ORIGINAL ARTICLE
Effects of adrenolytic mitotane on drug elimination pathways assessed in vitro
Dirk Theile . Walter Emil Haefeli . Johanna Weiss
Received: 29 September 2014/ Accepted: 18 December 2014 @ Springer Science+Business Media New York 2014
Abstract Mitotane (1,1-dichloro-2-(o-chlorophenyl)-2- (p-chlorophenyl)ethane, o,p’-DDD) represents one of the most active drugs for the treatment of adrenocortical car- cinoma. Its metabolites 1,1-(o,p’-dichlorodiphenyl) acetic acid (=0,p’-DDA) and 1,1-(o,p’-dichlorodiphenyl)-2,2 di- chloroethene (=0,p’-DDE) partly contribute to its pharma- cological effects. Because mitotane has a narrow therapeutic index and causes pharmacokinetic drug-drug interactions, knowledge about these compounds’ effects on drug metabolizing and transporting proteins is crucial. Using quantitative real-time polymerase chain reaction, our study confirmed the strong inducing effects of o,p’-DDD on mRNA expression of cytochrome P450 3A4 (CYP3A4, 30-fold) and demonstrated that other enzymes and trans- porters are also induced (e.g., CYP1A2, 8.4-fold; ABCG2 (encoding breast resistance cancer protein, BCRP), 4.2- fold; ABCB1 (encoding P-glycoprotein, P-gp) 3.4-fold). P-gp induction was confirmed at the protein level. o,p’- DDE revealed a similar induction profile, however, with less potency and o,p’-DDA had only minor effects. Reporter gene assays clearly confirmed o,p’-DDD to be a PXR activator and for the first time demonstrated that o,p’- DDE and o,p’-DDA also activate PXR albeit with lower potency. Using isolated, recombinant CYP enzymes, o,p’- DDD and o,p’-DDE were shown to strongly inhibit
CYP2C19 (IC50 = 0.05 and 0.09 µM). o,p’-DDA exhibited only minor inhibitory effects. In addition, o,p’-DDD, o,p’- DDE, and o,p’-DDA are demonstrated to be neither sub- strates nor inhibitors of BCRP or P-gp function. In sum- mary, o,p’-DDD and o,p’-DDE might be potential perpetrators in pharmacokinetic drug-drug interactions through induction of drug-metabolizing enzymes or drug transporters and by potent inhibition of CYP2C19. In tumors over-expressing BCRP or P-gp, o,p’-DDD and its metabolites should retain their efficacy due to a lack of substrate characteristics.
Keywords Mitotane . o,p’-DDD . o,p’-DDE . o,p’-DDA . Drug transporters · Drug metabolizing enzymes
Introduction
When adrenocortical carcinoma cannot be treated surgi- cally, chemotherapy is often recommended, especially for recurrent, metastasising, or hormonally active tumors. Besides cisplatin and etoposide, mitotane (1,1-dichloro-2- (o-chlorophenyl)-2-(p-chlorophenyl)ethane, o,p’-DDD) is the drug of choice [1]. Mitotane is also the most frequently applied drug in adjuvant therapy of adrenal cancer [2] and it remains one of the standard therapies for steroidogenesis inhibition in Cushing’s disease when initial surgery has failed [3, 4]. Mitotane combines both antitumoral and an- tihormonal properties, whereas the latter bases upon its effects on steroidogenesis including enzyme inhibition of cholesterol side chain cleavage (cytochrome P450 (CYP) isozyme 11A1, CYP11A1), 11ß-hydroxylase (CYP11B1), and 18ß-hydroxylase (CYP11B2), as well as CYP-inde- pendent enzymes such as 3ß-hydroxysteroid dehydroge- nase [5]. CYP11A1 and CYP17A1 are additionally
Electronic supplementary material The online version of this article (doi:10.1007/s12020-014-0517-2) contains supplementary material, which is available to authorized users.
D. Theile . W. E. Haefeli . J. Weiss ☒ Department of Clinical Pharmacology and Pharmacoepidemiology, University Hospital Heidelberg, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany e-mail: johanna.weiss@med.uni-heidelberg.de
inhibited by suppression of their mRNA expression [6, 7]. The exact mechanism of action is still unknown, however, mitotane’s pharmacological effects appear to depend on its intraadrenal metabolic activation [8]. Mitotane is hydrox- ylated at the ß-carbon and quickly transformed by dehy- drochlorination into an acyl chloride that covalently binds to bionucleophiles in the target cells or is transformed into renal excreted acetic acid metabolite called 1,1-(o,p’-di- chlorodiphenyl) acetic acid (=0,p’-DDA) representing the major metabolite found in plasma [5, 8, 9]. Besides, a-hydroxylation leads to the formation of the minor inactive metabolite 1,1-(o,p’-dichlorodiphenyl)-2,2 dichloroethene (=0,p’-DDE) [9]. The first metabolic step of the o,p’-DDD activation, i.e., the hydroxylation of o,p’-DDD at the B-carbon is catalyzed by a CYP presumably being responsible for the adrenal selectivity of mitotane [10]. First data indicate that CYP2W1 being highly expressed in adrenal cells might be the responsible enzyme [11].
A positive correlation of mitotane’s clinical efficacy with plasma levels has been suggested early on [12]. Indeed, response rates were significantly higher and recurrence-free and overall survival were longer when o,p’-DDD plasma concentrations were ≥ 14 µg/ml [9, 13, 14]. Combining o,p’- DDD levels of 14 µg/ml or greater with o,p’-DDA levels of 92 µg/ml was associated with an additionally increased specificity for tumor response prediction [9]. On the other hand, hematological, gastrointestinal, and central nervous system toxicity is dose-limiting underlining the necessity for therapeutic drug monitoring [15] and the importance of knowing mitotane’s pharmacokinetic fate. Because patients suffering from adrenocortical carcinoma or Cushing’s dis- ease are usually treated with combination chemotherapy [16] or with adjunctive pharmacotherapy (e.g., antiemetics, an- tihypertensives, or steroid replacement therapy) [4, 5], drug- drug interactions are very probable. Pharmacokinetic drug- drug interactions are of special significance, because mito- tane increases the expression and activity of CYP3A4, the most important drug-metabolizing enzyme [17, 18], as well as of CYP2B6, and uridinediphosphate-glucuronosyl- transferase 1A1 (UGT1A1) [19]. Induction of additional CYPs such as CYP1A1, CYP2C9, and CYP2C19 can be expected as suggested by a case report documenting increased warfarin dose requirements during co-adminis- tration of mitotane [20]. However, thorough evaluation of mitotane as a perpetrator drug in pharmacokinetic drug-drug interactions and its potency to influence pharmacokinetically important proteins beyond CYP3A4, CYP2B6, and UGT1A1 is missing. Moreover, there is no data about the potential perpetrator characteristics of the metabolites o,p’- DDA and o,p’-DDE. Thus, this study aimed at comprehen- sively characterizing the multiple ways of mitotane, o,p’- DDA, and o,p’-DDE to influence expression and activity of important drug-metabolizing enzymes, uptake and efflux
transporters, and their substrate properties for two important drug transporters mediating chemoresistance (P-glycopro- tein (P-gp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2). Moreover, the main underlying regulation pathways were evaluated by reporter gene assays for aryl hydrocarbon receptor (AhR) and pregnane x receptor (PXR), two important xenobiotic-sensing nuclear receptors.
Materials and methods
Materials
The materials used can be found in the Online Resource.
Stock solutions
Stock solutions of o,p’-DDD, o,p’-DDE, o,p’-DDA, and rifampicin (all 100 mM) were prepared in DMSO.
Growth inhibition assay for assessing maximum concentrations for the induction assay
Growth inhibition assays in LS180 cells were conducted to determine suitable maximum concentrations for the induction assay without profound antiproliferative effects. Proliferation was quantified by crystal violet staining as described previously [21]. Each experiment was performed at least in triplicate with n = 8 wells for each concentration (0.005-100 µM).
Induction assay
The human colon adenocarcinoma cell line LS180 (avail- able at ATCC, Manassas, USA) was used for induction experiments as a surrogate for the intestine being a major site of drug interactions and being an ideal model for investigating PXR and AhR-mediated induction [22-25]. Cells were cultured as described previously [26]. For the induction experiments, LS180 cells were seeded in 75 cm2 culturing flasks and incubated for 3 days. Cells were then treated with culture medium containing o,p’-DDD, o,p’- DDA, or o,p’-DDE with different concentrations in qua- druplicate for 4 consecutive days. Despite the fact that the plasma Cmax of o,p’-DDE is about 2 uM only [9], drug exposure was performed with the same concentrations (2, 4, 20, and 40 µM, respectively) for all compounds to ensure comparability. Rifampicin (20 µM) served as a positive control and culture medium as a negative control. All incubation solutions were adjusted to 0.3 % DMSO. After harvesting, cell samples were divided for RNA and protein extraction and subjected to RT-PCR or western blot analysis as described below.
For o,p’-DDD, long-term induction effects were also evaluated, because in vivo increased CYP3A4 activity was recorded even 2 years after drug cessation [17]. Therefore, LS180 cells were seeded and incubated for 3 days. Med- ium was then replaced by either medium with 0.3 % DMSO, medium containing 20 µM rifampicin, or medium containing 40 µM o,p’-DDD. After 4 days, cells were either harvested for RNA extraction or the old medium was replaced by new medium without any inducing compounds and incubated until day 3, 7, or 14 before harvesting and RNA extraction.
Quantification of mRNA expression by real-time RT-PCR
We evaluated a broad set of genes that covers phase I through phase III reactions of drug disposition and con- currently belong to the most important genes in the field of clinical pharmacology. RNA was isolated using the RNeasy Mini-Kit and cDNA was synthesized with the RevertAid™M H Minus First Strand cDNA Synthesis Kit according to the manufacturer’s instructions. mRNA expression was quantified by real-time RT-PCR with the LightCycler® 480 (Roche Applied Science, Mannheim, Germany) as described previously [26, 27]. Primer sequences were published previously [26, 28], CYP1A1 [29], CYP1A2 [30]. The most suitable housekeeping gene for normalization in LS180 cells was identified using geNorm (version 3.4, Center for Medical Genetics, Ghent, Belgium), which determines most stable reference genes from a set of tested genes in a given cDNA sample panel [31]. Among a panel of 8 housekeeping genes tested, ribosomal protein L13 (RPL13) was the most stable housekeeping gene in LS180 cells for the induction with o,p’-DDD and o,p’-DDE, and ß-glucuronidase (GU) for o,p’-DDA. Data were evaluated by calibrator-normalized relative quantification with efficiency correction using the LightCycler® 480 software version 1.5 (Roche Applied Science, Mannheim, Germany). All samples were ampli- fied in duplicate. Quantification of target genes focussed on PXR-activated genes (CYP3A4, ABCB1, ABCC2 (coding for multidrug resistance-associated protein, MRP2), UGT1A3) and AhR-regulated genes (ABCG2, CYP1A1, CYP1A2) as well as some other transporters/enzymes being important for drug-drug interactions or drug resistance (ABCC1, SLCO1B1 (coding for organic anion transporting polypeptide 1B1 (OATP1B1)), SLCO1B3, UGT1A9, and UGT2B7).
Western blot analysis of P-gp expression
P-gp protein expression was analyzed in triplicate by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and western
blotting as described previously [30]. Immunoblot analysis was carried out with a murine monoclonal antibody raised against human P-gp (clone C219) or B-actin (Clone AC-74). Bands were visualized by enhanced chemiluminescence using the SuperSignal®West Pico Chemiluminescent Substrate Kit and semi-quantified by FluorChem Q SA Alpha View Version 3.2.2, Cell Biosciences (Santa Clara, USA).
PXR reporter gene assay
A PXR reporter gene assay using the reporter plasmid pGL4.21-CYP3A4-Luc, the PXR expression vector, and the pGL4.74 [hRluc/TK] renilla vector was conducted as described previously [32] and applied to investigate whe- ther mitotane and its metabolites can activate PXR. Rif- ampicin (0.1-20 µM) served as a positive control. The reporter gene assay was performed with the Dual-Glo™M Luciferase Assay System according to the manufacturer’s instructions. Compound-induced increases of PXR activity were calculated by division of firefly luminescence by re- nilla luminescence (transfection efficiency control) and normalized to the PXR activity of non-drug-treated con- trols set to 1 (=100 %). EC50 values are expressed as mean ± SD for n = 3 experiments with triplicates for each concentration tested.
AhR reporter gene assay
A reporter gene assay for AhR was applied to investigate whether mitotane and its metabolites are AhR activators. The used stably transfected HepG2 reporter cell line AZ- AhR was described previously [33] and was kindly pro- vided by Dr. Zdenek Dvorak (Olomouc, Czech Republic). Cells were treated for 24 h with o,p’-DDD, o,p’-DDE, o,p’- DDA (0.04-40 µM), or vehicle control. The assay was performed with the Steady-Glo™M Luciferase Assay Sys- tem according to the manufacturer’s instructions. Drug- induced increases of AhR activity were normalized to activity of non-drug treated controls.
Cytotoxicity assay
o,p’-DDD, o,p’-DDE, and o,p’-DDA were tested for cytotox- icity prior to P-gp, BCRP, and OATP inhibition assays with the Cytotoxicity Detection Kit (Roche Applied Science, Mann- heim, Germany) according to the manufacturer’s instructions in order to rule out experimental bias by cytotoxic side effects. Respective cells were exposed to the compounds for 2 h.
P-gp inhibition assay (calcein uptake assay)
The calcein assay was performed with P-gp over-express- ing L-MDR1 cells and its wild-type counterpart LLC-PK1
[34]. These cell lines originate from porcine kidney epi- thelial cells and were stably transfected with human P-gp encoding vectors leading to L-MDR1 cells. Thus, these cells only differ by P-gp expression and differences between the over-expressing and the parental cells can be attributed to P-gp. To validate the experimental results, a second P-gp model was used. P388 cells originate from murine leucocytes that were stimulated for high (murine) P-gp expression through long-term exposure to doxorubi- cin leading to the P388/dx called sub-cell line [35]. The cell lines were obtained and cultivated and the assay was conducted and validated as described previously [36, 37]. The L-MDR1 cell line was kindly provided by Dr. A. H. Schinkel (The Netherlands Cancer Institute, Division of Experimental Therapy, Amsterdam, The Netherlands) and the P388/dx cells by Dr. D. Ballinari (Pharmacia & Up- john, Milano, Italy). Each concentration (0.005-100 µM) was tested in octuplet and each experiment was performed in triplicate.
BCRP-inhibition assay (pheophorbide A flow cytometry efflux assay)
To test whether o,p’-DDD, o,p’-DDA, and o,p’-DDE inhibit BCRP efflux function, MDCKII wild-type and BCRP-over- expressing MDCKII-BCRP cells were used. These canine madin-darby kidney cells were stably transfected with a human BCRP-encoding vector to generate MDCKII-BCRP cells. As for the P-gp models, the differences between MDCKII and MDCKII-BCRP cells can be attributed to BCRP expression making them an ideal model to study BCRP activity or inhibition. Cells were kindly provided by Dr. A. H. Schinkel (The Netherlands Cancer Institute, Division of Experimental Therapy, Amsterdam, The Netherlands). Flow cytometric BCRP-inhibition assays were conducted as described and validated previously [38] and performed in triplicate. o,p’-DDD, o,p’-DDA, and o,p’- DDE were tested from 0.05 up to 100 µM.
OATP inhibition assay (8-FcA flow cytometry uptake assay)
Inhibition of OATP1B1 and OATP1B3 was analyzed by flow cytometric uptake of 8-FcA into, respectively, over- expressing human embryonic kidney cell lines (HEK293) over-expressing the respective OATP [39, 40] as described previously [32]. Cells transfected with the empty control vector (HEK293-VC G418) were served as a control. Differences between the cell lines are only made up by the expression of the respective OATPs making them an ideal model to study OATP function or inhibition. Cell lines were kindly provided by Dr. D. Keppler (German Cancer Research Centre, Heidelberg, Germany). Each experiment
was performed at least in triplicate and compounds were tested from 0.05 up to 100 µM.
Inhibition of CYP3A4, CYP2C19, and CYP2D6
Inhibition studies were performed with the CYP2D6/ AMMC, the CYP2C19/CEC, and the CYP3A4/BFC High Throughput Inhibitor Screening Kit (Becton-Dickinson Biosciences, Heidelberg, Germany) according to the man- ufacturer’s instructions. The Screening Kits contain fluo- rogenic substrates and the respective recombinant CYP. The fluorogenic substrates are blocked dyes yielding a minimal fluorescence signal until cleaved by the enzyme. o,p’-DDD, o,p’-DDA, and o,p’-DDE were analyzed for their capacity to inhibit the production of the fluorescent signal. Eight concentrations (0.045-100 µM) were tested and each experiment was conducted in triplicate.
Growth inhibition assay for assessing substrate characteristics
Growth inhibition assays in MDCKII cells and their ABC- transporter over-expressing counterparts (MDCKII-MDR1 [41], MDCKII-BCRP [42]) were conducted to evaluate whether these drug transporters mediate resistance to o,p’- DDD, o,p’-DDA, or o,p’-DDE. This approach therefore indicates whether the drugs are transported (exported) by the respective ABC-transporters. The specific P-gp inhibi- tor LY335979 was used to confirm possible transport by P-gp and the specific BCRP inhibitor FTC was used to confirm possible transport by BCRP. Over-expressing cell lines were kindly provided by Dr. A. H. Schinkel and Dr. P. Borst (The Netherlands Cancer Institute, Amsterdam, The Netherlands) and cultured as published previously [43].
Proliferation was quantified by crystal violet staining as described previously [21]. Each experiment was performed at least in triplicate with n = 8 wells for each concentration (0.005-100 µM).
Statistical analysis
Data were analyzed using GraphPad Prism Version 6.02, InStat Version 3.06 (GraphPad Software, San Diego, USA) or using the R software/environment version 3.1.0 (R Foundation for statistical computing, Vienna, Austria). The differences between mRNA/protein expression following incubation with the investigated compounds and the respective vehicle controls were tested using Kruskal- Wallis test with Dunn post hoc test and Wilcoxon signed rank test with continuity correction, respectively. A p value <0.05 was considered significant.
Results
Induction of drug metabolizing and transporting proteins
LS180 cells were used to investigate the transcriptional effects of mitotane and its metabolites on genes encoding CYPs, UGTs, or drug transporters. Prior to the induction, IC20 values for antiproliferative effects of all compounds were assessed in growth inhibition assays to determine maximum possible concentrations without profound anti- proliferative. IC20 values were 45.6 ± 3.4 uM for o,p’- DDD and 90.8 ± 23 uM for o,p’-DDE. o,p’-DDA showed no antiproliferative effects up to 100 uM. To ensure comparability, the maximum concentration for the induc- tion assay was set to 40 uM for all compounds, which perfectly matches the maximum plasma concentrations of o,p’-DDD.
Most potent effects were observed for 40 uM o,p’-DDD and 40 µM o,p’-DDE (Fig. 1). For instance, CYP3A4 was significantly induced 30-fold by o,p’-DDD and 13-fold by the minor metabolite o,p’-DDE (albeit not significant), whereas the positive control rifampicin significantly increased CYP3A4 mRNA level 19-fold. Moreover, clear inductions by 40 uM of o,p’-DDD or o,p’-DDE were observed for CYP1A1 (7.0-fold, 4.4-fold) and CYP1A2 (8.4-fold, 6.3-fold). With respect to drug transporters, 40 µM o,p’-DDD and o,p’-DDE up-regulated ABCG2 (encoding BCRP) 4.2-fold and 2.2-fold, respectively. ABCB1 (encoding P-gp) expression was increased 3.5-fold and 2.2-fold by 40 uM of o,p’-DDD and o,p’-DDE. Using western blotting methodology, the latter inductions were confirmed at the protein level showing a significant induction of P-gp protein expression for o,p’-DDD (p=0.004) and o,p’-DDE (p=0.0006) being about threefold at 40 µM (Fig. 2).
In contrast, the major active metabolite o,p’-DDA had only minor effects on mRNA expression of drug-metabo- lizing enzymes and drug transporters (data not shown). At 40 µM o,p’-DDA only induced ABCB1 1.8-fold, SLCO1B1 1.6-fold, and UGT1A9 1.7-fold. The mRNAs of ABCC1, SLCO1B1, and SLCO1B3 were not significantly induced by all compounds tested. In summary, expression of genes that encode proteins restricting systemic drug exposure (CYP3A4, CYP1A1, CYP1A2, ABCG2, ABCB1) was clearly enhanced by o,p’-DDD and o,p’-DDE.
Because inductive effects of mitotane therapy can endure for a long time after drug withdrawal, long-term effects of o,p’-DDD on mRNA expression were also investigated. As exemplarily depicted for ABCB1, mRNA expression, however, rapidly returns to baseline levels when the inducer is removed (Fig. 3).
Activation of xenobiotic-sensing nuclear receptors
Several xenobiotic-sensing nuclear receptors are known, which enhance transcription of their target genes after binding an activating drug (inducing drug). Thus, to mechanistically scrutinize the observed enhancements of mRNA and protein expressions, the activation of two of the most important nuclear receptors was recorded.
AhR was not activated by any compound (data not shown). In contrast, PXR activity was increased by all compounds with a different potency and efficacy (Fig. 4). For o,p’-DDA, calculation of an EC50 value was not pos- sible, because plateau effects were not reached.
Cytotoxicity on the cell lines used for the inhibition assays
To exclude possible cytotoxic effects of the compounds influencing the inhibition assay, all compounds were tested in all cell lines used for their toxicity after 2 h. For all compounds, cytotoxicity did not exceed 10 % at 100 µM excluding an important effect on the assays applied.
Inhibition of drug metabolizing and transporting proteins
As a counterpart to gene induction (translating into increased activity), CYPs and transporters can also be inhibited by a given drug. Measurement of intracellular calcein (P-gp) or pheophorbide A (BCRP) accumulation is validated surrogates for P-gp or BCRP activity, respectively.
None of the drugs enhanced calcein fluorescence in the P-gp-inhibition assay (neither in the LLC-MDR1 cells nor in the P388/dx cells) or pheophorbide A accumulation in the BCRP-inhibition assay (Table 1) indicating that these two efflux transporters are not inhibited by those com- pounds. For influx transporters such as OATP1B1 and OATP1B3, 8-FcA is one possible prototype substrate. Consequently, impact of mitotane and its metabolites on 8-FcA uptake (=OATP1B1/3 function) was recorded. o,p’- DDA weakly inhibited OATP1B1 function but not OATP1B3, whereas o,p’-DDD and o,p’-DDE had no effect on OATP function up to 100 µM.
In contrast, CYP function was clearly influenced. Using recombinant proteins, CYP3A4 was shown to be inhibited by o,p’-DDD and o,p’-DDE, but not by o,p’-DDA. CYP2D6 was inhibited by o,p’-DDD, whereas o,p’-DDE and o,p’-DDA had only minor effects. CYP2C19 was strongly inhibited by o,p’-DDD, o,p’-DDE, and to a minor extent by o,p’-DDA (Table 1).
Induction of CYP1A1 mRNA
8
7
6
5
$
3
?
CYP1A1
7
0
Medium
Rifampicin [20 [M]
Induction of CYP3A4 mRNA
o,p’-DDD [2 [M]
35
o,p’-DDD [4 µM]
30
25
o,p’-DDD [20 [M]
20
15
o,p’-DDD [40 [M]
10
S
0
o,p’-DDE [2 [M]
Medium
o,p’-DDE [4 [M]
o,p’-DDE [20 [M]
10
Rifampicin [20 [M]
Induction of UGT1A9 mRNA
CYP3A4
Induction of CYP1A2 mRNA
o,p’-DDE [40 [M]
0
>
o,p’-DDD [2 [M]
Z
J
4
o,p’-DDD [4 [M]
0
7
CYP1A2
1
o,p’-DDD [20 [M]
o,p’-DDD [40 [M]
Medium
7
Rifampicin [20 [M]
0
UGT1A9
o,p’-DDE [2 [M]
Induction of UGT1A3 mRNA
Medium
o,p’-DDE [4 [M]
o,p’-DDD [2 μM]
3
Rifampicin [20 [M]
o,p’-DDE [20 [M]
o,p’-DDD [4 [M]
Induction of ABCB1 mRNA
o,p’-DDE [40 µM]
?
o,p’-DDD [20 [M]
Endocrine
o,p’-DDD [40 [M]
6
o,p’-DDD [2 [M]
7
S
o,p’-DDD [4 [M]
8
o,p’-DDD [20 μM]
0
UGT1A3
o,p’-DDE [2 [M]
3
Medium
o,p’-DDE [4 [M]
P
o,p’-DDD [40 µM]
7
Rifampicin [20 [M]
0
ABCB1=P-gp
o,p’-DDE [20 [M] o,p’-DDE [40 [M]
o,p’-DDE [2 [M]
Induction of UGT2B7 mRNA
Medium
o,p’-DDE [4 [M]
o,p’-DDD [2 [M]
Rifampicin [20 [M]
o,p’-DDE [20 [M]
0
o,p’-DDD [4 [M]
Induction of ABCG2 mRNA
o,p’-DDE [40 [M]
?
o,p’-DDD [20 [M]
o,p’-DDD [2 [M]
o,p’-DDD [40 μM]
S
7
8
o,p’-DDD [4 [M]
3
o,p’-DDD [20 [M]
0
o,p’-DDE [2 [M]
₹
o,p’-DDD [40 [M]
Medium
ABCG2=BCRP
Induction of ABCC2 mRNA
UGT2B7
o,p’-DDE [4 [M]
7
Rifampicin [20 [M]
o,p’-DDE [20 [M]
0
o,p’-DDE [2 [M]
o,p’-DDE [40 [M]
Medium
o,p’-DDE [4 [M]
Rifampicin [20 [M]
3
o,p’-DDD [2 [M]
o,p’-DDE [20 [M]
o,p’-DDD [4 µM]
o,p’-DDE [40 [M]
-
o,p’-DDD [20 [M]
o,p’-DDD [2 µM]
7
o,p’-DDD [40 μM]
o,p’-DDD [4 [M]
o,p’-DDD [20 [M]
0
ABCC2=MRP2
o,p’-DDE [2 [M]
o,p’-DDD [40 [M]
Medium
o,p’-DDE [4 [M]
Rifampicin [20 [M]
o,p’-DDE [20 [M]
o,p’-DDE [2 [M]
o,p’-DDE [40 µM]
o,p’-DDE [4 [M]
o,p’-DDD [2 [M]
o,p’-DDE [20 [M]
o,p’-DDD [4 [M]
o,p’-DDE [40 [M]
o,p’-DDD [20 µM]
o,p’-DDD [40 [M]
o,p’-DDE [2 [M] o,p’-DDE [4 [M] o,p’-DDE [20 [M] o,p’-DDE [40 µM]
Springer
4Fig. 1 Concentration-dependent effect of o,p’-DDD, and o,p’-DDE (2-40 µM) and 20 µM rifampicin (positive control) after 4 days on mRNA expression in LS180 cells compared to untreated medium control. Expression data were normalized to the housekeeping RPL13 (o,p’-DDD, o,p’-DDE). Data are expressed as mean ± SEM for n = 8 (four biological replicates and two PCR runs for every sample). Data were analyzed by Kruskal-Wallis test with Dunn post hoc test *p< 0.05, ** p < 0.01, *** p < 0.001
Transport of o,p’-DDD, o,p’-DDE, and o,p’-DDA by ABC-transporters
Because mitotane and its metabolites are antiproliferative drugs, cell proliferation should be inhibited concentration dependently. However, when mitotane or its metabolites can be transported out of the cell by P-gp or BCRP, the respective cell line over-expressing this efflux transporter should withstand higher drug concentrations (right shift of the concentration-response curve). Here, MDCK cells selectively over-expressing human P-gp or human BCRP were however not more resistant to o,p’-DDD than the wild-type counterparts (without human drug transporter expression) as exemplarily shown for P-gp in Fig. 5. Moreover, the specific inhibitors LY335979 and FTC did not decrease resistance in the respective over-expressing cell lines. This indicates irrelevance of P-gp and BCRP for the efflux of o,p’-DDD. o.p’-DDE and o,p’-DDA had no profound antiproliferative effects in the cell lines used up to 100 µM precluding evaluation of potential substrate characteristics with this assay.
Discussion
Mitotane is often used as a chemotherapeutic agent, when adrenocortical carcinoma cannot be treated surgically and especially in patients with metastatic or progressive dis- ease. Recently, it has been demonstrated in vivo and in vitro that mitotane can lead to profound drug-drug interactions by inducing cytochrome P450 (CYP) 3A4 via PXR [17-19, 44]. However, data on other important drug- metabolizing enzymes and drug transporters were missing as were data on potential perpetrator characteristics of the two mitotane metabolites. Such information is, however, crucial for an individualized pharmacotherapy. We there- fore evaluated their interaction potential with major phar- macokinetic targets in vitro.
As expected, mitotane activated PXR and consequently strongly induced mRNA expression of CYP3A4, the most important drug-metabolizing enzyme. In agreement with this mechanism, mitotane also induced the mRNA of other
(a)
170 kDa P-gp
42 kDa ß-Actin
Medium control
20 µM rifampicin
2 µM o,p’-DDD
4 µM o,p‘-DDD
20 µM o,p’-DDD
40 µM o,p’-DDD
(b)
170 kDa P-gp
42 kDa ß-Actin
Medium control
20 µM rifampicin
2 µM o,p’-DDE
4 µM o,p’-DDE
20 µM o,p’-DDE
40 µM o,p’-DDE
(c)
170 kDa P-gp
42 kDa
ß-Actin
Medium control
20 µM rifampicin
2 µM o,p’-DDA
4 µM o,p’-DDA
20 µM o,p’-DDA
40 µM o,p’-DDA
Induction of ABCB1 mRNA
after 4 days of induction
after drug cessation
6
5
4
3
2
1
0
Medium do
Rifampicin [20 [M] do
o,p’-DDD [40 [M] do
Medium d3
Rifampicin [20 [M] d3
o,p’-DDD [40 [M] d3
Medium d7
Rifampicin [20 [M] d7
o,p’-DDD [40 [M] d7
Medium d14
Rifampicin [20 [M] d14 o,p’-DDD [40 [M] d14
PXR activity normalised to control
4
o,p’-DDD (EC50 5.6 µM)
3
o,p’-DDE (EC50 7.7 UM)
2
o,p’-DDA
PHXH
1
1
0
0.01
0.1
1
10
100
Compound [uM]
125
100
% Proliferation
75
50
MDCK-Par
- MDCK-Par + LY335979
25
MDCK-MDR1
MDCK-MDR1 + LY335979
0
0.01
0.1
1
10
100
1000
o,p’-DDD concentration [uM]
PXR-regulated genes such as ABCB1, ABCC2, and UGT1A3 [45-48]. Interestingly, this effect was not restricted to the parent drug and also involved-albeit to a lesser extent-the minor metabolite o,p’-DDE. In contrast, the major metabolite o,p’-DDA revealed to be only a weak PXR activator with nearly no effects on the mRNA expression of PXR-regulated genes.
The strong induction of P-gp is an important finding, because this ABC-transporter influences intestinal absorp- tion, drug distribution, and hepatic/renal excretion pro- cesses and thus plays an important role for drug-drug interactions [49]. For instance, digoxin absorption mainly depends on P-gp expression/activity [50]. In consequence, such increased P-gp expression through mitotane therapy might lower drug exposure [50]. Because dioxin has a narrow therapeutic index, such an interaction can be of clinical relevance [51].
Because increased CYP3A4 activity can last for more than 2 years after end of mitotane therapy [17], we investigated the reversal of the induction process after cessation of mitotane in vitro. Theoretically, irreversible effects unrelated to local drug exposure could lead to long- term ‘induction’ effects. However, induced mRNA levels
| Protein inhibited | o,pMDCK-Par-DDD IC50 (LM) | o,pMDCK-Par-DDE IC50 (LM) | o,pMDCK-Par-DDA IC50 (LM) |
|---|---|---|---|
| BCRP | No inhibition | No inhibition | No inhibition |
| P-gp | No inhibition | No inhibition | No inhibition |
| OATP1B1 | No inhibition | No inhibition | 103.1 ± 27.3 |
| OATP1B3 | No inhibition | No inhibition | No inhibition |
| CYP3A4 | 55.3 ± 14.0 | 18.6 ± 5.1 | No inhibition |
| CYP2C19 | 0.05 ± 0.01 | 0.09 ± 0.02 | 41.9 ±20.3 |
| CYP2D6 | 23.0 ± 11.5 | >50 | >200 |
returned to untreated levels already 3 days after removal of mitotane (day 3 Fig. 3). This suggests that the increased long-lasting CYP3A4 activity after mitotane cessation is likely related to the long plasma elimination half-life of mitotane, which is highly variable and ranges between 18 and 159 days [17, 18]. Because gene induction can thus last for months after mitotane cessation, monitoring of drug efficacy and safety seems reasonable.
Interestingly, despite the lack of AhR activation, its target genes CYP1A1 and CYP1A2 were significantly induced by o,p’-DDD and o,p’-DDE. Because CYP1A2 catabolises theophylline being a drug with a narrow ther- apeutic index [52], an induction of CYP1A2 can lead to lower plasma concentrations and thus diminished efficacy of theophylline when combined with mitotane. Since these isozymes also metabolize warfarin, increased expression and activity of CYP1A2 can also contribute to enhanced elimination of (R-) warfarin [53, 54] potentially requiring dose increases as suggested by a case report [20]. To pre- vent this interaction, heparin should be considered as an alternative.
In our in vitro system, CYP2C19 was very strongly inhibited by o,p’-DDD and o,p’-DDE at nanomolar con- centrations, which might also be of clinical relevance. For instance, pharmacokinetics and subsequent pharmacody- namics of clopidogrel are highly dependent on CYP2C19 functionality. In consequence, strong inhibition of CYP2C19 by o,p’-DDD and o,p’-DDE might lead to insufficient transformation of clopidogrel (prodrug) to its active form harboring the risk of antithrombotic unre- sponsiveness [55, 56]. However, o,p’-DDD and o,p’-DDE might also induce CYP2C19 via PXR, which we did not investigate, because this gene can only be slightly induced in LS180 even with the prototypical inducer rifampicin [26]. Further interaction studies are needed to definitively clarify, which effects predominate in vivo.
We also investigated possible inhibition of drug trans- porters by mitotane and its metabolites. OATP1B1 was only weakly inhibited by o,p’-DDA, but not by o,p’-DDD and o,p’-DDE. P-gp and BCRP efflux function and OATP1B3 uptake function were not influenced by any of the compounds tested. Data for P-gp inhibition are con- tradictory so far. Accumulation and cytotoxicity of vin- blastine and actinomycin D, two typical P-gp substrates, were enhanced in vitro by mitotane-indicating P-gp inhi- bition [57]. Recently, P-gp-inhibiting properties of mito- tane were again suggested by demonstrating that the retention of calcein was increased by 50 % with mitotane in a P-gp expressing cell line. In addition, this cell line was more resistant to mitotane compared to a non-P-gp expressing counterpart [58]. In this particular study, validity of the conclusion, however, remained question- able, because the two cells lines used were not related.
Thus, observed effects might be attributed to different cellular background physiology, and expression of further transporters was not investigated. Moreover, calcein and calcein-AM can also be transported by MRPs [59] and verapamil also unspecifically inhibits both BCRP and MRP1 [60, 61]. This raises the question whether the effects were rather caused by MRP inhibition. In contrast, there is also data confirming our recent results. Ex-vivo studies with CD56+ mononuclear cells investigating rhodamine transport by flow cytometry demonstrated a lack of P-gp inhibition by mitotane [62]. We here used two separated, unrelated cell systems for calcein extrusion test, both did not indicate any inhibition of P-gp. Thus, we are convinced that mitotane is at least no strong inhibitor of P-gp.
From an oncological point of view, it is also of interest to know whether mitotane or its metabolites are transported by P-gp or BCRP potentially mediating resistance through active efflux out of the adrenocortical carcinoma cell. We could demonstrate at least for the parent compound mito- tane that cells selectively over-expressing P-gp or BCRP were not more resistant to the compounds than the wild- type cells. This implies that mitotane will retain its anti- neoplastic and adrenolytic efficacy even in tumors over- expressing these drug transporters. Interestingly, the con- centration required to inhibit cell proliferation of these MDCK cell lines (~50 µM) was much higher than those needed in adrenal carcinoma cell lines (as low as 10 µM, [58]), possibly underlining a certain tissue-specific efficacy of mitotane. Nevertheless, mitotane also exhibits a non- specific general cell-damaging effect being the prerequisite for this transporter-substrate assay.
When collecting in vitro data, the question of clinical relevance always arises. Often, in vitro studies document effects at drug concentrations never achieved in vivo. In case of mitotane, the target plasma concentration of 14 µg/ml [9] corresponds to 43.7 uM. Bearing in mind that mitotane is 6 % bound to plasma proteins [63], free plasma concentra- tions are about 41 µM. Thus, our approach of using 40 uM as the maximum exposure concentration is likely clinically relevant. The clinically relevant induction of CYP3A4 has in fact be demonstrated in vivo [17, 18, 44], but we now dem- onstrated for the first time that the minor metabolite o,p’- DDE exerts similar inducing and inhibiting properties as the parent compound. However, o,p’-DDE reaches concentra- tions of only about 2.2 uM in plasma [9] making systemic in vivo effects most likely less pronounced than those of the parent drug mitotane. In contrast, the major metabolite o,p’- DDA reaching higher plasma concentrations than the parent compound (209 µM; [9] is nearly inactive concerning its perpetrator characteristics.
This study has some limitations. First, we used an immortalized cell line and not primary cells for the induction assay. However, LS180 cells are a standard
model for enzyme and drug transporter induction experi- ments via PXR and AhR, and thus adequately represent in vivo conditions [22-25]. In contrast, other induction pathways (e.g., mediated by constitutive androstane receptor) are less pronounced in this cell line [23]. Second, not all inductions observed at the mRNA level were con- firmed at the protein level. However, in the majority of cases, mRNA changes are associated with changes in the corresponding protein level or altered function [26, 28, 63- 67]. For CYP3A4, it has already previously been demon- strated that mitotane not only induces the mRNA but also the protein level [19] and we now have confirmed it for P-gp.
In conclusion, o,p’-DDD and o,p’-DDE might be potent perpetrator compounds potentially causing pharmacoki- netic drug-drug interactions through gene induction of CYP3A4, CYP1A2, or ABCB1 and by potent inhibition of CYP2C19. When mitotane is used against adrenocortical carcinoma, thorough monitoring of pharmacotherapy’s efficacy and safety is recommended. In tumors over- expressing BCRP or P-gp, mitotane and its metabolites should retain their efficacy due to a lack of substrate characteristics.
Acknowledgments We would like to thank Dr. Johannes Pöschl for providing access to the flow cytometer. We also thank Corina Mueller, Jutta Kocher, and Stephanie Rosenzweig for excellent technical assistance and Dr. Andreas Meid for his help with statistics.
Conflict of interest The authors declare that they have no conflict of interest.
References
1. A.P. Fay, A. Elfiky, G.H. Teló, R.R. Mckay, M. Kaymakcalan, P.L. Nguyen, A. Vaidya, D.T. Ruan, J. Bellmunt, T.K. Choueiri, Adrenocortical carcinoma: the management of metastatic disease. Crit. Rev. Oncol. Hematol. 92, 123-132 (2014)
2. M. Terzolo, A. Ardito, B. Zaggia, F. Laino, A. Germano, S. De Francia, F. Daffara, A. Berruti, Management of adjuvant mitotane therapy following resection of adrenal cancer. Endocrine 42, 521-525 (2012)
3. A. Colao, M. Boscaro, D. Ferone, F.F. Casanueva, Managing Cushing’s disease: the state of the art. Endocrine 47, 9-20 (2014)
4. D. Ferone, C. Pivonello, G. Vitale, M.C. Zatelli, A. Colao, R. Pivonello, Molecular basis of pharmacological therapy in Cush- ing’s disease. Endocrine 46, 181-198 (2014)
5. I. Veytsman, L. Nieman, T. Fojo, Management of endocrine manifestations and the use of mitotane as a chemotherapeutic agent for adrenocortical carcinoma. J. Clin. Oncol. 27, 4619-4629 (2009)
6. C.W. Lin, Y.H. Chang, H.F. Pu, Mitotane exhibits dual effects on steroidogenic enzymes gene transcription under basal and cAMP- stimulating microenvironments in NCI-H295 cells. Toxicology 298, 14-23 (2012)
7. T.P. Lehmann, T. Wrzesiński, P.P. Jagodziński, The effect of mitotane on viability, steroidogenesis and gene expression in
NCI-H295R adrenocortical cells. Mol. Med. Rep. 7, 893-900 (2013)
8. D.E. Schteingart, Adjuvant mitotane therapy of adrenal cancer- use and controversy. N. Eng. J. Med. 356, 2415-2418 (2007)
9. I.G. Hermsen, M. Fassnacht, M. Terzolo, S. Houterman, J. den Hartigh, S. Leboulleux, F. Daffara, A. Berruti, R. Chadarevian, M. Schlumberger, B. Allolio, H.R. Haak, E. Baudin, Plasma concentrations of o, p’DDD, o, p’DDA, and o, p’-DDE as pre- dictors of tumor response to mitotane in adrenocortical carci- noma: results of a retrospective ENS@T multicenter study. J. Clin. Endocrinol. Metabol. 96, 1844-1851 (2011)
10. F. Martz, J.A. Straw, The in vitro metabolism of 1-(o-chloro- phenyl)-1-(p-chlorophenyl)-2,2-dichloroethane (o, p’-DDD) by dog adrenal mitochondria and metabolite covalent binding to mitochondrial macromolecules: a possible mechanism for the adrenocorticolytic effect. Drug Metab. Dispos. 5, 482-486 (1977)
11. C.L. Ronchi, S. Sbiera, M. Volante, S. Steinhauer, V. Scott-Wild, B. Altieri, M. Kroiss, M. Bala, M. Papotti, T. Deutschbein, M. Terzolo, M. Fassnacht, B. Allolio, CYP2W1 is highly expressed in adrenal glands and is positively associated with the response to mitotane in adrenocortical carcinoma. PLoS One 9, e105855 (2014)
12. H.R. Haak, J. Hermans, C.J. van de Velde, E.G. Lentjes, B.M. Goslings, G.J. Fleuren, H.M. Krans, Optimal treatment of adre- nocortical carcinoma with mitotane: results in a consecutive series of 96 patients. Br. J. Cancer 69, 947-951 (1994)
13. M. Terzolo, A.E. Baudin, A. Ardito, M. Kroiss, S. Leboulleux, F. Daffara, P. Perotti, R.A. Feelders, J.H. de Vries, B. Zaggia, S. De Francia, M. Volante, H.R. Haak, B. Allolio, A. Ghuzlan, M. Fassnacht, A. Berruti, Mitotane levels predict the outcome of patients with adrenocortical carcinoma treated adjuvantly fol- lowing radical resection. Eur. J. Endocrinol. 169, 263-270 (2013)
14. E. Baudin, G. Pellegriti, M. Bonnay, A. Penfornis, A. Laplanche, G. Vassal, M. Schlumberger, Impact of monitoring plasma 1,1-dichlor- odiphenildichloroethane (o, p’DDD) levels on the treatment of patients with adrenocortical carcinoma. Cancer 92, 1385-1392 (2001)
15. M. Terzolo, A. Pia, A. Berruti, G. Osella, A. Alì, V. Carbone, E. Testa, L. Dogliotti, A. Angeli, Low-dose monitored mitotane treatment achieves the therapeutic range with manageable side effects in patients with adrenocortical cancer. J. Clin. Endocrinol. Metabol. 85, 2234-2238 (2000)
16. M. Fassnacht, M. Terzolo, B. Allolio, E. Baudin, H. Haak, A. Berruti, S. Welin, C. Schade-Brittinger, A. Lacroix, B. Jarzab, H. Sorbye, D.J. Torpy, V. Stepan, D.E. Schteingart, W. Arlt, M. Kroiss, S. Leboulleux, P. Sperone, A. Sundin, I. Hermsen, S. Hahner, H.S. Willenberg, A. Tabarin, M. Quinkler, C. de la Fouchardière, M. Schlumberger, F. Mantero, D. Weismann, F. Beuschlein, H. Gelderblom, H. Wilmink, M. Sender, M. Edgerly, W. Kenn, T. Fojo, H.H. Müller, B. Skogseid, FIRM-ACT Study Group. Combination chemotherapy in advanced adrenocortical carcinoma. N. Engl. J. Med. 366, 2189-2197 (2012)
17. V. Chortis, A.E. Taylor, P. Schneider, J.W. Tomlinson, B.A. Hughes, D.M. O’Neil, R. Libé, B. Allolio, X. Bertagna, J. Bertherat, F. Beuschlein, M. Fassnacht, N. Karavitaki, M. Man- nelli, F. Mantero, G. Opocher, E. Porfiri, M. Quinkler, M. Sherlock, M. Terzolo, P. Nightingale, C.H. Shackleton, P.M. Stewart, S. Hahner, W. Arlt, Mitotane therapy in adrenocortical cancer induces CYP3A4 and inhibits 5a-reductase, explaining the need for personalized glucocorticoid and androgen replacement. J. Clin. Endocrinol. Metabol. 98, 161-171 (2013)
18. N.P. van Erp, H.J. Guchelaar, B.A. Ploeger, J.A. Romijn, J.D. Hartigh, H. Gelderblom, Mitotane has a strong and a durable inducing effect on CYP3A4 activity. Eur. J. Endocrinol. 164, 621-626 (2011)
19. A. Takeshita, J. Igarashi-Migitaka, N. Koibuchi, Y. Takeuchi, Mitotane induces CYP3A4 expression via activation of the ste- roid and xenobiotic receptor. J. Endocrinol. 216, 297-305 (2013)
20. P.G. Cuddy, L.S. Loftus, Influence of mitotane on the hypo- prothrombinemic effect of warfarin. South. Med. J. 79, 387-388 (1986)
21. T. Peters, H. Lindenmaier, W.E. Haefeli, J. Weiss, Interaction of the mitotic kinesin Eg5 inhibitor monastrol with P-glycoprotein. Naunyn Schmiedebergs Arch. Pharmacol. 372, 291-299 (2006)
22. S. Harmsen, A.S. Koster, J.H. Beijnen, J.H. Schellens, I. Meij- erman, Comparison of two immortalized human cell lines to study nuclear receptor-mediated CYP3A4 induction. Drug Metab. Dispos. 36, 1166-1171 (2008)
23. A. Gupta, G.M. Mugundu, P.B. Desai, K.E. Thummel, J.D. Un- adkat, Intestinal human colon adenocarcinoma cell line LS180 is an excellent model to study pregnane X receptor, but not con- stitutive androstane receptor, mediated CYP3A4 and multidrug resistance transporter 1 induction: studies with anti-human immunodeficiency virus protease inhibitors. Drug Metab. Dispos. 36, 1172-1180 (2008)
24. H. Brandin, E. Viitanen, O. Myrberg, A.K. Arvidsson, Effects of herbal medicinal products and food supplements on induction of CYP1A2, CYP3A4 and MDR1 in the human colon carcinoma cell line LS180. Phytother. Res. 21, 239-244 (2007)
25. W. Li, P.A. Harper, B.K. Tang, A.B. Okey, Regulation of cyto- chrome P450 enzymes by aryl hydrocarbon receptor in human cells: CYP1A2 expression in the LS180 colon carcinoma cell line after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin or 3-methylcholanthrene. Biochem. Pharmacol. 56, 599-612 (1998)
26. J. Weiss, M. Herzog, W.E. Haefeli, Differential modulation of the expression of important drug metabolising enzymes and trans- porters by endothelin-1 receptor antagonists ambrisentan and bosentan in vitro. Eur. J. Pharmacol. 660, 298-304 (2011)
27. N. Albermann, F.H. Schmitz-Winnenthal, K. Z’graggen, C. Volk, M.M. Hoffmann, W.E. Haefeli, J. Weiss, Expression of the drug transporters MDR1/ABCB1, MRP1/ABCC1, MRP2/ABCC2, BCRP/ABCG2, and PXR in peripheral blood mononuclear cells and their relationship with the expression in intestine and liver. Biochem. Pharmacol. 70, 949-958 (2005)
28. S.J. König, M. Herzog, D. Theile, N. Zembruski, W.E. Haefeli, J. Weiss, Impact of drug transporters for the cellular resistance towards saquinavir and darunavir. J. Antimic. Chemother. 65, 2319-2328 (2010)
29. Z. Dvorak, R. Vrzal, P. Henklova, P. Jancova, E. Anzenbacher- ova, P. Maurel, L. Svecova, P. Pavek, J. Ehrmann, R. Havlik, P. Bednar, K. Lemr, J. Ulrichova, JNK inhibitor SP600125 is a partial agonist of human aryl hydrocarbon receptor and induces CYP1A1 and CYP1A2 genes in primary human hepatocytes. Biochem. Pharmacol. 75, 580-588 (2008)
30. I. Ayed-Boussema, J.M. Pascussi, P. Maurel, H. Bacha, W. Hassen, Zearalenone activates pregnane X receptor, constitutive androstane receptor and aryl hydrocarbon receptor and corre- sponding phase I target genes mRNA in primary cultures of human hepatocytes. Environ. Toxicol. Pharmacol. 31, 79-87 (2011)
31. J. Vandesompele, K. De Preter, F. Pattyn, B. Poppe, N. Van Roy, A. De Paepe, F. Speleman, Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, Research0034 (2002)
32. J. Weiss, D. Theile, A. Spalwisz, J. Burhenne, K.D. Riedel, W.E. Haefeli, Influence of sildenafil and tadalafil on the enzyme- and transporter-inducing effects of bosentan and ambrisentan in LS180 cells. Biochem. Pharmacol. 85, 265-273 (2013)
33. A. Novotna, P. Pavek, Z. Dvorak, Novel stably transfected gene reporter human hepatoma cell line for assessment of aryl hydrocarbon receptor transcriptional activity: construction and characterization. Environ. Sci. Technol. 45, 10133-10139 (2011)
34. A.H. Schinkel, E. Wagenaar, C.A. Mol, L. van Deemter, P-gly- coprotein in the blood-brain barrier of mice influences the brain
penetration and pharmacological activity of many drugs. J. Clin. Invest. 97, 2517-2524 (1996)
35. D. Boesch, C. Gavériaux, B. Jachez, A. Pourtier-Manzanedo, P. Bollinger, F. Loor, In vivo circumvention of P-glycoprotein - mediated multidrug resistance of tumor cells with SDZ PSC 833. Cancer Res. 51, 4226-4233 (1991)
36. J. Weiss, S.M. Dormann, M. Martin-Facklam, C.J. Kerpen, N. Ketabi-Kiyanvash, W.E. Haefeli, Inhibition of P-glycoprotein by newer antidepressants. J. Pharmacol. Exp. Ther. 305, 197-204 (2003)
37. M. Fröhlich, N. Albermann, A. Sauer, I. Walter-Sack, W.E. Haefeli, J. Weiss, In vitro and ex vivo evidence for modulation of P-glycoprotein activity by progestins. Biochem. Pharmacol. 68, 2409-2416 (2004)
38. J. Weiss, J. Rose, C.H. Storch, N. Ketabi-Kiyanvash, A. Sauer, W.E. Haefeli, T. Efferth, Modulation of human BCRP (ABCG2) activity by anti-HIV drugs. J. Antimicrob. Chemother. 59, 238-245 (2007)
39. J. König, Y. Cui, A.T. Nies, D. Keppler, Localization and genomic organization of a new hepatocellular organic anion transporting polypeptide. J. Biol. Chem. 275, 23161-23168 (2000)
40. J. König, Y. Cui, A.T. Nies, D. Keppler, A novel human organic anion transporting polypeptide localized to the basolateral hepa- tocyte membrane. Am. J. Physiol. Gastrointest. Liver Physiol. 278, G156-G164 (2000)
41. A.H. Schinkel, E. Wagenaar, L. van Deemter, C.A. Mol, P. Borst, Absence of the mdr1a P-glycoprotein in mice affects tissue dis- tribution and pharmacokinetics of dexamethasone, digoxin, and cyclosporin A. J. Clin. Invest. 96, 1698-1705 (1995)
42. P. Pavek, G. Merino, E. Wagenaar, E. Bolscher, M. Novotna, J.W. Jonker, A.H. Schinkel, Human breast cancer resistance protein: interactions with steroid drugs, hormones, the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, and transport of cimetidine. J. Pharmacol. Exp. Ther. 312, 144-152 (2005)
43. N.C. Zembruski, G. Büchel, L. Jödicke, M. Herzog, W.E. Hae- feli, J. Weiss, Potential of novel antiretrovirals to modulate expression and function of drug transporters in vitro. J. Antimic- rob. Chemother. 66, 802-812 (2011)
44. M. Kroiss, M. Quinkler, W.K. Lutz, B. Allolio, M. Fassnacht, Drug interactions with mitotane by induction of CYP3A4 metabolism in the clinical management of adrenocortical carci- noma. Clin. Endocrinol. (Oxf) 75, 585-591 (2011)
45. A. Geick, M. Eichelbaum, O. Burk, Nuclear receptor response elements mediate induction of intestinal MDR1 by rifampin. J. Biol. Chem. 276, 14581-14587 (2001)
46. W. Xie, M.F. Yeuh, A. Radominska-Pandya, S.P. Saini, Y. Negishi, B.S. Bottroff, G.Y. Cabrera, R.H. Tukey, R.M. Evans, Control of steroid, heme, and carcinogen metabolism by nuclear pregnane X receptor and constitutive androstane receptor. Proc. Natl. Acad. Sci. USA 100, 4150-4155 (2003)
47. D. Gardner-Stephen, J.M. Heydel, A. Goyal, Y. Lu, W. Xie, T. Lindblom, P. Mackenzie, A. Radominska-Pandya, Human PXR variants and their differential effects on the regulation of human UDP-glucuronosyltransferase gene expression. Drug Metab. Dispos. 32, 340-347 (2004)
48. H.R. Kast, B. Goodwin, P.T. Tarr, S.A. Jones, A.M. Anisfeld, C.M. Stoltz, P. Tontonoz, S. Kliewer, T.M. Willson, P.A. Edwards, Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor. J. Biol. Chem. 277, 2908-2915 (2002)
49. J. König, F. Müller, M.F. Fromm, Transporters and drug-drug interactions: important determinants of drug disposition and effects. Pharmacol. Rev. 65, 944-966 (2013)
50. B. Greiner, M. Eichelbaum, P. Fritz, H .- P. Kreichgauer, O. von Richter, J. Zundler, H.K. Kroemer, The role of intestinal P-gly- coprotein in the interaction of digoxin and rifampin. J. Clin. Invest. 104, 147-153 (1999)
51. M. Ehle, C. Patel, R.P. Giugliano, Digoxin: clinical highlights: a review of digoxin and its use in contemporary medicine. Crit. Pathw. Cardiol. 10, 93-98 (2011)
52. A. Ohnishi, M. Kato, J. Kojima, H. Ushiama, M. Yoneko, H. Kawai, Differential pharmacokinetics of theophylline in elderly patients. Drugs Aging 20, 71-84 (2003)
53. L.S. Kaminsky, Z.Y. Zhang, Human P450 metabolism of war- farin. Pharmacol. Ther. 73, 67-74 (1997)
54. M. Ufer, Comparative pharmacokinetics of vitamin K antago- nists: warfarin, phenprocoumon and acenocoumarol. Clin. Phar- macokinet. 44, 1227-1246 (2005)
55. K. Kim, O. Park, S. Hong, J.Y. Park, The effect of CYP2C19 polymorphism on the pharmacokinetics and pharmacodynamics of clopidogrel: a possible mechanism for clopidogrel resistance. Clin. Pharmacol. Ther. 84, 236-242 (2008)
56. T. Simon, C. Verstuyft, M. Mary-Krause, L. Quteineh, E. Drouet, N. Méneveau, P.G. Steg, J. Ferrières, N. Danchin, L. Becque- mont, French registry of acute ST-elevation and non-ST-eleva- tion myocardial infarction (FAST-MI) investigators. Genetic determinants of response to clopidogrel and cardiovascular events. N. Engl. J. Med. 360, 363-375 (2009)
57. S.E. Bates, C.Y. Shieh, L.A. Mickley, H.L. Dichek, A. Gazdar, D.L. Loriaux, A.T. Fojo, Mitotane enhances cytotoxicity of chemotherapy in cell lines expressing a multidrug resistance gene (mdr-1/P-glycoprotein) which is also expressed by adrenocortical carcinomas. J. Clin. Endocrinol. Metabol. 73, 18-29 (1991)
58. T. Gagliano, E. Gentilin, K. Benfini, C. Di Pasquale, M. Tassi- nari, S. Falletta, C. Feo, F. Tagliati, E.D. Uberti, M.C. Zatelli, Mitotane enhances doxorubicin cytotoxic activity by inhibiting P-gp in human adrenocortical carcinoma cells. Endocrine 2014 (Epub ahead of print)
59. M. Essodaigui, H.J. Broxterman, A. Garnier-Suillerot, Kinetic analysis of calcein and calcein-acetoxymethylester efflux medi- ated by the multidrug resistance protein and P-glycoprotein. Biochemistry 37, 2243-2250 (1998)
60. S.P. Cole, K.E. Sparks, K. Fraser, D.W. Loe, C.E. Grant, G.M. Wilson, R.G. Deeley, Pharmacological characterization of mul- tidrug resistant MRP-transfected human tumor cells. Cancer Res. 54, 5902-5910 (1994)
61. P. Matsson, J.M. Pedersen, U. Norinder, C.A. Bergström, P. Arturs- son, Identification of novel specific and general inhibitors of the three major human ATP-binding cassette transporters P-gp, BCRP and MRP2 among registered drugs. Pharm. Res. 26, 1816-1831 (2009)
62. J. Abraham, S. Bakke, A. Rutt, B. Meadows, M. Merino, R. Alexander, D. Schrump, D. Bartlett, P. Choyke, R. Robey, E. Hung, S.M. Steinberg, S. Bates, T. Fojo, A phase II trial of combination chemotherapy and surgical resection for the treat- ment of metastatic adrenocortical carcinoma: continuous infusion doxorubicin, vincristine, and etoposide with daily mitotane as a P-glycoprotein antagonist. Cancer 94, 2333-2343 (2002)
63. A. D’Avolio, S. De Francia, V. Basile, J. Cusato, F. De Martino, E. Pirro, F. Piccione, A. Ardito, B. Zaggia, M. Volante, G. Di Perri, M. Terzolo, Influence of the CYP2B6 polymorphism on the pharmacokinetics of mitotane. Pharmacogenet. Genomics 23, 293-300 (2013)
64. S.L. Plasschaert, E.S. de Bont, M. Boezen, D.M. vander Kolk, S.M. Daenen, K.N. Faber, W.A. Kamps, E.G. de Vries, E. Vel- lenga, Expression of multidrug resistance-associated proteins predicts prognosis in childhood and adult acute lymphoblastic leukemia. Clin. Cancer Res. 11, 8661-8668 (2005)
65. E. Jigorel, M. Le Vee, C. Boursier-Neyret, Y. Parmentier, O. Fardel, Differential regulation of sinusoidal and canalicular hepatic drug transporter expression by xenobiotics activating drug-sensing receptors in primary human hepatocytes. Drug Metab. Dispos. 34, 1756-1763 (2006)
66. N. Hariparsad, S.C. Nallani, R.S. Sane, D.J. Buckley, A.R. Buckley, P.B. Desai, Induction of CYP3A4 by efavirenz in pri- mary human hepatocytes: comparison with rifampin and pheno- barbital. J. Clin. Pharmacol. 44, 1273-1281 (2004)
67. V. Dixit, N. Hariparsad, F. Li, P. Desai, K.E. Thummel, J.D. Unadkat, Cytochrome P450 enzymes and transporters induced by anti-human immunodeficiency virus protease inhibitors in human hepatocytes: implications for predicting clinical drug interactions. Drug Metab. Dispos. 35, 1853-1859 (2007)