1521-009X/44/12/1872–1880$25.00 http://dx.doi.org/10.1124/dmd.116.071688DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:1872–1880, December 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Development of an Ecofriendly Anticoagulant Rodenticide Basedon the Stereochemistry of Difenacoum

Marlène Damin-Pernik, Bernadette Espana, Stéphane Besse, Isabelle Fourel, Hervé Caruel,Florence Popowycz, Etienne Benoit, and Virginie Lattard

USC 1233 INRA-VetAgro Sup, Veterinary School of Lyon, Marcy l’Etoile, France (M.D.-P., B.E., S.B., I.F., E.B., V.L.); Liphatech,Bonnel, Pont du Casse, France (M.D.-P., H.C.); and Laboratoire de Chimie Organique et Bio-organique, Institut National des

Sciences Appliquées (INSA-Lyon), ICBMS-CNRS-UMR 5246, Villeurbanne Cedex, France (F.P.)

Received May 19, 2016; accepted September 9, 2016

ABSTRACT

Difenacoum, an antivitamin K anticoagulant, has been widely usedas rodenticide to manage populations of rodents. Difenacoumbelongs to the second generation of anticoagulant, and, as all themolecules belonging to the second generation of anticoagulant,difenacoum is often involved in primary poisonings of domesticanimals and secondary poisonings of wildlife by feeding contam-inated rodents. To develop a new and ecofriendly difenacoum, weexplored in this study the differences in properties betweendiastereomers of difenacoum. Indeed, the currently commercialdifenacoum is a mixture of 57% of cis-isomers and 43% of trans-isomers. Cis- and trans-isomers were thus purified on a C18

column, and their respective pharmacokinetic properties and theirefficiency to inhibit the coagulation of rodents were explored.Tissue persistence of trans-isomers was shown to be shorter thanthat of cis-isomers with a half-life fivefold shorter. Efficiency toinhibit the vitamin K epoxide reductase activity involved in thecoagulation process was shown to be similar between cis- andtrans-isomers. The use of trans-isomers of difenacoum allowed todrastically reduce difenacoum residues in liver and other tissues ofrodents when the rodent is moribund. Therefore, secondarypoisonings of wildlife should be decreased by the use of difenacoumlargely enriched in trans-isomers.

Introduction

Anticoagulant rodenticides (ARs) are used worldwide since the1940s to control infestations of rats and mice, which cause importantagricultural and structural damage and are associated with publichealth issues. The death of rodents occurs 3–7 days after consumptionof baits. This delay of action eludes the alimentary aversion problem,which is a very important behavioral trait among rodents. The firstcommercial rodenticide was dicoumarin, replaced a couple of yearslater by warfarin, a more efficient molecule (Hadler and Buckle,1992). These products with coumatetralyl, chlorophacinone, andother molecules developed from 1950s compose the first generationof ARs (FGARs), which require multiple ingestions to cause death ofrodents (Rattner et al., 2014).ARs, derivatives of either 4-hydroxycoumarin or indane-1,3-dione,

are noncompetitive inhibitors of the vitamin K epoxide reductaseenzyme (VKOR)C1 described for the first time in 2004 (Li et al.,2004; Rost et al., 2004). VKORC1 is essential to reduce vitamin K 2,3epoxide to vitamin K hydroquinone. Vitamin K hydroquinone is theessential cofactor for the g-glutamyl carboxylase, which catalyzes thepost-translational modifications of the clotting factors II, VII, IX, and Xand other vitamin K–dependent proteins (Suttie, 1985; Furie and Furie,1988). ARs acting as VKORC1 inhibitors stop the vitamin K–dependentclotting factor activation, impairing the coagulation function, and thuslead to the death of rodents by hemorrhages.

After 10 years using FGARs, the first case of apparent resistance toFGARs was discovered in the United Kingdom in 1958 (Boyle, 1960). Inthe following years, resistant rodents have been described everywhere inEurope (Dodsworth, 1961) and also in the United States (Jackson andKaukeinen, 1972), Canada (Siddiq andBlaine, 1982), Australia (Saunders,1978), and Japan. Since then, two main resistance mechanisms have beendescribed, a metabolic resistance due to an overexpression of CYP450 anda target resistance due to a decrease of the susceptibility of the VKORactivity to ARs. This target resistance was definitively associated withsingle-nucleotide polymorphisms in the Vkorc1 gene in 2004 after thediscovery of this gene (Pelz et al., 2005; Rost et al., 2009). The occurrenceof resistance in all of the major commensal species to all the FGARs led tothe development of a second generation of ARs (SGARs) in the 1970s.SGARs are usually more toxic and more persistent in animal tissues

than FGARs, and they are thus active after a single bait feeding (Rattneret al., 2014). Because of this increased persistence in animal tissues,especially in liver (Langford et al., 2013), the use of SGARs is associatedwith an increased risk of secondary poisoning for predators and scavengersfeeding on contaminated rodents. Evidence of secondary poisoning wasconfirmed in predatory bird species such as barn owl (Tyto alba), buzzard(B. buteo), kestrel (Falco tinnunculus), Red kyte (Milvus milvus), andtawny owl (Strix aluco) (Christensen et al., 2012; Hughes et al., 2013;Geduhn et al., 2015), and in predatory mammals such as red foxes (Vulpesvulpes) (Sage et al., 2010), European mink (Mustela lutreola) (Fournier-Chambrillon et al., 2004), and weasels (Mustela nivalis) (Elmeros et al.,2011). Therefore, ARs were identified by the European Union ascandidates for future comparative risk assessment and substitution in viewof their unacceptable risk of secondary poisoning for wildlife. However, in

This work was supported by Bpi France [Grants ISI n�I1301001W “NEORAMUS”].dx.doi.org/10.1124/dmd.116.071688.

ABBREVIATIONS: AR, anticoagulant rodenticide; AUC, area under the curve; FGAR, first generation of AR; HPLC, high-performance liquidchromatography; PT, prothrombin time; SGAR, second generation of AR; VKOR, vitamin K epoxide reductase enzyme.

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the absence of an alternative, AR molecules were included in Annex 1 ofEuropean Union Biocidal Products Directive 98/8/EC, and their use is stilltolerated until a more appropriate solution is found.Difenacoum (i.e., 2-hydroxy-3-[3-(4-phenylphenyl)-1-tetralinyl]-4-

chromenone), described for the first time in 1975, belongs to SGAR.It is efficient to control warfarin-resistant rodent populations (Hadler andShadbolt, 1975; Hadler et al., 1975; RRAC, 2015) and would be lesstoxic for nontarget species than for target species (Buckle and Smith,2015). This molecule is widely used worldwide (Atterby et al., 2005;Buckle et al., 2013; Hughes et al., 2013). For example, difenacoum wasreported to be used on approximately 45% of agricultural premises inGreat Britain (Atterby et al., 2005; Buckle et al., 2013). It is used tocontrol rodent pests in and around buildings, in waste sites, and in sewers(European Parliament, 2009), but, according to the European countries,not always authorized in open areas because of its associated ecotoxicity.Difenacoum has two asymmetric carbons in its chemical structure (Kellyet al., 1993; Buckle and Smith, 2015), conferring this product to exist intwo diastereomeric forms, (1R,3R)(1S,3S) or (1R,3S)(1S,3R), withpotentially different chemical and physical properties. The difenacoumcurrently available on the market is a mixture of both diastereomersreported to contain from 50 to 80% of one major diastereomer.This study aims to develop a new difenacoum based on the concepts of

stereochemistry. Indeed, diastereomers of difenacoum might have differ-ent tissue persistence. The change of the composition of the difenacoumcurrently available, either by decreasing or by increasing the proportion ofthe major diastereomer, might result in a way to diminish its tissuepersistence and thus its ecotoxicity, while keeping its inhibiting activity.For this, an appropriate analytical method was developed to separatediastereomers of difenacoum. After characterization and assignment ofeach diastereomer of difenacoum by nuclear magnetic resonance,pharmacokinetics studies were performed to evaluate the tissuepersistence of each diastereomer. Thus, comparative evaluation ofefficacy of each diastereomer as inhibitor of the VKOR activity wasperformed in vitro and in vivo to compare their anticoagulant activity.

Materials and Methods

Chemicals. Difenacoum was supplied by Hangzhou Ich Biofarm (Hangzhou,China). The (1R,3R)(1S,3S) isomers of difenacoum (called in the following trans-isomers) and (1R,3S)(1S,3R) isomers of difenacoum (called in the following cis-isomers) were separated and purified by means of flash column chromatography onsilica gel in our laboratory. Brodifacoum was obtained from Sigma-Aldrich (l’Isled’Abeau,Chesnes, France); dimethylsulfoxide, acetonitrile,methanol, acetone, diethylether, and orthophosphoric acid were obtained from VWR International (Fontenaysous bois, France); and Vetflurane and vitamin K1 from Alcyon, (Miribel, France).Vitamin K1 was converted to vitamin K epoxide, according to the method describedby Tishler et al. (1940). Purity was estimated by LC/MS (Liquid chromatography–mass spectrometry) using an analytical standard (Cayman Chemical, Ann Arbor, MI)and was higher than 99%. High-performance liquid chromatography (HPLC) gradewaterwas prepared using amilli-Q plus system (Millipore, Saint-Quentin enYvelines,France) and used for preparation of HPLC eluents.

Animals. Experimental research on animals was performed according to anexperimental protocol following international guidelines and with approval fromthe ethics committee of the Veterinary School of Lyon.

Eight-week-old male OFA-Sprague Dawley rats (each weighing 175–200 g)were obtained from a commercial breeder (Charles Rivers, l’Arbresle, France) andwere acclimated for a minimum period of 5 days. The rats were housed four percage under a constant photoperiod and ambient temperature. The animals werekept in standard cages (Eurostandard, type IV; Tecniplast, Limonest, France) andreceived standard feed (Scientific Animal Food and Engineering; reference A04,SAFE, Augy, France) and water ad libitum.

Male OFA-Sprague Dawley rats received through peros administration eithercommercial difenacoum (i.e., corresponding to a mixture of 57% of cis-isomers and43%of trans-isomers) or silica gel column–purified cis-isomers or silica gel column–purified trans-isomers. Anticoagulantswere dissolved in 10%dimethylsulfoxide and

90% vegetable oil and were administered by force feeding. Rats were maintained inlife by daily s.c. administration of vitaminK1 (5mg/kg). Next, rats were anesthetizedwith isoflurane, and blood was taken by cardiac puncture into citrated tubes. Finally,rats were euthanized with CO2, and organs of each rat were immediately collectedand stored at 220�C until analysis.

Prothrombin Time Determination. After blood collection into citrated tubes,blood sample was centrifuged immediately at 3000 rpm for 10 minutes. Theprothrombin time (PT; in seconds) was assessed immediately in duplicate using aBiomerieux Option 2 Plus (Behnk Electronick, Norderstedt, Germany) with theNeoplastin CI, INR Determination kit (Diagnostica Stago, Asniere, France). ThePT was determined in 100 ml samples of plasma with thromboplastin, accordingto the manufacturer’s instructions. PT was determined as the clotting time of acitrated plasma sample to which thromboplastin had been added. Each value wasdetermined as the mean of two measurements.

Extraction of Difenacoum in Plasma. An aliquot of 1 ml sample supple-mented with 1.9 mM internal standard (i.e., brodifacoum) was placed in a tubewith 8 ml diethyl ether. The extract was mixed and centrifuged at 3000 rpm for5 minutes. The diethyl ether layer was evaporated to dryness under a gentlenitrogen flow at ambient temperature. The final purified sample was dissolved in200 ml methanol, and difenacoum was analyzed by HPLC, as described below.

Extraction of Difenacoum in Tissues. The samples (1 g tissue) wereextracted with 40 ml acetone using an Ultra Turrax tissue disperser (IKALabortechnick, VWR International, Strasbourg, France) for 1minute after additionof 1.9 mM internal standard (i.e., brodifacoum). The extract was centrifuged at3000 rpm for 5 minutes. The supernatant was transferred in a flask and evaporatedat 50�C with a rotary evaporator. The flask was rinsed three times with 1 mlmethanol, which were put together and evaporated to dryness under a gentlenitrogen flow. The final purified sample was dissolved in 200 ml methanol, anddifenacoum was analyzed by HPLC, as described below.

Difenacoum Analysis by HPLC. Analysis of cis- and trans-difenacoum wasdetermined by HPLC. Brodifacoum was used as internal standard. The HPLCsystem (JASCOLC-Net II/ADC) consists of a PU2089 JASCOpump, anAS2051sampler, a UV2075 detector (258 nm), and a LACHROM start software (VWRInternational, Strasbourg, France). A total of 50 ml extracted sample or standardsolution was injected in a LIChrospher 100RP18 encapped column (5mmparticlesize, 250 � 4.6 mm; VWR International, Strasbourg, France). A gradientelution system was used with a flow rate of 1 ml/min as follows: from 60%acetonitrile/40% water acidified with 0.2% H3PO4 to 80% acetonitrile/20% wateracidified with 0.2% H3PO4 at 5 minutes.

The method was fully validated according to the guideline on bioanalyticalmethod validation published by the European Medicines Agency (http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_ guideline/2011/08/WC500109686.pdf) with respect to specificity, carryover, limit of detection (i.e.,65, 119, 56, 228, 80, 64, and 34 ng/g for liver, small intestine, kidney, feces, colon,plasma, and urine, respectively), limit of quantification (i.e., 162, 298, 140, 570,201, 161, and 107 ng/g for liver, small intestine, kidney, feces, colon, plasma, andurine, respectively), calibration curve, accuracy, precision, dilution integrity,matrix effect, and stability. The recovery rate of diastereomers of difenacoumfrom tissues was between 74 and 90% in liver and 80–100% in plasma, with aprecision below 15%.

Preparation of Liver Microsomes. Liver microsomes were prepared fromfresh livers by differential centrifugation. Briefly, livers were resuspended in 50mMphosphate buffer (pH 7.4) containing 1.15% (w/v) of KCl. Liver cells werehomogenized in buffer by using amotor-driven Potter glass homogenizer and furthersubmitted to differential centrifugation at 4�C. The 100,000g pellet corresponding tothe membrane fraction was resuspended by Potter homogenization in HEPESglycerol buffer (50mMHEPES, 20% glycerol, pH 7.4). Protein concentrations wereevaluated by the method of Bradford using bovine serum albumin as a standard.Microsomes were frozen at 280�C and used for kinetic analysis.

VKOR Activity Assay and Kinetics. Microsomal VKOR activity wasassayed according to the protocol described by Hodroge et al. (2011, 2012). Theinhibiting effect of the silica gel column–purified cis- or trans-isomers ofdifenacoum was evaluated by the determination of Ki. Ki was determined afteraddition of various concentrations of the anticoagulant to the standard reaction inthe presence of increasing amounts of vitamin K epoxide (from 0.001 to 0.2 mM),using anticoagulant concentrations from about 0.05 to 20 � Ki (Ki of commercialdifenacoum is 0.03 mM) (Hodroge et al., 2011). The Ki values were obtained fromat least three separate experiments performed on two different batches of proteins.

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Analysis of Data. Pharmacokinetic calculations were performed usingthe noncompartmental approach on the mean results per group. The cis-difenacoum and total difenacoum, that is, cis-isomer plus trans-isomer,concentrations used to calculate the elimination rate constant and half-lifewere selected from the 12- to 336-hour time points. For trans-difenacoum, theconcentrations used to calculate the elimination rate constant and half life wereselected from 12 to 72 h, because concentrations 72 h and more afteradministration were too weak to be accurately quantified (,0.25 mg/kg).The half-life of elimination (t1/2(el)) was calculated using a linear regressionlinear in GraphPad Prism 6 (La Jolla, CA). The total area under the curve(AUC) was calculated using the linear trapezoidal method and adding theestimated terminal portion of the curve (AUC 0→‘). The three models weredrawn using Berkeley Madonna Software.

Statistical analysis of pharmacokinetic parameters and PT was done usingGraphPad Prism 6 software. A Mann–Whitney test was used with a , 0.01 tocompare statistically the results between the two groups.

For kinetic analysis of the VKOR activity, data were fitted by nonlinearregression to the noncompetitive or competitive inhibition model using GraphPadPrism 6. The choice of the best model was based on the Corrected AkaikeInformation Criterion.

Results

Separation of Diastereomers of Difenacoum and Assignment ofthe Stereochemistry. When pure difenacoum was analyzed by HPLCon a C18 reversed-phase column, as described in the experimentalprocedure, two different peaks were obtained with respective retentiontime of 11.2 6 0.1 minutes and 12.1 6 0.1 minutes (Fig. 1A). Bothpeaks presented the same absorption spectrum with a maximalabsorbance at 258 nm. Both peaks were thus identified as difenacoumexisting in two diastereomeric forms, cis and trans (Fig. 2A).

Fig. 1. Chromatograms of cis- and trans-isomers of difena-coum, in HPLC on a C18 reversed-phase column. Chromato-gram (A) corresponds to the commercial difenacoum at 10 mMwith cis-isomers at 11.2 minutes and trans-isomers at 12.1 min-utes. Chromatograms (B) and (C) are residues on the liver aftera single oral administration of 5.2 mg/kg21 difenacoum (ratio57/43) in warfarin-susceptible rats at 24 and 120 hours,respectively.

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To assign the stereochemistry of diastereomers, they were purified onsilica column and analyzed by NMR. Assignment of the stereochemistrywas validated in accordance with NMR experiments already reported inthe literature for H1 proton of decalin in C6D6 (Fig. 2B) (Van Heerdenet al., 1997a,b). Molecules eluted at 12.1 minutes corresponded to trans-isomers of difenacoum with d 4.85 ppm (dd, J = 6 Hz and J = 2 Hz).Molecules eluted at 11.2 minutes corresponded to cis-isomers ofdifenacoum with d 4.96 ppm (broad singlet). In CDCl3, the resultsprovided slightly different signals at 300 MHz: trans-isomers, d 4.56ppm (t, J = 7.0 Hz); cis-isomers, d 4.84 ppm (dd, J = 6.0 and J =11.3 Hz), but also confirmed the assignment described in this solvent(Fig. 2C) (Kelly et al., 1993).Comparative Analysis of the Persistence of Trans-isomers and

Cis-isomers of Difenacoum in Liver. To study the hepatic and plasmapersistence of trans- and cis-isomers of difenacoum, 42 male OFA-Sprague Dawley rats received by peros administration 5.2mg/kg21 (i.e.,8 � ED50 of difenacoum determined for Rattus norvegicus) ofcommercial difenacoum corresponding to a mixture of 57% of cis-isomers of difenacoum and 43% of trans-isomers of difenacoum. Ratswere killed 1, 2, 4, 6, 8, 10, 12, 24, 72, 120, 168, 240, 336, and 504 hoursafter administration of difenacoum and liver concentrations of cis-isomers (Fig. 3, B and C) and trans-isomers (Fig. 3, B and C), but alsototal difenacoum (by adding cis- and trans-isomers) was determined foreach rat. Representative UV chromatograms of the analysis ofdifenacoum at different time points of the pharmacokinetics (i.e., 24 hand 120 h after the administration of 5.2 mg/kg21 commercial

difenacoum) are shown in Fig. 1, B and C, with elution of cis-isomers at11.2 minutes and trans-isomer at 12.1 minutes. At 120 hours after theadministration, trans-isomers disappeared. Moreover, production of twomajor metabolites was detected with retention time of 6.8 and7.3 minutes, respectively, and a UV spectrum similar to that obtainedfor difenacoum, proportion of these metabolites varying during thepharmacokinetics.The pharmacokinetic profile of total difenacoum liver concentra-

tion (calculated by addition of concentrations of cis- and trans-isomers) is presented in Fig. 3A. It increased rapidly in liver, reached amaximum (Cmax) in 6 hours after the administration, and thendecreased. Pharmacokinetic parameters of total difenacoum in liverwere calculated using a noncompartmental approach and are pre-sented in Table 1. The pharmacokinetic profile of cis- and trans-isomers in liver is presented in Fig. 3B. Cmax were reached 6 hoursafter the administration for both isomers. When Cmax were reached,ratio between cis- and trans-isomers was similar to that of thecommercial difenacoum (i.e., mix of 57% cis- and 43% trans-isomers) (Fig. 3C). Trans-isomers of difenacoum were more rapidlyeliminated than cis-isomers. AUC of trans-isomers was sevenfoldsmaller than that of cis-isomers. Half-lives of cis-isomers and trans-isomers in liver calculated were about 83 hours and 14 hours,respectively. The cis-isomers represented 76% of the total amountof difenacoum present in liver 24 hours after the administration of thecommercial difenacoum (i.e., mix of 57% cis- and 43% trans-isomers)and 99%, 168 hours after the administration.

Fig. 2. Chemical structure of trans-isomers and cis-isomers of difena-coum (A) and the assignment of the stereochemistry with NMR experimentsusing H19 proton of decalin in C6D6 (B) or in CDCl3 (C). The

1H NMRspectra in C6D6 (B) or in CDCl3 (C) of molecules eluted at 10.9 minutes (onthe right) and at 11.7 minutes (on the left) are presented plotted as signalintensity (vertical axis) versus chemical shift (in ppm on the horizontal axis).

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To avoid competitive effects between cis- and trans-isomers, phar-macokinetic studies were repeated with silica gel column–purified trans-isomers or silica gel column–purified cis-isomers. Male OFA-SpragueDawley rats received by peros administration 3.0 mg/kg21 trans-isomersor cis-isomers. The pharmacokinetic profile of cis-isomer concentrationand trans-isomers in liver after administration of purified isomers ispresented in Fig. 4. Trans-isomers of difenacoum were still more rapidlyeliminated than cis-isomers. Half-lives of cis-isomers and trans-isomers

in liver were calculated using a linear regression model and were similarto those calculated after administration of a commercial mixture ofdifenacoum (Table 1).Comparative Analysis of the Persistence of Trans-isomers and

Cis-isomers of Difenacoum in Rat Tissues. A comparative analysis ofpersistence of isomers was performed in different tissues of rat toconfirm that the liver is the major organ of storage for both isomers andto evaluate the pharmacokinetics of cis- or trans-isomers in extrahepatic

Fig. 3. Liver concentration time profiles of total difenacoum(A) and cis- or trans-isomers (B) after a single oral adminis-tration of 5.2 mg/kg21 difenacoum (ratio 57/43) in warfarin-susceptible rats. The results of each time are the mean 6 S.D.of three rats per time. The total difenacoum is the sum ofconcentration of cis- and trans-isomers of difenacoum at eachtime. Modellings of the experimental points corresponding tothe cis-isomers (modeling cis-isomers), trans-isomers (mod-eling trans-isomers), and total difenacoum (modeling totaldifenacoum) were obtained using a mono-compartmentallymodel (with the software Berkeley Madonna). The differenttimes are 1, 2, 4, 6, 8, 10, 12, 24, 72, 120, 168, 240, 336, and504 hours. Ratio of cis- and trans-isomers of difenacoum isshown in (C).

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tissues. For that, 3 days after the administration of 3.0 mg/kg21 cis- ortrans-isomers, concentration of cis- and trans-isomers was determined inliver, but also in plasma, feces, intestine, kidney, mesenteric fat, andurine of rats. Results are presented in Fig. 5. Concentrations ofdifenacoum after administration of cis-isomers were systematicallysimilar or higher than those obtained after administration of trans-isomers in all of the tissues investigated in this study. After adminis-tration of trans-isomers, difenacoum was detected almost only in liver,whereas when cis-isomers were administered, difenacoum was found inliver, plasma, intestine, and kidney. Nevertheless, whatever the isomersused, liver was the major tissue of storage.Analysis of the In Vitro Inhibiting Effect of Trans-isomers and

Cis-isomers of Difenacoum on the Vitamin K Epoxide ReductaseActivity. The ability to inhibit the VKOR activity catalyzed bysusceptible rat liver microsomes was evaluated for cis- and trans-isomer by determination of inhibition constants (Ki). The plots of thevelocity of the VKOR activity catalyzed by rat liver microsomes, versusthe substrate concentration in the presence of different concentrations oftrans-isomers, are presented in Fig. 6. Trans-isomers of difenacoumwereable to inhibit VKOR activity. Apparent Km was not modified by theaddition of trans-isomers. On the contrary, apparent Vmax was loweredby the use of trans-isomers. This result revealed that trans-isomersinhibited VKOR activity catalyzed by rat liver microsomes in anoncompetitive manner, as previously reported for commercial difena-coum corresponding to a mixture of cis- and trans-isomers (57%/43%)(Hodroge et al., 2011). Data were fitted to theMichaelis–Menten model,which takes into account the presence of competitive, noncompetitive, oruncompetitive inhibitor by nonlinear regression. The best fit, the higherR2, and the lower corrected Akaike information criterion were obtainedwhen the model that takes into account a noncompetitive inhibitor was

used. Finally, Ki toward trans-isomers for rat liver microsomes was20.96 1.56 nM. Same experiment was performedwith cis-isomers. Cis-isomers were also able to inhibit VKOR activity catalyzed by rat livermicrosomes in a noncompetitive manner with Ki similar to that obtainedwith trans-isomers (17.4 6 7.9 nM).Analysis of the Ability of Trans-isomers and Cis-isomers of

Difenacoum to Inhibit In Vivo Blood Coagulation. The ability of cis-isomers or trans-isomers of difenacoum to inhibit the blood coagulationwas determined by their respective ability to increase in vivo the PT. Thenormal PT was determined to be 17.9 6 2.5 seconds in male OFA-Sprague Dawley rats. Effect of cis- or trans-isomers on PT wasmonitored 1, 3, and 5 days after peros administration to male OFA-Sprague Dawley rats of 4 ED50 (i.e., 2.6 mg/kg21 for Rattus norvegicus)of difenacoum containing only cis-isomers or only trans-isomers. ED50

is the dose that creates an increase of PT in the international normalizedratio higher than 50% of the tested population. Results corresponding tothe mean of four rats are presented in Fig. 7A. Oral administration of2.6 mg/kg21 trans-isomers of difenacoum led to significant increase ofthe PT for 24 hours. Three days later, it was almost normal again. On thecontrary, oral administration of 2.6 mg/kg21 cis-isomers of difenacoumresulted in a considerable increase in PT for at least 5 days.To maintain the effect of trans-isomers for at least 3 days, the dose was

increased to 3.9 mg/kg21 (corresponding to 6 ED50). This dose wasadministered as a single dose on day 0 or in three successive doses of1.3 mg/kg21/day on days 0, 1, and 2. The PT was thus evaluated on day 4.Results are presented in Fig. 7B. Administration of 3.9 mg/kg21 trans-isomers as a single dose did not lead to significant increase of the PT 3 days

TABLE 1

Pharmacokinetics parameters of difenacoum in liver after a single oral administration of difenacoum (ratio 57/43 cis-/trans-isomers) at 5.2 mg/kg21 or after administration ofcis- or trans-isomers purified in silica gel column at 3 mg/kg21

Peros Administration of Molecules AUC (mg.h/g) Cmax (mg/g) t1/2 (h) (95% CI) Ke (95% CI)

Cis- + trans-isomers 969.6 6 232.7 14.35 6 2.71 75.8 (66.5–88.3) 0,0091 (0.0078–0.0104)5.2 mg/kg21 commercial difenacoum

(57% cis/43% trans)Cis-isomers 872.1 6 181.2* 8.82 6 1.71 97.2* ( 84.9–113.8) 0.0071* (0.0061–0.0082)

Trans-isomers 118.3 6 24.7* 5.96 6 0.77 15.6* ( 14.0–17.6) 0.0444* (0.0393–0.0495)3 mg/kg21 cis-isomers Cis-isomers 709.4 6 87.7* 9.05 6 2.83* 70.6* ( 60.6–84.4) 0.0098* (0.0082–0.0114)3 mg/kg21 trans-isomers Trans-isomers 236.4 6 87.7* 3.43 6 1.14* 24.2* (14.7–68.6) 0.0287* (0.0101–0.0472)

*Denotes a significant difference between two groups (cis- or trans-isomers in a concomitant administration or cis- or trans-isomers in isolated administration) with a , 0.01 by using a Mann–Whitney test.

Fig. 4. Time-dependent concentrations of cis- and trans-isomers of difenacoum afterperos administration of, respectively, 3 mg/kg21 silica gel column–purified cis- ortrans-isomers of difenacoum to warfarin-susceptible rats. Results are the mean 6S.D. of four rats. The different times are 24, 72, 168, 336, and 504 hours.

Fig. 5. Concentrations of difenacoum in different tissues 3 days after a single oraladministration of 3 mg/kg21 silica gel column purified of cis-isomers or trans-isomers of difenacoum in warfarin-susceptible rats. Results are the mean 6 S.D. offour rats. *Denotes a significant difference between cis- and trans-isomeradministration with a , 0.01 by using a Mann–Whitney test.

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later. On the contrary, the administration of the same total dose of trans-isomers (3.9 mg/kg21) three times (1.3 mg/kg21/day during 3 days) led todrastic increase of the PT associated with a major anticoagulant effect.

Discussion

Development of SGAR able to kill rodents after a unique administra-tion was achieved by increasing the tissue persistence of AR enabling theinhibition of VKOR activity and thus blood clotting for several weeksinstead of several hours for FGAR. However, this increased persistenceled to molecules with excessive half-lives, whereas they kill rodents inonly 3–7 days. For example, half-life of brodifacoum has been reportedas being 307 days in mice (Vandenbroucke et al., 2008). Although thesemolecules have been used without any report of cases of secondarypoisoning for more than 30 years after the beginning of their marketing(Kaukeinen, 1982; Vandenbroucke et al., 2008), the ecological impact ofthese molecules on wildlife is now obvious. In spite of this obvious

ecological impact, there has been no improvement of these ARmoleculessince the patent of difethialone in 1986.A new generation of ARs is nowadays required to overcome the

ecotoxicity associated with the SGARs. In this study, we explored a wayto develop an ecofriendly anticoagulant rodenticide able to efficientlyinhibit VKOR activity and less persistent into animal tissues to avoidsecondary poisoning. The explored solution is an improvement of theexisting difenacoum based on the concept of the stereochemistry. Indeed,many studies report differences in the pharmacological, toxicological, orpharmacokinetic properties between stereoisomers of the same molecule(Nguyen et al., 2006; Cort et al., 2012; Chhabra et al., 2013).The pharmacokinetic studies performed in this work showed that cis-

and trans-isomers of difenacoum are similarly absorbed after oraladministration. Indeed, after administration of a mixture of 57% ofcis- and 43% of trans-isomers, Cmax for cis- and trans-isomers arereached at the same time, and proportions of cis- and trans-isomers in theliver at Tmax remain unchanged comparedwith the initial proportions of the

Fig. 6. Plots of vitamin K epoxide reductase activity versusvitamin K epoxide (from 1.25 to 200 mM), in the presenceof 0, 10, 20, and 50 nM trans-isomers of difenacoumincubated with microsomes from warfarin-susceptible rats.Each data point represents the mean 6 S.D. of twodeterminations. The experimental results were fitted bynonlinear regression using the noncompetitive inhibitionmodel.

Fig. 7. PT after single or multiple oral administrations of cis-or trans-isomers of difenacoum in warfarin-susceptible rats. In(A), a comparison of PT between cis- and trans-isomers wasperformed. For this, 2.6 mg/kg21 cis- or trans-isomers wasadministered at day 0, and PT was evaluated 1, 3, or 5 daysafter the oral administration. In (B), a comparison of PT after asingle or a multiple oral administration of trans-isomers ofdifenacoum was performed. For this, 3.9 mg/kg21 trans-isomers was administered in a single oral administration at day0 or in three oral administrations (1.3 mg per day) at days 0, 1,and 2, and PT was evaluated at day 3. Each point is the mean6S.D. of four rats. *Denotes a significant between two groupswith a , 0.01 by using a Mann–Whitney test. Value .500 seconds = value out of range.

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administered mixture. On the other side, these studies demonstrate a cleardifference in hepatic half-life between isomers with a sixfold shorter half-life for the trans-isomers compared with the cis-isomers. This difference inhepatic half-life results in an almost absence of trans-residues in liver 72 hafter a peros administration of 5.2 mg/kg21 current difenacoum.This difference in half-life does not result from a difference in storage

between diastereoisomers. Indeed, the determination of concentrationsof residues of difenacoum 72 hours after the administration of the currentdifenacoum did not show a preferential localization of trans-isomers inan extrahepatic tissue. In all of the tissues analyzed, the concentrations oftrans-isomers were systematically lower than those of cis-isomers. Thisdifference in half-life is not due to a chiral inversion of trans-isomers incis-isomers. Indeed, when only trans-isomers are administered to rats, noappearance of cis-isomers is detected in any tissue. This differenceseems to result from a difference in elimination with an elimination rateconstant of trans-isomers (i.e., calculated ke of 0.044 hour21) sixfoldgreater than that of cis-isomers (i.e., calculated ke of 0.0071 hour21).The accelerated elimination of trans-isomers could be due to an

accelerated metabolism of the trans-isomers compared with cis-isomers.Indeed, production of two metabolites with a UV spectrum similar todifenacoum was observed after administration of difenacoum. Becauseanticoagulants have been described to be oxidized by CYP450 (Moreauet al., 2011), these metabolites could result from the oxidation of cis- andtrans-isomers. These metabolites are then either excreted as hydroxymetabolites or could undergo conjugation into sulfates or glucuronides,as reported for warfarin in humans (Jones and Miller, 2011). Theabsence of other detected metabolites during the pharmacokinetic studysuggests that both metabolites are excreted as unchanged. Nevertheless,both metabolites seem to persist in tissues 120 hours after administrationeven if the proportion of one of the two metabolites clearly decreased.This persistence could be problematic if these metabolites retainedanticoagulant activity. However, 72 hours after administration of2.6 mg/kg21 trans-difenacoum, PT becomes normal, which suggeststhat metabolites produced from trans-isomers are no longer active. Tobetter understand the role of metabolism in the different eliminationobserved between cis- and trans-isomers, complete metabolic studieswill be necessary.Because of the slow elimination rate of cis-isomers, the ecotoxicity

associated with the difenacoum currently commercially available is dueto its composition rich in cis-isomers. Indeed, it is when rodent is dyingthat it becomes an easy prey for predators. During difenacoumpoisoning, rodent is weakened as it presents a hemorrhagic syndrome,that is, 3–7 days after consumption of difenacoum. At this moment,rodent tissues and especially rodent liver contain only cis-isomers, trans-isomers having been eliminated. Consequently, the use of difenacoumcontaining only the trans-isomers would greatly reduce the tissuepersistence of difenacoum and therefore the ecotoxicity associated withthis product. Indeed, 3 and 7 days after consumption of 3 mg/kg21 trans-isomers, there are 3.5- and 4.5-fold less residue in the liver than whenrodent consumes 3 mg/kg21 cis-isomers. Moreover, this decrease ofpersistence would facilitate the management of primary intoxication indomestic animals if the pharmacokinetic properties of cis- and trans-isomers are identical to those observed in rats. Currently, to treat dogs orcats poisoned with difenacoum currently commercially available, a dailyinjection of vitaminK1 for 2–5weeks is required (Pouliquen, 2001). Thedecrease of persistence of difenacoum would certainly reduce theduration of treatment, and therefore the cost associated with a primarypoisoning with difenacoum.To develop the trans-isomers of difenacoum as rodenticide, it must

be an inhibitor of the VKOR activity at least as effective as thecurrent difenacoum and inhibit the hepatic VKOR activity for at least3 consecutive days to deplete the pool of activated clotting factors. This is

why the ability of the trans-isomers of difenacoum to inhibit the VKORactivity was assessed in vitro and in vivo. The inhibition constant (i.e.,20.9 nM) obtained for trans-isomers of difenacoum using rat livermicrosomes was similar to that of cis-isomers or difenacoum currentlyavailable. This inhibition constant was even lower than those obtainedunder the same conditions for the other SGARs (70 nM, 30 nM, and40 nM for bromadiolone, brodifacoum, and difethialone, respectively)(Hodroge et al., 2011). Even if the inhibition constant obtained for trans-isomers is compatible with the use of trans-isomers of difenacoum asrodenticide, a single administration of 2.6 mg/kg21 trans-isomers (whichwould correspond to an ingestion of 20 g baits, the normal amountgenerally ingested by a 200 g–weighted rat, dosed at 25 ppm) failedin vivo in this study to maintain a prolonged inhibition of the VKORactivity. This is due to the low persistence of trans-isomers leading toliver concentration too low 3 days after the administration. Nevertheless,rodents consume usually several times the same bait when the bait isgiven in sufficient quantity. In this way, the inhibition of the VKORactivity by trans-isomers persists several days and enables to cause deathof rodents.Trans-isomers of difenacoum could thus be considered as an

alternative to the use of SGAR for avoiding secondary poisoningproblems. If a single-feeding use is necessary for such products, adding asmall proportion of cis-isomers would certainly maintain a prolongedinhibition of the VKOR activity to lead to animal death whilemaintaining a limited quantity of residues. Further studies will be neededto optimize the most favorable proportions between trans- and cis-isomers to develop a product with a good balance between efficiencyand tissue persistence.

Authorship ContributionsParticipated in research design: Besse, Benoit, Lattard.Conducted experiments: Damin-Pernik, Espana, Besse, Fourel, Benoit,

Lattard.Contributed new reagents or analytic tools: Espana, Besse, Caruel,

Popowycz.Performed data analysis: Damin-Pernik, Espana, Benoit, Lattard.Wrote or contributed to the writing of the manuscript: Damin-Pernik, Fourel,

Popowycz, Benoit, Lattard.

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