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1
The anthelmintic triclabendazole and its metabolites inhibit the 2
membrane transporter ABCG2/BCRP 3
4
Borja Barrera1,2, Jon A. Otero1,2, Estefanía Egido1,2,4, Julio G. Prieto1,3, Anna Seelig4, 5
Ana I. Álvarez1,2 and Gracia Merino1,2* 6
7 8
Departamento de Ciencias Biomédicas -Fisiología, Facultad de Veterinaria1, Instituto de 9
Desarrollo Ganadero y Sanidad Animal (INDEGSAL)2, Instituto de Biomedicina 10
(IBIOMED)3, Universidad de León, Campus de Vegazana, 24071 León, Spain; and 11
Biozentrum, Universitat Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland4. 12
13 14 15 16 17 18 19 20
Running title: ABCG2 interaction with triclabendazole metabolites 21
22
23 * Corresponding author. Mailing address: Departamento de Ciencias Biomédicas-24
Fisiología, Facultad de Veterinaria, Universidad de León, Campus de Vegazana, 24071 25
León, Spain. Phone: 34-987291263; Fax: 34-987291267; E-mail: [email protected] 26
27
28
29 30
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.06345-11 AAC Accepts, published online ahead of print on 16 April 2012
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ABSTRACT 31
ABCG2/BCRP is an ATP binding cassette transporter that extrudes compounds from cells 32
in the intestine, liver, kidney and other organs such as the mammary gland, affecting 33
pharmacokinetics and milk secretion of antibiotics, anticancer drugs and other compounds 34
and mediating drug-drug interactions. In addition, ABCG2 expression in cancer cells may 35
directly cause resistance by active efflux of anticancer drugs. The development of ABCG2 36
modulators is critical in order to improve drug pharmacokinetic properties, reduce milk 37
secretion of xenotoxins and/or increase the effective intracellular concentration of 38
substrates. Our purpose was to determine whether the anthelmintic triclabendazole (TCBZ) 39
and its main plasma metabolites triclabendazole sulfoxide (TCBZSO) and triclabendazole 40
sulfone (TCBZSO2) inhibit ABCG2 activity. ATPase assays using human ABCG2-enriched 41
membranes demonstrated a clear ABCG2-inhibition exerted by these compounds. 42
Mitoxantrone accumulation assays using murine Abcg2- and human ABCG2-transduced 43
MDCKII cells confirmed that TCBZSO and TCBZSO2 are ABCG2 inhibitors, reaching 44
inhibitory potencies between 40 and 55% for a concentration range from 5 to 25 μM. 45
Transepithelial transport assays of ABCG2 substrates in presence of both TCBZ 46
metabolites at 15 μM showed a very efficient inhibition of the Abcg2/ABCG2-mediated 47
transport of the antibacterial agents nitrofurantoin and danofloxacin. TCBZSO 48
administration also inhibited nitrofurantoin Abcg2-mediated secretion into milk by more 49
than 2-fold and increased plasma levels of the sulphonamide sulfasalazine by more than 50
1.5-fold in mice. These results support the potential role of TCBZSO and TCBZSO2 as 51
ABCG2 inhibitors to participate in drug interactions and modulate ABCG2-mediated 52
pharmacokinetic processes. 53
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INTRODUCTION 54
ABCG2/BCRP is a described member of the ABC transporter family, a group of 55
proteins that transport certain chemicals out of cells (29). These ABC drug efflux 56
transporters extrude a wide range of xenotoxins from cells in intestine, liver and other 57
organs and so affect the bioavailability of many compounds and participate in drug-drug 58
interactions. In addition, ABCG2 also mediates secretion into the milk of its substrates 59
(both therapeutic and toxic) such as antibiotics, antitumoral agents, carcinogens or vitamins 60
(31, 32). Recently, the International Transporter Consortium has included ABCG2 in the 61
group of transporters that are clinically relevant (10). Moreover, the overexpression of ABC 62
transporters has been associated with multidrug resistance (MDR), a major impediment to 63
successful cancer chemotherapy. Increasing interest has been given to the development of 64
inhibitors to overcome MDR and to increase oral bioavailability and tissue penetration or to 65
decrease milk secretion of its substrates (21, 28). 66
Some benzimidazole drugs such as the anthelmintics albendazole sulfoxide and 67
oxfendazole and the antacid pantoprazole have been reported to interact with ABCG2 (3, 68
19). In the case of pantoprazole, its use as an ABCG2 inhibitor to improve plasma 69
pharmacokinetics and brain penetration of ABCG2 substrates has been reported (2, 3). 70
Triclabendazole (TCBZ) is a flukicidal halogenated benzimidazole thiol derivative used for 71
treating liver fluke infections in livestock, and is the drug of choice against human 72
fascioliasis (6). TCBZ parent drug is not detected in plasma after its oral administration, 73
because it is rapidly metabolized into its metabolites triclabendazole sulfoxide (TCBZSO) 74
and triclabendazole sulfone (TCBZSO2), respectively (9) (Fig. 1). TCBZ and TCBZSO 75
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have been shown to interact with other ABC transporters in vitro (4); however, the 76
interaction of TCBZ and its metabolites with ABCG2 has not yet been investigated. 77
In this paper we studied whether TCBZ and its metabolites (TCBZSO and 78
TCBZSO2) in vitro inhibit ABCG2 transporter in ATPase assays using ABCG2-enriched 79
membranes and in mitoxantrone accumulation and transepithelial transport assays using 80
ABCG2-transduced cell lines. In vivo inhibition of the transporter was assessed by studying 81
the Abcg2-mediated effect of TCBZSO coadministration on the secretion into milk of the 82
antibacterial agent nitrofurantoin and on plasma levels of the sulphonamide sulfasalazine 83
using Abcg2 -/- and wild-type mice. Experiments with murine Abcg2-transduced cells and 84
mice are included in this study as mice are extensively used as experimental models to 85
study the transporter function in vivo. 86
87
88
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MATERIALS AND METHODS 90
Reagents and drugs. Mitoxantrone, sulfasalazine and nitrofurantoin were 91
purchased from Sigma-Aldrich (St. Louis, MO, USA), danofloxacin from Fluka Chemie 92
(Buchs, Switzerland), TCBZ from Sequoia Research Products (Pangbourne, United 93
Kingdom), TCBZSO and TCBZSO2 from LGC Standars (Barcelona, Spain), isoflurane 94
(Isovet®) from Schering-Plough (Madrid, Spain), oxytocin (Oxiton®) from Ovejero (León, 95
Spain) and Ko143 from Tocris (Bristol, UK). All the other chemicals were analytical grade 96
and available from commercial sources. 97
Animals. Animals were housed and handled according to procedures approved by 98
the Research Committee of Animal Use of the University of León (Spain) and carried out 99
according to the “Principles of Laboratory Animal Care” and the European guidelines 100
described in the EC Directive 86/609. Animals used were male or lactating female Abcg2-/- 101
and wild-type mice, all of >99% FVB genetic background between 9 and 13 weeks of age. 102
Animals were kindly provided by Dr. A.H. Schinkel (The Netherlands Cancer Institute, 103
Amsterdam, The Netherlands) and were kept in a temperature-controlled environment with 104
a 12-h light/12-h dark cycle and received a standard diet (Panlab; Barcelona, Spain) and 105
water ad libitum. 106
Cell cultures. MDCK-II cells and their human ABCG2- and murine Abcg2-107
transduced subclones were kindly provided by Dr. A.H. Schinkel (The Netherlands Cancer 108
Institute, Amsterdam, The Netherlands). Culture conditions were as previously described 109
(12, 23). 110
Transport studies. Transepithelial transport assays using Transwell plates were 111
carried out as previously described (19) with minor modifications. Transepithelial 112
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resistance was measured in each well using a Millicell ERS ohmmeter (Millipore, Bedford, 113
MA); wells registering a resistance of 150 ohms or greater, after correcting for the 114
resistance obtained in blank control wells, were used in the transport experiments. The 115
measurement was repeated at the end of the experiment to check the tightness of the 116
monolayer. Experiments were performed using Optimem medium, a reduced serum 117
medium that is a modification of Eagle's Minimum Essential Media, buffered with HEPES 118
and sodium bicarbonate. Active transport across MDCK-II monolayers was expressed by 119
the relative transport ratio, defined as the apically directed transport percentage divided by 120
the basolaterally directed translocation percentage, after 4 h (30). 121
ATPase assay. ABCG2 associated ATP hydrolysis was determined by quantifying 122
the release of inorganic phosphate (Pi) with a colorimetric assay with small modifications 123
(1). Experiments were carried out in 96-well microtiter plates (F96 Micro Well plate, 124
nontreated; Nalge Nunc, Rochester, NY, USA). Plasma membrane vesicle preparations 125
from isolated mammalian cells containing human ABCG2 (BCRP-M-ATPase) were 126
obtained from SOLVO Biotechnology (Budapest, Hungary) (8). Vesicles were diluted in 127
reaction volumes of 60 µL containing a protein concentration of 0.075 mg/mL, in ice-cold 128
phosphate release assay buffer (25 mM Tris-HCl including 50mM KCl, 3 mM ATP, 2.5 129
mM MgSO4, 3mM DTT, 0.5 mM EGTA, 2mM ouabain and 3 mM sodium azide) adjusted 130
to pH 7 at 37 ºC (1). Incubation of compounds and membranes was started by transferring 131
the plate from ice to a water bath kept at 37 ºC for 1 h and was terminated by rapidly 132
cooling the plate on ice. The phosphate release assays were performed in parallel in the 133
presence of vanadate to inhibit ABCG2 ATPase activity, and the vanadate values were 134
subtracted from the measurements. Al least two independent measurements in plasma 135
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membrane vesicles were performed. Each independent experiment consisted of one 96-136
well-plate with two measurements. 137
Accumulation assays. In vitro accumulation assays were carried out as previously 138
described (23). Mitoxantrone (MXR, 10 μM) was used as fluorescent substrate. Relative 139
cellular accumulation of MXR of at least 5,000 cells was determined by flow cytometry 140
using a CYAN cytometer (Beckman Coulter®, Fullerton, CA, USA). The fluorescence of 141
the accumulated substrate in tested populations was quantified from histogram plots using 142
the median of fluorescence (MF). Flow cytometry data were processed and analyzed using 143
SUMMIT version 4.3 software (Innovation Drive, Fort Collins, CO, USA). Inhibitory 144
potencies of compounds were calculated as previously described (23) in MCDKII-ABCG2 145
or MCDCKII-Abcg2 cells according to the following equation: Inhibitory potency = (MF 146
with tested compound – MF without inhibitor) / (MF with Ko143 – MF without inhibitor) 147
X 100 %. 148
Plasma levels of sulfasalazine. Sulfasalazine (20 mg/Kg) was intragastrically 149
administered to wild-type and Abcg2−/− male mice by oral gavage feeding in 4-h-fasted 150
mice, as a solution of 6 % ethanol, 42 % PEG400 and 52 % water. Oral administration 151
consisted of 300 μl of solution per 30 g body weight. TCBZSO (50 mg/Kg) or vehicle (6 % 152
ethanol, 42 % PEG400 and 52 % water) were orally administered 15 min before oral 153
administration of sulfasalazine (20 mg/kg). Blood was collected after 30 min of 154
administration of sulfasalazine by cardiac puncture after anesthesia with isoflurane. At the 155
end of the experiment the mice were killed by cervical dislocation. Heparinized blood 156
samples were centrifuged immediately at 1500 x g for 10 min and collected plasma was 157
stored at –20ºC until HPLC analysis. Between 4 and 7 animals were used for each 158
experimental group. 159
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Milk secretion experiments. Pups of approximately 10 days old were separated 160
from their mother approximately 4 h before the start of the experiment. Nitrofurantoin (5 161
mg/Kg) was administered in the tail vein to wild-type and Abcg2−/− lactating female mice 162
as a solution of 6 % ethanol, 42 % PEG400 and 52 % water. The intravenous administration 163
consisted of 150 μl of solution per 30 g body weight. TCBZSO (50 mg/Kg or 100 mg/Kg) 164
or vehicle (6 % ethanol, 42 % PEG400 and 52 % water) were administered intraperitoneally 165
(500 µl of solution per 30 g body weight) 5 min before i.v. administration of nitrofurantoin. 166
Oxytocin (200 μl of 1 I.U./ml solution) was administered subcutaneously to lactating dams 167
in order to stimulate milk secretion 20 min after the administration of nitrofurantoin. Blood 168
and milk were collected 30 min after substrate administration under anesthesia with 169
isoflurane. Blood was collected by orbital bleeding and heparinized blood samples were 170
centrifuged immediately at 1500 x g for 10 min. Milk was collected from the mammary 171
glands by gentle pinching. At the end of the experiment mice were subsequently killed by 172
cervical dislocation. Collected plasma and milk samples were stored at –20ºC until HPLC 173
analysis. Between 4 and 7 animals were used for each experimental group. 174
HPLC Analysis. The chromatographic system consisted of a Waters 2695 175
separation module and a Waters 2998 UV photodiode array detector. 176
The conditions for HPLC analysis of danofloxacin were modified according to 177
previously published methods (17, 18). Samples from the transport assays were not 178
processed and 50 μl of the culture media was injected directly into the HPLC system. 179
Separation of the samples was performed on a reversed-phase column (Phenomenex 180
Synergi 4 μm Hydro-RP 80A). The mobile phase consisted of 25 mM orthophosphoric acid 181
(pH 3.0)/acetonitrile (75:25), the flow rate of the mobile phase was set to 1.5 ml/min and 182
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UV absorbance was measured at 278 nm. The temperature of the samples was 4ºC. 183
Standard samples were prepared in the appropriate drug-free matrix, yielding a 184
concentration range from 0.02 μg/ml to 5 μg/ml. 185
The conditions for HPLC analysis of nitrofurantoin were modified according to a 186
previously published method (20). Samples from the transport assays were not processed 187
and 50 μl of the culture media was injected directly into the HPLC system. For the mouse 188
samples of nitrofurantoin, to each 50 μl aliquot of plasma or milk, 5 µl of furazolidone 189
(12.5 μg/ml) was incorporated as internal standard and 50 μl of cold methanol was added. 190
Samples were shaken and kept at -20ºC for 15 minutes and the organic and water phases 191
were separated by centrifugation at 16000 x g for 5 min and 50 µl of the supernatant was 192
injected into the HPLC system. Separation of the samples was performed on a reversed-193
phase column (Phenomenex Synergi 4 μm Hydro-RP 80A). The mobile phase consisted of 194
25 mM potassium phosphate buffer (pH 3)/ acetonitrile (75:25), the flow rate of the mobile 195
phase was set to 1.2 ml/min and UV absorbance was measured at 366 nm. The temperature 196
of the samples was 4ºC and the temperature of the column was 30ºC. Standard samples in 197
the appropriate drug-free matrix were prepared, yielding a concentration range from 0.039 198
μg/ml to 5 μg/ml for transport samples; 0.125 μg/ml to 4 μg/ml for plasma mouse samples 199
and 0.0312 μg/ml to 4 μg/ml for milk mouse samples. 200
The conditions for HPLC analysis of sulfasalazine were modified according to 201
previously published methods (13). For the mouse samples of sulfasalazine, to each 100 μl 202
aliquot of plasma, 10 µl of probenecid (37.5 μg/ml in methanol) was incorporated as 203
internal standard and 300 μl of methanol was added. Samples were shaken and kept at -204
20ºC for 15 min and the organic and water phases were separated by centrifugation at 1500 205
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x g for 2 min. The supernatant was collected in a new eppendorf tube and evaporated to 206
dryness under a nitrogen stream. The samples were resuspended in 100 μl of methanol and 207
injected into the HPLC system. Separation of the samples was performed on a reversed-208
phase column (Chemcobond 5-ODS-H 5 μm particle size 4.6 x 250mm). The mobile phase 209
consisted of 12 mM phosphate buffer containing 0.06% tetrabutylammonium hydrogen 210
sulphate (pH 7.4)/ methanol (50:50), the flow rate of the mobile phase was set to 1 ml/min 211
and UV absorbance was measured at 260 nm. The temperature of the samples was 4ºC and 212
the temperature of the column was 40ºC. Standard samples in the appropriate drug-free 213
matrix were prepared yielding a concentration range from 0.04 µg/ml to 40 µg/ml. 214
Integration was performed using Empower® software (Waters). 215
Statistical Analysis. The two-sided unpaired Student’s t test was used throughout to 216
assess the statistical significance of differences between the two sets of data. Results are 217
presented as means ± S.D. Differences were considered to be statistically significant when 218
p < 0.05. 219
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RESULTS 227
Effect of TCBZ and its metabolites TCBZSO and TCBZSO2 on ABCG2 228
ATPase activity. To characterize the interaction of TCBZ and its metabolites (TCBZSO 229
and TCBZSO2) with ABCG2, drug-stimulated ATPase activity in inside-out plasma 230
membranes vesicles from isolated mammalian cells containing human ABCG2 was 231
measured by monitoring the phosphate release rate at pH 7 and T = 37 °C. Figure 2 shows 232
the rate of ABCG2 ATPase activity as a function of compound concentration (Log scale). 233
Drug-stimulated ABCG2 ATPase activity is expressed as a percentage of the basal activity 234
(taken as 100%). ABCG2 titration curves of the three compounds showed typical bell-235
shaped curves previously observed for P-glycoprotein (1), with an activation at lower drug 236
concentrations and a clear inhibition at higher drug concentrations, indicating an important 237
interaction with the transporter. Maximum activity increases in the order TCBZ < TCBZSO 238
< TCBZSO2 and the concentration of half-maximum inhibition increases in the same order. 239
The higher the half-maximum inhibition, the lower the inhibitory power of the compound. 240
Note that, in all cases, the inhibition in ABCG2 ATPase activity is achieved at rather low 241
concentrations. As has been seen for ATPase activity, all three compounds are probably 242
effectively transported by ABCG2, the best activation curve being for TCBZSO2. 243
Mitoxantrone accumulation assays. To further study the Abcg2/ABCG2 244
inhibitory effect of the major plasma metabolites TCBZSO and TCBZSO2, the ability of 245
these compounds to reverse the reduced mitoxantrone accumulation in murine Abcg2- and 246
human ABCG2-expressing cell lines was tested in flow cytometry experiments. 247
Abcg2/ABCG2 inhibition with the model inhibitor Ko143 increased the accumulation of 248
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mitoxantrone in Abcg2- and ABCG2-transduced cells and thus increased the median of 249
fluorescence (MF) to levels similar to those in the parental cells. 250
Our results showed that the addition of TCBZSO or TCBZSO2 at different 251
concentrations (0.01 to 25 µM, higher concentrations were cytotoxic) (Fig. 3) increased, in 252
a concentration-dependent manner, the accumulation of mitoxantrone (10 µM) in 253
Abcg2/ABCG2-transduced cells. The strongest inhibitory potency for TCBZSO was 254
reached at 25 µM for murine Abcg2-transduced cells (40%) and at 10 µM in the human 255
ABCG2-transduced cells (55%). In the case of TCBZSO2, the strongest inhibitory potency 256
was reached at 25 µM for Abcg2- and 5 µM for ABCG2-transduced cells with values of 55 257
% in both cases. All these data indicate that TCBZSO and TCBZSO2 are inhibitors of 258
Abcg2/ABCG2. 259
In vitro transport of nitrofurantoin and danofloxacin in presence of TCBZSO 260
and TCBZSO2. To complete the characterization of the inhibitory behaviour of the TCBZ 261
metabolites on Abcg2/ABCG2 using other assays and Abcg2/ABCG2 substrates, we tested 262
the effect of these compounds (TCBZSO 15 µM and TCBZSO2 15 µM) on the 263
Abcg2/ABCG2-mediated in vitro transport of two known Abcg2/ABCG2 substrates, the 264
antibacterial agents nitrofurantoin (10 µM) and danofloxacin (10 µM). As has already been 265
reported (20, 26), we observed for nitrofurantoin (Fig. 4) and danofloxacin (Fig. 5) that in 266
the MDCK-II parental cell line, apically and basolaterally directed translocations were 267
similar (Figs. 4A and 5A), but in the Abcg2/ABCG2-transduced MDCK-II cell lines, 268
apically directed translocation was highly increased and basolaterally directed translocation 269
dramatically decreased (Figs. 4D, 4G, 5D and 5G), since these drugs are excellent 270
Abcg2/ABCG2 substrates. When we added TCBZSO (15 µM) and TCBZSO2 (15 µM) as 271
inhibitors, apically directed translocation decreased and subsequently basolaterally directed 272
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translocation increased as compared to the control situation without inhibitor in 273
Abcg2/ABCG2-transduced cells (Figs. 4E, 4F, 4H, 4I, 5E, 5F, 5H and 5I). Murine Abcg2-274
mediated transport was moderately inhibited, and in the case of the human ABCG2, 275
transport was almost completely inhibited in both cases, with relative transport ratios 276
similar to the parental cells. 277
These results therefore showed that TCBZSO (15 µM) and TCBSZO2 (15 µM) very 278
efficiently inhibit the Abcg2/ABCG2-mediated transport of antibacterial substrates such as 279
nitrofurantoin and danofloxacin. 280
Effect of coadministration of TCBZSO on plasma levels of sulfasalazine. To 281
assess whether the in vitro Abcg2/ABCG2 inhibitory role of the major plasma metabolites 282
TCBZSO and TCBZSO2 were also relevant in vivo, we studied the effect of the 283
coadministration of TCBZSO on plasma levels of the sulphonamide sulfasalazine, a model 284
ABCG2 substrate (35). Danofloxacin was not used for these pharmacokinetic experiments 285
because Abcg2 does not affect plasma levels of danofloxacin in mice (26) and therefore this 286
antibacterial cannot be considered as an in vivo model to study Abcg2-mediated effects on 287
plasma levels. 288
TCBZSO (50 mg/kg) or vehicle was orally administered to wild-type and Abcg2 -/- 289
male mice 15 min prior to oral administration of sulfasalazine (20 mg/kg) and plasma 290
samples were collected 30 min after sulfasalazine administration. Plasma concentration of 291
sulfasalazine was more than 1.5-fold higher in wild-type mice coadministered with 292
TCBZSO compared to control wild-type mice (0.63 ± 0.11 µg/ml versus 0.40 ± 0.13 µg/ml, 293
p<0.05) (Fig. 6A). No significant differences in plasma concentration of sulfasalazine were 294
observed with TCBZSO treatment in the Abcg2-/- mice (4.91 ± 1.67 vs. 5.83 ± 1.70 µg/ml, 295
control and TCBZSO-treated animals, respectively), indicating that the TCBZSO effect is 296
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Abcg2 specific. Plasma concentrations of sulfasalazine in Abcg2-/- mice were 297
approximately 10-fold higher than the wild-type animals (4.91 ± 1.67 vs. 0.40 ± 0.13 µg/ml 298
µg/ml) according to the results obtained by Zaher et al. (35), confirming that this compound 299
is a very good in vivo substrate of Abcg2. We thus demonstrated that the coadministration 300
of TCBZSO affects oral plasma levels of sulfasalazine through inhibition of Abcg2 at the 301
dosage used. 302
Effect of TCBZSO coadministration on plasma and milk levels of 303
nitrofurantoin. To further demonstrate an in vivo Abcg2/ABCG2 inhibitory role of TCBZ 304
metabolites in other relevant drug-drug interactions and biological processes, the effect of 305
the coadministration of TCBZSO on the secretion of the antibacterial nitrofurantoin into 306
milk, an in vivo Abcg2/ABCG2 model substrate, was studied. Nitrofurantoin transfer into 307
milk has been previously used as an experimental setting to test the in vivo effect of 308
ABCG2 inhibitors (21, 33). 309
TCBZSO (50 and 100 mg/kg) was administered i.p. to lactating Abcg2-/- and wild-310
type females 5 min prior to an intravenous administration of nitrofurantoin (5 mg/Kg). 311
Thirty minutes after nitrofurantoin administration, milk and blood were collected. No 312
significant differences were observed in plasma concentrations in wild-type mice after 313
coadministration of TCBZSO at both doses (Fig. 6A). Plasma concentrations of 314
nitrofurantoin in Abcg2 -/- mice were approximately 3-fold higher than in wild-type animals 315
(1.70 ± 0.71 vs 0.59 ± 0.25 µg/ml, p<0.05), confirming that this compound is a very good 316
in vivo substrate of Abcg2. The milk concentration of nitrofurantoin (Fig. 6B) was more 317
than 2-fold lower in wild-type mice treated with TCBZSO (50 mg/Kg) (0.74 ± 0.44 µg/ml) 318
and more than 4-fold lower in wild-type mice treated with TCBZSO (100 mg/Kg) (0.38 ± 319
0.18 µg/ml) compared to control wild-type mice (1.61 ± 0.53 µg/ml, p<0.05). No 320
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differences were observed after TCBZSO treatment in Abcg2-/- mice, indicating that the 321
TCBZSO effect is Abcg2-specific. Consequently, TCBZSO inhibits Abcg2-mediated 322
secretion of nitrofurantoin into milk since the milk-to-plasma ratio of this compound (Fig. 323
6C) was 3-fold lower in wild-type mice treated with TCBZSO (50 mg/Kg) (0.93 ± 0.25) 324
and almost 4-fold lower for wild-type mice treated with TCBZSO (100 mg/Kg) (0.75 ± 325
0.49) compared to control wild-type mice (2.79 ± 1.42, p<0.05). 326
Our results show that coadministration of TCBZSO inhibits Abcg2/ABCG2-327
mediated secretion of nitrofurantoin into milk at the dosage used. 328
329
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DISCUSSION 330
The concomitant administration of multiple drugs is often used in pharmacotherapy 331
and may affect their kinetics and pharmacological activity. There is increasing evidence to 332
suggest that interference between drugs and ATP binding cassette (ABC) proteins is a key 333
mechanism underpinning clinically important drug interactions (17). It is therefore of 334
interest to study the potential effect of the major active plasma metabolites of the widely 335
used fasciolicide TCBZ (TCBZSO and TCBSZO2) in drug interactions with 336
Abcg2/ABCG2 substrates affecting pharmacokinetics and milk secretion. In this study, we 337
have shown that TCBZSO and TCBZSO2 efficiently inhibit in vitro and in vivo ABCG2 338
transporter activity using different in vitro and in vivo assays with different substrates. 339
In ATPase assays (Fig. 2), ABCG2 inhibition was observed for all three compounds 340
studied, TCBZ, TCBZSO and TCBZSO2, at concentrations higher than 1 μM, with the 341
strongest inhibition observed in the case of TCBZ, the most hydrophobic compound. 342
Subsequent inhibition studies were performed with the major plasma metabolites TCBZSO 343
and TCBZSO2, since due to its high metabolism the TCBZ parent drug is not detected in 344
plasma. In mitoxantrone accumulation assays, a concentration range from 5 to 25 μM of 345
both compounds show inhibitory potencies between 40 and 55% for murine/human 346
Abcg2/ABCG2. Some drugs considered good ABCG2 inhibitors showed IC50 values in the 347
same range for the same cell line (34): lopinavir (7.66 µM), nelfinavir (13.50 µM), 348
saquinavir (27.40 µM) and delavirdine (18.60 µM). For other benzimidazole drugs 349
considered to interact with ABCG2 such as pantoprazole and omeprazole, IC50 values were 350
13 μM y 36 μM, respectively (3). Our concentration values with an inhibitory potency 351
close to 50% are in the same range as the plasma concentrations of the active metabolite 352
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TCBZSO reported in humans (25 µM ≈ 9.4 μg/ml) (5) and in veterinary species (30 µM ≈ 353
11.3 μg/ml) (7) after treatment at the therapeutic dose. 354
The Abcg2/ABCG2 inhibitory potential of the TCBZ metabolites was also 355
confirmed for other known Abcg2/ABCG2 substrates such as the antibacterial agents 356
nitrofurantoin and danofloxacin in transepithelial transport experiments at a concentration 357
of 15 μM, showing a moderate inhibition for murine Abcg2 and a complete inhibition for 358
human ABCG2 (Figs 4 and 5). The 15 μM concentration was chosen based on the stronger 359
inhibition observed in the mitoxantrone accumulation assays for human ABCG2. Inhibition 360
of the in vitro transepithelial transport of both compounds at concentrations of TCBZ 361
metabolites below 15 μM could not be excluded. Inhibition in transepithelial transport 362
experiments can be expected as long as the concentration of drug is higher than the 363
concentration at maximum activity in ATPase assay (for all three compounds, in the 364
ABCG2 ATPase activity profiles the maximum activity was reported around 1 µM) (27). 365
The similar inhibitory power of TBCZSO and TBCZSO2 observed in transport assays is 366
due to the similar concentration of half-maximum inhibition in ATPase assays (Fig. 2). 367
Although interaction of these compounds with other ABC transporters, such as P-368
glycoprotein has been previously reported (4), lack of effect of these compounds on 369
vectorial transport in parental cells (Figs 4A, 4B, 4C, 5A, 5B and 5C) indicates that this 370
interaction is probably Abcg2 specific in our experimental setting. All these data indicate 371
that both TCBZ metabolites are good in vitro inhibitors of Abcg2/ABCG2. 372
Furthermore, we demonstrated the relevance of the ABCG2 inhibition properties of 373
these compounds in mice using two different ABCG2 substrates in two different 374
pharmacokinetic processes. Plasma levels of sulfasalazine and milk levels of nitrofurantoin 375
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(Fig. 6) were significantly affected by the coadministration of TCBZSO only in wild-type 376
animals with no effect on Abcg2-/- mice indicating the Abcg2-specific effect. This effect is 377
most likely not only due to the inhibition exerted by TCBZSO itself but also by its 378
metabolite TCBZSO2. TCBZSO coadministration did not affect nitrofurantoin plasma 379
levels. Some authors have reported local effects mediated by Abcg2 (fetal distribution and 380
milk secretion) but no difference in plasma systemic profile between wild-type and 381
knockout mice for some substrates (24, 30, 36). Unlike the nitrofurantoin experiment, there 382
seems to have been an Abcg2-mediated effect of TCBZSO coadministration on plasma 383
levels of sulfasalazine since the difference in plasma concentrations of this compound after 384
oral administration between untreated Abcg2 -/- and wild-type mice was approximately 10-385
fold, whereas in the case of nitrofurantoin (i.v. administration) it was only 3-fold, thus 386
indicating a higher effect of Abcg2 on the systemic disposition of sulfasalazine after oral 387
administration. In addition, the different routes of TCBZSO administration (oral for 388
sulfasalazine experiment and intraperitoneal for nitrofurantoin experiment) and/or the 389
gender or physiological status of the animals may influence the TCBZSO inhibitory effect. 390
This in vivo interaction between drugs resulting in higher plasma levels or lower 391
secretion of the substrate into milk could be applied not only to the substrates tested but 392
also to other ABCG2 substrates. This finding is highly relevant considering that concurrent 393
administration of different drugs is a usual clinical practice. In addition, TCBZ is marketed 394
in combination with other anthelmintics to improve efficacy, to broaden spectrum of 395
activity and to limit resistance emergence (4). Some of these drug combinations include 396
drugs known to interact with ABC transporters such as ivermectin (15) or oxfendazole (19). 397
It will therefore be of interest to further study the possible in vivo effect of these TCBZ 398
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metabolites in the potential drug interactions with other known Abcg2/ABCG2 substrates 399
in therapeutic-target species (humans, livestock). 400
ABCG2 inhibitors can be used in combination therapy with substrates of the 401
transporter in order to modulate their pharmacokinetics, brain penetration and milk 402
secretion and thus their efficacy. Several studies have managed to increase bioavailability 403
and milk secretion of antibacterial agents such as nitrofurantoin or antitumorals such as 404
topotecan or to improve brain penetration of the antitumoral imatinib with the use of 405
ABCG2 and P-glycoprotein inhibitors such as elacridar, the benzimidazole pantoprazole or 406
isoflavones (2, 11, 14, 21, 25). However, it has to be noted that the use of TCBZ for this 407
purpose could be controversial in animals whose products are destined for human 408
consumption or in endemic parasite areas due to the potential development of resistance. 409
In addition, inhibitors of ABCG2 may be useful in other application fields, e.g. for 410
reversal resistance in chemotherapy (22). Further studies are needed to show the application 411
of these compounds in this field. 412
In summary, in this study we have shown a clear in vitro and in vivo interaction of the 413
major plasma metabolites of TCBZ with ABCG2. These compounds are excellent ABCG2 414
inhibitors, and their relevance could be important for ABCG2-mediated drug-drug 415
interactions affecting drug bioavailability. 416
417
418
419
420
421
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ACKNOWLEDGMENTS 422
This work was supported by the Research project grant AGL2009-11730 and Ramon 423
y Cajal grant (to G.M.) from the Ministry of Science and Technology and the European 424
Regional Development Fund (Spain) and by Predoctoral grant (FPU) (to B.B.) from 425
Ministry of Education (Spain). 426
We thank Dr. A.H. Schinkel (The Netherlands Cancer Institute, Amsterdam, The 427
Netherlands) who provided MDCK-II cells and their transduced cell lines, and Abcg2 -/- 428
mice. We are grateful to Prof. James McCue for assistance in language editing. 429
430
431
432
433
434
435
436
437
438
439
440
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and University of Washington Specialized Center of Research Study. Mol. Pharmacol. 564
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566
567
568
569
570
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FIGURE LEGENDS 571
FIG. 1. Chemical structures of triclabendazole (TCBZ) and its metabolites triclabendazole 572
sulfoxide (TCBZSO) and triclabendazole sulfone (TCBZSO2). Molecular weight (M.W.) 573
for each compound is shown. 574
575
FIG. 2. ATPase activity in inside-out plasma membrane vesicles as a function of the 576
compound concentration for ABCG2. The titration curves shown represent the average of 577
two-four measurements; standard deviations are given. Solid lines are fits to the modified 578
Michaelis-Menten equation proposed by Litman et al. (16). 579
580
FIG. 3. Effect of TCBZSO (A) and TCBZSO2 (B) on accumulation of mitoxantrone (10 581
µM) at different concentrations in parent MDCK-II cells and in their murine Abcg2- and 582
human ABCG2-transduced derivatives. Cells were preincubated with or without Ko143 (1 583
µM). Results (units of fluorescence, median) are expressed as means of at least three 584
experiments; error bars indicate S.D. In addition, inhibitory potencies of the different 585
concentrations of the tested compounds for Abcg2 and ABCG2 were represented at the top 586
of each graph. Inhibitory potency was related to the effect of reference inhibitor Ko143 (set 587
at 100 % inhibition of Abcg2/ABCG2). 588
589
FIG. 4. Transepithelial transport of nitrofurantoin (10 µM) in parent MDCK-II (A) and in 590
their murine Abcg2- and human ABCG2-transduced derivatives (D and G) in absence or 591
presence of TCBZSO (15 µM) or TCBZSO2 (15 µM). The experiment was started with the 592
addition of nitrofurantoin to one compartment (basolateral or apical). After 2 and 4 h, the 593
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percentage of drug appearing in the opposite compartment was measured by HPLC and 594
plotted. TCBZSO (B, E and H) and TCBZSO2 (C, F and I) were present as indicated. 595
Results are means; error bars (sometimes smaller than the symbols) indicate S.D. (n = 3). 596
●, translocation from the basolateral to the apical compartment; ○, translocation from the 597
apical to the basolateral compartment. r represents the relative transport ratio (i.e., the 598
apically directed translocation divided by the basolaterally directed translocation) at t = 4 h. 599
600
FIG. 5. Transepithelial transport of danofloxacin (10 µM) in parent MDCK-II (A) and in 601
their murine Abcg2- and human ABCG2-transduced derivatives (D and G) in absence or 602
presence of TCBZSO (15 µM) or TCBZSO2 (15 µM). The experiment was started with the 603
addition of danofloxacin to one compartment (basolateral or apical). After 2 and 4 h, the 604
percentage of drug appearing in the opposite compartment was measured by HPLC and 605
plotted. TCBZSO (B, E and H) and TCBZSO2 (C, F and I) were present as indicated. 606
Results are means; error bars (sometimes smaller than the symbols) indicate S.D. (n = 3). 607
●, translocation from the basolateral to the apical compartment; ○, translocation from the 608
apical to the basolateral compartment. r represents the relative transport ratio (i.e., the 609
apically directed translocation divided by the basolaterally directed translocation) at t = 4 h. 610
611
FIG. 6. In vivo effect of TCBZSO coadministration. (A) Plasma concentration of 612
sulfasalazine and nitrofurantoin in wild-type mice. TCBZSO (50 mg/Kg) or vehicle was 613
administered orally to males 15 min prior to oral administration of sulfasalazine (20 614
mg/Kg). TCBZSO (50 or 100 mg/Kg) or vehicle was administered i.p. to lactating females 615
5 min prior to i.v. administration of nitrofurantoin (5 mg/Kg). (B and C) Milk concentration 616
(B) and milk/plasma ratio (C) of nitrofurantoin in wild-type and Abcg2-/- lactating females. 617
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TCBZSO (50 or 100 mg/Kg) or vehicle was administered i.p. to mice 5 min prior to i.v. 618
administration of nitrofurantoin (5 mg/Kg). Plasma and milk were collected after 30 min of 619
drug administration and analyzed by HPLC. Results are means; error bars indicate S.D. 620
(n=4-7; * p< 0.05 significant differences between control and TCBZSO treatment in wild-621
type mice). 622
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CH3CH3
Triclabendazole(TCBZ) M.W. 359.7
CH3
O
Triclabendazole(TCBZ) M.W. 359.7
CH3
O
Triclabendazole sulfoxide(TCBZSO) M.W. 375.7
CH3
O
Triclabendazole sulfoxide(TCBZSO) M.W. 375.7
CH3
O
OTriclabendazole sulfone(TCBZSO2) M.W. 391.7
OTriclabendazole sulfone(TCBZSO2) M.W. 391.7
FIG. 1.
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TCBZSO2
TCBZSOTCBZ
200
250
y (%
)
0.01 0.1 1 10 100
50
100
150
AT
Pa
se a
ctiv
ity
Concentration (µM)
FIG. 2.
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350
400
e
A
0
20
40
60
80
100
0.01 0.1 1 10 100
[TCBZSO]
Inh
ibit
ory
Po
ten
cy
(%)
0
20
40
60
80
100
0.01 0.1 1 10 100[TCBZSO]
Inh
ibit
ory
Po
ten
cy
(%)
50
100
150
200
250
300
Units
of fluore
scen
ce
0
Contr
ol
Ko 1
µM
0.01
µM
0.05
µM
0.1
µM
1 µM
5 µM
10 µ
M
15 µ
M
25 µ
M
Contr
ol
Ko 1
µM
0.01
µM
0.05
µM
0.1
µM
1 µM
5 µM
10 µ
M
15 µ
M
25 µ
M
Contr
ol
Ko1
µM
0.01
µM
0.05
µM
0.1
µM
1 µM
5 µM
10 µ
M
15 µ
M
25 µ
M
B
Parental Abcg2 ABCG2
80
100
nc
y (%
)
80
100
nc
y (%
)
400
500
600
ore
scen
ce
0
20
40
60
0.01 0.1 1 10 100[TCBZSO2]
Inh
ibit
ory
Po
ten
0
20
40
60
0.01 0.1 1 10 100[TCBZSO2]
Inh
ibit
ory
Po
ten
0
100
200
300
ontr
ol
o 1
µM
01 µ
M
05 µ
M
.1 µ
M
1 µM
5 µM
10 µ
M
15 µ
M
25 µ
M
ontr
ol
o 1
µM
01 µ
M
05 µ
M
.1 µ
M
1 µM
5 µM
10 µ
M
15 µ
M
25 µ
M
ontr
ol
o 1
µM
01 µ
M
05 µ
M
.1 µ
M
1 µM
5 µM
10 µ
M
15 µ
M
25 µ
M
Units
of fluo
Co Ko
0.0
0.0 0. 1 1 2
Co Ko
0.0
0.0 0. 1 1 2
Co Ko
0.0
0.0 0. 1 1 2
Parental Abcg2 ABCG2
FIG 3FIG. 3.
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20
30
40
50
60
ran
spo
rt (
%)
20
30
40
50
60
ran
spo
rt (
%)
20
30
40
50
60
ran
spo
rt (
%)
A B CPARENTAL
r = 0.6 ± 0.0
PARENTAL+TCBZSO2
r = 0.7 ± 0.0
PARENTAL+ TCBZSO
r = 0.7 ± 0.1
0
10
0 2 4
Time (h)
T
60
0
10
0 2 4
Time (h)T
60
0
10
0 2 4
Time (h)
T
60D E F
Abcg2 Abcg2+TCBZSO Abcg2+TCBZSO2
0
10
20
30
40
50
Tra
nsp
ort
(%
)
0
10
20
30
40
50
Tra
nsp
ort
(%
)
0
10
20
30
40
50
Tra
nsp
ort
(%
)
g
r = 8.3 ± 2.8 r = 2.2 ± 0.5r = 1.7 ± 0.1
0 2 4
Time (h)
40
50
60
t (%
)
0 2 4
Time (h)
40
50
60
t (%
)
0 2 4
Time (h)
40
50
60 (
%)
G HI
ABCG2
r = 5.0 ± 1.7
ABCG2+TCBZSO
r = 0.8 ± 0.1
ABCG2+TCBZSO2
r = 0.9 ± 0.1
0
10
20
30
0 2 4
Time (h)
Tra
nsp
ort
0
10
20
30
0 2 4
Time (h)
Tra
nsp
ort
0
10
20
30
0 2 4
Time (h)
Tra
nsp
ort
Time (h) Time (h) Time (h)
FIG. 4.
on April 22, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
20
30
40
50
60
ran
spo
rt (
%)
20
30
40
50
60
ran
spo
rt (
%)
20
30
40
50
60
ran
spo
rt (
%)
A B C
PARENTAL
r = 0.9 ± 0.1
PARENTAL+TCBZSO2
r = 1.0 ± 0.6
PARENTAL+ TCBZSO
r = 1.0 ± 0.1
0
10
0 2 4
Time (h)
Tr
60
0
10
0 2 4
Time (h)T
r
60
0
10
0 2 4
Time (h)
Tr
60D E F
Abcg2 Abcg2+TCBZSO Abcg2+TCBZSO2
0
10
20
30
40
50
Tra
nsp
ort
(%
)
0
10
20
30
40
50
Tra
nsp
ort
(%
)
0
10
20
30
40
50
Tra
nsp
ort
(%
)r = 10.2 ± 1.2 r = 1.4 ± 0.2 r = 1.5 ± 0.6
0
0 2 4
Time (h)
40
50
60
(%)
0
0 2 4
Time (h)
40
50
60
(%)
0
0 2 4
Time (h)
40
50
60
(%)
G H I
ABCG2
r = 3.2 ± 0.6
ABCG2+TCBZSO
r = 1.0 ± 0.2
ABCG2+TCBZSO2
r = 1.2 ± 0.1
0
10
20
30
0 2 4
Ti (h)
Tra
nsp
ort
0
10
20
30
0 2 4
Ti (h)
Tra
nsp
ort
0
10
20
30
0 2 4
Ti (h)
Tra
nsp
ort
Time (h) Time (h) Time (h)
FIG. 5.
on April 22, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
0.7
0.8
0.9
1
(µg
/mL
)
A
*
0.3
0.4
0.5
0.6
asm
a C
on
cen
trat
ion
Vehicle
TCBZSO (50 mg/Kg)
TCBZSO (100 mg/Kg)
0
0.1
0.2Pla
Sulfasalazine Nitrofurantoin
CB
2
2.5
ura
nto
in(µ
g/m
L)
3
3.5
4
4.5
tro
fura
nto
in
1
1.5
nce
ntr
atio
no
f n
itro
fu
1
1.5
2
2.5
k/p
lasm
a ra
tio
of
nit
* * *
*
0
0.5
Milk
con
0
0.5
1
Mil
WT Abcg2 -/-WT Abcg2 -/-
FIG. 6.
on April 22, 2018 by guest
http://aac.asm.org/
Dow
nloaded from