the anthelmintic triclabendazole and its metabolites...

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The anthelmintic triclabendazole and its metabolites inhibit the 2

membrane transporter ABCG2/BCRP 3

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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

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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

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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

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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

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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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methoxy-9-oxo-4-acridine carboxamide (GF120918) as a chemical ATP-binding cassette 552

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Breast cancer resistance protein (Bcrp/abcg2) is a major determinant of sulfasalazine 559

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36. Zhou, L., S. B. Naraharisetti, H. Wang, J. D. Unadkat, M. F. Hebert, and Q. Mao. 561

2008. The breast cancer resistance protein (Bcrp1/Abcg2) limits fetal distribution of 562

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and University of Washington Specialized Center of Research Study. Mol. Pharmacol. 564

73:949-959. 565

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.


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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 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.


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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.


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the anthelmintic triclabendazole and its metabolites...

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