because rifampin does indeed up-regulate ABCC2 mRNA expression but not the protein abundance of MRP2. MRP2 protein translation seems to be prevented by up-regulation of specific miRNA expression. Chronic treatment with carbamazepine leads, in major difference to rifampin, to an increase of the duodenal mRNA expression of the nuclear receptor CAR, and the mRNA expression of all studied efflux carriers, but not to a respective increase in their protein abundance. The reason for the discrepancy between mRNA expression and protein abundance of P-gp, MRP2 and BCRP might be due to up-regulation of specific miRNA. Lowering of talinolol plasma exposure after


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carbamazepine treatment is, contrarily to our previous discussion, obviously due to other reasons rather than induction of intestinal P-gp, e.g. up-regulation of renal efflux transporters as the renal clearance of talinolol is increased. Repeated-dose administration with carbamazepine leads to nearly non-fluctuating steady-state plasma levels above the in vitro concentration for CARinduction.10 All carbamazepine effects in the clinical study, however, were within the accepted range of bioequivalence. In summary, differences in the pharmacokinetics of rifampin and carbamazepine, tissue specific expression and regulation of the intestinal nuclear receptors PXR and CAR, as well as epigenetic regulation by miRNAs, are likely major factors in the regulation of drug efflux transporter function as measured using pharmacokinetic outcome criteria of the probe drugs ezetimibe and talinolol. Limitations of the study 1.

Pharmacokinetics of ezetimibe was studied after single dose administration before and

after chronic treatment with rifampin whereas pharmacokinetics of talinolol was measured during multiple-dose administrations before and after chronic co-medication of carbamazepine. Therefore, potential effects of talinolol on transporter expression cannot be excluded. 2.

With regard to the expression of the nuclear receptors PXR and CAR, we could only

provide mRNA data but not protein levels 3.

After chronic treatment with rifampin and carbamazepine, the protein abundance of P-gp,

MRP2 and BCRP were weakly, but significantly correlated to the changes in expression of the miRNAs miR-485-3p, miR-26a-5p and miR-577, respectively. Significant elevation of the


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miRNA-contents could not be confirmed, which was likely caused by the low statistical power of our assessments which involved samples sizes of n = 12 (rifampin-ezetimibe study) and n = 8 (carbamazepine-talinolol study).7,10 4.

The LC-MS/MS method to measure protein abundance of the efflux transporters in our

study was performed with 5 - 10 mg duodenal biopsy tissue. The assay had been validated for tissue samples of 50 - 100 mg wet weight. For the purpose of this study, the method has been optimized for samples of 5 - 10 mg of wet weight without re-validation of the assay. However, the expression pattern of P-gp, MRP2 and BCRP was generally the same as in our previous study in which 50-100 mg wet weight were available for the validated LC-MS/MS assay.37


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ASSOCIATED CONTENT Supporting Information Additional data on change in mRNA expression; Information on assays used for mRNA expression analysis; Information on in silico prediction and reporter gene assays; Information on altered expression of miRNAs; Data on miRNA – drug transporter correlation analysis (PDF)


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AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Author Contributions Susanne Brueck: wrote manuscript, designed research, performed research, analyzed data; Henrike Bruckmueller: designed research, performed research, analyzed data; Danilo Wegner: analyzed data; Diana Busch: performed research; Paul Martin: performed research; Stefan Oswald: designed research, performed research, analyzed data; Ingolf Cascorbi: designed research, performed research, analyzed data; Werner Siegmund: wrote manuscript, designed research, analyzed data. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENTS The authors are very grateful to Anja Moll for technical assistance and Dr. Anna Derr (Dr. R. Pfleger GmbH, Bamberg, Germany) for proof-reading the manuscript. Funding Sources This study was supported by the German Federal Ministry for Education and Research (InnoProfile grant COM-DAT [03IPT612X]).


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REFERENCES (1) Giacomini, K. M.; Huang, S.-M.; Tweedie, D. J.; Benet, L. Z.; Brouwer, K. L. R.; Chu, X.; Dahlin, A.; Evers, R.; Fischer, V.; Hillgren, K. M. et al. Membrane transporters in drug development. Nature reviews. Drug discovery 2010, 9, 215–236. (2) Terada, T.; Hira, D. Intestinal and hepatic drug transporters: pharmacokinetic, pathophysiological, and pharmacogenetic roles. Journal of gastroenterology 2015, 50, 508–519. (3) Urquhart, B. L.; Tirona, R. G.; Kim, R. B. Nuclear receptors and the regulation of drugmetabolizing enzymes and drug transporters: implications for interindividual variability in response to drugs. Journal of clinical pharmacology 2007, 47, 566–578. (4) Ingelman-Sundberg, M.; Zhong, X.-B.; Hankinson, O.; Beedanagari, S.; Yu, A.-M.; Peng, L.; Osawa, Y. Potential role of epigenetic mechanisms in the regulation of drug metabolism and transport. Drug metabolism and disposition: the biological fate of chemicals 2013, 41, 1725– 1731. (5) Dresser, G. K.; Schwarz, U. I.; Wilkinson, G. R.; Kim, R. B. Coordinate induction of both cytochrome P4503A and MDR1 by St John's wort in healthy subjects. Clinical pharmacology and therapeutics 2003, 73, 41–50. (6) Greiner, B.; Eichelbaum, M.; Fritz, P.; Kreichgauer, H. P.; Richter, O. von; Zundler, J.; Kroemer, H. K. The role of intestinal P-glycoprotein in the interaction of digoxin and rifampin. The Journal of clinical investigation 1999, 104, 147–153. (7) Oswald, S.; Haenisch, S.; Fricke, C.; Sudhop, T.; Remmler, C.; Giessmann, T.; Jedlitschky, G.; Adam, U.; Dazert, E.; Warzok, R. et al. Intestinal expression of P-glycoprotein (ABCB1), multidrug resistance associated protein 2 (ABCC2), and uridine diphosphateglucuronosyltransferase 1A1 predicts the disposition and modulates the effects of the cholesterol absorption inhibitor ezetimibe in humans. Clinical pharmacology and therapeutics 2006, 79, 206–217. (8) Schwarz, U. I.; Hanso, H.; Oertel, R.; Miehlke, S.; Kuhlisch, E.; Glaeser, H.; Hitzl, M.; Dresser, G. K.; Kim, R. B.; Kirch, W. Induction of intestinal P-glycoprotein by St John's wort reduces the oral bioavailability of talinolol. Clinical pharmacology and therapeutics 2007, 81, 669–678. (9) Westphal, K.; Weinbrenner, A.; Zschiesche, M.; Franke, G.; Knoke, M.; Oertel, R.; Fritz, P.; Richter, O. von; Warzok, R.; Hachenberg, T. et al. Induction of P-glycoprotein by rifampin increases intestinal secretion of talinolol in human beings: A new type of drug/drug interaction. Clinical pharmacology and therapeutics 2000, 68, 345–355. (10) Giessmann, T.; May, K.; Modess, C.; Wegner, D.; Hecker, U.; Zschiesche, M.; Dazert, P.; Grube, M.; Schroeder, E.; Warzok, R. et al. Carbamazepine regulates intestinal P-glycoprotein and multidrug resistance protein MRP2 and influences disposition of talinolol in humans. Clinical pharmacology and therapeutics 2004, 76, 192–200. (11) Meyer zu Schwabedissen, H. E.; Oswald, S.; Bresser, C.; Nassif, A.; Modess, C.; Desta, Z.; Ogburn, E. T.; Marinova, M.; Lütjohann, D.; Spielhagen, C. et al. Compartment-specific gene regulation of the CAR inducer efavirenz in vivo. Clinical pharmacology and therapeutics 2012, 92, 103–111. (12) Mouly, S.; Lown, K. S.; Kornhauser, D.; Joseph, J. L.; Fiske, W. D.; Benedek, I. H.; Watkins, P. B. Hepatic but not intestinal CYP3A4 displays dose-dependent induction by efavirenz in humans. Clinical pharmacology and therapeutics 2002, 72, 1–9.


27


Page 28 of 29

(13) Faucette, S. R.; Zhang, T.-C.; Moore, R.; Sueyoshi, T.; Omiecinski, C. J.; Lecluyse, E. L.; Negishi, M.; Wang, H. Relative activation of human pregnane X receptor versus constitutive androstane receptor defines distinct classes of CYP2B6 and CYP3A4 inducers. The Journal of pharmacology and experimental therapeutics 2007, 320, 72–80. (14) Moore, L. B.; Goodwin, B.; Jones, S. A.; Wisely, G. B.; Serabjit-Singh, C. J.; Willson, T. M.; Collins, J. L.; Kliewer, S. A. St. John's wort induces hepatic drug metabolism through activation of the pregnane X receptor. Proceedings of the National Academy of Sciences of the United States of America 2000, 97, 7500–7502. (15) Nishimura, M.; Naito, S.; Yokoi, T. Tissue-specific mRNA Expression Profiles of Human Nuclear Receptor Subfamilies. Drug metabolism and pharmacokinetics 2004, 19, 135–149. (16) Acocella, G. Clinical pharmacokinetics of rifampicin. Clinical pharmacokinetics 1978, 3, 108–127. (17) Scheuch, E.; Hoffmann, C.; Franke, G.; Rabending, G.; Zschiesche, M.; Siegmund, W. A bioequivalence study on retarded formulations of carbamazepine tablets. International journal of clinical pharmacology, therapy, and toxicology 1992, 30, 486–487. (18) Oswald, S.; Terhaag, B.; Siegmund, W. In vivo probes of drug transport: commonly used probe drugs to assess function of intestinal P-glycoprotein (ABCB1) in humans. Handbook of experimental pharmacology 2011, 403–447. (19) Oswald, S.; May, K.; Rosin, J.; Lütjohann, D.; Siegmund, W. Synergistic influence of Abcb1 and Abcc2 on disposition and sterol lowering effects of ezetimibe in rats. Journal of pharmaceutical sciences 2010, 99, 422–429. (20) Oswald, S.; Westrup, S.; Grube, M.; Kroemer, H. K.; Weitschies, W.; Siegmund, W. Disposition and sterol-lowering effect of ezetimibe in multidrug resistance-associated protein 2deficient rats. The Journal of pharmacology and experimental therapeutics 2006, 318, 1293– 1299. (21) Pfaffl, M. W.; Tichopad, A.; Prgomet, C.; Neuvians, T. P. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations. Biotechnology letters 2004, 26, 509–515. (22) Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif.) 2001, 25, 402–408. (23) Gröer, C.; Brück, S.; Lai, Y.; Paulick, A.; Busemann, A.; Heidecke, C. D.; Siegmund, W.; Oswald, S. LC-MS/MS-based quantification of clinically relevant intestinal uptake and efflux transporter proteins. Journal of pharmaceutical and biomedical analysis 2013, 85, 253–261. (24) Betel, D.; Wilson, M.; Gabow, A.; Marks, D. S.; Sander, C. The microRNA.org resource: Targets and expression. Nucleic acids research 2008, 36, D149-53. (25) Grimson, A.; Farh, K. K.-H.; Johnston, W. K.; Garrett-Engele, P.; Lim, L. P.; Bartel, D. P. MicroRNA targeting specificity in mammals: Determinants beyond seed pairing. Molecular cell 2007, 27, 91–105. (26) Paraskevopoulou, M. D.; Georgakilas, G.; Kostoulas, N.; Vlachos, I. S.; Vergoulis, T.; Reczko, M.; Filippidis, C.; Dalamagas, T.; Hatzigeorgiou, A. G. DIANA-microT web server v5.0: Service integration into miRNA functional analysis workflows. Nucleic acids research 2013, 41, W169-73. (27) Haenisch, S.; Zhao, Y.; Chhibber, A.; Kaiboriboon, K.; Do, L. V.; Vogelgesang, S.; Barbaro, N. M.; Alldredge, B. K.; Lowenstein, D. H.; Cascorbi, I. et al. SOX11 identified by


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target gene evaluation of miRNAs differentially expressed in focal and non-focal brain tissue of therapy-resistant epilepsy patients. Neurobiology of disease 2015, 77, 127–140. (28) Strolin Benedetti, M.; Ruty, B.; Baltes, E. Induction of endogenous pathways by antiepileptics and clinical implications. Fundamental & clinical pharmacology 2005, 19, 511– 529. (29) Jung, K.; Scholz, D.; Schreiber, G. Improved determination of D-glucaric acid in urine. Clinical chemistry 1981, 27, 422–426. (30) Jung, K.; Pergande M. D-glucarate. In Methods of Enzymatic Analysis Vol. VI. Metabolites 1: carbohydrates; Bergmeyer H.U., Graßl M., Eds.; Verlag Chemie: Weinheim (Germany), 1984; pp 228–238. (31) U.S. Food and Drug Administration. Draft Guidance for Industry. In Vitro Metabolism- and Transporter- Mediated Drug-Drug Interaction Studies Guidance for Industry Recommendations (October 2017). https://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/ Guidances/ucm064982.htm (accessed April 11, 2019). (32) Vavricka, S. R.; van Montfoort, J.; Ha, H. R.; Meier, P. J.; Fattinger, K. Interactions of rifamycin SV and rifampicin with organic anion uptake systems of human liver. Hepatology (Baltimore, Md.) 2002, 36, 164–172. (33) Pasquinelli, A. E. MicroRNAs and their targets: Recognition, regulation and an emerging reciprocal relationship. Nature reviews. Genetics 2012, 13, 271–282. (34) Baek, D.; Villén, J.; Shin, C.; Camargo, F. D.; Gygi, S. P.; Bartel, D. P. The impact of microRNAs on protein output. Nature 2008, 455, 64–71. (35) Gebert, L. F. R.; MacRae, I. J. Regulation of microRNA function in animals. Nature reviews. Molecular cell biology 2019, 20, 21–37. (36) Fromm, M. F.; Kauffmann, H. M.; Fritz, P.; Burk, O.; Kroemer, H. K.; Warzok, R. W.; Eichelbaum, M.; Siegmund, W.; Schrenk, D. The effect of rifampin treatment on intestinal expression of human MRP transporters. The American journal of pathology 2000, 157, 1575– 1580. (37) Drozdzik, M.; Gröer, C.; Penski, J.; Lapczuk, J.; Ostrowski, M.; Lai, Y.; Prasad, B.; Unadkat, J. D.; Siegmund, W.; Oswald, S. Protein abundance of clinically relevant multidrug transporters along the entire length of the human intestine. Molecular Pharmaceutics 2014, 11, 3547–3555.


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